Fusarium Head Blight Outbreak Frequency Under a Changing Climate Environment

Willingthon Pavan

In the hard winter wheat region of the Great Plains, Fusarium head blight (FHB) is a sporadic disease. Drought is more common than rain at flowering, however, within the region, rain at and after flowering often occurs in some parts of the region leading to severe FHB in localized areas. Hence the need to identify lines with FHB tolerance is needed, but most plant breeding programs have insufficient plant pathology support and financial support to actively pursue FHB tolerance. The Regional Germplasm Observation Nursery (RGON) is a USDA coordinated nursery that screens early generation experimental lines for a number of diseases, insects, and abiotic stresses. Every public and private wheat breeding effort in the Great Plains submits between 10 and 40 lines for this collaborative screening effort. The USDA-University of Nebraska wheat improvement effort, with support of US Scab Initiative efforts, proposes testing this nursery for FHB tolerance. The advantages of screening this nurs...

Fusarium head blight (FHB), also known as head scab, is a devastating fungal disease that affects small grain cereal crops such as wheat (Triticum aestivum L.). The predominant causal agent, Fusarium graminearum Schwabe (teleomorph: Gibberella zeae (Schwein.) Petch), is ranked the fourth most important fungal plant pathogen worldwide. Apart from yield and quality losses, mycotoxin production can occur from FHB infection, resulting in harmful effects on human and animal health. Some level of disease control may be achieved by using certain fungicides and agronomic practices plus host resistance. In South Africa, there are currently no registered fungicides or bio-fungicides, no resistant wheat cultivars and only limited control is achieved by cultural practices. Because effective disease reduction cannot be achieved by using a single strategy, the integration of multiple management strategies can enhance disease control. We review possible strategies for reducing the risk for FHB inf...

Wheat and Barley is the most production and consumption grains in the world. The necrotrophic Fusarium spp is pathogen caused many diseases on plants, the major two disease caused by Fusarium on wheat is Fusarium Crown rot (FCR) and head blight (FHB), also known as scab. These both disease caused severe damage on yield quality and quantity, the produced seed seem to be small, shriveled pale white appearance with low contain of protein, that reflected on reduce yield up to 61%. Mycotoxins contamination in the grain, will lead to contamination the products that manufactured from these crops, as well as the contamination yield will be lower price than the healthy yield, which leads to a significant loss to the producer. The major FCR and FHB mycotoxins as DON, NIV, ZEN can caused many disease symptoms to consumers who consumes contaminate food, whether is human or animal, such as cancers, immunosuppression, fertilization problem, abortion and etc.

Proceedings of the 2007 National Fusarium Head Blight Forum he Westin Crown Center • Kansas City, Missouri 2-4 December, 2007 Proceedings compiled and edited by: Susan Canty, Anthony Clark, Donna Ellis and David Van Sanford Proceedings Cover designed by: Kris Versdahl of Kris Versdahl & Associates Red Lake Falls, MN University of Kentucky ©Copyright 2007 by individual authors. All rights reserved. No part of this publication may be reproduced without prior permission from the applicable author(s). Printed in the United States by ASAP Printing, Inc., Okemos, MI 48864 Copies of this publication can be viewed at http://www.scabusa.org. REFERENCING ITEMS IN THE FORUM PROCEEDINGS When referencing abstracts or papers included in these proceedings, we recommend using the following format: Last Name and Initial(s) of Author, [followed by last names and initials of other authors, if any]. Year of Publication. Title of paper. In: Description of proceedings and Title of Conference; Year Month and Days of Conference; Location of Conference. Place of Publication: Publisher. Page Numbers. Sample Reference: Buerstmayr, H., Steiner, B., Hart, L., Griesser, M., Angerer, N., Lengauer, D. and Lemmens, M. 2002. “Molecular Mapping of QTLs for Fusarium Head Blight Resistance in Spring Wheat.” In: Canty, S.M., Lewis, J., Siler, L. and Ward, R.W (Eds.), Proceedings of the National Fusarium Head Blight Forum; 2002 Dec 7-9; Erlanger, KY. East Lansing: Michigan State University. pp. 22-25. Proceedings of the 2007 National Fusarium Head Blight Forum The Westin Crown Center Kansas City, Missouri 2-4 December, 2007 Dedication The 2007 National Fusarium Head Blight Forum and these Proceedings are dedicated to the memory of Thomas E. Anderson, who served as Co-Chair of the U. S. Wheat and Barley Scab Initiative from 1997 until his death in July 2007. His passion for research led Tom to become an activist in the arena of agricultural research, and he served on many commodity and research boards during the last twenty years of his life. As Co-Chair of the Initiative, Tom’s steady hand, great sense of humor and penchant for asking hard questions earned him the respect, friendship and admiration of the entire USWBSI community. The tenth annual National FHB Forum coincides with the implementation of the USWBSI Action Plan, and a rededication of our efforts to develop and implement “control measures that minimize the threat of Fusarium Head Blight (Scab) to the producers, processors, and consumers of wheat and barley”. As we undertake this huge task it is appropriate to remember and draw inspiration from the example of our colleague, friend and leader, Tom Anderson. FORUM ORGANIZING COMMITTEE Co-Chairs: Mike Davis, American Malting Barley Association, Milwaukee, WI, USA Jane DeMarchi, North American Millers’ Association, Washington, DC, USA Members: Gary Bergstrom, Cornell University, Ithaca, NY, USA Gina Brown-Guedira, USDA-ARS, Raleigh, NC, USA Blake Cooper, Busch Agricutural Resources, Inc., Fort Collins, CO, USA Marty Draper, USDA-CSREES, Washington, DC, USA Mohamed Mergoum, North Dakota State University, Fargo, ND, USA Jim Pestka, Michigan State University, East Lansing, MI, USA Frances Trail, Michigan State University, East Lansing, MI, USA Dave Van Sanford, University of Kentucky, Lexington, KY, USA ORGANIZED AND HOSTED BY: We would like to acknowledge our sponsors and partners for their generous contributions to the 2007 National Fusarium Head Blight Forum. American Malting Barley Association Anheuser-Busch Companies BASF Bayer CropScience Boulevard Brewing Company Michigan State University Miller Brewing Company Sierra Nevada Brewing Company U.S. Department of Agriculture-Agricultural Research Service (USDA-ARS) University of Kentucky WestBred, L.L.C. TABLE OF CONTENTS KEYNOTE PRESENTATION Fusarium Head Blight Outbreak Frequency under a Changing Climate Environment. J.M. Fernandes, W. Pavan and E.M. Del Ponte ................................................................................ 3 SESSION 1: FOOD SAFETY, TOXICOLOGY AND UTILIZATION OF MYCOTOXIN-CONTAMINATED GRAIN Quantification of the Tri5 gene, Expression and Deoxynivalenol Production during the Malting of Barley. Anuradha Boddeda, Paul Schwarz and Charlene E. Wolf-Hall .............................Poster #1 ........... 9 High-Throughput Homogenization of Grain Samples for DON Testing. D.M. Reaver and D.G. Schmale III .......................................................................Poster #2 ......... 10 Analysis of Deoxynivalenol, Masked Deoxynivalenol and Fusarium graminearum Pigment in Grain Cultures using a New LC-UV/MS Method. Sasanya, J.J, Hall, C. and Wolf-Hall, C. ...............................................................Poster #3 ......... 11 Production and Purification of Deoxynivalenol from Rice Culture and Analysis using Liquid Chromatography-Ultraviolet-Mass Spectrometry. Sasanya, J.J, Hall, C and Wolf-Hall, C. ................................................................Poster #4 ......... 12 Deoxynivalenol Measurement: Sources of Error and Sampling Recommendations. Paul Schwarz, Yanhong Dong and Ruth Dill-Macky ....................................... Invited Talk ......... 13 Development of a Multiplex Real-Time PCR Assay for Rapid Detection and Quantification of Fusarium spp. in Barley. D.J. Tobias, A. Vashisht, A. Boddeda, C.E. Wolf-Hall and P.B. Schwarz .............Poster # 5 ......... 14 Ozone as an Antimycotic Agent in Malting Barley. Tobias D.J., C. Wolf-Hall and P.B, Schwarz .........................................................Poster #6 ......... 15 Economic Perspectives of Growers Facing the Challenges of FHB and DON. Felicia Wu ...................................................................................................... Plenary Talk ......... 16 Sex Differences in Apparent Adaptation to Immunotoxicity of Deoxynivalenol. Xianai Wu, Marian Kohut, Joan Cunnick and Suzanne Hendrich .........................Poster #7 ......... 17 Doehlert Matrix Design for Optimization of the Determination of Bound Deoxynivalenol in Barley Grain with TFA. Bing Zhou, Yin Li, James Gillespie, Richard Horsley and Paul Schwarz ..............Poster #8 ......... 18 Effect of Enzyme Pretreatments on the Determination of Deoxynivalenol in Barley. Bing Zhou, James Gillespie, Richard Horsley and Paul Schwarz ..........................Poster #9 ......... 19 Table of Contents SESSION 2: PATHOGEN BIOLOGY AND GENETICS Quantitative Expression of Fusarium sporotrichioides Genes in the Presence of Xanthotoxin. N.J. Alexander and S.P. McCormick ...................................................................Poster #10 ......... 23 Genetic Diversity of Fusarium graminearum Populations from Cereal and Noncereal Hosts. Rishi R. Burlakoti, Shaukat Ali, Gary A. Secor, Stephen M. Neate, Marcia P. McMullen and Tika B. Adhikari ......................................................... Poster #11 ......... 24 Identify and Characterize Genes Regulated by the FMK1 MAP Kinase in Fusarium graminearum. Sheng-Li Ding, Xiaoying Zhou, H. Corby Kistler and Jin-Rong Xu ...................Poster #12 ......... 25 Diversity in Fusarium graminearum sensu stricto from the U.S.: An Update. Liane R. Gale, Stephen A. Harrison, Eugene A. Milus, Jerry E. Ochocki, Kerry O’Donnell, Todd J. Ward and H. Corby Kistler ........................................Poster #13 ......... 26 Phenotypic and Molecular Diversity of Fusarium graminearum sensu stricto from the U.S. Liane R. Gale and H. Corby Kistler ................................................................. Invited Talk ......... 27 Structural and Functional Studies of Trichothecene Biosynthetic Enzymes: A Novel Approach to Combating Fusarium Head Blight. Garvey, G., S.P. McCormick and I. Rayment ......................................................Poster #14 ......... 29 Functions of the Sex Pheromones of Gibberella zeae. J. Lee, J.F. Leslie and R.L. Bowden ...................................................................Poster # 15 ......... 30 Isolation of Two Xylanase from Fusarium graminearum. S.W. Meinhardt, X. Dong and P.B. Schwarz .......................................................Poster #16 ......... 31 Spore Development and Trichothecene Mutants of Fusarium graminearum. Matias Pasquali, Kye-Yong Seong, Jon Menke, Erik Lysøe, Karen Hilburn, Jin-Rong Xu and H. Corby Kistler ......................................................................Poster #17 ......... 32 Structural and Functional Studies of Trichothecene 3-O-Acetyltransferase: Progress towards Development of an Improved Enzyme for Controlling FHB. Ivan Rayment .................................................................................................. Invited Talk ......... 33 Trichothecene Chemotypes of Isolates of Gibberella zeae Recovered from Wheat in Argentina. M.M. Reynoso, M.L Ramirez, J.F. Leslie and S.N. Chulze .................................Poster #18 ......... 34 Trichothecene Mycotoxin Genotypes of Gibberella zeae in Brazilian Wheat. L.B. Scoz, P. Astolfi, D.S. Reartes, D.G. Schmale III, M.G. Moraes and E.M. Del Ponte ............................................................................................Poster #19 ......... 35 Population of Fusarium graminearum Schwabe associated with Head and Seedling Blight in Slovakia. A. Šrobárová and N. Alexander .........................................................................Poster # 20 ......... 36 Life Cycle and Survival of Fusarium graminearum. Frances Trail .................................................................................................... Invited Talk ......... 40 Update on the Life Cycle of Fusarium graminearum. Frances Trail, John C. Guenther, Heather Hallen, Brad Cavinder and Lilly Yu ......................................................................................................Poster # 21 ......... 41 ii Table of Contents Trichothecene Chemotype Composition of Fusarium graminearum and Related Species in Finland and Russia. T. Yli-Mattila, K. O’Donnell, T. Ward and T. Gagkaeva ....................................Poster #22 ......... 42 SESSION 3: GENE DISCOVERY AND ENGINEERING RESISTANCE Studies on Barley Spikes Treated with the Trichothecene, Deoxynivalenol: Insight into Barley-Fusarium graminearum Interaction. Jayanand Boddu and Gary J Muehlbauer ...........................................................Poster #23 ......... 47 Expression of a Truncated Form of Ribosomal Protein L3 in Transgenic Wheat Confers Resistance to Deoxynivalenol and Fusarium Head Blight. Rong Di, Ann Blechl, Ruth Dill-Macky, Andrew Tortora and Nilgun E. Tumer.............................................................................................. Invited Talk ......... 48 Inhibition of Fusarium graminearum Germling Development caused by Combinatorially Selected Defense Peptides. N.W. Gross, Z.D. Fang, B. Cooper, F.J. Schmidt and J.T. English ......................Poster #24 ......... 49 Transgenic Wheat Expressing Antifungal Plant Defensin MtDef4 is Resistant to Fusarium Head Blight (FHB). Jagdeep Kaur, Thomas Clemente, Aron Allen and Dilip Shah ............................Poster #25 ......... 50 Reducing DON Potential in Virginia Hulless Barley Lines through Genetic Engineering. P.A. Khatibi, D.G. Schmale III, W.S. Brooks and C.A. Griffey ...........................Poster #26 ......... 51 Enhancing Fusarium Head Blight Resistance in Wheat by Manipulating Mechanisms Contributing to Host Resistance and Susceptibility. Ragiba Makandar, Vamsi Nalam, Juliane S. Essig, Melissa A. Schapaugh, Harold Trick, Ruth Dill-Macky and Jyoti Shah ..................................................Poster #27 ......... 52 Engineering Barley with Gastrodianin for Improved Resistance to Fusarium Head Blight. Eng-Hwa Ng, Tilahun Abebe, James E. Jurgenson and Ronald W. Skadsen ......................................................................................Poster #28 ......... 54 Genes that Confer Resistance to Fusarium. H. Saidasan and M. Lawton............................................................................. Invited Talk ......... 58 Genetic Studies Define Distinct Pathways of Resistance to Fusarium Head Blight. H. Saidasan and M. Lawton................................................................................Poster #29 ......... 59 Rapid Functional Identification of Genes Contributing to FHB Resistance. Steven Scofield and Megan Gillespie ............................................................... Invited Talk ......... 60 Engineering Resistance to Fusarium graminearum using Antifungal Plant Defensins. Dilip Shah, Mercy Thokala, Jagdeep Kaur, Tom Clemente and Anita Snyder ............................................................................................. Invited Talk ......... 61 Engineering Scab Resistance in Wheat with Plant Defense Signaling Genes. Jyoti Shah, Ragiba Makandar, Vamsi Nalam and Harold N. Trick .................. Invited Talk ......... 62 Transgenic Wheat with Enhanced Resistance to Fusarium Head Blight. S.H. Shin, J.M. Lewis, C.A. Mackintosh, A. Elakkad, K. Wennberg, S.J. Heinen, R. Dill-Macky and G.J. Muehlbauer ...............................................Poster #30 ......... 63 iii Table of Contents Comparative Analysis of FHB QTLs in the Mini Mano/Frontana and Frontana/Remus DH Populations. A. Szabo-Hever, B. Toth, Sz. Lehoczki-Krsjak, H. Buerstmayr, M. Lemmens and Á. Mesterházy ..............................................................................................Poster #31 ......... 64 Extra- and Intracellular Targeting of Antifungal Plant Defensins in Transgenic Arabidopsis for Resistance to Fusarium graminearum. Mercy Thokala, Aron Allen and Dilip Shah ........................................................Poster #32 ......... 65 SESSION 4: FHB MANAGEMENT Effects of Wheat Genotypes and Inoculation Timings on Fusarium Head Blight (FHB) Severity and Deoxynevalenol (DON) Production in the Field. Shaukat Ali and Tika B. Adhikari .......................................................................Poster #33 ......... 69 Aerobiology of Gibberella zeae: Whence Come the Spores for Fusarium Head Blight? Gary C. Bergstrom and David G. Schmale III .................................................. Invited Talk ......... 70 2007 Uniform Fungicide Trials on Soft White Winter Wheat in Michigan. D.E. Brown-Rytlewski, W.W. Kirk, R. Schafer and L. Liddell ............................Poster #34 ......... 72 Duration of Post-Flowering Moisture and Infection Timing Affect on FHB and DON in Wheat. C. Cowger and C. Medina-Mora .........................................................................Poster #35 ......... 73 Effect of Post Inoculation Moisture on Deoxynivalenol Accumulation in Fusarium graminearum-infected Wheat. Pravin Gautam and Ruth Dill-Macky .................................................................Poster #36 ......... 75 Prosaro® – A New Fungicide for Control of Fusarium and Mycotoxins in Cereals. I. Haeuser-Hahn, S. Dutzmann, R. Meissner and F. Goehlich .............................Poster #37 ......... 78 Addition of Adjuvant to Improve Coverage and Fungicide Efficacy on Barley, Langdon 2006 S. Halley, V. Hofman and G. Van Ee .............................................................................................. 79 Assessment of Air Stream Speed with Two Nozzle Types as a Tool to Improve Deposition of Fungicide for Control of FHB in Wheat. S. Halley, V. Hofman and G. Van Ee ............................................................................................. 82 Characterizing Parameters of Air Delivery Type Spray Systems to Maximize Fungicide Efficacy on Small Grain. S. Halley, K. Misek, V. Hofman and G. Van Ee ..................................................Poster #38 ......... 88 Evaluation of Fungicide for Control of Fusarium Head Blight with Aerial Application Technology. S. Halley and V. Hofman ............................................................................................................... 89 Relationships between Yield, Grain Quality Variables, and Fusarium Head Blight Intensity in Winter Wheat. John Hernandez Nopsa and Stephen N. Wegulo..................................................Poster #39 ......... 93 Outcomes of using Integrated FHB Management Strategies on Malting Barley Cultivars and Germplasm in Minnesota. C.R. Hollingsworth, L.G. Skoglund, D.B. Cooper, C.D. Motteberg and L.M. Atkinson ..............................................................................................Poster #40 ......... 94 iv Table of Contents Understanding Practical Outcomes from Implementing FHB Management Strategies on Spring Wheat. C.R. Hollingsworth, C.D. Motteberg, D.L. Holen and L.M. Atkinson ................Poster #41 ......... 96 Contribution of within-Field Inoculum Sources to Fusarium Head Blight in Wheat. M.D. Keller, K.D. Duttweiler, D.G. Schmale and G.C. Bergstrom ......................Poster #42 ......... 98 Time of Flowering in Wheat for Managing Fusarium Head Blight. Gregory S. McMaster ...................................................................................... Invited Talk ......... 99 Differential Effects of Infection Timing on Fusarium Head Blight and on DON and DON Derivatives in Three Spring Grains. Marcia McMullen, Jim Jordahl and Scott Meyer ................................................Poster #43 ....... 100 Effects of Fungicide Timing on Fusarium Head Blight and on DON and DON Derivatives in Three Spring Grains. Marcia McMullen, Scott Meyer and Jim Jordahl ................................................Poster #44 ....... 101 Experiences in Reducing Disease and DON through Components of FHB Management. Marcia McMullen............................................................................................ Invited Talk ....... 102 Comparison of Fungicides and Nozzle Types against FHB in Wheat at Farm Application. Á. Mesterházy, A. Szabo-Hever, B. Toth, G. Kaszonyi, and Sz. Lehoczki-Krsjak .....................................................................................Poster #45 ....... 104 Field and Laboratory Studies to Monitor Cell Populations, Lipopeptides and Lipopeptide Genes of Bacillus 1BA, a Biocontrol Agent Active against Fusarium Head Blight. J. Morgan, B.H. Bleakley and C.A. Dunlap ........................................................Poster #46 ....... 106 Effects of Solar Radiation on the Viability of Gibberella zeae Ascospores. Mizuho Nita, Erick De Wolf and Scott Isard ......................................................Poster #47 ....... 107 Mechanistic Simulation Models for Fusarium Head Blight and Deoxynivalenol. M. Nita, E. De Wolf, L. Madden, P. Paul, G. Shaner, T. Adhikari, S. Ali, J. Stein, L. Osborne and S. Wegulo .....................................................................Poster #48 ....... 108 Spore Load, Disease, and DON: A Four Year Variety by Residue Study for FHB Management. Lawrence E. Osborne, Jeffrey M. Stein and Christopher A. Nelson .....................Poster #49 ....... 109 Spore Load, Disease, and DON: An Inoculum Gradient Study using Sister Wheat Lines. Lawrence E. Osborne, Jeffrey M. Stein, Karl D. Glover and Christopher A. Nelson ..................................................................................Poster #50 ....... 114 A Quantitative Synthesis of the Relative Efficacy of Triazole-based Fungicides for FHB and DON Control in Wheat. Pierce Paul, Patrick Lipps, Don Hershman, Marcia McMullen, Martin Draper and Larry Madden ....................................................................................................................... 115 An Integrated Approach to Managing FHB and DON in Wheat: Uniform Trials 2007. P. Paul, L. Madden, M. McMullen, D. Hershman, L. Sweets, S. Wegulo, W. Bockus, S. Halley and K. Ruden ............................................................................................................... 117 Fungicide Effects on FHB and DON in Wheat across Multiple Locations and Wheat Classes: Uniform Fungicide Trials 2007. P. Paul, L. Madden, M. McMullen, D. Hershman, D. Brown-Rytlewski, L. Sweets, E. Adee, C. Bradley, B, Padgett and K. Ruden ............................................................................. 123 Influence of SRWW, HRSW, and HRWW Varieties on the Relationship between FHB and DON. Pierce A. Paul, Larry V. Madden, Stephen Wegulo, Tika Adhikari, Shaukat Ali and Erick De Wolf ..............................................................................................Poster #51 ....... 128 v Table of Contents DONcast: Seven Years of Predicting DON in Wheat on a Commercial Scale. R. Pitblado, D.C. Hooker, I. Nichols, R. Danford and A.W. Schaafsma ..............Poster #52 ....... 129 Effects of Fungicides on FHB Control and Yield of Winter Wheat Cultivars in North Dakota. J.K. Ransom, M.P. McMullen and S. Meyer .......................................................Poster #53 ....... 130 Effects of Fusarium Head Blight on Yield and Quality Parameters of Winter Wheat. K. Rehorova, O. Veskrna, P. Horcicka and T. Sedlacek ......................................Poster #54 ....... 131 2007 Uniform Fungicide Performance Trials for the Suppression of Fusarium Head Blight in South Dakota. K.R. Ruden, B.E. Ruden, K.D. Glover and J.L. Kleinjan....................................Poster #55 ....... 135 2007 Uniform Trials for the Performance of Biological Control Agents in the Suppression of Fusarium Head Blight in South Dakota. K.R. Ruden, B.H. Bleakley and B.E. Ruden .......................................................Poster #56 ....... 136 Characterization of DON Accumulation in SRWW Cultivars with Different Levels of Type II Resistance to FHB. Jorge D. Salgado, Gloria Broders, Larry Madden and Pierce Paul ......................Poster #57 ....... 137 Contribution of Local Inoculum Sources to Regional Atmospheric Populations of Gibberella zeae. D.G. Schmale III, B.R. Dingus, M.D. Keller and A.K. Wood-Jones ....................Poster #58 ....... 139 Environmental Factors Influencing FHB Severity and DON in Barley. J.M. Stein, L.E. Osborne, S. Neate and C. Hollingsworth ...................................Poster #59 ....... 140 Differential Sensitivity to Triazole-based Fungicides among Isolates of Fusarium graminearum . Matthew Wallhead, Larry Madden and Pierce Paul ............................................Poster #60 ....... 141 A Method for Quantifying Trichothecenes and Ergosterol in Single Wheat Florets using Gas Chromatography with Electron Capture Detection. K.T. Willyerd, K. Boroczky and G.A. Kuldau .....................................................Poster #61 ....... 142 Influence of Infection-Timing on Fusarium Head Blight Severity, Wheat Kernel Damage and Deoxynivalenol Accumulation during a 2007 Field Study. K.T. Willyerd, M. Nita, E.D. DeWolf and G.A. Kuldau ......................................Poster #62 ....... 143 Control of Fusarium Inoculum Production in Corn Residue by Mechanical, Biological, and Chemical Treatments. G.Y. Yuen, C.C. Jochum, J.E. Scott and S.Z. Knezevic .......................................Poster #63 ....... 144 Effects of Spray Application Methods on Biocontrol Agent Viability. G.Y.Yuen, C.C.Jochum, S. Halley, G. Van Ee, V. Hoffman and B.H. Bleakley ....Poster #64 ....... 149 Results from the 2007 Standardized Evaluation of Biological Agents for the Control of Fusarium Head Blight on Wheat and Barley. G.Y. Yuen, C.C. Jochum, K.R. Ruden, J. Morgan, B.H. Bleakley and L.E. Sweets ..................................................................................................Poster #65 ....... 153 SESSION 5: VARIETY DEVELOPMENT AND HOST RESISTANCE Air Separation and Digital Photo Analysis as Novel Methods to Measure the Percentage of Fusarium Damaged Kernels. Andres Agostinelli, Anthony Clark and D. Van Sanford ......................................Poster #66 ....... 161 vi Table of Contents Use of MAS for FHB Resistance: Is it working for Wheat Breeders? James A. Anderson, Sixin Liu and Shiaoman Chao .......................................... Invited Talk ....... 163 Marker-Assisted Transfer of 3BS QTL for FHB Resistance into Hard Winter Wheat. G.-H. Bai, P. St. Amand, D.-D. Zhang, A. Ibrahim, S. Baenziger, B. Bockus and A. Fritz .........................................................................................................Poster #67 ....... 164 Genetic Linkage Mapping with DArT Markers to Detect Scab Resistance QTLs in a ‘Sumai-3’ Derived Wheat Population. Bhoja. R. Basnet, Yang Yen, Shiaoman Chao and Karl D. Glover ......................Poster #68 ....... 165 Using the Affymetrix Array to Discover Single Nucleotide Polymorphisms in Wheat. A.N. Bernardo, S.-W. Hu, P.J. Bradbury, R.L. Bowden, E.S. Buckler and G-H. Bai ......................................................................................................Poster #69 ....... 166 Enhancing Host Resistance to Fusarium Head Blight: Pyramiding Genes in Spring Wheat. W.A. Berzonsky, E.L. Gamotin, G.D. Leach and T. Adhikari ..............................Poster #70 ....... 167 QTL associated with Reduced Kernel Damage and Resistance to Fusarium Head Blight in Wheat. C.M. Bonin, F.L. Kolb and E.A. Brucker ............................................................Poster #71 ....... 168 Resistance to Kernel Damage caused by Fusarium Head Blight in an RIL Population. C.M. Bonin and F.L. Kolb ..................................................................................Poster #72 ....... 169 Barley Chromosome 2(2H) Bin 10 Fusarium Head Blight Resistance QTL: Mapping and Development of Isolines. Christine N. Boyd, Richard Horsley and Andris Kleinhofs ..................................Poster #73 ....... 170 Haplotyping of Known FHB Resistance QTL in Pacific Northwest Wheat Genotypes. Jianli Chen, Juliet Windes, Robert Zemetra and Carl Griffey..............................Poster #74 ....... 173 Validation of Six QTLs associated with Fusarium Head Blight Resistance in Adapted Soft Red Winter Wheat. Jianli Chen, Carl Griffey, Shiaoman Chao and Gina Brown-Guedira .................Poster #75 ....... 174 Development of Scab Resistant Soft Red Winter Wheat Germplasm using MarkerAssisted Selection. Jose M. Costa, Leila Al-Tukhaim, Raquel Brown, Neely Gal-Edd, Alice Ku, Erin Wenger, David Van Sanford and Gina Brown-Guedira ...............................Poster #76 ....... 175 Applying Single Kernel Sorting Technology to Developing Scab Resistant Lines. F.E. Dowell and E.B. Maghirang ........................................................................Poster #77 ....... 176 Tunisian Durum Wheat as New Sources of Resistance to Fusarium Head Blight. Farhad Ghavami, Melissa Huhn, Elias Elias and Shahryar Kianian ....................Poster #78 ....... 177 Responding to Fusarium Head Blight for the Northern Rocky Mountains and Western Great Plains. W. Grey, A. Dyer and L. Talbert .................................................................................................. 179 Resistant Germplasm from Susceptible Parents: An Evolutionary Approach. Steve Haber and Jeannie Gilbert .........................................................................Poster #79 ....... 182 Resistance of Winter Wheat Lines to Deoxynivalenol and Nivalenol Chemotypes of Fusarium graminearum. P. Horevaj, E.A. Milus, L.R. Gale and H.C. Kistler ............................................Poster #80 ....... 183 Current Strategies for Breeding Fusarium Head Blight Resistant Wheat in Canada. G. Humphreys, D. Somers, S. Fox, D. Brown, H. Voldeng, A. Brule-Babel, F. Eudes and A. Comeau .................................................................................. Invited Talk ....... 188 vii Table of Contents Effects of Agronomic and Morphological Characters on FHB Severity, Deoxynivalenol and Ergosterol Concentrations in Near-isogenic Line Pairs of Barley. Haiyan Jia, Brian J. Steffenson and Gary J. Muehlbauer ....................................Poster #81 ....... 189 Fusarium Head Blight (FHB) Resistance into Soft Red Winter Wheat AGS2000. Jerry Johnson, Zhenbang Chen, James Buck and Mingli Wang ..........................Poster #82 ....... 190 Characterization of Resistance to Deoxynivalenol (DON) Accumulation in Different Wheat Lines. L. Kong, Y. Dong and H.W. Ohm .......................................................................Poster #83 ....... 191 Development of CIMMYT’s 11th Scab Resistance Screening Nursery. J.M. Lewis, T. Ban, R.Ward and E. Duveiller .....................................................Poster #84 ....... 192 Preliminary Examination of the Influence of Grain Color in FHB Resistance. J.M. Lewis, R. Trethowan, T. Ban, E. Duveiller and R. Ward .............................Poster #85 ....... 193 FHB Resistance and DON Contamination in Virginia Barley. S. Liu, D. G. Schmale III, W.S. Brooks, P.A. Khatibi and C.A. Griffey ...............Poster #86 ....... 194 Meta-Analyses of QTL associated with Fusarium Head Blight Resistance. Shuyu Liu, Carl A. Griffey, Anne L. McKendry and Marla D. Hall ....................Poster #87 ....... 195 Pyramiding FHB Resistance QTL using Marker-Assisted Selection in Wheat. S.Y. Liu, C.A. Griffey, J. Chen and G. Brown-Guedira .......................................Poster #88 ....... 200 Winter and Spring Wheat Parental Diallel Analysis for Scab Resistance. S. Malla, A.M.H. Ibrahim and K. Glover ............................................................Poster #89 ....... 201 Role of a Plasma Membrane Ca2+-ATPase in the Resistance of Potato Cells to Fusarium solani. A.M. Manadilova, G.T. Tuleeva and R.M. Kunaeva ...........................................Poster #90 ....... 202 Prospects for Identifying Fusarium Head Blight Resistance QTL by Association Mapping using Breeding Germplasm. Jon Massman and Kevin Smith ...........................................................................Poster #91 ....... 203 Breeding for FHB Resistance in Winter Wheat: What’s Ahead? Anne L. McKendry .......................................................................................... Invited Talk ....... 204 Scab Epidemic in Nebraska. Neway Mengistu, P. Stephen Baenziger, Stephen Wegulo, Julie Breathnach and Janelle Cousell .............................................................................................Poster #92 ....... 205 ‘Faller’: A New Hard Red Spring Wheat Cultivar with High Yield and Quality added to Combat Fusarium Head Blight Disease. Mohamed Mergoum, Richard C. Frohberg and Robert W. Stack ................................................. 206 Putative FHB Resistance Components Resistance to Kernel Infection and Tolerance in the SSRWW Nursery, 2005-2007. Á. Mesterházy, A. Szabo-Hever, B. Toth, G. Kaszonyi and Sz. Lehoczki-Krsjak .....................................................................................Poster #93 ....... 211 Evaluation of FHB Profiles of Advanced Wheat Breeding Lines Treated with a Fungicide. N. Mundell, D. Hershman, Chad Lee and D. Van Sanford ..................................Poster #94 ....... 212 The 2006-07 Southern Uniform Winter Wheat Scab Nursery. J.P. Murphy and R.A. Navarro ............................................................................Poster #95 ....... 213 Haplotype Structure and Genetic Diversity at Fusarium Head Blight Resistance QTLs in Soft Winter Wheat Germplasm. Leandro Perugini, Clay Sneller, Fred Kolb, David VanSanford, Carl Griffey, Herb Ohm and Gina Brown-Guedira ..................................................................Poster #96 ....... 214 viii Table of Contents Solving the FHB Problem: Growers, Export Market and Wheat Commodity Perspectives. Jim Peterson ................................................................................................... Plenary Talk ....... 218 Association Mapping of Complex Traits in a Diverse Durum Wheat Population. C.J. Pozniak, S. Reimer, D.J. Somers, F.R. Clarke, J.M. Clarke, R.E. Knox, A.K. Singh and T. Fetch................................................................................. Plenary Talk ......... 220 Molecular Characterization of a Wheat-Leymus Compensating Translocation Line Conferring Resistance to Fusarium Head Blight. L.L. Qi, M.O. Pumphrey, B. Friebe, P.D. Chen and B.S. Gill ........................... Poster #97 ......... 225 FHB Resistance of Wheat Lines Near-isogenic for Five Different FHB Resistance QTLs. Pilar Rojas-Barros, Zachary J. Blankenheim, Karen J. Wennberg, Amar M. Elakkad, Ruth Dill-Macky and David F. Garvin ............................................................. Poster #98 ......... 226 Family Based Mapping of FHB Resistance QTLs in Hexaploid Wheat. Rosyara, R.U., Maxson-Stein, K.L., Glover, K.D., Stein, J.M and Gonzalez-Hernandez, J.L. .......................................................................... Poster #99 ......... 227 Facing the FHB Challenges to Malting Barley and Brewing through Barley Breeding. L.G. Skoglund ................................................................................................ Invited Talk ......... 228 Development of Barley Variety Candidate M122 with Enhanced Resistance to FHB. Kevin P. Smith, Ed Schiefelbein and Guillermo Velasquez .......................................................... 232 Report on the 2006-07 Northern Uniform Winter Wheat Scab Nurseries (NUWWSN and PNUWWSN). C. Sneller, P. Paul, L. Herald, B. Sugerman and A. Johnston ....................................................... 237 Deoxynivalenol Accumulation and Fusarium Head Blight Severity in Winter Wheat after Spray-Inoculation with Mixture or Single Isolates of Fusarium graminearum. L. Tamburic-Ilincic and A.W. Schaafsma. .................................................................................... 243 The Effect of Fusarium solani Metabolites on Peroxidase Activity in Potato. A.Sh.Utarbayeva, O.A.Sapko and R.M. Kunaeva........................................... Poster #100 ......... 249 Searching for New Sources of FHB Resistance in the Relatives of Wheat. S.S. Xu, R.E. Oliver, X. Cai, T.L. Friesen, S. Halley and E.M. Elias .............. Invited Talk ......... 250 Comparison of Barley Seed Proteomic Profiles associated with Fusarium Head Blight Reaction. J. Zantinge, K. Kumar, K. Xi, A. Murray, M. Johns, J.H. Helm and P. Juskiw .................................................................................................. Poster #101 ......... 251 Novel Fusarium Head Blight Resistance in Triticum aestivum Revealed by Haplotyping with DNA Markers associated with a Known Resistance QTL. C. Zila, J.R. Recker, X. Shen, L. Kong and H.W. Ohm ................................... Poster #102 ......... 252 ix KEYNOTE PRESENTATION Keynote Presentation FUSARIUM HEAD BLIGHT OUTBREAK FREQUENCY UNDER A CHANGING CLIMATE ENVIRONMENT. J.M. Fernandes1*, W. Pavan2 and E.M. Del Ponte3 Embrapa Trigo, Rodovia BR 285, km 174,99001-970 Passo Fundo, RS, Brasil; 2Instituto de Ciências Exatas e Geociências, Universidade de Passo Fundo, BR 285 Km 171, 99001-970, Passo Fundo, RS, Brasil; and 3Departmento de Fitossanidade, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 7712, 91540000, Porto Alegre, RS, Brasil * Corresponding Author: PH: (55 54) 33165800; Email: mauricio@cnpt.embrapa.br 1 FHB pathogens, their efficacy is influenced by dose rate, application timing and spray quality for adequate spike coverage (Cromey et al., 2001). The strong dependence on environmental conditions and the relative narrow window of vulnerability to infection by the fungus makes FHB a suitable system for modeling and forecasting, which could lead to a rational and more effective disease control. EXECUTIVE SUMMARY Fusarium Head Blight (FHB) is a re-emerging disease of increasing concern to wheat and other small grains with devastating impacts worldwide (Goswami & Kistler, 2004). In Brazil, FHB epidemics have become more frequent and often resulting in significant yield losses (Panisson et al., 2003). FHB constitutes a disease complex in which several fungal species may cause largely indistinguishable symptoms, although the predominant causal agent worldwide is the fungus Gibberella zeae (Fusarium graminearum sensu stricto, anamorph) (Goswami & Kistler, 2004). Previous reports in Brazil showed that G. zeae was the principal causal agent of FHB in wheat (Angelotti et al., 2006). In the last few decades, modeling of plant diseases, especially computer modeling, has expanded very quickly and played an important role in integrated disease management. The recent advances in computer hardware and information technologies have assisted in this development, bringing operational advantages to speed up development and application of computer models representing complex processes. Modularity and generic are terms that describe the new and widely accepted methodology to surpass the complexities in the development and re-use of models. Our group has been applying novel software engineering techniques towards the development of linked crop-disease generic simulation models. These techniques revealed to be appropriate and robust to guide in the development of plant disease simulation models. In addition, combining a suite of technologies proved to be possible to use existing knowledge legacy. FHB is best known as a disease of flowering stage but evidences suggest that wheat may be susceptible at later stages of kernel development. In temperate climates, it has been reported that monoculture, reduced tillage, and maize-wheat rotations have greatly increased inoculum levels in soil (McMullen et al., 1997). In southern Brazil, inoculum is available the year round because of the abundant crop residues from other hosts, widespread no-till and the absence of freezing temperatures or dry seasons impairing fungal development (Fernandes, 1997). Critical epidemiological knowledge of a plant disease is of fundamental importance for developing disease models. Epidemiological aspects of FHB have been studied in southern Brazil since early 1980’s in both field and controlled environment. Climatic conditions are most suitable for the disease in that region and moderate to severe FHB epidemics show a periodical occurrence. Studies for developing an FHB model initiated by our group since late 1990s and the result- Even though progresses have been made over time, disease control is still challenging. Most of cultivars in use do not possess desirable levels of resistance that could lead to a good genetic control. Breeding for wheat scab resistance is a difficult task but some progress has been achieved in the recent years (Mesterhazy et al. 2005). Although a range of fungicides have been identified with good activity against 3 Keynote Presentation Second, the web-based platform has also the capability to forecast infection risks of FHB near real-time during the season. The system integrates hourly weather data collected in a network of weather stations and 5-days hourly forecast weather data generated by computer models. The user interacts with the system by selecting the nearest location and the crop heading date. The model then calculates risk of infections and warns the user whenever a risk level of concern is achieved, based on both actual and forecast weather. A user-friendly mapping interface is under development and weather data from other locations are being integrated into the system in order to generate regional risk maps, besides the site-specific preIn this presentation, two applications of the model us- dictions already available. ing an integrated systems approach for the development of a web-based information technology platform REFERENCES will be illustrated. First, the potential effect of climate changing/variability in a selected location in the main Angelloti F., Tessmann D.J., Alburquerque T.C., Vida J.B., Filho D.S.J., Harakava R., 2006. Caracterização morfológica e wheat regions of the Argentinean, Brazilian and Uru- identificação molecular de isolados de Fusarium guayan Pampas was studied using the wheat-disease graminearum associados à giberela do trigo e triticale no sul model. Using daily weather data from a 50-year pe- do Brasil. Summa Phytopathologica 32:177-79. riod and assuming non-limiting inoculum conditions as Cromey M.G., Lauren D.R., Parkes R.A., Sinclair K.I., Shorter input into the wheat-FHB model, our results showed S.C., Wallace A.R. 2001. Control of Fusarium head blight of that climate at Passo Fundo, Brazil, for example, was wheat with fungicides. Australian J Plant Pathol. 30:301-308. very favorable for FHB outbreaks after 1990 followDel Ponte E.M., Fernandes J.M.C., Pavan W. 2005. A risk ing a period of less favorableness for epidemics in the infection simulation model for Fusarium head blight in wheat. 1970s and 1980s, when outbreaks were very spo- Fitopatologia Brasileira 30:634-642. radic. Outbreaks were more frequent in El Niño than Fernandes, J.M.C. 1997. As doenças das plantas e o sistema in La Niña years, especially for later planting dates in de plantio direto. Revisão Anual de Patologia de Plantas 5:317a crop year. These results suggest that, considering a 352. fixed amount of inoculum, a changing climate (espeGoswami R.S., Kistler C., 2004. Heading for disaster: Fusarium cially wetter) was associated with a higher frequency graminearum on cereal crops. Molecular Plant Pathology of outbreaks especially for the later plantings after the 5:515. 1990s. Earlier sowing and use of early maturing varieties with a shorter reproductive period would be good strategies for Brazilian growers to avoid more favorable environment later in a season. ing models has improved along the years. It has been built based on existing knowledge on disease cycle components and a series of local studies to empirically model host development and inoculum availability. The FHB model has been validated against observed epidemic data from Brazil and well explained the variation in FHB severity under distinct conditions (Del Ponte et al., 2005). The model was further coupled onto a wheat growth and development simulation model. The wheat-disease model has been validated with regards to prediction of wheat phenological stages and FHB severity observed in Passo Fundo location, Brazil. 4 SESSION 1: FOOD SAFETY, TOXICOLOGY AND UTILIZATION OF MYCOTOXIN-CONTAMINATED GRAIN Chairperson: Jim Pestka Session 1: Food Safety, Toxicology, and Utilization of Mycotoxin-contaminated Grain QUANTIFICATION OF THE TRI5 GENE, EXPRESSION AND DEOXYNIVALENOL PRODUCTION DURING THE MALTING OF BARLEY. Anuradha Boddeda1, Paul Schwarz2 and Charlene E. Wolf-Hall1* Department of Veterinary and Microbiological Sciences, North Dakota State University, Fargo, ND; and 2Department of Plant Sciences, North Dakota State University, Fargo, ND * Corresponding Author: PH: (701) 231-6387; Email: Charlene.Hall@ndsu.edu 1 ABSTRACT Barley quality and safety is affected by Fusarium both in the field and during post-harvest processes. Fusarium strains can survive, grow and produce mycotoxins during malting. We evaluated percentage of Fusarium infection (FI), and deoxynivalenol (DON) concentration in three, raw barley samples (high quality, naturallyinfected, F. graminearum inoculated barley) during various stages of malting. We also applied real-time PCR and real-time reverse transcriptase PCR (real-time RT-PCR) methods to quantify the Tri5 DNA concentration and expression respectively in the barley samples. We observed that FI significantly (P < 0.05) increased during the germination stage of malting in all the barley samples. Temperatures of 49°C and higher during kilning reduced the FI in high quality barley samples, but for inoculated samples more than 60°C during kilning was needed to reduce Fusarium infection. The average Tri5 DNA was found to be in the range of 0 to 3.9 ng/ 50 mg, 0.06 to 109.79 ng/50 mg and 3.38 to 397.55 ng/50 mg in malted high quality, inoculated and infected barley samples respectively. Strong gene expression (Tri5) in naturally infected barley samples was found during 3rd day of germination, however very low amounts of gene expression were observed in high quality and inoculated barley samples during malting. Deoxynivalenol was found to be present even at high kilning temperatures as DON is heat stable. The average DON concentration was found to be in the range of 0 to 0.13 μg/g, 0 to1.09μg/g and 1.53 to 45.86 μg/g during malting in high quality, inoculated and infected barley samples respectively. Overall, the last two days of germination and initial stages of kilning were peak processing stages for FI, Tri5 gene production, Tri5 gene expression and DON production. 9 Session 1: Food Safety, Toxicology, and Utilization of Mycotoxin-contaminated Grain HIGH-THROUGHPUT HOMOGENIZATION OF GRAIN SAMPLES FOR DON TESTING. D.M. Reaver and D.G. Schmale III* Virginia Polytechnic Institute and State University, Blacksburg, VA, 24061 * Corresponding Author: PH: (540) 231-6943; Email: dschmale@vt.edu ABSTRACT Concerns about deoxynivalenol (DON) continue to mount, and there is a growing need to develop new tools and techniques to enhance the speed, capacity, and uniformity of DON testing services in the United States. Tens of thousands of wheat and barley samples associated with USWBSI research projects are processed by DON testing labs every year. Many of these samples consist of 100 g kernel lots, and they must be cleaned, milled, and sieved before DON is extracted and quantified. The processing of a high number of grain samples in such a manner is extremely laborious and costly. We developed a rapid and affordable high-throughput homogenization protocol for DON testing that can homogenize twelve grain samples weighing from 0.1 to 2.5 g in as little as ten seconds. A Biospec MiniBeadBeater-96 ™operating at 2100 oscillations per minute was used to homogenize grain samples in individual 7 mL HDPE vials containing 13.7 mm chrome balls. Grain samples were homogenized into a fine flour of nearly uniform particle size, and DON extractions were conducted in the same vials that were used for the homogenization of the samples. The extraction solvent containing DON was passed through a clean-up column, and a measured fraction of the flow-through was dried down using a nitrogen evaporator at 55C. DON samples were derivatized using TMSI and quantified using a GC/ MS operating in a SIM/SIM scan mode for target and reference ions of DON. Over 800 grain samples originating from single inoculated or non-inoculated (control) wheat spikes from southern uniform FHB greenhouse trials in AR, NC, and VA were processed for DON testing using this new methodology. High-throughput homogenization protocols may assist in providing affordable and timely DON testing services for USWBSIassociated research projects in the future. 10 Session 1: Food Safety, Toxicology, and Utilization of Mycotoxin-contaminated Grain ANALYSIS OF DEOXYNIVALENOL, MASKED DEOXYNIVALENOL AND FUSARIUM GRAMINEARUM PIGMENT IN GRAIN CULTURES USING A NEW LC-UV/MS METHOD. Sasanya, J.J.1, Hall, C.1,2* and Wolf-Hall, C.1,3 Great Plains Institute of Food Safety, North Dakota State University (NDSU), Fargo, ND; 2 Department of Cereal and Food Sciences, NDSU, Fargo, ND; and 3Department of Veterinary and Microbiological Sciences, NDSU, Fargo, ND * Corresponding Author: PH: (701) 231-6359; Email: clifford.hall@ndsu.edu 1 ABSTRACT The presence of the mycotoxin deoxynivalenol (DON, 3,7,15-trihydroxy-12,13-epoxytrichothec-9-ene-8one) in grains presents a food safety risk. Also, DON may conjugate with sugars resulting in masked mycotoxins such as deoxynivalenol-3-glucoside (DON-3-glucoside) which may be metabolized in vivo to DON thus increasing the risk. Such masked mycotoxins and the potentially toxic Fusarium pigment are not routinely analyzed in grains. To promote continued understanding of the presence of masked mycotoxins in grains and their coexistence with DON, we analyzed rice and wheat culture samples inoculated with different Fusarium strains, using a new liquid chromatography (LC) - mass spectrometry (MS) method. We also quantified the Fusarium pigment. Grain samples cultured for 14 days were extracted by centrifugation with methanol:methylene chloride (50/50, v/v) followed by cleanup through C18 columns. Elution solvents included methanol, water, acetonitrile and acetic acid before analysis using LC-UV/MS. An isocratic mobile phase (70% methanol) was used. The DON average in rice culture was 15.2 (± 41.0) mg/kg while DON-3-glucoside averaged 5.4 (± 8.6) mg/kg in rice. Neither DON nor DON-3-glucoside were observed in wheat culture samples. The pigment averaged 142.2 ±256.3 mg/kg in wheat and 7.1±126 mg/kg in rice culture samples. Therefore, we report here how analytical tools such as this new LC-UV/MS method can be used to quantify masked and parent mycotoxins in rice plus potentially toxic pigment in rice and wheat culture for risk assessment studies. The coexistence of DON with DON-3-glucoside in grain cultures such as rice spiked with Fusarium graminearum emphasizes the conjugation of DON to form masked mycotoxins hence the need to regularly analyze grains for both parent and masked mycotoxins. Such studies can be optimized to further explore ways of producing and isolating masked mycotoxins in culture since standard masked mycotoxins are not commercially available. Potentially toxic pigments can also be studied further. 11 Session 1: Food Safety, Toxicology, and Utilization of Mycotoxin-contaminated Grain PRODUCTION AND PURIFICATION OF DEOXYNIVALENOL FROM RICE CULTURE AND ANALYSIS USING LIQUID CHROMATOGRAPHY-ULTRAVIOLET-MASS SPECTROMETRY. Sasanya, J.J.1, Hall, C.1,2* and Wolf-Hall, C.1,3 Great Plains Institute of Food Safety, North Dakota State University (NDSU), Fargo, ND; 2 Department of Cereal and Food Sciences, NDSU, Fargo, ND; and 3Department of Veterinary and Microbiological Sciences, NDSU, Fargo, ND * Corresponding Author: PH: (701) 231-6359; Email: clifford.hall@ndsu.edu. 1 ABSTRACT Mycotoxins such as trichothecenes represented by deoxynivalenol (DON) present a major global food safety problem with associated economic implications. Thus toxicological, analytical and detoxification studies relevant to industry and regulatory agencies are priority. Such studies require relatively large quantities of pure mycotoxins such as DON. Here we present a new method were we produced and purified (96-99%) large quantities of DON (403 μg/g) from rice (85g) inoculated with Fusarium graminearum (3x107 spores per ml) and incubated for 4 days at 30 °C. Purification was achieved using a combination of silica gel (32 g), alumina (7.2 g) and celite (4.8 g) in a glass column. This followed extraction by high speed centrifugation using methanol:methylene chloride (50/50, v/v). High recovery rates (100 ± 9.9%; CV=0.1) were also recorded. Elution of the mycotoxin was done using methanol, acetonitrile, water and acetic acid (60:30:10:0.1, v/v). Analysis of DON was done using thin layer chromatography and liquid chromatography connected to ultraviolet and mass spectrometer detectors. These findings present a new method to culture and purify DON in bulk using relatively cheaper clean up column followed by analysis using chromatographic and mass spectrometric techniques. Such findings also support toxicological, analytical and detoxification studies pertaining DON. 12 Session 1: Food Safety, Toxicology, and Utilization of Mycotoxin-contaminated Grain DEOXYNIVALENOL MEASUREMENT: SOURCES OF ERROR AND SAMPLING RECOMMENDATIONS. Paul Schwarz1*, Yanhong Dong2 and Ruth Dill-Macky2 1 Department of Plant Sciences, North Dakota State University, Fargo, ND 58105; and 2 Department of Plant Pathology, University of Minnesota, St Paul, MN 55108 * Corresponding Author: PH: (701) 231-7732; E-mail: Paul.Schwarz@ndsu.edu ABSTRACT For any type of analysis there is measurement error that is composed of various factors such as sampling, sample preparation, and the analytical instrument itself. In the determination of deoxynivalenol (DON) by gas chromatography (GC), sample preparation steps include grinding, extraction, clean-up and derivatization. Factors influencing derivatization are probably most critical, and have greater influence than instrument variability. Inter-laboratory check samples of ground wheat and barley are periodically analyzed by the USWBSI funded laboratories as a means of assuring consistency of results. The coefficients of variation (CV) on these analyses typically range from 5 to 15%, which is considered acceptable for analytical work in the mg/kg (ppm) range. As an example, the expected analytical range in DON results with a CV of 10% for a sample at 1.00 mg/kg would be 0.89- 1.11 mg/kg. However, it must be remembered that much of the variability observed in DON levels in grain is related to the biology of the disease, rather than the chemical analysis. This follows as DON accumulation in grain results from a complex host-pathogen interaction which is subject to environmental variability. The production of DON, like visible symptoms of FHB, varies greatly from spikelet to spikelet, spike to spike, and environment to environment. Grain sampling greatly affects the accuracy of DON analysis, and the responsibility of providing a representative sample rests with the individual researcher. Sampling considerations include the collection of a representative samples from the experimental unit (plot, field), and then the reduction of this material to a representative sub-sample with a sample divider. The sample provided to the DON analysis laboratories should be no more than 100 g, and ideally around 20 g. This follows, as the grinding of 10,000 100g samples, as opposed to 20 g samples, requires an additional 21 days of labor. USWBSI recommendations on the sampling of grain for DON are posted at http://www.scabusa.org/pdfs/ptt/ researchers_grain-sampling-protocols.pdf 13 Session 1: Food Safety, Toxicology, and Utilization of Mycotoxin-contaminated Grain DEVELOPMENT OF A MULTIPLEX REAL-TIME PCR ASSAY FOR RAPID DETECTION AND QUANTIFICATION OF FUSARIUM SPP. IN BARLEY. 1* D.J. Tobias , A. Vashisht1, A. Boddeda1, C.E. Wolf-Hall1 and P.B. Schwarz2 Dept. of Veterinary and Microbiological Sciences, and 2Dept. of Plant Sciences, North Dakota State University, Fargo, ND 58105 * Corresponding Author: PH: (701) 231-6386; Email: dennis.tobias@ndsu.edu 1 ABSTRACT The persistence of trichothecenes in Fusarium-infected stored grains and in processed food poses a great risk to human health and animals. The ability to rapidly detect Fusarium species and monitor their distribution in collected wheat and barley grains across the state of ND is important due to the significant number of grain samples and the differences in the toxicity of these secondary metabolites. This can be accomplished rapidly using a polymerase chain reaction (PCR)-based detection of FHB-associated Fusarium species. Our objective is to develop a multiplex real-time PCR assay to identify and quantify pathogenic Fusarium species based on primer pairs derived from Intergenic Spacer (IGS) of rDNA unit sequences. The selected primers for species-specific detection showed amplification products of 123, 418, 462, 293 and 186 bp using positive controls (template DNA) which were derived from F. graminearum (NRRL R-6574), F. avenaceum (FRC# R-04608), F. poae (FRC# T-0487) and F. sporotrichioides (FRC# T-0348), respectively. Multiplexing (3 to 4-species) resulted in amplifications for species-specific detection using naturally-infected malting barley, Robust, with 0 and 2 ppm DON levels. Five picograms of fungal DNA were found to be enough to obtain a visible amplification product. For reliability, the multiplex real-time PCR assay will also test several isolates of each Fusarium spp. This high-throughput assay will help screen the malting barley samples and accurately assess the distribution of Fusarium spp. which are predominant in the region. Malting barley collections in 2005-2006 from 3 ND districts (and 1 MN district) will be utilized for future assays. 14 Session 1: Food Safety, Toxicology, and Utilization of Mycotoxin-contaminated Grain OZONE AS AN ANTIMYCOTIC AGENT IN MALTING BARLEY. Tobias D.J.1, C. Wolf-Hall1* and P.B, Schwarz2 Department of Veterinary and Microbiological Sciences; and 2Department of Plant Sciences, North Dakota State University, Fargo, ND 58105 * Corresponding Author: PH: (701) 231-6387; Email: charlene.hall@ndsu.edu 1 ABSTRACT Fusarium spp. are known producers of important mycotoxins such as trichothecenes. The persistence of trichothecenes in infected stored barley grains and in processed food poses a great risk to human health and concern in the malting and brewing industry. At present, the only available effective control is testing and diversion or dilution. The effectiveness of ozone as an insecticidal fumigant in stored grains has been reported previously. The objective of this project is to evaluate the efficacy of gaseous ozone as an antifungal and antimycotoxin treatment for barley. Preliminary tests by gaseous ozone treatment (GOT) of pure Fusarium graminearum (FRC# R-06574) culture at 26 mg/g O3 for 90 min. on the broth surface showed a detrimental effect on the morphological structure (non-branching and breakage of hyphae) and a 30% decrease in fungal biomass. The fungus was not totally eliminated probably due to the presence of sugar and nutrients in the broth allowing recovery and slow growth. In the present study, an extended treatment of 120 min at 26 mg/g O3 were tested on five Fusarium spp. (F. graminearum, F. culmorum, F. poae, F. sporotrichioides and F. avenaceum) separately grown in PD broth. The same treatment was applied to malting barley before steeping and after 2 or 8 hrs steeping through a submerged gas sparger. We report on the effect of ozone on the growth and survival (FS) of five Fusarium spp., germinative energy (GE) and DON in malting barley. 15 Session 1: Food Safety, Toxicology, and Utilization of Mycotoxin-contaminated Grain ECONOMIC PERSPECTIVES OF GROWERS FACING THE CHALLENGES OF FHB AND DON. Felicia Wu Department of Environmental & Occupational Health, Graduate School of Public Health, University of Pittsburgh, 100 Technology Dr. Suite 560, Pittsburgh PA 15219 Corresponding Author: PH: 412-624-1306; Email: few8@pitt.edu ABSTRACT The economic problem of Fusarium Head Blight (FHB) and DON is extremely complex, yet can provide insight into how to focus research and development to prevent these conditions. We start with the guiding question: How can the economic perspective of wheat and barley growers help us decide how we should focus our research and initiatives? This talk describes four categories of economic consideration: 1) the sources of uncertainty and variability in wheat and barley production, FHB, and DON; 2) the cost-effectiveness of various control methods to reduce FHB and DON; 3) benefit-cost analysis of adopting FHB/DON control methods; and 4) the tradeoffs for growers planning to plant wheat or barley compared with competing crops such as corn. 16 Session 1: Food Safety, Toxicology, and Utilization of Mycotoxin-contaminated Grain SEX DIFFERENCES IN APPARENT ADAPTATION TO IMMUNOTOXICITY OF DEOXYNIVALENOL. Xianai Wu1, Marian Kohut2, Joan Cunnick3 and Suzanne Hendrich1* Food Science and Human Nutrition, 2Kinesiology, and 3Animal Science, Iowa State University, Ames, IA 50011 * Corresponding Author: PH: 515-294-4272; Email: shendric@iastate.edu 1 ABSTRACT Deoxynivalenol (DON) is a mycotoxin produced by Fusarium graminearum and F. culmorum commonly found in grains. We hypothesized that DON was immunotoxic in BALB/c mice, suppressing peripheral blood lymphocytes at 1 ppm but not at lesser doses. Groups of 10 female and 10 male BALB/c mice were fed DON at 0, 0.25, 0.5, 1, and 2 ppm over 14 days and 28 days. Peripheral blood and single spleen cells suspension were stained with fluorescently labeled antibodies for CD4, CD19, CD8a and CD11b leukocyte cell surface markers. Flow cytometry was used to detect leukocyte phenotypes. In peripheral blood, the percentage of T cytotoxic and B cells were inhibited in both sexes of BALB/c mice after 14 days of DON exposure, and toxic dose of DON varied by sex, whereas exposure to DON over 28 days did not inhibit these lymphocytes, compared with the control diet. Dietary DON did not influence hematology in males but red blood cells (RBC) at 0.5 and 1 ppm DON and hemoglobin (Hb) at all DON doses were suppressed in female mice by dietary DON over 14 days. The inhibition of RBCs by DON disappeared after 28 days compared with the control diet. The percentage of monocytes (CD11b+) was decreased in peripheral blood (at doses of 0.5-2 ppm DON) and spleen (2 ppm DON) only in BALB/c female mice after 28 days compared with control diet. These results indicate that BALB/c mice adapted to most signs of DON immunotoxicity and hematotoxicity after 28 days. At this time, the percentage of monocytes was decreased in peripheral blood and spleen by as little as 0.5 ppm DON in female mice, suggesting that female sex hormones potentiate DON immunotoxicity in BALB/c mice. ACKNOWLEDGEMENT AND DISCLAIMER This material is based upon work supported by the U.S. Department of Agriculture, under Agreement No. 590790-6-060. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the U.S. Department of Agriculture. 17 Session 1: Food Safety, Toxicology, and Utilization of Mycotoxin-contaminated Grain DOEHLERT MATRIX DESIGN FOR OPTIMIZATION OF THE DETERMINATION OF BOUND DEOXYNIVALENOL IN BARLEY GRAIN WITH TFA. 2 1 Bing Zhou , Yin Li , James Gillespie1, Richard Horsley1 and Paul Schwarz1* 1 Department of Plant Sciences, North Dakota State University, Fargo, ND 58105; and 2Department of Food Sciences and Nutrition, Zhejiang University, Hangzhou, 310029 P.R.China * Corresponding Author: PH: 701-231-7732; Email: Paul.Schwarz@ndsu.edu ABSTRACT Fusarium Head Blight (FHB) is an impediment to barley production in many regions of the world. Tricothecene toxins, associated with FHB infected grain, particularly deoxynivalenol (DON) pose a serious threat to human and animal health. Recent research has suggested that a portion of the DON present on grain is bound and escapes detection through conventional determination. The objective of this study was to optimize a method for determination of non-extractable DON in barley grain using TFA. A Doehlert matrix design was performed to determine the optimal conditions for time, temperature and TFA concentration. These conditions were treatment with 1.25 N TFA in 86:14 acetontrile:water for 54 min at 133°C. Clean-up, derivatization and determination of DON by GC-ECD was as normal. Treatment of the test sample resulted in release of an additional 58% DON under the optimized conditions, and an increase of 9 to 88% in a set of verification samples. 18 Session 1: Food Safety, Toxicology, and Utilization of Mycotoxin-contaminated Grain EFFECT OF ENZYME PRETREATMENTS ON THE DETERMINATION OF DEOXYNIVALENOL IN BARLEY. Bing Zhou2, James Gillespie1, Richard Horsley1 and Paul Schwarz1* Department of Plant Sciences, North Dakota State University, Fargo, ND 58105; and 2Biosystems Engineering and Food Sciences, Zhejiang University, Hangzhou 310029, People’s Republic of China * Corresponding Author: PH: 701-231-7732; Email: Paul.Schwarz@ndsu.edu 1 ABSTRACT The impact of particle size and enzymatic treatment was determined on the quantification of deoxynivalenol (DON) in FHB-infected barley samples. Particle size significantly affected DON determination in eight of ten samples analyzed. Significance of the barley sample x particle size interaction demonstrated that samples did not respond uniformly to the different particle sizes in terms of DON. The fine-grind samples often yielded higher results than medium and coarse grinds. This trend was most pronounced in samples with higher DON content. Enzyme treatments involved either amylolytic (alpha-amylase/amyloglucosidase), proteolytic (papain) or cell wall degrading (cellulase/xylanase) enzymes. The interaction between barley sample and enzyme treatment was significant, meaning that samples did respond uniformly to the three treatments. Papain treatment resulted in significant increases (16 to 28%) in the amount of DON detected in five of the seven samples tested when compared to the untreated samples or enzyme controls. Treatment with cellulase/xylanase resulted in increased DON in three of the seven samples, while amylase/amyloglucosidase resulted in increased DON in only a single sample. The results strongly indicated that FHB infected barley samples can contain bound DON which might not be determined in the routine quantification, but can be released by proteolytic or cell wall degrading activity. 19 SESSION 2: PATHOGEN BIOLOGY AND GENETICS Chairperson: Nancy Alexander Session 2: Pathogen Biology and Genetics QUANTITATIVE EXPRESSION OF FUSARIUM SPOROTRICHIOIDES GENES IN THE PRESENCE OF XANTHOTOXIN. N.J. Alexander* and S.P. McCormick Mycotoxin Research Unit, NCAUR, USDA/ARS, 1815 N. University St., Peoria, IL 61604 * Corresponding Author: PH (309) 681-6295; Email: nancy.alexander@ars.usda.gov ABSTRACT Xanthotoxin (8-methoxypsoralen) is a phototoxic furocoumarin that covalently binds to and crosslinks with DNA. It is also known to inhibit P450 oxygenases. To test the effect of xanthotoxin on gene expression in Fusarium sporotrichioides, we developed a reverse transcriptase, quantitative polymerase chain reaction (RTqPCR) method to measure the expression of genes involved in the biosynthetic pathway of trichothecenes. We found that xanthotoxin treatment of wild-type F. sporotrichioides blocked production of T-2 toxin and caused the accumulation of its hydrocarbon precursor, trichodiene. This suggested that FsTri5, the gene encoding trichodiene synthase, may be up-regulated and that FsTri4, a trichodiene oxygenase, may be downregulated. However, our RTqPCR results showed that 1 and 5 h after xanthotoxin treatment, both FsTri5 and FsTri4 were upregulated while FsTri101, encoding the trichothecene C-3 transacetylase, was downregulated. When FsTr4- mutants that accumulate trichodiene in culture were treated with xanthotoxin, trichodiene accumulation increased. Although the FsTRI4 protein is non-functional in these mutants, the RTqPCR showed that FsTri4 was transcribed and was up-regulated in the presence of xanthotoxin. These results suggest that xanthotoxin may be involved in the up-regulation of FsTri5 expression, but that factors other than gene regulation account for the increased accumulation of trichodiene. 23 Session 2: Pathogen Biology and Genetics GENETIC DIVERSITY OF FUSARIUM GRAMINEARUM POPULATIONS FROM CEREAL AND NON-CEREAL HOSTS. Rishi R. Burlakoti, Shaukat Ali, Gary A. Secor, Stephen M. Neate, Marcia P. McMullen and Tika B. Adhikari* Department of Plant Pathology, North Dakota State University, Fargo, ND 58105 * Corresponding Author: PH: (701) 231-7079; Email: tika.adhikari@ndsu.edu ABSTRACT Fusarium graminearum, a causal agent of Fusarium head blight (FHB) of wheat and barley, is of the most economically important pathogens of cereals worldwide. Although F. graminearum has been reported on non-cereal crops in the northern Great Plains, little is known about population structure of F. graminearum associated with non-cereal crops. We hypothesized that substantial genetic exchange has occurred between populations of F. graminearum across cereal and non-cereal hosts. In this study, we analyzed the genetic structure and the trichothecene diversity of four populations of F. graminearum collected from barley and wheat (cereals) and potato and sugar beet (non-cereals) hosts using ten variable number tandem repeat (VNTR) markers and primers designed from the genes involved in trichothecene biosynthesis. Both gene diversity (H = 0.449 to 0.616) and genotype diversity (GD = 0.984 to 0.998) were high, while estimates for linkage disequilibrium (r¯ d = 0.003 to 0.041) were low in F. graminearum populations, suggesting frequent recombination due to sexual reproduction. Our results further demonstrated that the deoxynivalenol (DON) genotype was the most frequently detected in the populations regardless of origin of host. The 3-acetyl (3-ADON) and 15acetyl DON (15-ADON) genotypes were commonly found in both cereal and non-cereal populations, however, the 15-ADON genotype was predominant. In addition, low genetic differentiation (Fst = 0.043) and genetic distance (D= 0.144) was observed between the cereal and non-cereal populations. Sequence analysis of the representative isolates from four hosts confirmed that F. graminearum populations belonged to phylogenetic lineage 7, further supporting the hypothesis of a single interbreeding population in the United States. 24 Session 2: Pathogen Biology and Genetics IDENTIFY AND CHARACTERIZE GENES REGULATED BY THE FMK1 MAP KINASE IN FUSARIUM GRAMINEARUM. Sheng-Li Ding1, Xiaoying Zhou1, H. Corby Kistler2 and Jin-Rong Xu1* Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907; and 2USDA-ARS, Cereal Disease Laboratory, Department of Plant Pathology, University of Minnesota, St. Paul, MN 55108 * Corresponding Author: PH: 765-496-6918; Email: jinrong@purdue.edu 1 ABSTRACT Fusarium graminearum is a devastating pathogen of wheat, barley, and maize throughout the world. The FMK1 gene encodes a well conserved MAP kinase that is essential for plant infection. To identify genes regulated by PMK1, in this study we conducted microarray experiments with the fmk1 mutant using the Fusarium graminearum Affimetrix GeneChip. In comparison with the wild-type strain, a total of 333 and 155 genes were down- and up-regulated (≥2-fold), respectively, in the fmk1 mutant. Functional classification of the probe sets revealed multiple processes were affected by the deletion of FMK1. Many of these genes were unique to F. graminearum. Forty four of them encoded putative transcription factors with DNAbinding motifs. We selected 12 genes with altered expression levels in the pmk1 for verification by qRT-PCR. Four of the genes verified by qRT-PCR were functionally characterized. While two other genes appeared to be dispensable for growth and pathogenesis in F. graminearum, deletion of the ATG8 homolog and a putative Zn2Cys6 transcription factor significantly reduced its virulence on flowering wheat heads. The ATG8 homolog in Magnaporthe grisea also was down-regulated in the pmk1 mutant, suggesting that this MAP kinase pathway may have a regulatory role in autophagy. Our results also were useful to determine the transcription regulatory network controlled by this well conserved MAP kinase pathway for fungal development and pathogenesis. 25 Session 2: Pathogen Biology and Genetics DIVERSITY IN FUSARIUM GRAMINEARUM SENSU STRICTO FROM THE U.S.: AN UPDATE. Liane R. Gale1*, Stephen A. Harrison2, Eugene A. Milus3, Jerry E. Ochocki4, Kerry O’Donnell5, Todd J. Ward5 and H. Corby Kistler1,4 Dept. of Plant Pathology, University of Minnesota, St. Paul, MN; 2Dept. of Agronomy, Louisiana State University, Baton Rouge, LA; 3Dept. of Plant Pathology, University of Arkansas, Fayetteville, AR; 4 USDA-ARS, Cereal Disease Laboratory, St. Paul, MN; and 5USDA-ARS, National Center for Agricultural Utilization Research Laboratory, Peoria, IL * Corresponding Author: PH: (612) 625-9266; Email: lianeg@umn.edu 1 ABSTRACT Efforts are ongoing to understand the population structure of Fusarium graminearum sensu stricto (Fg) in the U.S., its dynamics and its significance for small grain production. At previous FHB forums, we described the existence of genetically divergent populations of Fg in some regions of Minnesota and North Dakota (emergent populations) that were in the process of displacing the pre-existing population of Fg and that were also found to be more toxigenic, i.e. produced more deoxynivalenol (DON) on the susceptible variety Norm in greenhouse experiments. Recent population genetic analyses of 1,132 Fg strains from our 2006 collection indicated that the emergent populations are moving further south, as they were found to be present for the first time in South Dakota, at 3.5% of the total Fg population. Greenhouse experiments were conducted that assessed the toxigenic potential of these emergent populations on the commercially important cultivars Alsen, Knudson, Briggs, Freyr, Oklee and Granite that also represent various degrees of FHB susceptibility. Results from these experiments mirrored those from the initial experiments on Norm, i.e. substantially higher DON levels were obtained for all cultivars when inoculated with members of the emergent populations compared to when inoculated with member of the pre-existing Fg population. A second region that we also closely monitor is the southern United States. Previously, we reported that almost all Fg strains from Louisiana were nivalenol producers. Our 2007 collection from Louisiana originated from 17 commercial fields in three parishes. This collection was established to supplement Fg population information from nurseries. Very similar to population data from nurseries, nivalenol producers were predominant (79% of isolates). DON producers were mainly of a 3ADON trichothecene type (17% of isolates). Nivalenol producers also have been identified from Arkansas. From a limited sampling, 12% of isolates from Arkansas were nivalenol producers; among the DON producers the 15ADON trichothecene type was predominant (68% of isolates). Initial analyses of isolate genotypes established by using a PCR-RFLP marker system determined that overall the Fg population from Louisiana is genetically distinct from the Fg population that is commonly found in the Midwest. ACKNOWLEDGEMENT AND DISCLAIMER This material is based upon work supported by the U.S. Department of Agriculture. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture. 26 Session 2: Pathogen Biology and Genetics PHENOTYPIC AND MOLECULAR DIVERSITY OF FUSARIUM GRAMINEARUM SENSU STRICTO FROM THE U.S. Liane R. Gale1* and H. Corby Kistler1,2 Dept. of Plant Pathology, University of Minnesota, St. Paul, MN; and 2USDA-ARS, Cereal Disease Laboratory, St. Paul, MN * Corresponding Author: PH: (612) 625-9266; Email: lianeg@umn.edu 1 ABSTRACT Our long-term objectives are to accurately determine the composition and genetic structure of genetically coherent populations of FHB pathogens in the U.S., to evaluate their potential to change in composition and genetic structure over time and to determine the effect of such change on deployed host genotypes and/or other agricultural practices. To accomplish these goals we have established an extensive collection of Fusarium graminearum sensu stricto (Fg) strains from the U.S. gathered over eight years (1999-2007). Many of these strains originated from yearly disease surveys of the USDA-ARS Cereal Disease Laboratory, St. Paul, MN that cover routes of ca. 20,000 km of Midwestern and some Southern states. Other strains were contributed from collaborators or originated from directed sampling efforts of specific sites. To date, we have characterized about 7,500 U.S. Fg strains from 16 states using one or more markers or methods. Of particular usefulness is the molecular characterization of the trichothecene type. By using a multiplex PCR system developed by T. J. Ward (USDA-ARS, NCAUR, Peoria, IL) we can easily distinguish among the three trichothecene types of Fg, i.e. 15ADON, 3ADON or nivalenol (NIV). These trichothecene types accurately predict the specific chemotypes produced in host-pathogen interaction, i.e. 15ADON trichothecene type strains will produce [DON] > [15ADON] > [3ADON] and 3ADON trichothecene type strains will produce [DON] > [3ADON] > [15ADON], while the NIV trichothecene type will produce NIV. In the U.S. all three trichothecene types are present. While the NIV type of Fg has been identified from four states (LA, AR, MO, NC), it is currently common only in LA (ca. 80% of total strains) and AR (12% of strains). The likely presence of NIV in grain from these two states poses a problem insofar as NIV is currently not detected by commercial mycotoxin test kits. In addition to trichothecene type, we also have used a variety of molecular markers (RFLPs, VNTRs, PCR-RFLPs) to genotype strains. Genotyping allows us to further group strains into populations that are reproductively cohesive. Employing a population concept is important as each population may react to selective pressures in their own way. While most Midwestern states currently are populated by a genetically cohesive population of Fg with a predominant 15ADON trichothecene type, populations of Fg that are genetically distinct from this Midwestern 15ADON population have become very common in particular regions of MN and ND. These emerging populations are currently classified as the Upper Midwestern 3ADON population (UMW 3ADON) or as the Upper Midwestern 15ADON population (UMW 15ADON), depending on their trichothecene type. Members of both populations produce on average substantially more DON in greenhouse experiments on all cultivars tested compared to members of the Midwestern 15ADON population. Results of collections from 2006 indicate that the UMW 3ADON and UMW 15ADON populations are migrating further south and are now also present in South Dakota. Future proposed work will test the hypothesis of host genotype x pathogen chemotype/genotype interaction in field experiments. 27 Session 2: Pathogen Biology and Genetics ACKNOWLEDGEMENTAND DISCLAIMER This material is based upon work supported by the U.S. Department of Agriculture. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture. 28 Session 2: Pathogen Biology and Genetics STRUCTURAL AND FUNCTIONAL STUDIES OF TRICHOTHECENE BIOSYNTHETIC ENZYMES: A NOVEL APPROACH TO COMBATING FUSARIUM HEAD BLIGHT. Garvey, G.1, S.P. McCormick2 and I. Rayment1* University of Wisconsin, 453 Babcock Dr., Madison, WI 53706; and 2 USDA-ARS, NCAUR, 1815 N. University, Peoria, IL 61604 * Corresponding Author: PH: 608-262-0437; Email: ivan_rayment@biochem.wisc.edu 1 ABSTRACT Fusarium Head Blight (FHB) is a plant disease with serious economic and health impacts. Although it has proved difficult to combat this disease, one strategy that has been examined is the introduction of an indigenous fungal protective gene into cereals such as wheat, barley and rice. Thus far the gene of choice has been tri101 whose gene product catalyses the transfer of an acetyl group from acetyl Coenzyme A to the C3 hydroxyl moiety of several trichothecene mycotoxins. In vitro this has been shown to reduce the toxicity of the toxins by ~100 fold but has demonstrated limited resistance to FHB in transgenic cereal. In order to understand the molecular basis for the differences between in vitro and in vivo resistance the three-dimensional structures and kinetic properties of two TRI101 orthologs isolated from Fusarium sporotrichioides and Fusarium graminearum have been determined. The kinetic results reveal important differences in activity of these enzymes towards B-type trichothecenes such as deoxynivalenol. These differences in activity can be explained in part by the three dimensional structures for the ternary complexes for both these enzymes with Coenzyme A and trichothecene mycotoxins. The structural and kinetic results together emphasize that the choice of an enzymatic resistance gene in transgenic crop protection strategies must take into account the kinetic profile of the selected protein. Examination of the trichothecene biosynthetic pathway suggest that other enzymes might provide a more suitable scaffold for engineering new degradative activities for improved resistance. Therefore, it is planned to continue the biochemical characterization and three-dimensional structure determination of the remaining enzymes in the biosynthetic pathways for deoxynivalenol, nivalenol, and T-2 toxin. To date the crystal structure for FsTRI3 both apo and in complex with 15-decalonectrin have been determined and the kinetics of this enzyme towards native substrate and final toxins evaluated. This structural information will be used to create new enzymes by directed evolution utilizing a yeast selection system to detect new activities that degrade or inactivate the toxins. 29 Session 2: Pathogen Biology and Genetics FUNCTIONS OF THE SEX PHEROMONES OF GIBBERELLA ZEAE. J. Lee1, J.F. Leslie1 and R.L. Bowden2* Department of Plant Pathology, Kansas State University, Manhattan, KS; and 2 USDA-ARS Plant Science and Entomology Research Unit, Manhattan, KS * Corresponding Author: PH: (785) 532-2368; Email: robert.bowden@ars.usda.gov 1 ABSTRACT In heterothallic ascomycete fungi, idiomorphic alleles at the MAT locus control two sex pheromone/receptor pairs that function in recognition and attraction of strains with opposite mating types. In the ascomycete Gibberella zeae, the MAT locus is rearranged such that both alleles are adjacent on the same chromosome. Strains of G. zeae are self-fertile, but they can outcross facultatively. Our objective was to determine if pheromones retain a role in sexual reproduction in this homothallic fungus. Putative pheromone precursor genes (ppg1 and ppg2) and their corresponding pheromone receptor genes (pre2 and pre1) were identified in the genomic sequence of G. zeae by sequence similarity and microsynteny with other ascomycetes. ppg1, a homolog of the Saccharomyces α-factor pheromone precursor gene, was expressed in germinating conidia and mature ascospores. Expression of ppg2, a homolog of the a-factor pheromone precursor gene, was not detected in any cells. pre2 was expressed in all cells, but pre1 was expressed weakly and only in mature ascospores. Deletion mutations ∆ppg1 or ∆pre2 reduced fertility in self-fertilization tests. ∆ppg1 reduced male fertility and ∆pre2 reduce female fertility in outcrossing tests. In contrast, ∆ppg2 and ∆pre1 had no discernable effects on sexual function. ∆ppg1/∆ppg2 and ∆pre1/∆pre2 double mutants had the same phenotype as the ∆ppg1 or ∆pre2 single mutants. Thus, one of the putative pheromone/receptor pairs (ppg1/pre2) enhances, but is not essential for, selfing and outcrossing in G. zeae, whereas no functional role was found for the other pair (ppg2/ pre1). 30 Session 2: Pathogen Biology and Genetics ISOLATION OF TWO XYLANASE FROM FUSARIUM GRAMINEARUM. S.W. Meinhardt1*, X. Dong2 and P.B. Schwarz2 Department of Plant Pathology, and 2Department of Plant Sciences, North Dakota State University, Fargo, ND * Corresponding Author: PH: (701) 231-7944; Email: steven.meinhardt@ndsu.edu 1 ABSTRACT Fusarium head blight (FHB), caused by Fusarium graminearum (teleomorph Gibberella zeae), results in severe yield losses and crop quality reductions in wheat and barley, and is the predominant species of the FHB complex in North American. In addition to the toxins produced by the pathogen, cell wall degrading enzymes secreted by the pathogen may be involved in pathogenesis. The objective of this project was to purify and characterize xylanase(s) from F. graminearum. The in-vitro production of xylanase(s) by F. graminearum was obtained from cultures grown at 25°C for 5 days on modified synthetic media agar supplemented with sterile wheat bran. Xylanase activity was extracted by soaking one plate of the wheat bran agar in 100 ml of 100 mM sodium acetate buffer pH 4.5. Two xylanases have been purified 52- and 40- fold by a combination of ion-exchange, gel filtration, HPLC ion-exchange and HPLC hydrophobic interaction chromatography. The two xylanases were separated by the first ion-exchange step, and were then processed individually through subsequent steps. The purity and the relative molecular weights of the xylanases was estimated by SDS-PAGE to be 20 and 40 KDa, respectively. Only a single band was observed for each enzyme. The two xylanases were identified by trypsin digestion followed by LC-MS/MS as the gene products of FG03624 and FG06445. In the mass spectrometer, the high molecular weight xylanase, FG06445, 87% of the sequence was observed while for the low molecular weight xylanase, FG03624, 62% of the sequence was identified. After removal of the predicted signal sequence, the predicted molecular masses and iso-electric points were 22 and 38 KDa, and pH 9.2 and 8.5 for FG03624 and FG06445, respectively. 31 Session 2: Pathogen Biology and Genetics SPORE DEVELOPMENT AND TRICHOTHECENE MUTANTS OF FUSARIUM GRAMINEARUM. Matias Pasquali1, Kye-Yong Seong1, Jon Menke1, Erik Lysøe2, Karen Hilburn3, Jin-Rong Xu4 and H. Corby Kistler1,3* 1 Department of Plant Pathology, University of Minnesota, St. Paul, MN 55108, USA; 2Bioforsk - Norwegian Institute of Agricultural and Environmental Research, Ås, Norway; 3USDA ARS Cereal Disease Laboratory, University of Minnesota, St. Paul, MN 55108, USA; and 4Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907, USA * Corresponding Author: PH: (612) 625-9774; Email hckist@umn.edu. ABSTRACT To understand trichothecene accumulation and the infection cycle of the head blight pathogen F. graminearum sensu stricto, fungal gene expression profiles were monitored during germination of ascospores and during plant infection. A total of 328 genes were determined to be specifically expressed in ascospores. Among genes highly up-regulated in ascospores was one most closely related to FoStuA of F. oxysporum and StuA in Aspergillus. Mutants deleted for this gene in F. graminearum (FgStuA) are greatly decreased in sporulation and produce no perithecia. Unlike FoStuA mutants in F. oxysporum, FgStuA mutants are greatly reduced in pathogenicity. Reduced pathogenicity may be due to decreased levels of trichothecene toxins, which in the mutant are <1% the levels of wildtype. Levels of transcripts corresponding to Tri5, but not other genes involved trichothecene biosynthesis, were extremely diminished in the FgStuA mutant. Thus both sporulation and trichothecene synthesis may be regulated under the control of StuA. We are also developing isogenic lines of F. graminearum that differ only at the toxin biosynthesis cluster, in order to understand how DON and the chemical profile of trichothecene derivatives (trichothecene chemotype) influences fungal pathogenicity. The trichothecene biosynthetic gene cluster has been completely deleted from both a deoxynivalenol (DON) and a nivalenol producing strain of F. graminearum and will be replaced with the cluster from a different chemotype. Five separate genes from the cluster also have been individually deleted. Biological and regulatory characteristics of the mutant strains will be discussed. ACKNOWLEDGEMENT AND DISCLAIMER This material is based upon work supported by the U.S. Department of Agriculture. This is a cooperative project with the U.S. Wheat and Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed are those of the authors and do not necessarily reflect views of the U.S. Department of Agriculture. 32 Session 2: Pathogen Biology and Genetics STRUCTURAL AND FUNCTIONAL STUDIES OF TRICHOTHECENE 3O-ACETYLTRANSFERASE: PROGRESS TOWARDS DEVELOPMENT OF AN IMPROVED ENZYME FOR CONTROLLING FHB. Ivan Rayment Department of Biochemistry, 433 Babcock Drive, University of Wisconsin, Madison, WI 53706 Corresponding Author: PH: 608-262-0437; E-mail: ivan_rayment@biochem.wisc.edu ABSTRACT Biological control of Fusarium Head Blight (FHB) is a difficult and complex problem. One strategy that has been examined is the introduction of an indigenous fungal protective gene into cereals such as wheat, barley and rice. Thus far the gene of choice has been tri101 whose gene product catalyses the transfer of an acetyl group from acetyl Coenzyme A to the C3 hydroxyl moiety of several trichothecene mycotoxins. In vitro, this has been shown to reduce the toxicity of the toxins by ~100 fold but has demonstrated limited resistance to FHB in transgenic cereal. The reasons for this lack of success are unclear. Thus, a study to investigate the chemical framework that underlies the trichothecene biosynthetic pathway has been initiated with the goal of understanding the molecular basis for the differences between the in vitro and in vivo resistance. To this end the three-dimensional structures and kinetic properties of two TRI101 orthologs isolated from Fusarium sporotrichioides and Fusarium graminearum have been determined. The kinetic results reveal important differences in activity of these enzymes towards B-type trichothecenes such as deoxynivalenol. These differences in activity can be explained in part by the three dimensional structures for the ternary complexes for both these enzymes with Coenzyme A and trichothecene mycotoxins. The structural and kinetic results together emphasize that the choice of an enzymatic resistance gene in transgenic crop protection strategies must take into account the kinetic profile of the selected protein. The structural and functional studies now suggest that the enzymatic activity, stability, and solubility of Tri101 can be improved quite readily by protein engineering. This represents an exciting opportunity to utilize the fundamental knowledge of a pathogen’s biosynthetic pathway to modify the biochemical and biophysical characteristics an enzyme so that it can provide improved protection against FHB. 33 Session 2: Pathogen Biology and Genetics TRICHOTHECENE CHEMOTYPES OF ISOLATES OF GIBBERELLA ZEAE RECOVERED FROM WHEAT IN ARGENTINA. M.M. Reynoso1, M.L Ramirez1, J.F. Leslie2 and S.N. Chulze1* Departamento de Microbiología e Immunología, Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto, Ruta Nacional 36 Km 601, 5800 Río Cuarto, Códoba, Argentina; and 2Department of Plant Pathology, Kansas State University, Manhattan, KS 66506-5502, USA * Corresponding Author: PH: 54 358 4676429; Email: schulze@exa.unrc.edu.ar 1 ABSTRACT Wheat production in Argentina covers about 6.24 million hectares. Production reached 15 million tons during the 2006 harvest season, ranking Argentina as the fourth largest exporter in the world. The main pathogen associated with Fusarium Head Blight (FHB) in Argentina is Gibberella zeae (Schwein.) Petch (anamorph Fusarium graminearum Schwabe), which reduces both grain quality and yield. Wheat grains infected with G. zeae often are contaminated with a Type B trichothecene, usually deoxynivalenol (DON) or nivalenol (NIV), that is toxic to humans and domesticated animals. Strains of G. zeae usually express one of three sets of trichothecene metabolites (chemotypes): (i) nivalenol and acetylated derivatives (NIV chemotype), (ii) deoxynivalenol and 3-acetyldeoxynivalenol (3-ADON chemotype), and (iii) deoxynivalenol and 15acetyldeoxynivalenol (15-ADON chemotype). Other Fusarium isolates that can produce both deoxynivalenol and nivalenol (NIV/DON) have been described and can not be assigned to any of these three chemotypes. We used a multiplex PCR assay to identify the trichothecene chemotype of 123 strains of G. zeae lineage 7 (identified by AFLP) isolated from 3 localities (San Antonio de Areco, Alberti and Marcos Juarez) within the main Argentinean wheat production area. Most (> 92%) of the Argentinean isolates of G. zeae had the 15ADON chemotype, with the remainder having the NIV/DON chemotype. We did not detect the NIV or the 3-ADON chemotypes. Results from the PCR assays were consistent with those obtained by chemical analyses for all strains that produced trichothecenes. Knowledge of the chemotypes present in the G. zeae population is important when conducting mycotoxin surveys, implementing breeding programs, and identifying new and emerging populations of this fungal pathogen. 34 Session 2: Pathogen Biology and Genetics TRICHOTHECENE MYCOTOXIN GENOTYPES OF GIBBERELLA ZEAE IN BRAZILIAN WHEAT. L.B. Scoz1, P. Astolfi1, D.S. Reartes1, D.G. Schmale III2, M.G. Moraes1 and E.M. Del Ponte1* Departmento de Fitossanidade, Faculdade de Agronomia, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 7712, 91540000, Porto Alegre, RS, Brasil; and 2Department of Plant Pathology, Physiology, and Weed Science, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, 24061, USA * Corresponding Author: PH: (55-51) 33086908; Email: emerson.delponte@ufrgs.br 1 ABSTRACT Fusarium head blight (FHB), caused by Gibberella zeae, is a disease of increasing concern to wheat production in Brazil. Infested grain may be contaminated with trichothecene mycotoxins such as deoxynivalenol (DON) and nivalenol (NIV), posing a significant threat to the health of humans and domestic animals. Little is known about the mycotoxin potential of strains of G. zeae in Brazil. We obtained 82 single-spored strains of G. zeae from infected kernel samples originating from twenty locations in southern Brazil. Polymerase chain reaction (PCR) assays were used to characterize trichothecene mycotoxin genotypes of G. zeae (genetic profiles associated with the production of DON, NIV, and two acetylated derivatives of DON) and to assist in the identification of strains to species. To identify strains of G. zeae that may produce DON and NIV, we amplified portions of Tri5 and Tri7. To identify strains of G. zeae that produce 3-acetyl-deoxynivalenol (3-ADON) and 15-acetyl-deoxynivalenol (15-ADON), we amplified portions of Tri3 and Tri12. Nearly all of the strains studied (76/82) were of the DON/15-ADON genotype. Six of the strains were of the NIV genotype. We did not observe the 3-ADON genotype in our samples. The NIV genotype was observed in multiple samples from the same field and was present in all three southern states of Brazil studied. This is the first detailed report of trichothecene mycotoxin genotypes of G. zeae in southern Brazil. Additional information is needed to better determine the relative impact of different trichothecene mycotoxins in Brazilian wheat, and to employ appropriate methodologies for detecting mycotoxin contamination in the future. We are currently expanding our assays to screen for trichothecene mycotoxin genotypes in other geographic regions of Brazil, across additional growing seasons, and in other hosts such as barley and oats. 35 Session 2: Pathogen Biology and Genetics POPULATION OF FUSARIUM GRAMINEARUM SCHWABE ASSOCIATED WITH HEAD AND SEEDLING BLIGHT IN SLOVAKIA. A. Šrobárová1 and N. Alexander2* Institute of Botany Slovak Academy of Sciences, Dúbravská cesta 14, SK-845 23 Bratislava, Slovakia; and 2Mycotoxin Research Unit, National Center for Agricultural Utilization Research, 1815 N. University St., Peoria, IL 61604 USA * Corresponding Author: PH: 309-681-6295; E-mail:Nancy.Alexander@ars.usda.gov 1 MATERIALS AND METHODS OBJECTIVES To determine any significant differences among popu- Cultures: Fusarium spp. were isolated from the carylation isolates of F. graminearum from wheat in opses of wheat stands in Slovakia during 2000 and Slovakia in cultural and pathogenicity assays in vitro 2001. and in vivo. Culture conditions: The single-spore isolates from 2%- water agar (WA) were grown on potato-dexINTRODUCTION trose agar (PDA) from Difco Laboratories (Detroit, The growth of Fusarium species associated with MI) using 40 grams in 1L distilled H2O, pH 6 (± 0.2). Fusarium Head Blight (FHB) varies depending on ag- Radial growth rates of all isolates were determined ronomic characters and edaphic conditions (Bottalico by measuring colony diameters of single conidial culand Perrone, 2002). We have identified fifteen tures on PDA in 90-mm-diameter Petri dishes. The Fusarium species during the ten years of our investi- colony growth and sporulation were measured each gations in the Slovak Republic. The most commonly of three days. Cultures were incubated for 5 weeks in identified Fusarium species involved in FHB in wheat a 14-h photoperiod at 22°C by day and 15°C by night. were F. graminearum Schwabe and F. culmorum Measurements were made on three replicate cultures (W.G.Smith) Sacc. (Šrobárová and Vašková,1987). that each originated from single conidia per each strain. Both species produce mycotoxins, such as deoxynivalenol (DON) and zearalenone (ZEN) that Pathogenicity tests: Strains were assayed for pathocan reduce the quality of grain. A recent study we car- genicity by inoculating seedlings of wheat cv. Torysa, ried out demonstrated a drift in the populations from a wheat cultivar that is moderately resistant to F. culmorum (W. G. Smith) Sacc. to F. graminearum Fusarium infection (Pavlová and Šrobárová, 1998). Schwabe. Our hypothesis is that F. graminearum is Seeds were surface-sterilized with 1% sodium hya more aggressive species perhaps by producing more pochlorite (diluted 5% commercial bleach) for 2 min toxin as it invades the plant tissue, by adapting to cli- and rinsed three times in sterile distilled water for 2 matic conditions better, or by having some other se- min. After rinsing with sterile water, the seeds were lective advantage over F. culmorum. Strains of F. placed into Petri dishes (d = 90mm) on wet filters and graminearum harvested from infected wheat in kept in the dark for 2 days at 22 °C. The imbibed Slovakia during the years 2000 and 2001 were a seeds were transferred into test jars (150 by 100 mm) source for our study. containing 15 mL of solidified sterile 0.6% WA (ten uniform seedlings per jar). The jars were covered with sterile aluminum and incubated for 10 days with a 14h photoperiod at 22°C by day and 15°C by night. For 36 Session 2: Pathogen Biology and Genetics each of the 12 Fusarium isolated, inoculum was prepared and seedlings were inoculated with 0.5 mL of a 1x105spores/ml suspension and incubated for 10 additional days under the same conditions as those use for the initial growth of the seedlings. Controls were inoculated with 0.5 mL of sterile potato dextrose broth. Plants were rated visually for disease severity on a 0 to 5 scale reflecting the proportion of the root system with visual lesions as described in Wildermuth and McNamara (1994). Analysis of variance was performed and disease severity ratings were ranked. Fresh and dry weight (the seedlings were dried in an incubator at 105°C) were taken. Redascreen fast deoxynivalenol (DON) kit (R-Biopharm GmbH, Darmstadt, Germany) was used for semiquantity measurements of DON, according to the manufacturer’s instructions. Toxin levels: The highest levels of DON (Table1) were produced in vitro by isolates of F. graminearum #4 and #5. The lowest levels were produced by isolates #7 and #12. RESULTS DISCUSSION Vegetative growth: After 3 days of incubation at constant temperature, all F. graminearum isolates had grown significantly on PDA. Radial growth rates for all isolates (Table 1) were similar at 22°C, ranging from a mean of 22 mm (#2 isolate) to 46 mm (isolates 7 and 12) by 72 h The greatest differences were seen on the fourth day when the average difference between the slowest growth and the fastest growth was 3.1 cm. By day 6, almost all the isolates had reached the edge of the plate (9.0 cm) Low levels of genetic differentiation among geographic regions yet high levels of genetic variation within populations have been reported for the sexually reproducing wheat pathogen F. graminearum (Dusabenyagasani et al., 1999; Miedaner et al., 2001; Leslie et al. 2007). Our data also suggests genetic variation among populations isolated from distinct regions of Slovakia. Traditionally, species differentiation has been based on morphological characteristics. As the interest in Fusarium has increased during the last two decades as a result of the increased devastation of Fusarium Head Blight (FHB) worldwide, more efforts have been extended on using molecular techniques to characterize the populations of Fusarium. Although it has been suggested that F. graminearum consists of at least 9 separate species (O’Donnell et al. 2004), it appears that there is only a single species within F. graminearum (Leslie et al. 2007). Within this species, there is genetic variation in morphology, pathogenicity, and gene sequence variation. The pigmentation of the reverse side of the colony was usually carmine red for all the F. graminearum isolates while aerial mycelium was white to carmine red. No unusual colors or colony morphology were seen among the isolates. Perithecium were formed on WA in thirty to forty days except for two isolates, Michalovce #7 and Šariš #12, which did not form perithecium within the allotted time frame (Table 1 ). Pathogenicity: Relative pathogenicity was examined under laboratory conditions for all 12 isolates. All strains were pathogenic to wheat seedlings, as indicated by disease severity rankings (Fig.1). The highest degree of infection (DI) was measured for #4, #5, #6, #10, and #11 isolates of F. graminearum, but all isolates showed a degree of 3 or more. The DI of the controls ranged from 0.2 to 0.3. The isolates of F . graminearum may be said to be strongly aggressive but were not significantly different from one another. Based on mean values, they were significantly more virulent than the water controls to the seedlings. Fresh weights and dried weights of the plants infected with the Fusarium isolates were compared to control plants (Figure 2). Plants infected with isolates #7 and #8 had the lowest fresh weight while #9 and #12 had the highest. Almost all of the plants had a similar dry weight, except those inoculated with isolate #3 while the lowest were #2 and #7. In pathogenicity tests on wheat seedlings, Miedaner et al. (2000, 2001) found a variation of aggressiveness among F. graminearum isolates. Our results show there is no precise correlation in fresh and dry weight of infected seedlings among the Fusarium isolates. Variation in aggressiveness is associated with 37 Session 2: Pathogen Biology and Genetics the genetic diversity of this species and is most likely due to the amount of toxin produced by the isolate (Goswami and Kistler (2005). There is a positive correlation between head blight and DON (Proctor et al. 1995) however, mutants unable to produce toxin are still able to initiate infection (Bai et al., 2001) which suggests that aggressiveness is correlated with the amount of toxin produced. The results presented in this study show that all of our isolates are capable of producing DON in vitro, however, there was no precise correlation between the amount of DON produced and the degree of infection. REFERENCES Bai, G.-H., Desjardins, A.E. and Plattner, R.D. 2001.Deoxynivalenol – nonproducing Fusarium graminearum causes initial infection, but does not cause disease spread in wheat spikes. Mycopathologia 153: 91 – 98. Bottalico, A. and Perrone, G. 2002. Toxigenic Fusarium species and mycotoxins associated with head blight in smallgrain cereals in Europe. European Journal of Plant Pathology 108:611-624. Dusabenyagasani, D., Dostaler, D. and Hamelin, R.C. 1999. Genetic diversity among Fusarium graminearum strains from Ontario and Quebec. Can. J. Plant Pathol. 21: 308–314. 38 Goswami, R.S. and Kistler, H.C. 2005. Pathogenicity and in planta mycotoxin accumulation among members of the Fusarium graminearum species complex on wheat and rice. Phytopathology 95:1397-1404. Leslie, J.F., Anderson, L.L., Bowden, R.L., and Lee, Y-W. 2007. Inter-and intra-specific genetic variation in Fusarium. Int. J. Food Microbiol. Doi:10.1016/j.ijfoodmicro.2007.07.059. Miedaner, T., Reinbrecht, C., Schilling, A.G. 2000. Association among aggressiveness, fungal colonization, and mycotoxin production of 26 isolates of Fusarium graminearum in winter rye head blight.Z. Pflkrankh. Pflschutz 107:124-134. Miedaner, T., Schilling, A. G. and Geiger, H. H. 2001. Molecular genetic diversity and variation for aggressiveness in populations of Fusarium graminearum and Fusarium culmorum sampled from wheat fields in different countries. J. Phytopathology 149: 641-648. O’Donnell, K., Ward, T.J., Geiser, D.M., Kistler, H.C., and Aoki, T. 2004. Genealogical concordance between the mating type locus and seven other nuclear genes supports formal recognition of nine phylogenetically distinct species with the Fusarium graminearum clade. Fungal Genetics and Biology 41:600-623. Proctor, R.H., Hohn, T.M. and McCormick, S.P. 1995. Reduced virulence of Gibberella zeae caused by disruption of a trichothecene toxin biosynthetic gene. Mol. Plant-Microbe Interact. 8:593-601. Session 2: Pathogen Biology and Genetics Wildermuth, G.B.and Mc Namara, R.B.1994. Testing wheat seedling for resistance to crown rot caused by Fusarium graminearum group 1, Plant Dis. 78: 949-953. Pavlová, A., and Šrobárová, A.1998. Resistance of winter wheat cultivars after inoculation of the seedlings and heads by Fusarium culmorum (W.G. Sm.) Sacc. Agriculture 5/6: 432448. Šrobárová , A. and Vašková, M. 1987. Fusarium spp. associated with scab of wheat. Ochr. Rostl. 23/4: 279–282. 6 5 infection 4 3 2 1 0 1 2 3 4 5 7 8 9 10 11 12 C isolates Figure 1. Degree of infection of seedlings of wheat cv.Torysa by a water control (C) and isolates 1-12 of F .graminearum Schwabe. fresh weight drought weight 4,5 4 mg 3,5 3 2,5 2 1,5 1 0,5 0 1 2 3 4 5 6 7 8 9 10 11 12 isolates Figure 2. Fresh and dry weights of seedlings of cv. Torysa inoculated with F. graminearum isolates. 39 Session 2: Pathogen Biology and Genetics LIFE CYCLE AND SURVIVAL OF FUSARIUM GRAMINEARUM. Frances Trail Department of Plant Biology, Department of Plant Pathology, Michigan State University, E. Lansing, MI 48824 Corresponding Author: PH: (517) 432-2939; Email: trail@msu.edu ABSTRACT We have been studying the life cycle of F. graminearum in association with wheat. Each stage of the life cycle is intimately tied to the host life cycle. Infection occurs primarily through ascospores during host flowering. Following infection, the fungus must colonize the stalk and store lipids before the plant senesces. Stored lipids in hyphae are then used to fuel sexual development and spore production for the next disease cycle. In fungi, lipids are stored in vegetative hyphae and spores as lipid bodies. Lipid-filled hyphae produce perithecium initials in association with stomates along the stems and in association with silica cells at the notes. These initials go dormant and become competent to form perithecia during the final stages of grain maturation before harvest. After harvest, the dormant hyphae in the crop residue protect their resources by secreting antimicrobials. Consideration of these aspects of the life cycle of this pathogen will allow us to use controls such as fungicides and biological control agents in a more effective manner. 40 Session 2: Pathogen Biology and Genetics UPDATE ON THE LIFE CYCLE OF FUSARIUM GRAMINEARUM. Frances Trail1,2*, John C. Guenther1, Heather Hallen1, Brad Cavinder1 and Lilly Yu1 Department of Plant Biology, and 2Department of Plant Pathology, Michigan State University, E. Lansing, MI 48824 * Corresponding Author: PH: (517) 432-2939; Email:trail@msu.edu 1 ABSTRACT We have focused our studies on 3 stages of the life cycle that may provide opportunities for introducing novel controls. The process of forcible ascospore discharge launches the primary inoculum of the head blight disease from crop residues. The fungus must heavily colonize the crop tissue and store lipids in order to survive the winter and produce perithecia. We have characterized the process of lipid accumulation and utilization in association with perithecium development in culture and leading up to perithecium development in planta. Lipid-filled hyphae must protect their resources in crop residues until they use them to generate perithecia. We will present our latest findings on each of these stages as they are particularly vulnerable and may present targets for new controls. 41 Session 2: Pathogen Biology and Genetics TRICHOTHECENE CHEMOTYPE COMPOSITION OF FUSARIUM GRAMINEARUM AND RELATED SPECIESIN FINLAND AND RUSSIA. T. Yli-Mattila1*, K. O’Donnell2, T. Ward2 and T. Gagkaeva3 1 Lab. of Plant Phys. and Mol. Biol., Dept of Biology, Univ. of Turku, FIN-20014 Turku, Finland; 2Microbial Genomics Research Unit, USDA-ARS, Peoria, USA; and 3Dept. of Mycology and Phytopathology, All-Russian Institute of Plant Protection (VIZR), St. Petersburg, Russia * Corresponding Author: PH: 358-2-3336587; Email: tymat@utu.fi ABSTRACT Fusarium head blight (FHB) caused by Fusarium graminearum and related Fusarium species is an important fungal disease of cereals worldwide. FHB pathogens cause significant yield and quality losses and they pose a serious threat to food safety. F. graminearum isolates can be divided based on mycotoxin production into 3 main chemotypes (3ADON, 15ADON and NIV), which can be identified by SNP genotyping. These chemotypes are not species-specific. Isolates with the NIV chemotype are more toxigenic than those producing either 3ADON or 15ADON. The species and chemotype composition of 286 single-spore isolates causing FHB collected between 19862006 in different parts of Russia, Finland, China and Germany was investigated using a multilocus genotyping assay (MLGT) including multiplex PCR with six primer pairs followed by allele-specific primer extension (ASPE) utilizing 38 species- and chemotype-specific probes. Hybridization and detection were performed using a Luminex 100 flow cytometer. All F. graminearum isolates from Finland (15) and western Russian (23) possessed the 3ADON chemotype, while all isolates from southern Russia (43) except for one from barley and one from corn possessed the 15ADON chemotype. In other parts of Russia and northern China isolates with the 3ADON and 15ADON chemotype were both present. The only F. graminearum isolate with the NIV chemotype was from Germany. All (27) F. culmorum isolates (Finland and Russian Federation) possessed the 3ADON chemotype. In contrast, all six isolates of F. cerealis possessed the NIV chemotype. These results are in accordance with previous mycotoxin analyses of pure cultures of Finnish FHB isolates on rice and analyses of field samples. In Finland there were no differences in the F. graminearum chemotype composition between the years 1986-93 and 2001-2004, while in the Far East (85 isolates) the 3ADON chemotype frequency increased between the years 1998-2006. This apparent shift in trichothecene chemotype frequency is similar to recently observed shifts in FHB pathogen composition within North America. Two Russian F. graminearum isolates, one from southern Russia and one from Siberia, produced a positive signal with a 3ADON and 15ADON MLGT probe from opposite ends of the trichothecene gene cluster, suggesting that it may reflect recombination between isolates with these two chemotypes. Six single-spored isolates from this isolate gave the same result. Twelve isolates (ten from Far East and two from Siberia) produced unusually low positive signals for the F. graminearum probes, but they were all clearly positive for the B-clade (species producing B type trichothecenes) and the F. graminearum species complex probes. These isolates likely harbor previously unrecognized variation at the probe sites and will be sequenced to confirm the species identification and to inform additional probe design. 42 SESSION 3: GENE DISCOVERY AND ENGINEERING RESISTANCE Chairperson: Blake Cooper Session 3: Gene Discovery and Engineering Resistance STUDIES ON BARLEY SPIKES TREATED WITH THE TRICHOTHECENE, DEOXYNIVALENOL: INSIGHT INTO BARLEY-FUSARIUM GRAMINEARUM INTERACTION. Jayanand Boddu and Gary J Muehlbauer* Department of Agronomy and Plant Genetics, University of Minnesota, 1991 Buford Ave, St Paul, MN 55108 * Corresponding Author: PH: 612-625-6228; Email: muehl003@umn.edu ABSTRACT Fusarium head blight (FHB) of barley and wheat is a difficult disease to manage because of the complexity of the interactions. A serious problem associated with FHB is the accumulation of trichothecene mycotoxins such as deoxynivalenol (DON). Trichothecenes increase the virulence of the pathogen and reduce grain quality. A primary objective in our laboratory is to identify genes that reduce the impact of trichothecenes. Our laboratory has identified approximately 700 barley transcripts that respond to the invading pathogen and pathogenderived trichothecenes. In an effort to further understand the barley-F. graminearum interaction, a subset of 54 genes encoding transcription factors, regulatory proteins, UDP-glucosyltransferases, cytochrome-P450s, and proteins participating in ubiquitination and cell death were selected and tested for their response to DON treatment compared to mock water inoculation at 1, 6 and 12 hours after inoculation (hai). Twenty-one transcripts showed a qualitative response and 28 transcripts showed quantitative response to DON treatment. Seven of the qualitatively responding genes responded by 1 hai, while 14 genes responded by 6 hai. All the quantitatively responding genes showed differential expression from 1 hai through 12 hai. To develop markers for mapping and other genetic studies, some of these genes were sequenced from barley mapping population parents and genotypes exhibiting FHB resistance and susceptibility. In separate experiments, the fate of DON in planta was tested. In barley spikes treated with DON, over 30.0% was converted to DON-3-O-glucoside. In addition, our preliminary experiments show a cell death-like phenotype on DON-treated barley leaves progressed in a distal manner, indicating that either DON or the signal transduction induced by DON traveled to the tip of the treated leaves. 47 Session 3: Gene Discovery and Engineering Resistance EXPRESSION OF A TRUNCATED FORM OF RIBOSOMAL PROTEIN L3 IN TRANSGENIC WHEAT CONFERS RESISTANCE TO DEOXYNIVALENOL AND FUSARIUM HEAD BLIGHT. Rong Di1, Ann Blechl2, Ruth Dill-Macky3, Andrew Tortora1 and Nilgun E. Tumer1* Biotechnology Center, Cook College, Rutgers University, New Brunswick, NJ; 2 USDA-ARS, Western Regional Research Center, Albany, CA; and 3 Department of Plant Pathology, University of Minnesota, St. Paul, MN * Corresponding Author: PH: (732) 932-8165 ext. 215; Email: tumer@aesop.rutgers.edu 1 ABSTRACT DON belongs to the group of trichothecene toxins, which target ribosomal protein L3 at the peptidyltransferase site of eukaryotic ribosomes and inhibit protein synthesis. The goal of our work is to identify mutations in L3 that confer resistance to DON and to determine if FHB resistance can be engineered in transgenic wheat plants by expressing DON resistant L3 genes. In previous studies, we have demonstrated that overexpression of a truncated form of yeast ribosomal protein L3 (L3∆) in transgenic tobacco plants confers resistance to deoxynivalenol (DON). To determine if expression of the yeast L3∆ in transgenic wheat plants would provide resistance to FHB, the susceptible spring wheat cultivar, Bobwhite was transformed with the yeast L3∆ under the control of the barley floret-specific Lem1 or the maize constitutive Ubi1 promoter. Three homozygous Lem1::yeast L3∆ lines (771, 772 and 773) and two homozygous Ubi1::yeast L3∆ lines (8133 and 8153) were evaluated for resistance to FHB in greenhouse tests. The disease severity was reduced by 48-56% in four different transgenic wheat lines compared to the untransformed Bobwhite plants. The reduction in disease severity correlated well with the level of expression of L3∆ mRNA. These results demonstrated that transgenic wheat plants expressing the yeast L3∆ showed improved resistance to FHB over the untransformed Bobwhite plants. To determine if resistance to FHB would result in a reduction in DON levels, the mature kernels above and below the inoculated spikelets were analyzed for DON levels. There was a 63-76% reduction in DON levels in the four different FHB resistant transgenic lines. The DON levels in one transgenic line were lower than the DON levels in the resistant line, Alsen. These results provided evidence that resistance to DON correlates with resistance to FHB and results in reduced accumulation of DON in transgenic wheat plants. We have identified four more homozygouns lines containing Lem1::yeast L3∆, eight more homozygous lines containing Ubi1::yeast L3 and four new homozygous lines containing Lem1::yeast L3, which will be evaluated for resistance to FHB. The wheat RPL3A1 and RPL3B3 genes were cloned and wheat expression vectors were constructed with the L3∆ versions of these genes. Point mutations that confer a high degree of resistance to DON were introduced into the wheat RPL3A1. We have generated transgenic wheat plants containing the DON resistant forms of the wheat L3 genes to determine if their expression will lead to a higher level of resistance to FHB and a greater reduction in DON accumulation. 48 Session 3: Gene Discovery and Engineering Resistance INHIBITION OF FUSARIUM GRAMINEARUM GERMLING DEVELOPMENT CAUSED BY COMBINATORIALLY SELECTED DEFENSE PEPTIDES. 1* N.W. Gross , Z.D. Fang1, B. Cooper3, F.J. Schmidt2 and J.T. English1 Division of Plant Sciences, 2 Division of Biochemistry, University of Missouri, Columbia, MO 65211; and 3Soybean genomics and Improvement Laboratory, USDA-ARS, Beltsville, MD 20705 * Corresponding Author: PH: 573-884-6709; Email: nwghw2@mizzou.edu 1 ABSTRACT To address the problem of head blight in wheat, we are applying combinatorial peptide techniques to identify molecules that serve as antagonists to developing germlings of Fusarium graminearum. In this methodology, we mixed phage-display libraries that display 8-mer random peptides with F. graminearum germlings derived from macroconidia. Phage clones with binding affinity for germlings were recovered and amplified in E. coli. After additional rounds of incubation and amplification, we have recovered numerous peptides with affinity for surface molecules of germlings. We have sequenced an initial collection of selected peptides and are now evaluating their abilities to inhibit germling growth and development. At completion of these phenotype screens, we will test candidate peptides for inhibitory function when displayed on recently developed scaffold proteins. 49 Session 3: Gene Discovery and Engineering Resistance TRANSGENIC WHEAT EXPRESSING ANTIFUNGAL PLANT DEFENSIN MTDEF4 IS RESISTANT TO FUSARIUM HEAD BLIGHT (FHB). Jagdeep Kaur1, Thomas Clemente2, Aron Allen1 and Dilip Shah1* Donald Danforth Plant Science Center, Saint Louis, MO, USA; and 2University of Nebraska-Lincoln, NE, USA * Corresponding Author: PH: (314) 587-1481; Email: dshah@danforthcenter.org 1 ABSTRACT Plant defensin MtDef4 from Medicago truncatula is a potent inhibitor of F. graminearum in vitro. Transgenic wheat lines expressing MtDef4 were generated using Agrobacterium tumefaciens-mediated transformation of spring wheat cultivar Bobwhite and a Chinese cultivar Xin Chun 9 (XC9). Single floret inoculation method was used to evaluate Type II resistance of these transgenics in the greenhouse. Of the two lines tested thus far, one Bobwhite transgenic line expressing MtDef4 has reduced FHB severity when compared to nontransgenic Bobwhite. The level of resistance in this line is similar to that of FHB resistant cultivar Alsen. Two more transgenic lines are being evaluated for Type II resistance in the greenhouse. The results of this study will be presented. 50 Session 3: Gene Discovery and Engineering Resistance REDUCING DON POTENTIAL IN VIRGINIA HULLESS BARLEY LINES THROUGH GENETIC ENGINEERING. P.A. Khatibi, D.G. Schmale III*, W.S. Brooks and C.A. Griffey Virginia Polytechnic Institute and State Univeristy, Blacksburg, VA, 24061 * Corresponding Author: PH: (540) 231-6943; Email: dschmale@vt.edu ABSTRACT Hulless barley (HLSB) is a new and emerging crop in Virginia, and may be an important source of biofuels in the future. Dried distillers grains with solubles (DDGS), a byproduct of ethanol fermentation, are rapidly becoming one of the main sources of feed for domestic animals. Traditional ethanol production may concentrate trichothecene mycotoxins such as deoxynivalenol (DON) in DDGS, posing a significant threat to domestic animal health. Our work aims to genetically engineer Virginia HLSB lines with reduced DON potential and thus provide a safe supply of DDGS for animal feed. In 2006 and 2007, we determined the DON potential of 20 Virginia HLSB lines; a number of these lines demonstrated low levels of DON in both years. We generated callus from 17 HLSB lines, and five of the lines were selected for further tissue culturing analyses and genetic transformation. We amplified TRI101, a gene encoding a 3-O-acetyltransferase responsible for the conversion of DON to 3-acetyl-DON, from four different species of Fusarium. Preliminary expression studies using the yeast expression vector pYES2.1 suggested that these genes differ in their relative ability to reduce DON in vitro. We are currently developing an Agrobacterium transformation vector to move TRI101 into five selected HLSB lines, and we plan to monitor potential decreases in DON in both raw grain and DDGS following ethanol production using our genetically-engineered lines. We are currently exploring the function of additional genes that may detoxify DON (e.g., orthologs of TRI101 in Arabidopsis), and we hope to harness the potential of these genes to enhance food safety and security in the eastern U.S. 51 Session 3: Gene Discovery and Engineering Resistance ENHANCING FUSARIUM HEAD BLIGHT RESISTANCE IN WHEAT BY MANIPULATING MECHANISMS CONTRIBUTING TO HOST RESISTANCE AND SUSCEPTIBILITY. Ragiba Makandar1, Vamsi Nalam1, Juliane S. Essig2, Melissa A. Schapaugh2, Harold Trick2, Ruth Dill-Macky3 and Jyoti Shah1* 1 Department of Biological Sciences, University of North Texas, Denton, TX 76203; 2Department of Plant Pathology, Kansas State University, Manhattan, KS 66506; and 3Department of Plant Pathology, University of Minnesota, St Paul, MN 55108 * Corresponding Author: PH: (940) 565-3535; Email: Shah@unt.edu ABSTRACT Fusarium head blight (scab) caused by the fungal pathogen, Fusarium graminearum is a serious menace in wheat and barley, severely limiting crop productivity and quality. Previously, we had demonstrated that ectopic expression of the Arabidopsis thaliana AtNPR1 gene, which is a key regulator of salicylic acid (SA) signaling, enhanced FHB resistance in the hexaploid wheat cv. Bobwhite (Makandar et al. 2006). Similarly, FHB resistance is enhanced in transgenic AtNPR1 expressing durum cvs. Ben, Maier and Belzer. Three field trials to monitor the impact of AtNPR1 on FHB have been completed. Results of these trials will be presented. Genetic studies in Arabidopsis thaliana demonstrate that SA has an important role in plant resistance against F. graminearum. Pretreatment with SA enhances FHB resistance in wheat, also. Furthermore, SA accumulation in spikes correlates with FHB resistance in wheat. SA levels increase >200% in the fungus inoculated and distal spikelets of the resistant cv. Sumai-3, within 24 h of point inoculation with F. graminearum macroconidia. In contrast, a similar increase in SA content was not observed in the cv. Bobwhite, suggesting that SA accumulation can be targeted to enhance FHB resistance. Indeed, resistance against F. graminearum is enhanced in Arabidopsis plants that constitutively express the AtPAD4 gene, which modulates SA synthesis and signaling. We have initiated experiments to ectopically express AtPAD4 from the maize Ubi1 promoter in transgenic wheat. In addition, we have generated transgenic wheat plants that express a salicylate hydroxylase encoded by the bacterial nahG gene, to further test the involvement of SA in wheat defense against F. graminearum. In contrast to SA, our experiments in wheat and Arabidopsis indicate that jasmonic acid (JA) accumulation and the activation of JA signaling inversely correlates with resistance to F. graminearum, suggesting that JA or a related oxidized lipid (oxylipin) may be a susceptibility factor. Indeed, in Arabidopsis a lipoxygenase involved in oxylipin synthesis contributes to susceptibility to F. graminearum. Thus, oxylipin synthesis could provide another target to control FHB. In Arabidopsis, JA antagonizes SA accumulation. Similarly, JA accumulation could result in the suppression of SA accumulation in the spikes of FHB susceptible wheat cultivars. Alternatively, as was recently shown in maize (Gao et al., 2007), JA or a related oxylipin may contribute to fungal development, thereby contributing to susceptibility. ACKNOWLEDGEMENT AND DISCLAIMER This material is based upon work supported by the U.S. Department of Agriculture, under Agreement No. 590790-067. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. Any opinions, find52 Session 3: Gene Discovery and Engineering Resistance ings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture. REFERENCES Gao, X., Shim, W.-B., Göbel, C., Kunze, S., Feussner, I., Meeley, R., Balint-Kurti, P., and Kolomiets, M. (2007). Disruption of a maize 9-lipoxygenase results in increased resistance to fungal pathogens and reduced levels of contamination with mycotoxin fumonisin. Mol. Plant-Microbe Interact. 20: 922-933. Makandar, R., Essig, J. S., Schapaugh, M. A., Trick, H. N. and Shah, J. 2006. Genetically engineered resistance to Fusarium head blight in wheat by expression of Arabidopsis NPR1. Mol. Plant-Microbe Interact. 19:123-129 53 Session 3: Gene Discovery and Engineering Resistance ENGINEERING BARLEY WITH GASTRODIANIN FOR IMPROVED RESISTANCE TO FUSARIUM HEAD BLIGHT. Eng-Hwa Ng1, Tilahun Abebe1*, James E. Jurgenson1 and Ronald W. Skadsen2 1 Department of Biology, University of Northern Iowa, 144 McCollum Science Hall, Cedar Falls, IA 50614; and 2Cereal Crops Research Unit, USDA/ARS, 502 Walnut Street, Madison, WI 53726 * Corresponding Author: PH: (319) 273-7151; Email: Tilahun.Abebe@uni.edu OBJECTIVES Develop transgenic barley lines expressing the antifungal gene gastrodianin for resistance to Fusarium head blight (FHB). INTRODUCTION 2003). Gastrodianin is a non-agglutinating, monomeric, mannose and chitin-binding lectin that belongs to the superfamily of monocot mannose-specific lectins (Liu et al., 2005; Wang et al., 2001). Gastrodianin effectively inhibits hyphal growth of pathogenic and saprophytic fungi including Gibberella zeae, Armillaria mellea, , Rhizoctonia solani, Trichoderma viride and Valsa ambiens in vitro (Wang et al., 2001). In vivo studies have also demonstrated the importance of gastrodianin in fighting pathogens. In transgenic tobacco, gastrodianin reduces root diseases caused by fungal pathogens Rhizoctonia solani and Phytophthora nicotianae (Cox et al., 2006). In cotton, field tests showed that transgenic plants expressing gastrodianin are resistant to another fungal pathogen Verticillium wilt (Wang et al., 2004). Gastrodianin maintains inhibitory properties at fluctuating temperatures (Wang et al., 2001, Xu et al., 1998). This stability and its inhibitory effects on G. zeae makes gastrodianin protein an attractive candidate for engineering resistance to fungal diseases. Control of Fusarium head blight (FHB) infection in barley remains difficult because of lack of genetic resistance. One strategy that has great potential to reduce FHB infection is introduction of anti-Fusarium genes into barley through genetic engineering. Unfortunately, engineering resistance has been slow since common pathogenesis-related (PR) proteins are not effective against Fusarium graminearum. Transgenic wheat over-expressing combinations of chitinases, glucanases, and thaumatin-like proteins (TLPs) had partial resistance to FHB in green house testing. However, the greenhouse results were not reproducible under field conditions and no resistance was observed (Anand et al. 2003). Apparently, genes known to specifically inhibit F. graminearum are required to MATERIALS AND METHODS give adequate protection from FHB. We have developed barley lines expressing anti-Fusarium gene Expression vectors gastrodianin for resistance to FHB. Plasmids used for transformation are shown in Fig. 1. Gastrodianin is an anti-fungal gene isolated from a There are four gastrodianin genes in G. elata differtraditional Chinese herb, Gastrodia elata. G. elata ing only by 3 to 4 nucleotides (Wang et al., 1999). In is devoid of chlorophyll and leads a parasitic life on this study the variant VGM was used (GeneBank Acthe fungus Armillaria mellea. A. mellea hyphae usu- cession AJ277785). Gastrodianin has 513 nucleotides ally infect the nutritive corms of G. elata but are di- and encodes a polypeptide with 171 amino acids. The gested in the cortical cells. The released nutrients are coding region was amplified by PCR from a binary used by the host plant for growth and development. vector generously provided by the Plant Biology InExpression of gastrodianin and other anti-fungal pro- stitute, University of Ghent, Belgium. The PCR fragteins protects the developing terminal corm from in- ment was digested with restriction enzymes (EcoRV fection by A. mellea (Wang et al., 2007; Sa et al., and PstI) and fused to a Lem2 promoter (Abebe et 54 Session 3: Gene Discovery and Engineering Resistance al., 2005). The resulting fragment was ligated to pLem2gfp (Abebe et al, 2005) to get pLem2VGM2 (Fig. 1). Plasmid pLem2VGM2 also contains gfp under the control of the Lem2 promoter, making visual screening of transformed plants easier. transgenic plants was performed by inspecting tissuespecific fluorescence of the GFP protein in the spike and auricle. The sterile plants were bushy in appearance (had many tillers) and had thin stems and spikes (Fig. 2). We are screening plants for accumulation of the gastrodianin Transformation of barley protein using western blotting and ELISA. It will be Barley plants (Hordeum vulgare cv. Golden Prom- interesting to see if the phenotypes observed in sterile ise) were transformed as described in Wang and plants are due to high accumulation of the gastrodianin Lemaux (1994) with minor modification. Immature protein, disruption of spike-specific genes or kernels (approximately 14 days post-anthesis) were somaclonal variations introduced during tissue culture. surface sterilized with 70% ethanol (v/v) and 20% chlorox (v/v). After three washes with sterile water, To verify integration of gastrodianin in the genome, embryos were cut in half longitudinally and placed on we screened some transgenic plants expressing gfp callus induction medium (CIM), scutellum side down. by PCR. This showed that all the plants expressing After 3–5 days of incubation, embryos were bom- gfp also had the expected 0.5 kb gastrodianin insert barded with gold particles (0.6 μm) coated with an (Fig. 3). In the next phase of the study, resistance of equimolar amounts of plasmids pLem2VGM2 and transgenic plants to F. graminearum will be tested pAHC25 (contains the bar) using the He-driven PDS under greenhouse conditions using T1 and T2 genera1000 (Bio-Rad). The herbicide bialaphos was used tions. to select transgenic calli and plantlets. AKNOWLEDGEMENT Characterization of transgenic plants This research is supported by the U.S. Department of Integration of gastrodianin into the genome of Agriculture, under Agreement No. 59-0790-6-057. transgenic barley was verified by PCR. Genomic DNA This is a cooperative project with the U.S. Wheat & was isolated from wild type and transgenic barley Barley Scab Initiative. Funding was also received from plants using CTAB (Murray and Thompson, 1980). the College of Natural Sciences, University of NorthPCR was performed using 100 ng of genomic DNA, ern Iowa through the Student Opportunities for Acaalong with upstream and downstream VGM primers. demic Research (SOAR) award. We thank undergraduate students Lauren Alsager, Jay Burmeister, Ebony Jackson, Ryan Pape, Lindsay Smith, Diveena RESULTS AND DISCUSSION Vijayendran, Aaron Walck and Justin Wilkins for their We have recovered plants from 16 transformation help in tissue culture. We are grateful to Billie Hemmer events. Plants from four events were sterile, plants from and Stephanie Witt, University of Northern Iowa Botwo events were lost to fungal contamination and the tanical Center, for their assistance in growing plants. remaining ten plants produced seeds. At least two plants were regenerated from each transformation DISCLAIMER event. Recovery of T0 plants from tissue culture was significantly improved by visual screening of gfp ex- Any opinions, findings, conclusions, or recommendapression. Both gfp and gastrodianin were placed in tions expressed in this publication are those of the the same pLem2VGM2 plasmid (Fig. 1). By screen- author(s) and do not necessarily reflect the view of the ing for gfp expression we were able to indirectly se- U.S. Department of Agriculture. lect plants incorporating gasrodianin in their genome. The lem2 gene promoter directs tissue-specific expression (Abebe et al., 2005). Visual screening of 55 Session 3: Gene Discovery and Engineering Resistance REFERENCES Abebe, T., Skadsen, R.W., Kaeppler, H.F. 2005. A proximal upstream sequence controls tissue-specific expression of Lem2, a salicylate-inducible barley lectin-like gene. Planta 221:170-183. Anand, A., Zhou, T., Trick, H.N., Gill, B.S., Bockus, W.W., Muthukrishnan, S. 2003. Greenhouse and field testing of transgenic wheat plants stably expressing genes for thaumatin-like protein, chitinase and glucanase against Fusarium graminearum. Journal of Experimental Botany. 54:1101-1111. Cox, K.D., Layne, D.R., Scorza, R. and Schnabel, G. 2006. Gastrodia anti-fungal protein from the orchid Gastrodia elata confers disease resistance to root pathogens in transgenic tobacco. Planta 224: 1373-1383. Liu, W., Yang, N., Ding, J., Huang, R.H., Hu, Z. & Wang, D.C. 2005. Structural mechanism governing the quaternary organization of monocot mannose-binding lectin revealed by the novel monomeric structure of an orchid. Journal of Biological Chemistry 280: 14865-14876. Murray, H.G. and Thompson, W.F. 1980. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Research 8:4321–4326. Wang, X.C., Diaz, W.A., Bauw, G., Xu, Q., Van Montagu, M., Chen, Z.L. and Dillen, W. 1999. Molecular cloning of GAFP-1, an antifungal protein from Gastrodia elata. Acta Botanica Sinica 41:1041-1045. Wang, X., Bauw, G., Van Damme, E.J., Peumans, W.J., Chen, Z.L., Van Montagu, M., Angenon, G., Dillen, W. 2001. Gastrodianin-like mannose-binding proteins: a novel class of plant proteins with antifungal properties. The Plant Journal 25:651-61. Wang, Y.Q., Chen, D.J., Wang, D.M., Huang, Q.S., Yao, Z.P., Liu, F.J., Wei, X.W., Li, R.J., Zhang, Z.N. and Sun, Y.R. 2004. Over-expression of Gastrodia anti-fungal protein enhances Verticillium wilt resistance in coloured cotton. Plant Breeding 123: 454–459. Wang, Y. and Lemaux, P. 1994. Generation of large numbers of independent transformed fertile barley plants. Plant Physiol. 104: 37-48 Wang, H.X., Yang, T., Zeng, Y. and Hu, Z. 2007. Expression analysis of the Gastrodianin gene ga4B in an achlorophyllous plant Gastrodia elata Bl Plant Cell Reports 26: 253-259. Xu Q, Liu Y, Wang X, Gu H & Chen Z (1998) Purification and characterization of a novel anti-fungal protein from Gastrodia elata. Plant physiology biochemistry 36: 899-905. Sa, Q., Wang, Y., Li, W, Zhang, L. and Sun, Y. 2003. The promoter of an antifungal protein gene from Gastrodia elata confers tissue-specific and fungus-inducible expression patterns and responds to both salicylic acid and jasmonic acid. Plant Cell Rep 22:79–84 pLem2VGM2 (7.9 kb) Lem2 promoter gfp nos Lem2 promoter VGM nos GUS nos Ubi-1 promoter bar nos pAHC25 (9.7 kb) Ubi-1 promoter Fig.1. Map of plasmids used for transformation. Plasmid pLem2VGM2 contains gfp and gastrodianin (VGM) driven by the Lem2 promoter. Plasmid pAHC25 contains the bar gene for selection. It also contains the GUS reporter gene. 56 Session 3: Gene Discovery and Engineering Resistance (a) (b) (c) - Control 56C 52C 51E 50D 48F 47E 46A Wild GP λ/Pst Transgenic lines + Control Fig.2. Phenotype of T0 plants. Sterile T0 plants (a) have very thin spikes compared to fertile T0 (b) and no-transgenic (c) Golden promise plants. 1.1 kb 0.8 kb 0.5 kb Fig.3. PCR showing integration of the gastrodianin gene in the genome of Golden Promise (GP) barley. Positive control (+) for the PCR was plasmid pLem2VGM2 DNA and negative control (-) was water. 57 Session 3: Gene Discovery and Engineering Resistance GENES THAT CONFER RESISTANCE TO FUSARIUM. H. Saidasan1 and M. Lawton1,2* 1 Biotechnology Center for Agriculture and the Environment, and 2Department of Plant Biology and Plant Pathology, Rutgers University, New Brunswick, NJ, 08901-8520 * Corresponding Author: PH: (732) 932-8165 ext 223; Email: Lawton@aesop.rutgers.edu ABSTRACT There is a pressing need for sources of germplasm or genes that are effective against Fusarium graminearum, the causal agent of Fusarium Head Blight (FHB) on wheat and barley. Since sources of resistance from wheat and barley are limited, we have developed a functional assay system to evaluate genes from other sources for their efficacy against FHB. The assay system is based on the plant Physcomitrella patens, which serves as a ‘green yeast’ for the rapid evaluation of novel genes. This plant, uniquely, allows the contribution of individual genes to be assessed through either the creation of targeted genes knockouts or through the introduction and overexpression of transgenes. Importantly, the wild type plant is fully susceptible to F. graminearum and highly sensitive to mycotoxins, including DON. We have used this system to characterize genes that confer effective and robust resistance to FHB. The first set of genes acts through the plant programmed cell death pathway. Plants that contain knockouts for these genes are completely insensitive to DON and fully resistant to FHB. A similar level of FHB resistance can be conferred by overexpressing genes that suppress plant cell death. In these plants, FHB resistance is conferred by disabling a host susceptibility pathway (cell death) induced by mycotoxins. A second set of genes confers FHB resistance through a pathway that is independent of cell death. These plants, which overexpress nuclease genes, are still sensitive to DON and other mycotoxins, yet display significant resistance to FHB. One explanation, indirectly supported by our studies, is that the overexpressed protein is itself directly antifungal. In these plants, FHB resistance is conferred by enhancing existing plant defense mechanisms. A third set of genes that confer FHB resistance is associated with stress management, and in particular the response to reactive oxygen species (ROS), which are associated with the response to pathogen attack. Knockout and overexpressing lines for different genes associated with this response show enhanced resistance to both DON and to FHB but through a mechanism that acts downstream of the cell death pathway. These results show that FHB resistance can be introduced by manipulating a variety of cellular targets. By combining these approaches it should be possible to introduce an enduring FHB resistance into wheat and barley plants. The efficacy of these FHB-resistance genes in wheat is currently being tested by the Scofield lab using a VIGS-based assay. This will provide an early indicator of likely performance in transgenic wheat. ACKNOWLEDGEMENT AND DISCLAIMER This material is based upon work supported by the U.S. Department of Agriculture, under Agreement No. 590790-6-063. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the U.S. Department of Agriculture. 58 Session 3: Gene Discovery and Engineering Resistance GENETIC STUDIES DEFINE DISTINCT PATHWAYS OF RESISTANCE TO FUSARIUM HEAD BLIGHT. H. Saidasan1 and M. Lawton1,2* Biotechnology Center for Agriculture and the Environment, and 2Department of Plan Biology and Plant Pathology, Rutgers University, New Brunswick, NJ, 08901-8520 * Corresponding Author: PH: (732) 932-8165 ext 223; Email: Lawton@aesop.rutgers.edu 1 ABSTRACT We have used the Physcomitrella patens rapid assay system to characterize a number of genes for their ability to confer resistance to Fusarium graminearum, the causal agent of Fusarium head Blight (FHB). Using this approach we have screened several dozen genes for their ability to confer resistance to fungal mycotoxins and FHB. These studies have revealed that resistance to FHB can be achieved by manipulating multiple cellular pathways, including those involved in the regulation of programmed host cell death, the production and elimination of reactive oxygen species, the production of lytic enzymes and the expression of host defense responses. These mutant plants show distinct patterns of susceptibility to FHB and to various Fusarium-derived mycotoxins, compared to wild type plants, which are fully susceptible to FHB and highly sensitive to DON and T2 toxin. Plants that are mutated in the cell death pathway are highly resistant to FHB, and insensitive to DON and T-2 toxin. In contrast, plants that are mutated in the regulation of reactive oxygen species are highly resistant to FHB, insensitive to DON but partially sensitive to T-2 toxin. A further contrast is provided by plants that overexpress nuclease genes. These plants are resistant to FHB but fully sensitive to DON and T2toxin. These results illustrate that different FHB-derived toxins target different cellular pathways, and suggest that robust resistance to FHB in the field may require the concerted manipulation of more than one cellular pathway. Several of the genes we have manipulated are induced during the response to FHB inoculation. We tested whether these genes form part of a natural defense response by pre-treating plants with the defense response elicitor chitosan. Plants exposed to chitosan are highly resistant to subsequent inoculation with FHB. This indicates that Physcomitrella plants possess a natural and highly effective mechanism of induced FHB resistance. Presumably this response is suppressed during the interaction with F. graminearum. We will present data on this induced FHB-resistance response and discuss other approaches we have used to suppress virulence and enhance resistance to FHB in this system. ACKNOWLEDGEMENT AND DISCLAIMER This material is based upon work supported by the U.S. Department of Agriculture, under Agreement No. 590790-6-063. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the U.S. Department of Agriculture. 59 Session 3: Gene Discovery and Engineering Resistance RAPID FUNCTIONAL IDENTIFICATION OF GENES CONTRIBUTING TO FHB RESISTANCE. Steven Scofield1* and Megan Gillespie2 1 Crop Protection and Pest Control Unit, USDA-ARS, West Lafayette, IN 47907; and 2Department of Agronomy, Purdue University, West Lafayette, IN 47907 * Corresponding Author: PH: (765) 494-3674; Email: scofield@purdue.edu ABSTRACT This presentation will describe a new method being employed to rapidly identify genes that function in the Fusarium head blight (FHB) resistance mechanism of wheat. In this method, called virus-induced gene silencing (VIGS), genes thought to function in FHB resistance are switched-off, or silenced, and their role in FHB resistance is inferred if silencing results in resistant wheat plants becoming susceptible to FHB. This method utilizes the RNA virus, Barley stripe mosaic virus (BSMV), to activate RNA-mediated gene silencing in wheat. RNA-mediated gene silencing is an evolutionarily conserved defense mechanism in plants and animals that targets viral RNAs for sequence-specific degradation. In VIGS, the plant’s RNA-based defense response is exploited to cause plant genes selected by the experimenter to be silenced by inserting a piece of the chosen plant gene into the viral RNA. In this way, the messenger RNA from the chosen plant gene is targeted for degradation, thus silencing the expression of the gene, as the plant defense mechanism works to degrade all the viral RNA. This approach has several important advantages: 1) As it is homology-dependent, it can simultaneously silence multiple copies of genes, which are almost always present in hexaploid wheat. Without this capability the expression of any closely related genes would prevent observation of the effects of silencing. 2) It is rapid; an experiment can be accomplished in as little as 2 months from identification of a candidate gene to observing the effect of its silencing. Examples of the utility of this important new method will be presented. 60 Session 3: Gene Discovery and Engineering Resistance ENGINEERING RESISTANCE TO FUSARIUM GRAMINEARUM USING ANTIFUNGAL PLANT DEFENSINS. Dilip Shah1*, Mercy Thokala1, Jagdeep Kaur1, Tom Clemente2 and Anita Snyder1 Donald Danforth Plant Science Center, St Louis, MO 63132; and 2University of Nebraska-Lincoln, Lincoln, NE 68588 * Corresponding Author: PH: 314-587-1481; Email: dshah@danforthcenter.org 1 ABSTRACT Small cysteine-rich plants defensins have potential as antifungal agents in transgenic crops. Two such defensins, MsDef1 and MtDef4, from Medicago spp., share only 41% amino acid sequence identity, but potently inhibit the growth of Fusarium graminearum in vitro. These two defensins exhibit different modes of antifungal action. Using the Fusarium graminearum-Arabidopsis thaliana pathosystem, we have found that overexpression of either MsDef1 or MtDef4 extracellularly or intracellularly (in the vacuole or endoplasmic reticulum) conferred strong resistance to this pathogen in transgenic A. thaliana plants. Transgenic plants exhibited reduced foliar symptoms and growth of fungal hyphae. Moreover, growth of the pathogen-challenged transgenic plants was similar to that of non-inoculated wild-type plants. Since F. graminearum colonizes host tissue by both inter- and intra-cellular growth, we will develop and test transgenic A. thaliana lines co-expressing extraand intracellular defensins for more robust resistance to this pathogen. In parallel experiments, we have generated seven transgenic wheat lines over-expressing extracellular MtDef4. Of the two lines tested thus far, one line displayed improved FHB resistance when compared to non-transgenic Bobwhite. Furthermore, the level of resistance in this line was comparable to that of the disease resistant check, Alsen. No pleiotropic effects resulting from over-expression of defensins were observed in transgenic A. thaliana or wheat. 61 Session 3: Gene Discovery and Engineering Resistance ENGINEERING SCAB RESISTANCE IN WHEAT WITH PLANT DEFENSE SIGNALING GENES. Jyoti Shah1*, Ragiba Makandar1, Vamsi Nalam1 and Harold N. Trick2 1 Department of Biological Sciences, University of North Texas, Denton, TX 76203, and Department of Plant Pathology, Kansas State University, Manhattan, KS 66506, USA * Corresponding Author: PH: (940) 565-3535; Email: Jyoti Shah: shah@unt.edu 2 ABSTRACT Fusarium graminearum is the principal causative agent of Fusarium Head Blight (FHB)/scab, a devastating disease of wheat and barley that severely limits crop productivity and grain quality. Our approach has been to utilize plant genes that regulate defense responses for enhancing FHB resistance in wheat. A plant-pathogen system consisting of Arabidopsis thaliana and Fusarium graminearum has been developed to identify genes involved in regulating plant defense against F. graminearum. The Arabidopsis NPR1 (AtNPR1) gene was one of the promising genes identified in this screen. NPR1 is a key regulator of salicylic acid (SA) signaling in plant defense. Our studies in Arabidopsis and wheat have indicated that SA is also an important regulator of defense against F. graminearum. Expression of AtNPR1 gene (AtNPR1) was successfully engineered in the hexaploid wheat cultivar Bobwhite. In green house and growth chamber studies, AtNPR1 expression resulted in heightened FHB resistance in transgenic wheat. Furthermore, DON content was lower in the transgenic seeds. SA-regulated defense responses were turned on faster and to higher levels in the AtNPR1 expressing plants. Three field trials have been completed with AtNPR1 expressing transgenic Bobwhite plants. AtNPR1 expression has also been successfully engineered into the durum varieties Ben, Maier and Belzer. FHB evaluations of these transgenic plants are ongoing. Results on other promising genes identified in our Arabidopsis-F. graminearum screen and the status for engineering their expression in wheat will also be discussed. ACKNOWLEDGEMENT AND DISCLAIMER This material is based upon work supported by the U.S. Department of Agriculture, under Agreement No. 590790-067. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture. 62 Session 3: Gene Discovery and Engineering Resistance TRANSGENIC WHEAT WITH ENHANCED RESISTANCE TO FUSARIUM HEAD BLIGHT. S.H. Shin1, J.M. Lewis3, C.A. Mackintosh1, A. Elakkad2, K. Wennberg2, S.J. Heinen1, R. Dill-Macky2 and G.J. Muehlbauer1* Department of Agronomy and Plant Genetics, 411 Borlaug Hall, 1991 Upper Buford Circle, University of Minnesota, St. Paul, MN 55108; 2Department of Plant Pathology, 495 Borlaug Hall, 1991 Upper Buford Circle, University of Minnesota, St. Paul, MN 55108; and 3Department of Crop and Soil Sciences, Michigan State University, East Lansing, MI 48824 * Corresponding Author: PH: 612-625-6228, E-mail: muehl003@umn.edu 1 ABSTRACT We are developing and testing transgenic wheat for resistance to Fusarium Head Blight (FHB). We developed transgenic wheat carrying genes encoding chitinase, thaumatin-like protein 1 (tlp-1), ribosome-inactivating protein (RIP), lipid transfer protein (LTP), glutathione-S-transferase (GST), jasmonic acid inducible Myb transcription factor (JaMyb), germin-like protein1 (GLP1), and pathogenesis-related protein1 (PR1). Transgenic lines over-expressing these genes were generated using micro-projectile bombardment of the wheat cultivar ‘Bobwhite’. Both single and combinations of transgenes were generated. We developed 4, 4, 2, 2, 1, and 4 lines carrying LTP, RIP, RIP/tlp-1, TRI101/tlp-1, TRI101/ß-1,3-glucanase, and tlp-1/ß-1,3-glucanase, respectively. In multiple greenhouse screens of these lines, we identified five lines (one RIP, two TRI 101/tlp-1, and two tlp-1/ß-1,3-glucanase) that exhibited statistically significant reductions in FHB severity compared to the non-transgenic controls (p<0.05). Combined with our previous greenhouse screens, we identified and evaluated 24 lines (seven chitinase, two RIP, two chitinase/tlp-1, one chitinase/RIP, six RIP/tlp-1, two TRI 101/tlp-1, two tlp-1/ß-1,3-glucanase, and two LTP) in field trials in 2005 and/or 2007. Three lines (two chitinase and one RIP) exhibited statistically significant reductions in FHB severity and very scabby kernels (VSK) compared to the non-transgenic control (P<0.05) in 2005 and 2007. In 2007, four lines (one TRI 101/tlp-1, two tlp-1/ß-1,3-glucanase, and one RIP) showed reduced FHB severity and five (two TRI 101/ tlp-1, one tlp-1/ß-1,3-glucanase, two LTP) showed reduced VSK (p<0.05). We also crossed three transgenic wheat lines (two chitinase and one RIP), that exhibited statistically significant reductions in FHB severity in the field, to the type II resistant cv. Alsen. In addition, we developed 13, 10, 10, and 6 transgenic lines carrying GST, JaMyb, PR1, and GLP genes, respectively. Six lines (one GST, two JaMyb, and three GLP1) exhibited statistically significant reductions in FHB severity in compared to the non-transgenic Bobwhite in greenhouse screens (p<0.05). 63 Session 3: Gene Discovery and Engineering Resistance COMPARATIVE ANALYSIS OF FHB QTLS IN THE MINI MANO/ FRONTANA AND FRONTANA/REMUS DH POPULATIONS. A. Szabo-Hever1*, B. Toth1, Sz. Lehoczki-Krsjak1, H. Buerstmayr2, M. Lemmens2 and Á.. Mesterházy1 Cereal Research non-profit Company, Department of Biotechnology and Resistance Research, Szeged, Hungary, 2IFA Tulln, Austria * Corresponding Author: PH: (36) 62 435-235; Email: agnes.szabo@gabonakutato.hu 1 ABSTRACT Fusarium head blight (FHB) is one of the most important diseases in the aspects of food safety and yield quality also. The most effective strategy for controlling FHB in wheat is through the development of resistant cultivars. This can be reached by analyzing QTLs, and using them in a marker-assisted selection. Frontana is a Brazilian spring wheat cultivar that has small and medium effective QTLs. These types of QTLs are sensitive for the environmental factors and for the problems of heterogeneity. 220 DH lines from Frontana/Remus (IFA Tulln) /2005-2006/ and 110 DH lined of Mini Mano/Frontana (CRC Szeged) /2006-2007/ were inoculated with four Fusarium isolates of F. graminearum and F. culmorum. The Frontana/Remus population was developed traditionally, with about up to two weeks difference in flowering time and 60-70 cm differences in plant height. MM/Frontana was created by us so that too early and late DH lines were discarded and the remaining lines flowered within five days, so one inoculation time was enough to cover all genotypes and plant height differences were kept within 20-30 cm depending on season. The rest of the lines were discarded. In the Frontana/Remus population QTLs were identified on the chromosomes 2D, 3A, 5A, 5B, 3B, 6B, 7A/ 7D. The most consequent markers were found on 5A and 5B chromosomes (BARC197 and GWM156), the others gave positive signal seldom and the LOD values were around 2 and 2,8. In the MM/Frontana population 2B, 3B, 5A, 5B and 7B gave positive signal. The LOD values on 5B chromosome were the highest (BARC115), between 2,76 and 5,32. Even so in both populations Frontana was the resistant parent not the same markers gave positive signal. It seems that the more homogeneous population increases the accuracy of the QTL analysis. An increased morphologic homogeneity is necessary to decrease „noise” in QTL analyses and increase preciosity. Until now no QTL were found that gave positive signs for all epidemic situations. Therefore, the conditions to perform consequent MAS to identify superior genotypes in Frontana descendants are not yet in sight. ACKNOWLEDGEMENT The authors express their thanks to project FP5 FUCOMYR 2001-02044, the NKTH-KPI projects signed as OMFB 01286/2004 and OMFB 00313/2006 for financial support. 64 Session 3: Gene Discovery and Engineering Resistance EXTRA- AND INTRACELLULAR TARGETING OF ANTIFUNGAL PLANT DEFENSINS IN TRANSGENIC ARABIDOPSIS FOR RESISTANCE TO FUSARIUM GRAMINEARUM. Mercy Thokala1, Aron Allen1 and Dilip Shah1* Donald Danforth Plant Science Center, St. Louis, MO 63132, USA Corresponding Author: PH: (314) 587-1481; Email: dshah@danforthcenter.org 1 * ABSTRACT Recent studies have shown that Arabidopsis thaliana, a model host plant is susceptible to F. graminearum. Taking advantage of this foliar Fusarium-Arabidopsis pathosystem, we tested antifungal defensins, MsDef1 and MtDef4, from Medicago spp., for their ability to confer resistance to this pathogen. We generated chimeric defensin gene constructs that will result in over-expression of MsDef1 or MtDef4 either extra-cellularly or intra-cellularly (i.e., vacuole or endoplasmic reticulum) in transgenic A. thaliana ecotype Columbia (Col0). Here, we demonstrate that constitutive overexpression of MsDef1 and MtDef4 confers strong resistance to F.graminearum. Transgenic Arabidopsis lines overexpressing MsDef1 or MtDef4 either extra-cellularly or intra-cellularly showed 59-68 % reduction in disease severity (DS) index as compared to that of the wild type plants (100%) and supported significantly less fungal growth as evaluated by trypan blue staining. Transgenic inoculated plants also bolted normally like the mock inoculated wild-type plants, whereas the inoculated wildtype plants showed much delayed bolting. Since F. graminearum has both biotrophic and necrotrophic lifecycles, we hypothesize that MsDef1 and MtDef4 co-expressed extra- and intra-cellularly will confer much higher level of resistance to FHB. Hence, transgenic A. thaliana lines co-expressing extra- and intracellular defensins will be tested for increased resistance to this pathogen. 65 SESSION 4: FHB MANAGEMENT Chairperson: Gary Bergstrom Session 4: FHB Management EFFECTS OF WHEAT GENOTYPES AND INOCULATION TIMINGS ON FUSARIUM HEAD BLIGHT (FHB) SEVERITY AND DEOXYNEVALENOL (DON) PRODUCTION IN THE FIELD. Shaukat Ali and Tika B. Adhikari* Department of Plant Pathology, North Dakota State University, Fargo, ND 58105 * Corresponding Author: PH: (701) 231-7079; Email: tika.adhikari@ndsu.edu ABSTRACT Fusarium head blight (scab), caused primarily by Fusarium graminearum (teleomorph: Gibberella zeae), is an important disease of wheat and other cereals worldwide. The disease affects both yield and quality due to contamination of grains with various mycotoxins. Since 1993, the disease has caused billions of dollars loss to the wheat industry in the USA. Due to lack of effective resistant cultivars, FHB is managed through fungicide applications and cultural practices. New fungicides such as ‘Proline’ are effective in FHB management and DON reduction. It has been hypothesized that all wheat cultivars do not respond to fungicide applications in similar manner for DON production and yield increase. Research is in progress to make the FHB forecasting system more accurate. Information on wheat cultivars with various levels of resistance to FHB and their responses to the disease development are important parameters of accurate disease forecasting system. The main objectives of this study were to determine the effects of three hard red spring wheat cultivars, Glenn (FHB resistant), Steel-ND (moderately susceptible) and Trooper (susceptible), and two inoculation timings on FHB development, and to examine the correlation between FHB severity and DON production under field conditions. Wheat cultivars were planted on May 4 and May 14, 2007 at North Dakota State University Experimental Station, Fargo. The experiment was planted as a split-split plot design with 3 replications. Planting date (early and late), wheat cultivars (Glenn, Steel-ND, and Trooper), and inoculation timing (no inoculation, inoculation at early flowering, and inoculation at mid flowering) were assigned in main plot, sub-plot, and sub-sub plot, respectively. Plants were spray-inoculated with F. graminearum (~100,000 spores/ml). Two hundred-twenty-five heads from each sub-plot were examined for FHB incidence and severity, and 20-40 heads with disease severity of 0%, 7-21%, 22-50%, 51-79%, and 80-100% in each sub-plot were tagged at dough stage (Feekes GS 11.2). Wheat ear heads with each disease severity category were collected separately to estimate DON, and correlation between FHB severity and DON production. The cultivars differed significantly in FHB severity, but not in disease incidence and DON production. The resistant wheat cultivar Glenn has the lowest severity (20.6%) while the susceptible cultivar Trooper has the highest disease severity (28.12%). Inoculation timings also had significant effect on FHB incidence, severity, and DON production. All three disease components incidence (12.75%), severity (41%), and DON (2.45 ppm) were higher when the cultivars were inoculated at mid flowering stage (GS 10.52). A positive correlation (r = 0.98) was observed between FHB severity and DON concentration in all three cultivars. As expected, the susceptible cultivar Trooper had higher DON concentration in all five disease severity categories (ranged from 1.06 to 75.68 ppm) as compared to Steel-ND (1.39 to 56.86 ppm) and Glenn (0.91 to 64.63 ppm). The samples with high DON concentration also had with high amount of 3-ADON. Our results indicate that infection at mid flowering growth stage is crucial in FHB incidence, severity, and DON production. 69 Session 4: FHB Management AEROBIOLOGY OF GIBBERELLA ZEAE: WHENCE COME THE SPORES FOR FUSARIUM HEAD BLIGHT? Gary C. Bergstrom1* and David G. Schmale III2 1 Department of Plant Pathology, Cornell University, Ithaca, NY 14853; and 2Department of Plant Pathology, Physiology, and Weed Science, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061 * Corresponding Author: PH: (607) 255-7849; Email: gcb3@cornell.edu ABSTRACT Gibberella zeae (Fusarium graminearum sensu stricto) is the principal causal agent in North America of Fusarium head blight (FHB) of wheat and barley and in several regions is the predominant causal agent of stalk rot and ear rot of corn. Research done primarily in New York over the past decade on the aerobiology, epidemiology, and population biology of G. zeae was summarized in terms of its implications for the regional management of FHB. The fungus survives between crop seasons as a saprophyte in infected crop debris, especially in corn stalks and small grain residues on which it sporulates (both conidia and ascospores) profusely during warm, moist conditions. Viable spores of G. zeae rely on atmospheric motion systems for transport to the florets of wheat and barley where they initiate FHB. Ascospore liberation into turbulent air currents is favored by spore release during daylight hours when peak discharge from perithecia on corn residues also occurs. Ascospore survival on the surface of wheat spikes is on the order of hours to days. Using unmanned aerial vehicles (UAVs), we documented the abundance of viable spores of G. zeae 60 m above the surface of the earth at all times of the day and night under a broad range of meteorological conditions. Viable spores were deposited across cereal fields and other landscape areas by gravitational settling mainly at random and predominantly at night. The temporal uncoupling of peak spore release and deposition suggests that inoculum in cereal fields may originate from distant as well as within-field sources. Genotypic diversity was extremely high in atmospheric populations of G. zeae collected in central New York over a four-year period. The predominant trichothecene mycotoxin genotype of G. zeae found in New York in both infected grain and in atmospheric populations is one that produces deoxynivalenol (DON) plus smaller amounts of 15-acetyl-DON. Our findings suggest that atmospheric populations of G. zeae are an abundant, well-mixed, and diverse source of inoculum for regional epidemics of FHB. Model computations with the atmospheric transport model HYSPLIT suggest that ascospores of Gz may be dispersed kilometer distances from area sources of inoculum in a matter of minutes. The ability to predict the regional transport of Gz from local inoculum sources may help refine risk models for FHB. It is generally considered (but not proven) that airborne ascospores of G. zeae, constitute the principle inoculum for infection of wheat and barley florets. We provided evidence that viable ascospores are potentially transported at least kilometer distances from their site of discharge. Yet the conventional opinion among FHB researchers from Chester in 1890 to the present is that inoculum sources for FHB are mainly local and that long-distance dissemination of inoculum is of minor significance. For example, FHB risk forecasting models that predict local inoculum levels based on previous local weather are built, in part, on the assumption that local inoculum is derived exclusively or largely from nearby sources. This assumption awaits validation. Significant long-range dispersal would suggest that local management of overwintered inoculum (e.g., tillage, spraying of debris, etc.) may have negligible impact on the development of FHB in nearby cereal crops unless performed over extensive production areas. Long-range dispersal also implies that genotypes of G. zeae with novel toxin or virulence profiles could be rapidly disseminated across broad geographic regions. Published studies suggest that most rain-splash dispersal of spores to spikes occurs within 5 meters or less from inoculum sources on the 70 Session 4: FHB Management soil surface and that disease severity follows a similar gradient with distance from those inoculum sources. Various researchers have attempted to delineate spore dispersal gradients or disease gradients at linear distances from area sources of inoculum. Observations of 50% reduction in spore concentration or disease have ranged from 1 to 50 m distances from area inoculum sources with most studies indicating sharp gradients within 10 m of sources. In almost every study conducted, the background level of spores or disease has been at 50% or greater proportion of the level at the source area. Ascospores actively discharged from perithecia or even conidia caught in turbulent air may be deposited on local wheat spikes at a potentially much greater distance from debris than splash-dispersal. Spores may also escape the crop canopy, mix with spores over a wide area, and be transported in the atmosphere at least kilometer distances. Field survey-based studies of DON in grain have generally revealed that cereal cultivar and seasonal meteorological conditions were better quantitative predictors of toxin content than previous crop or tillage practice, strongly suggesting that regional inoculum plays a critical role in FHB epidemics. Based on several field studies with cereal debris level and crop sequence, within-field inoculum, where present, appears to be a significant source for local FHB, but regional inoculum appears to play an even greater role. There are no reliable estimates of the relative contributions of within-field, local inocula to spike infection compared to other airborne sources. In New York and Virginia, we are utilizing a marked isolate, releaserecapture experimental approach to assess relative contribution of localized clonal inoculum present in corn stalks to infection of wheat heads at varying distances from area sources of inoculum. Preliminary evidence from the first year of experimentation suggests that within-field sources of G. zeae provided a minor fraction of FHB inoculum compared to background atmospheric sources in a non-epidemic situation in New York and in a moderate epidemic situation in Virginia. ACKNOWLEDGEMENTS AND DISCLAIMER We acknowledge the financial support of Cornell University Hatch Projects NYC153433 and NYC153473 and the U.S. Department of Agriculture. This is a cooperative project with the U.S. Wheat and Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed are those of the authors and do not necessarily reflect views of the U.S. Department of Agriculture. REFERENCES Maldonado-Ramirez, S.L., Schmale, D.G., Shields, E.J., and Bergstrom, G.C. 2005. The relative abundance of viable spores of Gibberella zeae in the planetary boundary layer suggests the role of long-distance transport in regional epidemics of Fusarium head blight. Agric. For. Meteorol. 130:20-27. Schmale, D. G. III, and G.C. Bergstrom. 2007. The aerobiology and population genetic structure of Gibberella zeae. Plant Health Progress DOI:10.1094/PHP-2007-0726-04-RV. Schmale, D.G., Leslie, J.F., Zeller, K.A., Saleh, A.A., Shields, E.J., and Bergstrom, G.C. 2006. Genetic structure of atmospheric populations of Gibberella zeae. Phytopathology 96:1021-1026. Schmale, D. G., Shah, D. A., and Bergstrom, G. C. 2005. Spatial patterns of viable spore deposition of Gibberella zeae in wheat fields. Phytopathology 95: 472-479. Schmale, D.G., Shields, E.J., and Bergstrom, G.C. 2006. Night-time spore deposition of the Fusarium head blight pathogen, Gibberella zeae. Can. J. Plant Pathol. 28:100-108. 71 Session 4: FHB Management 2007 UNIFORM FUNGICIDE TRIALS ON SOFT WHITE WINTER WHEAT IN MICHIGAN. D.E. Brown-Rytlewski1*, W.W. Kirk1, R. Schafer1 and L. Liddell1 1 Department of Plant Pathology, Michigan State University, E. Lansing, MI 48824 * Corresponding Author: PH: (517) 432-0480; Email: rytlews1@msu.edu ABSTRACT The objective of this project was to evaluate the effectiveness of commercially available and experimental fungicides for the control of Fusarium head blight (FHB) and reduction of deoxynivalenol (DON) in white winter wheat in Michigan. Four trial locations were planted in with Caledonia white winter wheat during 8 – 31 Oct 2006. Plots at the East Lansing and Clarksville locations were artificially inoculated with F. graminearum infested corn kernels at a rate of 0.2 oz/sq. ft. a week prior to heading. Irrigation was begun after inoculation was completed. Plots were irrigated four times /day for 20 minute intervals beginning the week prior to flowering until two weeks after flowering. The remaining two sites in Sandusky and Saginaw relied on natural inoculum and rainfall. All treatments were applied at early flowering (Feekes 10.5.1) at 25gpa and 40 psi using a CO2-pressurized R&D tractor mounted spray boom with XR11003VS nozzles positioned forward and backward. Treatments consisted of: 1) Folicur (tebuconazole) 4 fl oz/a; 2) Proline (prothioconazole) 5 fl oz/a; 3) Caramba (metconazole) 13.5 fl oz/a; 4) Topguard (flutriafol) 14 fl oz/a; 5) Punch (flusiazole) 6 fl oz/a; 6) Proline 3 fl oz/a + Folicur 3 fl oz/a; and 7) untreated control. Disease pressure (FHB and foliar diseases) in Michigan was generally very low in 2007. Plots were rated for foliar diseases 7 days after treatment and again at the soft dough stage (Feekes 11.2). Saginaw did not develop sufficient foliar disease for rating. FHB incidence, severity and index were rated at the soft dough stage. Only the Clarksville and East Lansing (both inoculated and irrigated) locations developed sufficient FHB for field ratings. Yield, test weight, percent moisture, Fusarium damaged kernels (FDK) and thousand grain weights were determined post harvest. Sub samples from each plot were sent to the University of Minnesota for DON analysis. At the East Lansing location, FHB severity for all treatments was significantly lower (3.2-4.9%) than the control (21.9%). For FHB incidence, Punch (7.8%) was not significantly different from the control or other treatments, but other treatments (5.7-5.9%) were significantly lower than the control (21.9%). FHB index for all treatments (2.0-3.5%) was lower than the untreated control (21.9%). There were no significant differences in DON levels (1.1-1.9 ppm) among any treatments. Yields ranged from 72.1-87.9 bu/a. Proline + Folicur (87.9 bu/a) and Caramba (87.6 bu/a) were significantly higher than for Folicur alone (72.1 bu/a), but no treatments were significantly higher than the untreated control (73.7 bu/a). There were no significant differences among test weights or 1000 grain weights at the East Lansing location. At the Clarksville location, there were no significant differences in FHB incidence (55.0-98.1%), severity (12.9-26.4%) or index (8.7-26.3%). There were no significant differences among treatments for test weight, 1000 grain weights, or yield (73.9-85.4 bu/a). Average DON levels ranged from 3.6 -10.1 ppm, but none was significantly different from the untreated control (6.8 ppm). All the treatments resulted in significantly less stagonospora and leaf rust than the untreated control. No phytotoxicity was observed in any of the treatments at any of the sites. 72 Session 4: FHB Management DURATION OF POST-FLOWERING MOISTURE AND INFECTION TIMING AFFECT ON FHB AND DON IN WHEAT. C. Cowger1* and C. Medina-Mora2 USDA-ARS, Department of Plant Pathology, NCSU, Raleigh, NC; and 2Department of Plant Pathology, Michigan State University, East Lansing, MI * Corresponding Author: PH: (919) 513-7388; Email: Christina.Cowger@ars.usda.gov 1 ABSTRACT Our understanding of how environmental and host genetic influences interact to determine DON concentrations in small-grain spikes is incomplete. High levels of DON have sometimes been observed in the absence of abundant disease symptoms. This multi-year experiment explored the influences of post-flowering moisture duration, infection timing, and cultivar resistance differences on FHB and DON in winter wheat. The experiment had a split-plot design. Whole plots were four durations (0, 10, 20, or 30 days) of post-anthesis misting. Sub-plots were soft red winter wheat cultivars, of which one (2005) or two (2006 and 2007) were susceptible to FHB and six were moderately resistant. There were two plots of each cultivar under each duration of irrigation: one inoculated at anthesis with a backpack sprayer, and one in which individual funnel-isolated spikes were chosen at random and inoculated with a spray bottle at specific post-flowering intervals in order to study the effect of late infection. Inoculations utilized F. graminearum spore suspensions of 104 (2005) or 105 (2006 and 2007) macroconidia/ml. All treatments were replicated three times. In the backpack-inoculated plots, disease incidence and severity were assessed prior to the onset of senescence, and a DON time-course study was performed by collecting spike samples six times at 10-day intervals starting two weeks after flowering. Samples of all treatments were assayed for Fusarium-damaged kernels (FDK), percent infected kernels (using Komada’s medium), and DON concentration. Assays of F. graminearum DNA by tissue type (kernel, rachis, or glume) were performed on a limited sample in 2005, using real-time PCR, and these assays are being conducted for all treatments in 2006 and 2007. Preliminary results: 1) Under conditions conducive to disease (2006 and 2007), FHB incidence and severity and grain DON concentrations increased with increasing duration of post-flowering moisture (P d” 0.05). Cultivar grain DON rankings changed under longer moisture durations, suggesting that resistance to post-flowering moisture may be a distinct trait. 2) In 2006, spikes inoculated 10 days after flowering contained significantly less grain DON at harvest time than those inoculated at flowering (P < 0.0001). Spikes inoculated 20 days after flowering had still less harvest-time grain DON than those inoculated 10 days after flowering (P < 0.0001), and had the same level of grain DON as noninoculated spikes (P d” 0.48). Changes in DON rankings suggested that resistance to late infection may also be a distinct trait. (Data from 2007 not yet available.) 3) In 2006, for spikes inoculated 10 days after flowering, 0 and 10 days of post-flowering mist resulted in mean harvest-time grain DON levels of 0.6 and 1.2 ppm, respectively, while 20 and 30 days of postflowering mist resulted in harvest-time grain DON levels of 2.0 and 2.4 ppm, respectively. At the same time, the percentage of FDK from spikes inoculated 10 days after flowering was significantly lower than that from spikes inoculated at flowering (P < 0.0001), and not significantly different from the FDK percentage from 73 Session 4: FHB Management noninoculated spikes (P = 0.40). Thus, late infections coupled with extended post-flowering moisture may be one scenario accounting for observations of visually healthy grain with excessive DON at harvest. (Data from 2007 not yet available.) 4) In 2005 and 2006, the time-course study results showed a significant decline in grain DON between mid-May and early June, which is normal harvest time. In 2006, DON progression was evaluated under varying durations of post-flowering moisture. Prolonged moisture delayed the DON decline, and raised the DON levels from which decline commenced, but DON levels continued dropping in those treatments during the three weeks after normal harvest time. This suggests that DON may actually be reduced by delaying harvest if DON levels are high early in grain-fill. ACKNOWLEDGEMENTS We thank Dennis Fulbright, Candice Gatlin, Teagen Gray, Pat Hart, Erik Hrebenuyuk, Paige Langdon, Jonathan Lovett, Benjamin Munn, Jennifer Patton-Özkurt, Ji Hyung Yang, and Didem Yigit for technical assistance, including DON and RT-PCR analyses. This material is based upon work supported by the U.S. Department of Agriculture. This is a cooperative project with the U.S.Wheat & Barley Scab Initiative. DISCLAIMER Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author and do not necessarily reflect the view of the U.S. Department of Agriculture. 74 Session 4: FHB Management EFFECT OF POST INOCULATION MOISTURE ON DEOXYNIVALENOL ACCUMULATION IN FUSARIUM GRAMINEARUM-INFECTED WHEAT. Pravin Gautam and Ruth Dill-Macky* Department of Plant Pathology, University of Minnesota, St. Paul, MN 55108 * Corresponding Author: PH: (612) 625-2227; Email: ruthdm@umn.edu OBJECTIVE The objective of this study was to examine factors; including host genetics, pathogen aggressiveness and environmental moisture, affecting the production and accumulation of deoxynivalenol (DON) in wheat. INTRODUCTION tions. Main plots were days of mist-irrigation after inoculation (14, 21, 28 and 35 days after inoculation [DAI]), sub-plots were wheat genetics and sub-sub plots were individual F. graminearum isolates (49-3, B63A, Butte86-ADA11, 81-2, B45A) differing in their relative aggressiveness and a mock-inoculated (water) control. Two row plots (each 1.8 m long) of three wheat varieties; Alsen (moderately resistant, resistance source Sumai 3), 2375 (moderately resistant, unknown resistance source) and Wheaton (susceptible) were planted in mid-April each year of the study. All plots were inoculated at the anthesis (mid-June) and 3 days later with macroconidial inoculum (1 x 10 6 macroconidia ml-1) using a CO2-powered backpack sprayer dispensing inoculum at the rate of 30 ml per meter of row. Mist-irrigation was started immediately following the first inoculation. Disease was assessed visually 21 DAI by counting total infected spikelets in 20 arbitrarily selected heads in each plot (10 heads per plot row). Grain was harvested at maturity (late July), machine threshed and dried for 10 d at 950 C. The percentage of visually scabby kernels (VSK) analysis was assessed on a 25 g sub-sample of harvested grain following the procedures of Jones and Mirocha (1999). Following the assessment of VSK the sub-samples were analyzed for DON at the University of Minnesota’s Mycotoxin Laboratory. Data were analyzed by ANOVA and LSD tests and correlations performed using SAS. Fusarium head blight (FHB) or scab of wheat and other cereals, primarily caused by Fusarium graminearum, has reemerged as a devastating disease in the United States. The disease causes yield losses both from reduced grain number and grain weight, including the formation of tombstones (shriveled kernels with a chalky appearance). FHB also affects quality of the grain through the production of a range of mycotoxins, among which DON is the most commonly present and that which is regulated in the grain trade. Though the production and accumulation of DON in infected grain is generally positively correlated to FHB severity, the correlation is not consistent and predicting the DON content of grain in commercial wheat and barley crops or breeding nurseries based on visual disease assessments prior to harvest is generally unreliable. The production and accumulation of DON in infected grain is not well understood and likely results from the complex interactions of host and pathogen genetics which is modified by the prevailing environmental conditions. The objective of this multi-year study was to identify factors affecting the production RESULTS AND DISCUSSION and accumulation of DON in wheat. In 2006, the average FHB severity was 22.3%, the MATERIALS AND METHODS average VSK was 4.9% and the average DON accumulation was 0.62 ppm. Overall, FHB severity, VSK Experiments were conducted at the St. Paul Experi- and DON was higher in 2007 than 2006. In 2007, the mental Station of University of Minnesota in 2006 and average FHB severity was 37.5%, and the average 2007 as split-split plot designs, each with five replica- VSK was 27.8% while the average DON concentration was 10.5 ppm. The effects of F. graminearum 75 Session 4: FHB Management isolate, wheat genetics and mist-irrigation were significant in both years of the study. While the isolate 49-3 generated the highest FHB severities, VSK and DON production in 2006, B63A had the lowest DON levels despite inciting high FHB severities and VSK. In 2007, isolates B63A and 49-3 produced the highest levels of DON and were associated also with higher FHB severities and VSK. Isolates 81-2 and Butte86ADA11 generally were associated with lower FHB severities, VSK and DON. In 2006 the severity of FHB and the percent VSK was significantly higher in Wheaton (42.5% and 11.5%, respectively) than the other two varieties (FHB severity < 15.5%, VSK < 2.9%). The DON concentration of Wheaton, across all isolates, was significantly higher (1.2 ppm) than for the other two wheat varieties tested (< 0.4 ppm). FHB severity and VSK was significantly lower in the treatments receiving the least amount of mist-irrigation (14 DAI; FHB severity 19%; VSK 4%) than longer mist-irrigation treatments (FHB severity 22.6-25.4%: VSK > 5%). The DON concentration was however significantly lower in the longest mist-irrigation treatment (35 DAI; DON 0.5 ppm) than in treatments where the mist-irrigation was applied for shorter periods of time (DON 0.6 – 1 ppm). The Spearman’s rank correlations of DON with FHB severity and VSK were 0.78 (P < 0.0001) and 0.85 (P < 0.0001), respectively. Similarly, in 2007 Wheaton had significantly higher FHB severity (59%) VSK (53.87%) and DON (17.64 ppm), than the other wheat genotypes examined (FHB severity < 27.51%; VSK < 19.4%; DON < 7.45 ppm). The severity of FHB was highest in mist-irrigation treatments applying supplemental water for 28 DAI (40.3%) than the other mist-irrigation treatments (36.1-36.8%). VSK readings were significantly higher (37.7%) for the longest mist-irrigation treatment (35 DAI) than the others (19-33.2%). DON was significantly lower (7.95 ppm) in the 35 DAI mist-irrigation treatment compared to the other irrigation treatments (9.9-13.3 ppm). The Spearman’s rank correlations of DON with FHB severity and VSK were 0.78 (P < 0.0001) and 0.78 (P < 0.0001), respectively. Our results show that FHB severity, VSK and DON level increases in more susceptible wheat cultivars. It also varies with the fungal isolates aggressiveness with respect to disease and DON production. DON level increased with mist-irrigation applied till 28 DAI but was reduced in the 35 DAI irrigation treatments. These results are in concordance with several researches which reported a reduction in DON levels with the long irrigation treatments. Similarly, it has been reported that DON accumulation in Fusarium-infected tissues peaks approximately six weeks after infection and then declines prior to harvest. In our case, the peak DON level was observed between four and five weeks after inoculation. Since DON is water soluble, the decline in DON levels might have been accelerated by the mist-irrigation, perhaps from leaching of the DON. The observed increase in DON levels despite mist-irrigation until 28 DAI was likely due ongoing production of DON as the fungus continues to grow and infect new tissues under conditions favorable for the pathogen. Thus, any DON leached by mist-irrigation water before 28 DAI would likely have been replaced by that produced by the spreading fungus. As the plant begins senescence, growth of the fungus and the production of DON may be reduced or even stop, and thus the leaching effect of irrigation on reduction of DON was readily detectable. Based on our results it may be concluded that longer durations of wetting, from either mist-irrigation or rainfall, after infection will increase the severity of FHB and VSK and thus the damage to grain, although DON concentrations may be reduced. ACKNOWLEDGEMENTS We would like to thank Amar M. Elakkad, Karen J Wennberg, Beheshteh Zagaran and Janne H. Kvame for technical assistance with the experiment and Dr. Yanhong Dong for conducting DON analysis. This material is based upon work supported by the U.S. Department of Agriculture, under agreement No. 76 Session 4: FHB Management 59-0790-4-096. This is a cooperative project with thors and do not necessarily reflect the view of the the U.S. Wheat and Barley Scab Initiative. U.S. Department of Agriculture. DISCLAIMER REFERENCES Any opinions, findings, conclusions, or recommenda- Jones R.K. and Mirocha C.J. 1999. Quality parameters in small grains from Minnesota affected by Fusarium head blight. tions expressed in this publication are those of the au- Plant Dis. 83:506-511. 77 Session 4: FHB Management PROSARO® – A NEW FUNGICIDE FOR CONTROL OF FUSARIUM AND MYCOTOXINS IN CEREALS. I. Haeuser-Hahn, S. Dutzmann, R. Meissner* and F. Goehlich Bayer CropScience AG, Alfred-Nobel Str. 50, 41789 Monheim, Germany Corresponding Author: PH: 49-2173385683; Email:Ruth.Meissner@Bayercropscience.com * ABSTRACT Fusarium head blight (FHB) and mycotoxins can be major challenges in cereal production. FHB is a disease caused by different Fusarium species. Under field conditions, all the predominant Fusarium species may produce mycotoxins with the exception of M. nivale. Quality and quantity of grain harvest are affected by FHB. Combatting FHB is demanding because many factors may influence the severity of the infection. Agricultural practices (cultivars, cropping methods, crop rotation etc.) and environmental conditions are all contributing factors to any epidemic. Chemical control with Fusarium active compounds such as the triazole fungicides Prothioconazole and Tebuconazole contribute significantly to a reduction of FHB and mycotoxin production. Suppression of the mycotoxins with these fungicides can reach values of higher than 70%. Success of the fungicide treatment is dependent on optimal application timing and spray coverage. In general, smaller droplet sizes provide more effective Fusarium control. With the mixture of Tebuconazole & Prothioconazole – Prosaro ®- the application window can be broadened due to the combination of preventive and curative strengths of the two active ingredients involved. In addition to FHB control, Prosaro controls a broad range of fungal pathogens in cereals and thus contributes to higher yield. 78 Session 4: FHB Management ADDITION OF ADJUVANT TO IMPROVE COVERAGE AND FUNGICIDE EFFICACY ON BARLEY, LANGDON 2006 S. Halley1*, V. Hofman1 and G. Van Ee2 1 Langdon Research Extension Center and Dept. of Agricultural and Biosystems Engineering, North Dakota State University, Fargo, ND 58105; and 2Biosystems and Agricultural Engineering Dept., Michigan State University, East Lansing, MI * Corresponding Author: PH: (701) 256-2582; Email: Scott.Halley@ndsu.edu INTRODUCTION Most fungicide application technology studies have focused on maximizing deposition on the entire small grain head. Observations in the field would indicate that the Fusarium head blight (FHB) ascospore can infect the awn part of the grain head but generally will not move down the awn and infect the other parts of the spike or kernel. The awns are a very effective structure for collecting both ascospores that cause the initial infection for FHB and unfortunately the fungicide solutions we apply to protect against FHB. Most fungicides applied to control FHB have a localized systemic type of activity meaning that there is little translocation within the grain head. In addition, most of the translocation is in an upward and outward movement from the initial point of deposition. Studies have been initiated to determine if we can increase the amount of fungicide solution collected on the whole head and reduce the percentage of fungicide solution collected on the awns relative to the spike. This study reports results of those efforts. It includes evaluation of the addition of several adjuvants, suggested by adjuvant manufacturers, and two adjuvant compounds previously reported to significantly increase coverage on the other parts of the spike or kernel portion of the grain head. MATERIALS AND METHODS A study was initiated in 2006 at the North Dakota State University Langdon Research Extension Center, Langdon North Dakota. The objective of the study was to determine if an adjuvant included with fungicide could increase deposition on the grain head as a whole, the developing kernel portion of the spike and subsequently reduce deposition on the awn portion of the head decreasing FHB disease incidence and deoxynivalenol concentration in the harvest sample. The study was designed as a randomized complete block with five replicates. A block of barley was planted with a double-disk type drill, rows spaced 7inches apart to cultivar ‘Stellar’ in mid May. After emergence and after application of weed control, the block was divided into plots 12 x 30 ft. Prosaro fungicide, prothioconazole, was applied at 3.25 fl oz/ acre which is one-half the rate recommended by manufacturer Bayer CropScience. The one-half rate was used with expectation that we could maximize and measure the beneficial effect of the adjuvant. A food grade dye, FD&C blue #1, was mixed with each of the fungicide solutions at a rate of 44 grams per acre. The dye was included as an indirect type measurement to determine differences in coverage on the parts of the grain head. After delineation of the plots, a Fusarium inoculum was hand-broadcast on each plot to encourage development of disease. Fungicides were applied with a tractor using a side-mounted spray boom. The tractor traveled 6 mph delivering the solution at 10 GPA and 40 psi with Spraying Systems XR8002 nozzles angled 30 degrees downward from horizontal and oriented to spray forward or the same direction of travel as the tractor. The spray system was equipped with a CO2 type delivery system instead of a standard pump. After applying the treatments, a sample of 10 heads were collected from each plot, deposited in Ziploc type bags and placed on ice. The awns of each head were individually clipped from the kernels. The awns and the remainder of the heads were deposited in separate 250 ml Erlenmeyer type flasks and sealed with a rubber stopper. A solution of 80 ml 95% ethyl alcohol was added to each flask and shaken for three minutes with a Burrell wrist-type action shaker. A sub sample of the solution was placed in a cuvette 79 Session 4: FHB Management and placed in a Jenway photospectrometer to determine the absorbance of the solution. Each absorbance reading was indirectly used to determine differences in the amount of dye collected on the grain head parts. A whole head sample was the sum of the parts. After the fungicide was applied, a sprinkler irrigation system was installed to modify the environment as needed and encourage the development of disease to determine differences among treatments. North Dakota State University Extension recommended production practices for barley in Northeast North Dakota were followed. A visual estimation was made from 20 samples per plot collected 20 days after fungicide application to estimate the incidence (number of spikes infected) and field severity (number of FHB infected kernels per head divided by total kernels per individual spike) of FHB in each plot. A rotary mower removed the front and back five feet from each plot prior to harvest to minimize any chance of interference by drift from the tractor when stopping or starting. Each plot was harvested with a Hege plot combine and the grain sample cleaned and processed for yield, protein, plump, and test weight. A sub sample was ground and analyzed for deoxynivalenol (DON) by North Dakota State University. Data was analyzed with the general linear model (GLM) in SAS. Fisher’s protected least significant differences (LSD) were used to compare means at the 95% probability level. Fusarium head blight developed later in the season and may have negated some of the beneficial effects of the adjuvants and the fungicide. The fungicide reduced FHB field severity over the untreated but no differences were measured among treatments (Table 1). Although DON levels were reduced by more than 50% from the untreated by WECO 6065 and AG 6470, they were not statistically significant. Several adjuvants increased the deposition on the whole head and there were differences recorded among adjuvants. The most notable was the adjuvant In-Place which is an encapsulating compound that could be used with an additional adjuvant to further increase deposition, distribution on the head, and fungicide efficacy. Also of note was the low deposition value of the Silkin adjuvant. Silkin is an organosilicate type adjuvant. The results may be a rate related effect and may be improved with the addition of another type adjuvant. SylTac is also a silicon type adjuvant that includes a penetrator and performed considerably better. No differences were measured on the kernel portion of the spike. Significant correlations were determined between FHB incidence and yield, test weight and deposition on the awns, and most notably DON levels with deposition on the spike, awns, and whole head indicating that these efforts are focusing in the right areas. Significant negative correlations were measured between coverage parameters and DON levels. DISCUSSION AND RESULTS The environment at the LREC was warm and dry both before and after fungicide application in 2006. 80 Session 4: FHB Management Table 1. FHB incidence and field severity, yield, test weight, plump, coverage, and deoxynivalenol concentration (DON) by treatment, Langdon 2006. FHB Treatment/ Adjuvant Syl-Tac Untreated AG06038 no adjuvant WECO5036-7 Triton X405 Silkin Preference Alfonic 1412-80 In-Place AG 5004 Induce WECO6065 AG06470 LSD(0.05) % C.V. Pr>F Adjuvant Rate 0.5% v/v Incidence (%) 100 100 0.5% v/v 100 100 0.25% v/v 99 0.25% v/v 99 0.25 pint/100 100 0.25% v/v 99 0.25% v/v 100 1/4 (adj./fung) 100 8 fl oz/a 100 0.125% v/v 100 0.25% v/v 99 1%v/v 100 NS 1 0.6947 Field Severity (%) 10.4 14.8 11.9 10.5 10.6 10.1 10.0 11.1 11.2 10.0 10.2 10.7 10.6 10.8 2.1 15 0.0056 Yield (bu/a) 132.5 116.4 126.7 130.9 117.9 131.7 127.6 124.4 128.7 121.4 124.4 127.2 124.3 126.6 NS 9 0.5656 Test Weight (lb/bu) 46.7 47.2 47.0 47.3 47.6 47.0 47.3 47.4 47.5 46.9 47.2 47.4 47.6 47.3 NS 1 0.1053 Plump (%) 97 98 98 98 98 98 98 98 98 98 98 98 98 98 NS 1 0.5381 Absorbance Spike Awns .114 .336 .054 .143 .130 .321 .116 .347 .105 .296 .123 .335 .089 .281 .108 .339 .108 .406 .137 .413 .121 .379 .120 .322 .084 .297 .139 .301 NS .079 36 19 0.1193 <0.0001 Whole .450 .197 .451 .463 .401 .458 .370 .448 .514 .551 .500 .441 .381 .441 .103 19 <0.0001 DON (ppm) 1.84 1.80 1.64 1.62 1.34 1.20 1.12 1.10 1.08 1.02 1.02 0.96 0.86 0.76 NS 65 0.5012 Table 2. Pearson correlation coefficients for FHB incidence and field severity, yield, test weight, plump, coverage, and deoxynivalenol concentration (DON) Langdon, 2006. Incidence Field Severity Yield Test Weight Field Incidence Severity 1.00 0.124 0.308 1.00 Yield -0.255 0.033 -0.025 0.840 1.00 Test Weight -0.133 0.272 0.138 0.253 -0.124 0.306 1.00 Plump -0.153 0.204 -0.162 0.181 -0.275 0.021 0.193 0.109 1.00 Plump Spike Awns Whole DON 81 Absorbance Spike Awns -0.045 -0.067 0.714 0.584 -0.106 -0.212 0.381 0.078 0.113 0.234 0.352 0.051 -0.106 0.143 0.381 0.239 -0.185 -0.143 0.125 0.239 1.00 0.448 <0.001 1.00 Whole -0.068 0.545 -0.204 0.090 0.234 0.063 0.072 0.5560 -0.180 0.137 0.719 <0.001 0.943 <0.001 1.00 DON 0.048 0.692 0.221 0.066 0.111 0.361 -0.256 0.032 -0.123 0.310 -0.315 0.008 -0.235 0.050 -0.300 0.012 1.00 Session 4: FHB Management ASSESSMENT OF AIR STREAM SPEED WITH TWO NOZZLE TYPES AS A TOOL TO IMPROVE DEPOSITION OF FUNGICIDE FOR CONTROL OF FHB IN WHEAT. S. Halley1*, V. Hofman1 and G. Van Ee2 1 Langdon Research Extension Center and Dept. of Agricultural and Biosystems Engineering, North Dakota State University, Fargo, ND 58105; and 2Biosystems and Agricultural Engineering Dept., Michigan State University, East Lansing, MI * Corresponding Author: PH: (701) 256-2582; Email: Scott.Halley@ndsu.edu OBJECTIVES To determine most effective air stream speed using two contrasting nozzle types to maximize deposition on the grain spike and improve efficacy of fungicide on hard red spring wheat. MATERIALS AND METHODS A field was selected near Esmond, North Dakota that was previously cropped corn. The field was planted to ‘Alsen’ spring wheat in an east/west direction with an air seeder with tramlines every 80 feet. The study was arranged as a factorial (nozzle type x air stream speed) in a randomized complete block design laid out in four replicated blocks, split into plots 40 x 500 ft. to accommodate one spray boom and the grower’s combine straight cut header. Plots were arranged in an east/west direction between tramlines and the plot length measured with a global positioning unit mounted on an all terrain vehicle after all herbicide applications had been completed. The sprayer was a Hardi-Twin (Hardi, Davenport, IA 58206) modified to accommodate the tramlines and to spray one half of the area between the trams, 40 feet width, beginning at the center of the tractor. The sprayer contained a diaphragm type pump and traditional flat fan hydraulic nozzles. The spray nozzles are mounted to direct the spray into the air stream which carried the spray solution to the grain. Before the field trial were completed, the spray booms were equipped with Teejet XR11003 and TT11003 nozzles (Spraying Systems Co, Wheaton, IL 60189)on each boom, respectively and calibrated. The nozzles were directed to spray forward from vertical at the maximum of 30 degrees forward. Both nozzles were calibrated at 40 psi to determine the output of the specific nozzles. The air stream speed was determined by setting the rpm on the fan at 1800, 2400, or 2900. The air stream velocities were about 23, 35 and 50 mph respectively, measured at oneinch from the air stream orifice. This was measured with a ‘Kestrel’ 2000 wind velocity meter (Niche Retail, Sylvan Lake, MI 48302). The spray drop size measurement application parameters were characterized by mounting two water sensitive papers (WSP) 1" vertically x 30" horizontally” side by side on a piece of flat iron at canopy height and spraying across the WSP. Each combination of the respective factors was measured. Volume median diameter (VMD) of the spray drops formed on the WSP, spray volume, and % area coverage was completed with a WRK ‘Droplet Scan analyzer’ Cabot, Arkansas. The fungicide was applied at Feekes growth stage 10.51. The fungicide solution included Folicur (tebuconazole) fungicide 4 fl oz/acre + Induce adjuvant at 0.125% v/v and a food-grade tracer dye (FD & C Blue #1) added at 44grams/acre. Folicur is manufactured by Bayer CropScience and Induce by Helena Chemical Co. Immediately after the fungicide was applied, ten heads were sampled from each plot in each of three locations and placed in 250 ml Erlenmeyer flasks, sealed with a stopper, and placed on ice for transport to the laboratory to measure the relative volume of solution collected for each of the treatments. Barley production recommendations from the North Dakota State University Extension Service for northeast North Dakota were followed. Eighty ml of 95% ethyl alcohol was added to each flask and shaken for three minutes with a wrist-action mechanical shaker (Burrell Scientific Instruments and 82 Session 4: FHB Management Laboratory Supplies, Model BT, Pittsburgh, Pennsylvania 15219). A sub sample of the wash solution was measured with a Jenway spectrophotometer (Jenway, Model 6300, Dunmow, Essex CM6 3LB England) and an absorbance of the tracer dye recorded to determine differences among application parameters. The absorbance reading quantifies differences in the amount of tracer dye deposited on the grain spike (a larger absorbance value is the result of more tracer dye in the solution). A visual estimation of disease incidence (number of spikes infected) and field severity was made from 20 heads per plot at early dough stage. Field severity rating is the number of FHB infected kernels per head divided by total kernels per individual spike. All plots were harvested with a Caterpillar Lexion combine on 7 August and weighed with a weigh wagon and a grain sample collected. The yield was determined from the grain collected from the harvested plot. The grain sample was cleaned and processed to determine plump, test weight, and protein. A sub sample was ground and analyzed for the toxin deoxynivalenol (DON) by North Dakota State University. Data was analyzed with the general linear model (GLM) in SAS. Fischer’s protected least significant differences (LSD) were used to compare means at the 5% probability level. RESULTS The VMD measured with water sensitive paper describes the relative drop size of the spray deposits from each of the nozzles. Half of the spray volume is in drops larger than this size in microns and half of the spray volume is in drops smaller than this value. The XR11003 nozzles had a smaller VMD than the TT11003 nozzle. Increasing air speed increased the VMD by spreading the drop over a larger area. Adding air as a spray solution carrier increased the area of coverage on the cards and increasing air stream speed increased area of coverage further. air stream speed was statistically the same as no air but greater than the 1800 and 2400 air stream speed when averaged across both nozzle types indicating a benefit to the high speed air stream. A characterization of the nozzles (F025_110) supplied with the Hardi-Twin and operating parameters traditionally used by the grower, indicated a reduction in VMD when nozzle pressure was increased and a large reduction in coverage of the cards when nozzle pressure was 90 psi. This indicates that the sprayer may provide better consistency of fungicide application due to increased deposition on the wheat head when operated with lower nozzle pressure and increased air stream speed. The growing season was very dry during flowering and no measurable disease developed. Only the disease levels on the untreated plots were assessed, FHB incidence and field severity were found to be 6.3 and 0.5 percent, respectively. There were no differences among treatments in yield, test weight, plump, or deoxynivalenol concentration. ACKNOWLEDGEMENTS The authors wish to acknowledge the cooperation of Bill and Louis Arnold, Esmond, ND. This material is based upon work supported by the U.S. Department of Agriculture, under Agreement No. 59-0790-3-079. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. DISCLAIMER Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture. REFERENCES ASAE Standards, 50th Ed. 2003 St. Joseph, Mich.: ASAE Deposition (Absorbance) on the grain heads were the same with both nozzles when no air was used to assist in deposition. The XR11003 nozzle deposited more tracer dye than the TT11003 nozzle. The 2900 rpm Fritz, B.K, Kirk, I.W., Hoffmann, W.C., Martin, D.E., Hofman, V.L., Hollingsworth, C, McMullen, M., and S. Halley. 2006. Aerial application methods for increasing spray deposition 83 Session 4: FHB Management on wheat heads. ASAE Applied Engineering in Agriculture. 22 (3): 357-364. Halley, S., Van Ee, G., Hofman, V., Panigrahi, S. and H. Gu. 2003. Ground Spray Systems and Spray Parameter Evaluation for Control of Fusarium Head Blight on a Field Scale Basis. 2003 National Fusarium Head Blight Forum Proceedings. p. 69-75. Hofman, V., McMullen, M., Panigrahi, S., Gregoire, T., Halley, S., and D. Gu. 1999. Application Equipment for the Control of Fusarium Head Blight (Scab), ASAE and CSAE North Central Intersectional Conference Paper No. MBSK 99-119, ASAE, St. Joseph, MI. Ledebuhr, R.L., Van Ee, G.R., Resmer, R., Forbush, T., and H. S. Potter. 1986. Field comparison of the effectiveness of air assisted rotary atomizers vs. conventional hydraulic nozzles for disease control and vine kill in potatoes. Engineering for potatoes / B.F. (Burt) Cargill editor and coordinator for development of the “Engineering for Potatoes” Program. [St. Joseph, Mich.?] : Joint publishers, Mich St Univ and American Soc of Agri Engineers p. 107-119. McMullen, M., Halley, S., Pederson, J., Moos, J., Hofman, V., Panigrahi, S., Gu, H., and T. Gregoire. 1999. Improved fungicide spraying for wheat/barley head scab control. North Dakota State University Extension Service Circular Extension Report 56. Nordbo, E., Kristensen, K., and Kirknell, E. 1993. Effects of wind direction, wind speed and travel speed on spray deposition. Pesticide Science. 38:33-41. Panneton, B. 2002. Image analysis of water-sensitive cards for spray coverage experiments. ASAE Applied Engineering in Agriculture. 18(2):179-182. SAS. 2005. SAS version 9.1, SAS Institute Inc. Cary, NC, USA. Teejet Mobile System Products Catalogue. Catalogue 49A. Spraying Systems Company. Wheaton, Illinois. 106. Zhu, H., Reichard, D.L., Fox, R.D., Brazee, R.D., and H.E. Ozkan. 1996. Collection efficiency of spray droplets on vertical targets. Transactions of the ASAE 39:415-422. 14 XR11003 TT11003 12 11.5 10.2 10.1 10 GPA 8 6 7.6 7.6 5.9 4.5 4 2.5 2 0 1800 2400 2900 0 Air speed (rpm) Figure 1. Volume Median Diameter (VMD) drop size produced using XR11003 and TT11003 nozzles at varying spray air delivery speeds. 84 Session 4: FHB Management 700 635 XR11003 TT11003 600 578 547 524 511 500 467 462 432 VMD 400 300 200 100 0 1800 2400 0 2900 Air speed (rpm) Figure 2. Estimated spray volume in GPA using XR11003 and TT11003 nozzles with varying spray air delivery speeds. 45.0 41.7 XR11003 TT11003 40.0 37.3 35.0 32.8 30.0 % 25.0 20.0 15.0 21.2 19.8 13.0 13.0 10.0 7.3 5.0 0.0 1800 2400 2900 0 Air speed (rpm) Figure 3. Relative percent area of water sensitive paper coverage using XR11003 and TT11003 nozzles with varying spray air delivery speeds. 85 Session 4: FHB Management . 0.200 XR111003 0.175 TT11003 LSD=.027 0.154 0.160 0.150 0.140 Absorbance 0.135 0.118 0.120 0.141 0.118 0.080 0.040 0.000 1800 2400 0 2900 Relative air stream speed (rpm) Figure 4. Relative deposition of spray on wheat heads using XR11003 and TT11003 nozzles in varying spray air delivery streams. 600 555 474 500 400 VMD 310 300 279 294 90 and 2400 back card 90 and 2400 front card 200 100 0 50 and 1800 50 and 2400 62 and 2400 Nozzle pressure (psi) and relative air stream speed (rpm) Figure 5. Volume Median Diameter (VMD) drop size measured with water sensitive paper using Hardi FO25_110 nozzles at varying pressures and spray air delivery speeds. 86 Session 4: FHB Management Table 1. Effects of fungicide on absorbance, yield, test weight and protein by nozzle and air stream speed, Esmond 2006. Sources of Nozzle or Air Variation Stream Speed Nozzle Air Stream Speed Noz*Air %C.V. Absorbance 0.0444 0.0771 0.4275 18 Nozzles averaged across air stream speeds XR11003 .151 TT11003 .132 .02 LSD (0.05) Air stream speeds averaged across nozzles Fast .163 Medium .136 Slow .129 None .138 .03Z LSD (0.05) Z Significant at 0.10 level. 87 Yield (bu/a) 0.1438 0.9207 0.7027 8 Test Weight (lb/bu) 0.0867 0.6719 0.6749 1 Protein (%) 0.1075 0.6693 0.8804 3 30.3 28.8 NS 58.0 57.4 00.6Z 15.8 16.1 NS 29.0 29.5 29.2 29.7 NS 57.4 57.7 57.7 57.9 NS 16.1 16.0 15.8 15.9 NS Session 4: FHB Management CHARACTERIZING PARAMETERS OF AIR DELIVERY TYPE SPRAY SYSTEMS TO MAXIMIZE FUNGICIDE EFFICACY ON SMALL GRAIN. S. Halley1*, K. Misek1, V. Hofman2 and G. Van Ee3 Langdon Research Extension Center, and 2Dept. of Agricultural and Biosystems Engineering, North Dakota State University, Fargo, ND 58105; and 3Biosystems and Agricultural Engineering Dept., Michigan State University, East Lansing, MI * Corresponding Author: PH: (701) 256-2582; Email: Scott.Halley@ndsu.edu 1 ABSTRACT Several major manufacturers of ground application equipment (e.g. Hardi Spray Systems and Spray-Air Technologies Inc.), manufacture and sell sprayers that use an air stream to assist in delivering the spray solution to the plant canopy. These sprayers have been shown to offer several unique performance characteristics. First, the air stream minimizes spray drift by overpowering the ambient wind and carrying the smaller spray droplets to the target plant material. Second, the energy of the air stream tends to carry the small droplets (less than 200 microns) deeper into the plant canopy. Third, the turbulence of the air stream assists in more uniformly depositing the spray drops in the hard-to-reach areas of the canopy. The second and third characteristics would be important in controlling foliar diseases. The air stream, depending on velocity, also would be able to alter the orientation of the grain head and change potential deposition. Our objective is to characterize the effects of varying the speed of the air stream, drop sizes and application angles for improved fungicide efficiency to control Fusarium head blight on spring barley and hard red spring wheat (HRSW). The two studies were randomized complete block designs with factorial arrangements and replication. Factors included three drop sizes, three air speeds, and three spray angles. Prosaro fungicide and Induce adjuvant were applied at 6.5 fl. oz/acre and 0.125% v/v to control FHB. RESULTS Fungicide coverages were different among sprayer factor combinations on HRSW but not barley indicating the uniqueness of architecture of the individual crop. Fungicide applied with a ‘large’ fine drop at 60º angle had the lowest incidence and field severity on the HRSW. HRSW yield was greatest when a median air speed was used, 55.8 vs 52.4 and 51.3 bu/acre. On barley a smaller yield was measured when a coarse drop was used in combination with near vertical orientation and minimum air speed. Several sprayer configurations increased plump. The untreated control was included in the trials but was not included in the statistical calculations because it did not fit with the factorial arrangement. ACKNOWLEDGEMENT This material is based upon work supported by the U.S. Department of Agriculture, under Agreement No. 590790-3-079. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. DISCLAIMER Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture. 88 Session 4: FHB Management EVALUATION OF FUNGICIDE FOR CONTROL OF FUSARIUM HEAD BLIGHT WITH AERIAL APPLICATION TECHNOLOGY. S. Halley* and V. Hofman Langdon Research Extension Center and Dept. of Agricultural and Biosystems Engineering, North Dakota State University, Fargo, ND 58105 * Corresponding Author: PH: (701) 256-2582; Email: Scott.Halley@ndsu.edu INTRODUCTION Fusarium head blight (FHB) has been a major problem for cereal grain producers during the past decade. To combat this disease, growers have applied fungicide by both aerial and ground application. About 50% of the small grains acreage sprayed with fungicide in the Dakota-Minnesota region of the Great Plains is applied with spray planes. Aerial application has several advantages over ground application. The planes travel at speeds greater than 100 mph so large acreages can be sprayed in relatively short periods of time, the planes can make applications when surface conditions do not permit use of ground application equipment, and there is less damage to the crop due to tracking. Most aerial applications are applied at 3 to 7 GPA depending on the fungicide label. Applicators use a variety of nozzle types which often include deflectors to discharge spray perpendicular to the air stream. Spray discharge angles perpendicular to the air stream create a smaller drop size than nozzles directed parallel to the air stream. Faster travel speeds will decrease drop size and increasing liquid operating pressure will increase drop size. Spray volumes can be increased by increasing orifice size or by adding additional orifices along the spray boom. Aerial application spray drop size is determined by orifice size, nozzle orientation to the air stream, operating pressure and flying speed. MATERIALS AND METHODS An aerial application study was conducted near Esmond, North Dakota in 2006 to evaluate fungicide application for control of FHB on ‘Tradition’ cultivar barley. A site was selected on the Bill and Louis Arnold farm. The study team included Bill Arnold, farm operator, Dakota Aviation, Don Hutson-owner/pilot Grafton, ND, Vern Hofman and Scott Halley-North Dakota State University, Extension Engineer and Crop Protection Scientist, respectively. Several additional summer staff completed the team. The study was designed as a randomized complete block with four replicates. The plots were 150 ft wide (three application passes) by 450 to 850 ft long. Plots in blocks for replicate one and two were north/south and replicates three and four east/west. The treatments included Folicur 3.6 F (tebuconazole) fungicide (Bayer CropScience manufacturer) at 4 fl oz/acre applied with spray volumes of 3 or 7 GPA applied with a fine and a ‘small’ medium size drop and one volume of 5 GPA applied with a ‘small’ medium size drop (the 5 GPA treatment is a typical application standard of commercial aerial applicators). The applications were applied to heading barley (greater than 50% of main stem heads fully extended from the boot). The fungicide was applied with a fixed-wing Cessna Ag Truck aircraft equipped with CP-03 nozzles flying at 125 mph with an operating pressure of 40 psi. The different spray volumes were obtained by changing orifice size across the spray boom and the drop size adjustment was made by using the 30 or 90 degree deflector, large and smaller drop size, respectively. The treatments were applied on 30 June between 10:00 a.m. to 2:00 pm after the dew had dried from the plants. Wind conditions were WNW at speeds of 8.5 to 10.4 mph. This is a typical wind speed for the region at this time of year. The fungicide was applied with Induce adjuvant (Helena Chemical Co.) at 0.125% v/v and F D&C Blue #1 dye added at 44 grams per acre. The dye is a food grade type used in coloring food products. Water sensitive cards were placed on stands at grain head height in the center of each plot to replicate a head. The most commonly used method to evaluate spray technology is the use of water and oil sensitive paper (WSP Spraying Systems Co. ®, Wheaton, Illi89 Session 4: FHB Management nois 60189). Cards, 26 x76 mm, were placed at grain head height on stands (Panneton, 2002). One card was placed horizontal (Wolf and Caldwell, 2004). Applied stain size was determined with WRK DropletScan system (WRK, Cabot, Arkansas 72023) and presented as volume median diameter (VMD) which indicates that ½ of the spray volume is in drops smaller than this drop size and ½ of the spray volume is in drops larger than this size. The area of coverage is presented as percent of the card area analyzed. Three 50 ft spray passes were made side by side (150 ft.) on each plot. All data were collected from the center of the plot. Additionally, three samples of ten heads were collected at 3 points across the center swath and placed in glass Erlenmeyer flasks for determination of head coverage of the spray solution. The collected heads were stored on ice until they could be measured for dye coverage. The spray coverage of the heads was determined by washing the dye from the heads by wrist action shaking for three minutes with 80 ml of 95% ethyl alcohol and determining the absorbance with a Jenway spectrophotometer (model 6300). Differences among treatments were determined by a visual assessment of FHB and foliar disease at mid dough growth stage by assessing twenty heads per plot and determining the incidence of the disease (present or not) and the severity of the individual head. The summation of the incidence times the severity of the twenty heads gave a field severity per plot. Foliar disease differences were determined by estimating the infected area on five leaves at two locations. The field was harvested on 5 August. One pass of the combine was made through the center of each plot with a Caterpillar Lexion combine with a straight cut header. The grain from the harvested area of each plot was measured with a weigh wagon and a sub sample saved to determine yield, test weight, protein, plump and deoxynivalenol (DON) from the processed grain sample. Data were analyzed with the general linear model (GLM) in SAS. Fisher’s protected least significant differences (LSD) were used to compare means at the 5% probability level. RESULTS AND DISCUSSION The environmental conditions were in contrast in 2006 compared to 2005 when a duplicate trial was conducted. The crop in 2005 was devastated with FHB. In 2006 low relative humidity levels and little precipitation kept both FHB and foliar diseases from causing an economic loss. The limited available soil water also limited yields and reduced test weight and plump to levels so that malting barley standards were not met. No differences were determined among yield, test weight, protein and absorbance. Plump was increased 10% with a 3 GPA spray volume. The benefit was a result of increased amount of fungicide active ingredient collected on the spike th the larger fungicide concentration of the spray solution. Some fungicies extend the growing period of the plant before senescence and it is the authors’ perspective that this may have occurred. A trend was established showing greater deposition with the finer type drop size. The awns of the barley are efficient collectors of fine drops and also spores. This trend is different from a typical application with ground equipment where a drop size of 300 to 350 microns will deposit in greater quantities than a fine drop size. The ASABE standard S572 spray drop classification system for the two applications should have been about 240 and 300 microns. The WSP card showed a larger stain size than the reported limits of the technology. The differences show a drop size difference of about 50 microns between the two stain sizes and show one of the limitations of using WSP and field spray applications. The untreated in each replicate was not included in the statistical analysis because of the lack of fit with the factorial arrangement used to compare the mean volumes and drop sizes but is presented as a reference to overall fungicide efficacy. ACKNOWLEDGEMENTS The authors wish to acknowledge the cooperation of Bill and Louis Arnold, Esmond, ND. This material is based upon work supported by the U.S. Department of Agriculture, under Agreement No. 59-0790-3-079. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. 90 Session 4: FHB Management Panneton, B. 2002. Image analysis of water-sensitive cards for spray coverage experiments. ASAE Applied Engineering in Agriculture. 18(2):179-182 DISCLAIMER Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the Wolf, T.M., and B.C. Caldwell. 2004. Evaluation of double author(s) and do not necessarily reflect the view of the spray deposits on vertical targets. Aspects of Applied Biology 71:99-106. U.S. Department of Agriculture. REFERENCES http://apmru.usda.gov/downloads/AERIAL%20 SPRAY%20NOZZLE%20MODEL%20TABLES%202004.html. Table 1. Yield, Test Weight, Plump, Protein, and Head Coverage (Absorbance) by Spray Volume and Drop Size Esmond 2006. Yield Test Plump Protein Weight Spray Volume Drop Size (bu/ac) (lb/bu) (%) (%) Absorbance untreated 66.3 11.8 0.057 Spray volume averaged across drop sizes 62.0 45.8 58.3 64.8 45.4 52.8 NS NS 5.3 11.8 12.7 NS 0.255 0.263 NS Drop size averaged across spray volumes Fine 62.3 45.6 55.3 Medium 66.6 45.6 55.9 11.9 11.9 0.289 0.229 Fine Medium Fine Medium 3 7 LSD (0.05) 3 7 Sources of variation Rep Volume Drop Size Vol*Drop %C.V. 45.1 48.9 58.5 65.4 61.9 67.7 46.0 45.5 45.2 45.6 58.6 58.0 51.9 53.8 11.8 11.9 12.1 12.0 0.286 0.225 0.292 0.235 0.0474 0.8153 0.2256 0.6634 10 <0.0001 0.1592 0.8025 0.1146 1 0.0019 0.0430 0.7976 0.6099 9 0.1765 0.3871 0.9459 0.5453 3 0.0431 0.8911 0.3390 0.9735 45 91 Session 4: FHB Management Table 2. Volume Median Diameter (VMD), GPA, and Coverage determined Spray Solution Collected on Horizontal Placed Water Sensitive Cards, Esmond 2006. Spray Volume Drop Size VMD GPA Coverage (%) 3 3 5 7 7 Untreated Fine Medium Medium Fine Medium 360 404 370 401 451 4.2 2.0 4.9 7.5 4.7 10.1 4.7 12.0 17.8 11.9 200 0.07 0.2 92 Session 4: FHB Management RELATIONSHIPS BETWEEN YIELD, GRAIN QUALITY VARIABLES, AND FUSARIUM HEAD BLIGHT INTENSITY IN WINTER WHEAT. John Hernandez Nopsa and Stephen N. Wegulo* Department of Plant Pathology, University of Nebraska, Lincoln, NE, 68583-0722 * Corresponding Author: PH: 402-472-8735; Email: swegulo2@unl.edu ABSTRACT Fusarium head blight (FHB) of wheat, caused by Fusarium graminearum, can cause significant losses resulting from yield reduction, kernel damage, and presence of deoxynivalenol (DON), an important mycotoxin with serious food safety implications. In 2007, two experiments were conducted to identify relationships between (i) yield, grain quality variables, and FHB intensity and (ii) visual assessments of FHB and DON. In the first experiment, three winter wheat varieties (Jagalene, Harry and 2137) were planted on two planting dates, 5 and 27 October 2006. Plots were inoculated with conidia and ascospores of F. graminearum (1 x 105 spores/ml) at early and mid anthesis, or were not inoculated. Experimental design was a split-split-plot in randomized complete blocks with three replications. Planting date was the main plot, variety the subplot, and inoculation timing the sub-subplot. FHB severity was determined 21 and 25 days after inoculation on 20 heads in each of five arbitrarily selected locations in each plot. There was a significant positive correlation (0.48 < r < 0.76, P < 0.05) between FHB incidence and FHB severity in each variety (N = 18), first planting date (N = 27), and all varieties and planting dates combined (N = 54). Correlation between FHB index and yield was negative but not significant at P = 0.05. Correlation between FHB index and 1000 kernel weight was significant for the first planting date (r = 0.45, P = 0.0191) but positive, contrary to what as was expected. Correlation between FHB index and Fusarium damaged kernels (FDK) was significant for the first planting date (r = -0.47, P = 0.0132) but negative, contrary to what was expected. Correlation between FHB index and DON was not significant at P = 0.05. Correlation between FDK and 1000 kernel weight was not significant at P = 0.05 for Jagalene and Harry, but was significant for 2137 (r = -0.47, P = 0.0507), first planting date (r = -0.65, P = 0.0002), second planting date (r = -0.63, P = 0.0005), and all varieties and planting dates combined (r = -0.64, P < 0.0001). Correlation between FDK and DON was not significant at P = 0.05, as was correlation between FDK and yield. In the second experiment, two varieties (Harry and 2137) were planted on 9 October 2006. Plots were inoculated at early anthesis as described above. Varieties were arranged in randomized complete blocks with three replications. In mid June 2007, 20 heads were randomly tagged in each of 11 disease severity categories in each plot: 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50%. There was a significant positive correlation between FHB severity in the 11 severity categories and DON for both Harry (r = 0.74. P = 0.0092) and 2137 (r = 0.70, P = 0.0157). However, DON levels were higher in Harry than in 2137. The results from this study indicate that (i) relationships between yield, grain quality variables, and FHB intensity may not be clear cut, (ii) there is a positive association between DON levels and FHB severity, and (iii) wheat varieties differ in the levels of DON they accumulate. ACKNOWLEDGEMENT AND DISCLAIMER This material is based upon work supported by the U.S. Department of Agriculture, under Agreement No. 590790-7-080. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture. 93 Session 4: FHB Management OUTCOMES OF USING INTEGRATED FHB MANAGEMENT STRATEGIES ON MALTING BARLEY CULTIVARS AND GERMPLASM IN MINNESOTA. C.R. Hollingsworth1*, L.G. Skoglund2, D.B. Cooper2, C.D. Motteberg1 and L.M. Atkinson3 1 Northwest Research & Outreach Center and Dept. of Plant Pathology, University of Minnesota, Crookston, MN 56716; 2Busch Agricultural Resources, Inc., Fort Collins, CO 80524; and 3 Dept. of Earth System Science and Policy, Northern Great Plains Center for People & Environment, University of North Dakota, Grand Forks, ND 58202 * Corresponding Author: PH: (218) 281-8627; Email: holli030@umn.edu ABSTRACT The objective of our trial was to determine grain and malt quality responses from four commercially-available malting cultivars and four advanced malting germplasm lines following exposure to four Fusarium head blight (FHB) disease management strategies. The experiment was planted on 9 May 2007 into soybean residue and was situated in a commercial production field within Marshall County, in northwest Minnesota. Barley entries included Tradition, a 2004 Busch Agricultural Resources, Inc. (BARI) release; Legacy, a 2000 BARI release; Drummond, a 2000 North Dakota State University (NDSU) release; B2218 and B2513, BARI advanced germplasm lines; ND20448, NDSU advanced germplasm line; and M122, University of Minnesota advanced germplasm line. Treatments were replicated four times and exposed to one of four fungicide strategies (Table 1). The test area was neither misted nor inoculated. Environmental conditions did not support normal plant growth or stand establishment. Frequent rain events caused soil saturation for an extended period of time prior to Feekes growth stage (FGS) 10.5 (early heading). This resulted in plant stress which caused severe plant stunting, tiller abortion, and low yield (entry means for yield ranged from 21 bu/a to 37 bu/a). Split-plot analyses from PROC GLM in SAS were conducted where fungicide treatment represents whole plots and entry represents subplots. FHB symptoms were assessed at approximately FGS 14. Barley entry and fungicide treatment were significant for FHB incidence, while barley entry was also significant for FHB severity and FHB index (P<0.05). While FHB symptom expression was relatively low (entry means for FHB index ranged from 0.2% to 1.8%), resistant germplasm lines (B2218, M122, and B2513) had significantly lower index values than current commercial cultivars (Legacy, Tradition, and Robust). Deoxynivalenol (DON) levels in grain were miniscule to below detectable limits with an overall test mean of 0.1 ppm. The nontreated control fungicide treatment was not different from tebuconazole (4 fl. oz./a), but had significantly higher DON levels compared to either rate of Prosaro (P<0.05). Three replicates of grain samples from treatments #1 and #4 (Table 1) were micro-malted in the BARI Seed Research Quality Lab located at Fort Collins, CO. Resulting malt was analyzed for alpha amylase, beta glucan, diastatic power, free amine nitrogen, percent fine extract, predicted extract, malt protein, wort protein, and turbidity. Differences between barley entries were significant across all quality traits (P<0.05). Responses of malt to fungicide were significant for alpha amylase (P=0.03). Treatment #4 resulted in larger levels (75.0) of alpha amylase than the nontreated control (69.7). There were no significant fungicide*entry interactions at P<0.05. 94 Session 4: FHB Management Data produced from a single growing season in northwest Minnesota indicate that interactions between fungicide treatment and barley entries generally did not influence FHB disease symptoms, grain yield, or kernel and malt quality traits. However, additional data is needed from a typical growing season before further conclusions can be drawn. ACKNOWLEDGEMENTS The authors would like to thank Busch Agricultural Resources, Inc. for supporting this research; Bayer CropScience for supplying fungicide products; UM and NDSU breeders for providing germplasm; Busch Agricultural Resources, Inc. Seed Research Quality Lab and the University of Minnesota Mycotoxin Lab for providing malt quality analyses and DON results, respectively. Table 1. Fusarium head blight disease management strategies tested on eight malting barley entries near Warren in the northwest Minnesota Red River Valley. Fungicide applications were made at Feekes growth stage 10.5 (early heading). Rate* Trt Product Active ingredient (fl. oz./a) 1 Nontreated control... 2 Folicur...................... tebuconazole 4.0 3 Prosaro..................... tebuconazole and prothioconazole 6.5 4 Prosaro..................... tebuconazole and prothioconazole 8.2 *Treatments 2 through 4 included 0.125% Induce, a nonionic surfactant. 95 Session 4: FHB Management UNDERSTANDING PRACTICAL OUTCOMES FROM IMPLEMENTING FHB MANAGEMENT STRATEGIES ON SPRING WHEAT. C.R. Hollingsworth1*, C.D. Motteberg1, D.L. Holen2 and L.M. Atkinson3 Northwest Research & Outreach Center and Dept. of Plant Pathology, University of Minnesota, Crookston, MN 56716; 2University of Minnesota Fergus Falls Extension Regional Center, Fergus Falls, MN 56537; and 3Dept. of Earth System Science and Policy, Northern Great Plains Center for People & Environment, University of North Dakota, Grand Forks, ND 58202 * Corresponding Author: PH: (218) 281-8627; Email: holli030@umn.edu 1 ABSTRACT The objective of our trial was to determine grain yield and quality responses, as well as economic outcomes from 13 hard red spring wheat cultivars when exposed to six different disease management strategies. This research represents Minnesota’s participation in the multi-state, multi-year integrated disease management cooperative research which is meant to identify the most practical means in managing Fusarium head blight (FHB) across states and wheat classes. The test included four replicates at each of two experiment locations. Planted into soybean residue, a site was located near Oklee in northwest Minnesota and another was near Fergus Falls in west central Minnesota. The Oklee site was planted on 27 April 2007 and the Fergus Falls site on 2 May 2007. Spring wheat cultivars included Ada, Alsen, Banton, Bigg Red, Briggs, Freyr, Glenn, Knudson, Oklee, Samson, Steele-ND, Ulen, and Walworth which were exposed to one of six disease management strategies (Table 1). The test areas were neither misted nor inoculated. Environmental conditions varied substantially between locations. Split spilt-plot analyses using PROC GLM in SAS were made where ‘location’ represented the whole plot factor, ‘fungicide’ the subplot factor, and ‘cultivar’ the sub-subplot factor. Transformations were conducted on data identified with non-normal distributions. Fergus Falls had lower test weights, kernel protein, and FHB index ratings than the Oklee site (P<0.05). Deoxynivalenol (DON) levels in grain were miniscule to below detectable limits at Fergus Falls (d”0.13 ppm). Oklee location DON results are not yet available. Cultivar and disease management strategy were both significant for net revenue, yield, test weight, protein, and FHB incidence, while FHB severity and FHB index were significant for cultivar (P<0.05). Knudson (77.8 bu/a), Samson (77.6 bu/a), and Steele-ND (74.9 bu/ a) had the largest yields, while Bigg Red (62.4 bu/a) and Alsen (63.3 bu/a) had the smallest. Cultivars Samson, Ulen, Steele-ND, and Oklee had the highest ratings for FHB incidence, FHB severity, and FHB index while Bigg Red, Alsen, Glenn, and Knudson had the lowest. Knudson ($611.27/a), Samson ($608.26/a), and Steele-ND ($591.48/a) had the greatest net return while Bigg Red ($482.89/a) and Alsen ($497.13/a) returned the least (P<0.05). Across all cultivars, yield, protein, and test weight were significantly increased with disease management strategies #3, #4, and #5, compared with strategy #1 (Table 1). Strategy #4 resulted in the largest net return and strategies #1, #2, #5, #6 the least returns (P<0.05). Disease-associated limitations to yield were offset by timely fungicide application. Cultivars known for susceptibility to disease responded well to the growing environment, producing excellent yields of high quality grain. Fungicide application increased net returns compared with no fungicide even during a year of relatively low disease pressure. Economically-speaking, spring wheat growers in the Minnesota Red River Valley who benefited the most during 2007 grew cultivars that were moderately susceptible to FHB. 96 Session 4: FHB Management ACKNOWLEDGEMENTS AND DISCLAIMER We would like to thank the Minnesota Wheat Research and Promotion Council for supporting this research; BASF Corp., Bayer CropScience, and Syngenta for supplying fungicide products; the University of Minnesota Mycotoxin lab for providing DON results; Tom and Deb Jennen (Fergus Falls) and Ray and Barbara Swenson (Oklee) for cooperating with us. This material is based upon work supported by the U.S. Department of Agriculture, under Agreement No. 590790-3-080. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the U.S. Department of Agriculture. Table 1. Disease management strategies tested on 13 cultivars of hard red spring wheat at two locations in the Minnesota Red River Valley. Strategy Product 1 Nontreated control.. 2 Dividend Extreme.. Active ingredient difenoconazole and mefenoxam 3 Application Rate* Timing** 3 fl. oz./100 lbs. Headline................. pyraclostrobin 3 fl. oz./a Folicur/Proline....... tebuconazole & prothioconazole 3 + 3 fl. oz./a 3 fl. oz./100 lbs. difenoconazole & mefenoxam 4 Dividend Extreme.. 3 fl. oz./a pyraclostrobin Headline................. 3 + 3 fl. oz./a tebuconazole/prothioconazole Folicur/Proline....... 5 Dividend Extreme.. difenoconazole & mefenoxam 3 fl. oz./100 lbs. Folicur/Proline....... tebuconazole & prothioconazole 3 + 3 fl. oz./a 6 Folicur/Proline....... tebuconazole & prothioconazole 3 + 3 fl. oz./a *Treatments 3 through 6 included 0.125% Induce, a nonionic surfactant. ** Feekes growth stage (FGS) 2 = 4 to 5 leaf, and FGS 10.51 = early anthesis. 97 Seed applied preplant FGS 2 FGS 10.51 Seed applied FGS 2 FGS 10.51 Seed applied FGS 10.51 FGS 10.51 Session 4: FHB Management CONTRIBUTION OF WITHIN-FIELD INOCULUM SOURCES TO FUSARIUM HEAD BLIGHT IN WHEAT. M.D. Keller1, K.D. Duttweiler2, D.G. Schmale1 and G.C. Bergstrom2* 1 Department of Plant Pathology, Physiology, and Weed Science, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061; and 2Department of Plant Pathology, Cornell University, Ithaca, NY 14853 * Corresponding Author: PH: (607) 255-7849; Email:gcb3@cornell.edu ABSTRACT Knowledge of the relative contribution of within-field inoculum sources of Gibberella zeae to infection of local wheat and barley is important for developing and/or excluding strategies for managing Fusarium head blight (FHB). Our research is based on the hypothesis that spores of G. zeae that are deposited on wheat spikes and that result in Fusarium head blight come primarily from well-mixed, atmospheric populations in an area. Our experimental objective was to determine the relative contribution of within-field, clonal inoculum sources of G. zeae to FHB in susceptible wheat cultivars. In 2007, corn stalks and corn kernels infested with clonal, fingerprinted isolates of G. zeae containing rare alleles (relative to background populations) were released in replicated 1 m diameter circular plots in single wheat fields in New York and Virginia. We collected mature wheat spikes at the inoculum source, at a radius of 10 feet from the source, at a radius of 20 feet from the source, and in more distant parts of each field. We used amplified fragment length polymorphisms (AFLPs) to genotype isolates recovered from these spikes and to determine the contribution of released isolates to FHB at various distances from those sources. Since our inoculum sources contained clonal isolates that have unique AFLP haplotypes, we were able to observe these clones in a mixed/diverse background population containing numerous AFLP haplotypes. Nearly 500 isolates of G. zeae were collected and single-spored from NY and VA. Preliminary AFLP data from the first year of experimentation suggests that within-field sources of G. zeae provided a minor fraction of FHB inoculum compared to background atmospheric sources in a non-epidemic environment in New York and in a moderate epidemic environment in Virginia. ACKNOWLEDGEMENT AND DISCLAIMER This material is based upon work supported by the U.S. Department of Agriculture, under Agreement No. 590790-4-093. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed are those of the authors and do not necessarily reflect views of the U.S. Department of Agriculture. 98 Session 4: FHB Management TIME OF FLOWERING IN WHEAT FOR MANAGING FUSARIUM HEAD BLIGHT. Gregory S. McMaster USDA-ARS, Agricultural Systems Research Unit, Fort Colllins, CO Corresponding Author: PH: (970) 492-7340; Email: Greg.McMaster@ars.usda.gov ABSTRACT Efficacy of management is increasingly being timed based on crop developmental stage and consideration of crop developmental physiology. In the case of Fusarium head blight, it has often been managed by one pesticide application timed to the developmental stage of anthesis (i.e., flowering). However, flowering is controlled by the interaction of genotype, environment, and management and can occur over an extended period of time, confounding when to make the application. The objective of this talk is to discuss wheat development to provide information for improving the current management practice and exploring alternative management options. The presentation will first present a brief overview of wheat development and highlight when the various yield components are being formed. Wheat development follows a few general principles beginning with development being an orderly and predictable process. The genetics provides the “blueprint” for the orderly sequence of events leading to flowering. Temperature, reflecting thermal time, is then used to predict when flowering will occur. Sources of variation in flowering time are identified including a) within a shoot, b) among shoots on a plant, c) among plants within small areas/plots, and d) across landscapes. Other sources of variation exist among genotypes and variable planting/emergence dates. Management options to reduce the period of flowering are discussed, along with the risks of doing so 99 Session 4: FHB Management DIFFERENTIAL EFFECTS OF INFECTION TIMING ON FUSARIUM HEAD BLIGHT AND ON DON AND DON DERIVATIVES IN THREE SPRING GRAINS. Marcia McMullen*, Jim Jordahl and Scott Meyer * Dept. of Plant Pathology, North Dakota State University, Fargo, ND 58105 Corresponding Author: PH: (701) 231-7627; Email: marcia.mcmullen@ndsu.edu ABSTRACT The effect of infection timing on the development of Fusarium head blight (FHB) and DON and DON derivatives was evaluated under a controlled greenhouse environment. Test plants were susceptible and resistant or moderately resistant lines of three spring grains - hard red spring wheat (HRSW) (‘Grandin’ and ‘Glenn’), durum wheat (‘Munich’ and ‘Divide’), and six-rowed spring barley (‘Robust’ and ND20448 [a line from R. Horsley’s breeding program]). Infection timings included head half-emerged (Feekes 10.3), full head emergence (Feekes 10.5), anthesis in wheat (Feekes 10.51), and kernel watery ripe (Feekes 10.54), or dual infections at the two later growth stages. Infections were initiated by atomizing a mixture of four isolates of Fusarium graminearum, at 20,000 spores/ml, 20 ml/pot, with a DeVilbiss atomizer, followed by 24 hours of misting. FHB incidence and severity were determined at 21-25 days after inoculation. At maturity, kernels were hand threshed for subsequent mycotoxin analysis. DON, 3ADON, 15ADON and nivalenol (NIV) analyses were done using gas chromatography and electron capture techniques. FHB indices [(incidence x severity)/100] and mycotoxin levels (ppm) indicated that differential responses to single infection timings occurred among spring grain classes: a) in barley, values of these parameters were highest with infection at the watery ripe stage; b) in HRSW, at anthesis; and 3) in durum, about equally high at anthesis or watery ripe stage infections. In all three crops, infections at head half-emerged resulted in the lowest FHB severities and DON levels. The dual infections at the two latter growth stages, generally resulted in the highest FHB index and DON values in all grain classes. DON, 15ADON and 3ADON accumulations were highly correlated with FHB index in all spring grain classes. 15ADON and 3ADON levels also were highly correlated with DON levels. 15ADON was more frequently recovered and at higher ppm than 3ADON (highest average 15ADON was 4.5 ppm in barley, vs 1.1 ppm 3ADON in barley). 3ADON generally was detected only when the average DON levels were high: 22 ppm in barley, 45 ppm in HRSW and 37 ppm in durum, with average 3ADON levels well under 1.0 ppm. Resistant lines generally had much lower DON levels than susceptible cultivars, across all infection timings. 3ADON was not detected in the resistant HRSW or the moderately resistant durum cultivars. NIV was not detected in any of the grain classes. ACKNOWLEDGEMENT AND DISCLAIMER This material is based upon work supported by the U.S. Department of Agriculture, under Agreement No. 590790-4-114. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture. 100 Session 4: FHB Management EFFECTS OF FUNGICIDE TIMING ON FUSARIUM HEAD BLIGHT AND ON DON AND DON DERIVATIVES IN THREE SPRING GRAINS. Marcia McMullen*, Scott Meyer and Jim Jordahl Dept. of Plant Pathology, North Dakota State University, Fargo, ND 58105 Corresponding Author: PH: (701) 231-7627; Email: marcia.mcmullen@ndsu.edu * ABSTRACT The effects of fungicide timing on the reduction of Fusarium head blight (FHB) and mycotoxins were evaluated under a controlled greenhouse environment. Fungicides were tested on susceptible and moderately resistant to resistant lines of three spring grains - hard red spring wheat (HRSW), durum wheat, and six-rowed spring barley. Fungicide timings included head half-emerged (Feekes 10.3), full head emergence (Feekes 10.5), anthesis in wheat (Feekes 10.51), and kernel watery ripe (Feekes 10.54) or a dual treatment at full head emergence in barley or anthesis in wheat, followed by treatment at kernel watery ripe. Fungicide was applied with a greenhouse track sprayer with XR8001 forward and backward flat fan nozzles, 18 gpa, at appropriate growth stages. Treatments were either Prosaro (tebuconazole + prothioconazole) at 6.5 fl oz/A, or Proline (prothioconazole) at 5 fl oz/A. Fungicide treatments were applied 4 hours after infection initiations. Infections were initiated by atomizing a mixture of four isolates of Fusarium graminearum, at 20,000 spores/ml, 20 ml/ pot, with a DeVilbiss atomizer, followed by 24 hours of misting. FHB incidence and severity were determined at 21-25 days after treatment. At plant maturity, kernels were hand threshed for subsequent mycotoxin analysis. DON, 3ADON, 15ADON and nivalenol (NIV) analyses were done using gas chromatography and electron capture techniques. FHB indices [(incidence x severity)/100] and DON values (ppm) were significantly reduced by fungicide treatments in all grain classes and cultivars. In the most susceptible lines of all 3 grain classes, a single fungicide treatment, applied at optimal growth stage for infection, resulted in 90-98% reductions of FHB indices and DON levels (example: 24.4 ppm DON in durum wheat infected at anthesis, vs 0.56 ppm DON with fungicide treatment added at anthesis). With the dual timings of application, FHB indices and DON levels were reduced by 86 to 97%. Similar percent reductions were observed in the more resistant lines, but overall FHB and DON levels were lower in the more resistant lines. Fungicide treatment at any of the tested timings in HRSW and durum resulted in zero detection of 15ADON and 3ADON. In barley, fungicide treatment resulted in 100% reduction of 3ADON, and 91-94.4% reduction of 15A DON. These fungicide treatments were very effective in reducing FHB, DON and DON derivatives under greenhouse conditions. ACKNOWLEDGEMENT AND DISCLAIMER This material is based upon work supported by the U.S. Department of Agriculture, under Agreement No. 590790-4-114. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture. 101 Session 4: FHB Management EXPERIENCES IN REDUCING DISEASE AND DON THROUGH COMPONENTS OF FHB MANAGEMENT. Marcia McMullen Dept. of Plant Pathology, North Dakota State University, Fargo, ND 58105 Corresponding Author: PH: (701) 231-7627; Email: marcia.mcmullen@ndsu.edu ABSTRACT Few would argue that a favorable climate during vulnerable crop growth stages often is the key factor resulting in severe Fusarium head blight (FHB). However, as unfavorable weather is hard to avoid, researchers and producers have looked for implementable strategies for managing FHB. Champeil et al., 2004 (Fusarium head blight: epidemiological origin of the effects of cultural practices on head blight attacks and the production of mycotoxins by Fusarium in wheat grains. Plant Science 166:1389-1415) provided an extensive review of studies of cultural practices that may affect FHB severity and mycotoxin production, and more recent papers also have been published. Key strategies that have been extensively researched include: crop rotation, tolerant cultivars, and fungicide use. From 2003-2005, various regions in the US had severe FHB outbreaks, and individual FHB management strategies used alone did not necessarily reduce disease severity and DON to levels required by the grain industry. Several 2005 research trials in eastern ND provided quantitative evidence that a combination of crop rotation, variety choice, and fungicide treatment reduced FHB severity and DON levels in an additive manner, ie 10 ppm DON levels in spring wheat with no strategy; 5 ppm DON with soybean rotation added; 2.0 ppm with soybean + resistant variety; and 1.2 ppm with soybean + resistant variety + fungicide. Members of the management group of the US Wheat and Barley Scab Initiative (USWBSI) met in 2006 and decided to implement studies, across multiple states and grain classes, to quantify the value of additive strategies for FHB and DON management. These cropping systems studies were to be done under natural field conditions and the objectives were to: 1) demonstrate that integrated management is the most effective means of reducing losses to FHB/ DON; and 2) increase grower adoption of integrated strategies by demonstration of their effectiveness in a wide range of environments. Funded USWBSI cropping system studies were in place in 2007, a year in which some locations again had FHB. The Forum’s presentation for this abstract will provide examples of 2007 results from these studies. Dr. Pierce Paul has statistically analyzed results from these cropping system studies, and his poster will be presented at the 2007 FHB Forum. Others also may be presenting their individual state’s data. ND results will be published in the 2007 Proceedings of the 5th Canadian Fusarium Head Blight Workshop, Winnipeg. Nov. 27-30. 102 Session 4: FHB Management ACKNOWLEDGEMENT AND DISCLAIMER This material is based upon work supported by the U.S. Department of Agriculture, under Agreement No. 590790-4-114. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture. 103 Session 4: FHB Management COMPARISON OF FUNGICIDES AND NOZZLE TYPES AGAINST FHB IN WHEAT AT FARM APPLICATION. Á. Mesterházy1*, A. Szabo-Hever1, B. Toth1, G. Kaszonyi2, and Sz. Lehoczki-Krsjak2 Cereal Research non-profit Company; and 2Department of Biotechnology and Resistance Research, Szeged, Hungary * Corresponding Author: PH: (36) 30 915430; Email: akos.mesterhazy@gabonakutato.hu 1 ABSTRACT In 2006 the Turbo FloodJet and the TeeJet XR nozzles were compared in three winter wheat cultivars Petur (MR), Kapos (MS) and Miska (S). In 2007 the AIC TeeJet and Turbo TeeJet Duo nozzles were added to have a wider spectrum of comparison. All fungicides were run at 250 L/ha water and 8 km/hr speed. 17 m wide boom was used, on both sides mounted with a different nozzle type. A plot was 7 m wide and 300 m long. Technologies were evaluated across cultivars and fungicides, the fungicides were rated across cultivars and technologies. In both years natural epidemic occurred. In cv Miska, the most susceptible genotype, about 30 infected heads/m2 were recorded. In 2006 eight, in 2008 10 different fungicides were used: Prospect (200 g/L carbendazim, 80 g/L propiconazole) 1.5 L/ha; Falcon 460 EC (167 g/L tebuconazole 250 g/L spiroxamine 43 g/L triadimenol) 0.8 L/ha; Prosaro (125 g/L prothioconazole, 125 g/L tebuconazole) 1.0 L/ha; Tango Star (84 g/L epoxyconazole, 250 g/L fenpropimorph) 1.0 L/ha; Eminent 125 SL (125 g/L tetraconazole) 1.0 L/ha; Amistar Xtra (200 g/L azoxystrobin, 80 g/L ciproconazole) 1.0 L/ha; Coronet (Nativo) (200 g/L tebuconazole és 100 g trifloxystrobin) 1.0 L/ha; Artea 330 EC (250 g/L propiconazole, 80 g/L ciproconazole) 1.0 L/ha; and Juwel (125 g/L epoxyconazole, 125 g/L krezoxim-metil) 1.0 L/ha. In 2006 the mean efficacy of the TeeJet XR nozzle across fungicides was 44 %. The TurboFloodJet nozzles gave 58.60 % reduction of the natural head infection. At the Turbo FloodJet nozzle the lowest efficacy was measured for Eminent (16 %) and 91.5 % for Prosaro. At the traditional nozzles 14.42 and 79 % were the corresponding values. For DON the mean efficacy for TeeJet XR nozzles was 51 %, the reduction for Turbo FloodJet 65 %. In 2007 four nozzles were compared, the two nozzles types from 2006 were supplemented by AIC TeeJet and the Turbo TeeJet Duo nozzles as the Turbo FloodJet nozzles need very uniform soil level to keep the boom constantly at 20-30 cm above stand. This is not always at hand. The data for the FHB data showed a 60.7 % efficacy for AIC TeeJet and 62.8 % for TeeJet XR across fungicides. The Turbo TeeJet Duo reached 70.2 % and the Turbo FloodJet finished at 79.2 %. Prosaro was again the best with 95 % efficacy at the Turbo FloodJet nozzle type. The results provide several important conclusions. The traditional nozzles can reach with the best fungicides up to 70 % reduction when technology, timing is optimal. The difference is very large between fungicides, in this test 42 % for Eminent and 92 for Prosaro across technologies. We believe therefore that the successful chemical control needs both better technology and better fungicides. 104 Session 4: FHB Management ACKNOWLEDGEMENTS The authors express their thanks for financial support to NKTH-KPI projects signed as OMFB 01286/2004 and OMFB 00313/2006. 105 Session 4: FHB Management FIELD AND LABORATORY STUDIES TO MONITOR CELL POPULATIONS, LIPOPEPTIDES AND LIPOPEPTIDE GENES OF BACILLUS 1BA, A BIOCONTROL AGENT. ACTIVE AGAINST FUSARIUM HEAD BLIGHT. J. Morgan1, B.H. Bleakley1,2* and C.A. Dunlap3 Biology/Microbiology Department, South Dakota State University, Brookings, SD; 2Plant Science Department, South Dakota State University, Brookings, SD; and 3USDA-ARS, National Center for Agricultural Utilization Research, Peoria, IL * Corresponding Author: PH: (605) 688-5498; Email: bruce.bleakley@sdstate.edu 1 ABSTRACT Fusarium graminearum causes Fusarium Head Blight (FHB) on wheat, barley and other small grains. Biocontrol agents (BCAs), such as certain Bacillus sp., can be used to control FHB and/or reduce deoxynivalenol (DON) levels in grain. Population studies of the BCAs on inoculated grain heads show how BCA numbers fluctuate over time, and if there are differences in BCA populations between wheat and barley. In previous work to count populations of Bacillus 1BA in the field, we found that this bacterium is thermotolerant and salt tolerant, able to grow on Tryptic Soy Agar (TSA) at elevated salt and temperature conditions that inhibit most micrflora native to the grain heads. Field plot treatments in 2007 were 1BA by itself; and 1BA + Prosaro (a fungicide) + Induce NIS (nonionic surfactant). Population counts were done over a 20 day period on both the wheat and barley. Endospore formation was examined by pasteurizing the samples at 85°C for 10 minutes, then plating. 1BA was isolated using TSA containing 10% NaCl, with incubation at 50°C for 48 hours. Controls for wheat and barley that did not receive 1BA inoculation gave low cell counts throughout the experiment, no higher than about 3.5 X 102 CFU/g fresh plant weight. For wheat, in the treatment with 1BA alone, vegetative cell counts peaked around day 10, then declined sharply, followed by a second increase in numbers by day 20, with a concurrent increase in endospore numbers. Compared to the treatment with 1BA alone, in the wheat treatment combining 1BA with Prosaro and Induce NIS, the population peaks shifted in time. Peak numbers of vegetative cells occurred at about day 6 then declined, with a smaller second peak on day 17. As vegetative numbers of 1BA in this treatment declined, endospore numbers increased. Numbers of 1BA on inoculated barley were much lower than on wheat, not being much different from the uninoculated control and not fluctuating much over time. It appears that 1BA, originally isolated from wheat material, is much better able to colonize and grow on grain heads of wheat than barley. The biocontrol effect of 1BA may be due to its production of lipopeptides including surfactin. Attempts to directly detect presence of Bacillus lipopeptides in extracts from inoculated grain heads were not successful, but efforts to detect these molecules directly on inoculated plant material will continue. Direct or indirect detection of surfactin or other lipopeptide genes/ products on inoculated grain heads will be checked by methods including PCR. As a step leading to this, DNA of 1BA was isolated, then PCR was performed using primers for the surfactin production genetic locus (sfp). Good yield of PCR product was observed on an agarose gel, verifying that the primers and PCR method may be useful in detecting lipopeptide genes on inoculated grain heads. 106 Session 4: FHB Management EFFECTS OF SOLAR RADIATION ON THE VIABILITY OF GIBBERELLA ZEAE ASCOSPORES. Mizuho Nita1, Erick De Wolf1* and Scott Isard2 1 Department of Plant Pathology, Kansas State University, Manhattan, KS 66506; and 2Department of Plant Pathology, The Pennsylvania State University, State College, PA 16802 * Corresponding Author: PH: (785) 532-3968; Email: dewolf@ksu.edu ABSTRACT Ascospores of Gibberella zeae are considered to be an epidemiologically important type of inoculum for Fusarium head blight of wheat and barley. The objectives of this study were to evaluate the effects of solar radiation, temperature, and relative humidity on the survival of ascospores in the environment at Rock Springs, PA and Manhattan, KS. In each experiment, ascospores of G. zeae were collected by inverting cultures containing mature perithecia over glass cover slips for several hours. After deposition, the ascospores were placed on glass Petri dishes and exposed to solar radiation or shaded conditions for predetermined lengths of time. The temperature of the exposed and shaded ascospores was kept constant by allowing the dishes to contact a circulating source of water. Total solar radiation, UV radiation, air temperature, relative humidity, and temperature of the water in the spore exposure apparatus were recorded during each experiment. Following exposure, the spores were washed from the cover glass, placed on water agar and incubated for 8-10 h. The percentage of germinating spores in five sub-samples of 100 ascospores was recorded for each exposure period, and germination was expressed as a ratio of the initial germination of ascospores for that experimental run. The preliminary results of these experiments indicate that the mean initial germination rate of the ascospores produced by isolates considered in this study was 55.6% with a standard deviation of nearly 20%. Regression analysis suggests that total solar radiation and the dose of UV radiation significantly impacted spore survival, but that temperature and relative humidity may also be important variables. The dose of solar radiation resulting in 100% ascospore mortality was 19.8 MJ/m2 at the KS location, but was significantly greater at the PA location. Differences between the locations may be explained in part by differences in the isolate considered and range of temperatures experienced during the exposure periods. 107 Session 4: FHB Management MECHANISTIC SIMULATION MODELS FOR FUSARIUM HEAD BLIGHT AND DEOXYNIVALENOL. M. Nita1, E. De Wolf1*, L. Madden2, P. Paul2, G. Shaner3, T. Adhikari4, S. Ali4, J. Stein5, L. Osborne5 and S. Wegulo6 Department of Plant Pathology, Kansas State University, Manhattan KS 66506; 2Department of Plant Pathology, The Ohio State University, Wooster, OH 44691; 3Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907; 4Department of Plant Pathology, North Dakota State University, Fargo, ND 58105; 5Plant Science Department, South Dakota State University, Brookings, SD 57007; and 6Department of Plant Pathology, University of Nebraska, Lincoln, NE 68583 * Corresponding Author: PH: (785) 532-3968; Email: dewolf1@ksu.edu 1 ABSTRACT An empirical model with a single mathematical equation is commonly used as a part of disease forecasting systems. A web-based Fusarium Head Blight (FHB) forecasting tool (http://www.wheatscab.psu.edu/) is one of example of this approach to disease forecasting. These models are relatively easy to implement, and provide reasonably accurate forecasts in many cases. However, it may be difficult to derive biological interpretation from these empirical models. Other modeling approaches, such as mechanistic modeling, can be considered as an alternative. In this study, an object-oriented language STELLA (isee systems, Lebanon, NH) was utilized to create a mechanistic simulation models for FHB and deoxynivalenol (DON) based on the results of past studies on disease development and pathogen biology. Several candidate models have been developed with different scopes of interests, and one of candidate models will be discussed this presentation. This model estimates a distribution of Fusarium damaged kernels among heads of wheat and DON accumulation of the grain based on environmental conditions. Major steps in the disease cycle, such as perithecia development by Gibberella zeae and infection events were expressed as differential equations that use environmental conditions as input variables. These equations were connected in a logical manner, and using past weather data, a theoretical disease cycle of FHB was simulated over time. The model development process, preliminary results, as well as potential usage of this modeling approach as a tool for hypothesis testing for future studies will be discussed. 108 Session 4: FHB Management SPORE LOAD, DISEASE, AND DON: A FOUR YEAR VARIETY BY RESIDUE STUDY FOR FHB MANAGEMENT. Lawrence E. Osborne*, Jeffrey M. Stein and Christopher A. Nelson * Plant Science Dept., South Dakota State University, Brookings, SD 57007 Corresponding Author: PH: (605) 688-5158; Email: Lawrence.Osborne@sdstate.edu This variety, along with ‘Norm’, a HRSW with high susceptibility to FHB, were used in this study to A four year field study was established near Brookings, investigate the interaction of host resistance and ‘local’ SD in the years 2003 through 2006 to examine the maize residues in a region with abundant airborne impact of maize residues, host susceptibility and timing inoculum. of planting on disease and mycotoxin accumulation in hard red spring wheat due to Fusarium head blight. METHODS These factors were also examined to determine their impact on inoculum concentrations on spikes from Field plots were established in Brookings, SD in 2003 emergence through mid-milk stage. Finally, inoculum through 2006. Each year, two planting dates (PD1 concentration was compared with DON in grain at and PD2), timed 10-13 days apart, were utilized creharvest to determine if any consistent relationships ating two identical studies upon which all treatments were applied and all measurements were collected. existed. Planting date 1 represented the typical planting time for area wheat producers, whereas PD2 represented INTRODUCTION late-planted fields. In general, plots consisted of resiFusarium head blight, caused primarily by Gibberella due treatment (0, 30, and 80% soil coverage, by linezeae (Schwein.) Petch (anamorph: Fusarium transect method) to generate corresponding low, megraminearum Schwabe) in the Northern Great Plains dium and high levels of ‘local’ inoculum (local inocu(including South Dakota), is usually the greatest threat lum being defined as that produced from within the to wheat quality and production in the humid areas of plot area, in contrast to inoculum produced outside the region. The fungus survives and over-winters in the plot area). The medium level was discontinued afplant tissue residues including small grain stems and ter 2004. Sub-plots consisted of spring wheat varietroots as well as maize stalks and ear pieces (Sutton ies ‘Alsen’ and ‘Norm’. Plot size varied slightly from 1982). Recent research suggests that for South year to year due to space restrictions; however, final Dakota, ascospores and conidia of G. zeae are plot disease measurements were collected from areas somewhat ubiquitous in the air, thought to be a result no smaller that 3.1m by 4.6m. In each year, wholeof extensive acreage of spore-bearing residues in the plots (residue treatments) were buffered on all sides region coupled with extensive air mixing and medium by 8m of a tall wheat variety to mitigate inter-plot into long-distance spore movement (Osborne 2006). terference. The study was dependant on inoculum Management of FHB in South Dakota involves the formed locally (i.e. within or beneath the crop canopy use of alternative rotations (wheat not to follow maize), in plant tissues or residues), or externally (i.e. from fungicide application at flowering, and the use of adjacent fields with corn or small grain residue) and resistant varieties to reduce risk. Over the past five received no additional inoculum in the form of spore years, several moderately resistant varieties have been suspension or colonized grain (for ascospore spawn). released which are adapted to South Dakota growing No environmental modification was implemented to conditions. In 2003, however, only one variety, ‘Alsen’, alter the conditions for disease development. was both a recommended variety for SD and classified as ‘moderately resistant’ to FHB (Hall et al., 2003). OBJECTIVES 109 Session 4: FHB Management Within all sub-plots, a designated area was sampled daily after spike emergence by collecting five spikes per sub-plot for enumeration of spike-borne inoculum as described by Francl, et al. (1999). At three weeks post-flowering, disease assessments were performed on all sub-plots as described by Stack and McMullen, (1995) and included incidence and severity estimates on 100 spikes. Incidence is defined as proportion of 100 rated spikes exhibiting disease symptoms. Severity is the mean severity per infected spike. Disease index, often called ‘field severity’, is the product of incidence and severity and represents the overall ‘amount’ of disease in a given area. Harvest data collected included plot yield, test weight and moisture content. Harvested grain was sent to the NDSU Veterinary Toxicology Lab for assessment of mycotoxin concentration in grain following Tacke and Caspers (1996). A Burkard volumetric spore collector was placed for daily monitoring of airborne inoculum in the study area. Weather data was collected using a Campbell Scientific CR10X data logger and peripheral sensors. Parameters measured included temperatures and relative humidity in and above the crop canopy, wind, solar radiation, precipitation, soil temperature, soil wetness and leaf wetness estimations. RESULTS AND DISCUSSION The growing seasons 2003 through 2006 in eastern South Dakota were each distinct in terms of FHB levels on spring wheat across East-Central and Northeastern South Dakota. These differences were mirrored in the overall disease levels observed within this study each year, summarized in Table 1. Years within this study represented four distinct categories: very low disease (2006), low disease (2003), moderate disease (2005), and high disease (2004) seasons. The variation across years and PDs in this study established a range of environments that allowed for a more broad examination of treatment effects and disease parameters than if environments had been consistently favorable or unfavorable for disease over years. sity. Treatments were compared in each of eight yearPD environments to determine the effect of residue level on spike-borne inoculum for this study. Table 2 shows the average cfu’s per day washed from heads in each treatment. Residue treatments significantly affected cumulative inoculum load within two of the environments for ‘Alsen’ (2004-PD1 and 2004-PD2), and only one for ‘Norm’ (2004-PD2). In each case there was a significantly higher level of inoculum on heads within the 80% residue treatment compared to 0% or 30% residue treatments. In all other environments, residue treatments had no significant effect on inoculum load. Therefore, it cannot be said that distinct levels of spike-borne inoculum resulted from maize residue treatments. The whole-plot area may have been too small to overcome problems of fetch (upwind distance from major confounding inoculum sources) and edge-effects. These problems could have led to inter-plot interference. The 2004 experiments give the best example of local inoculum dynamics under a high disease-pressure environment. In 2004, local inoculum apparently did contribute significantly to total inoculum concentration on spikes and therefore the influence of maize residue under the wheat canopy cannot be ignored. However, their influence under most environments is less obvious because of the relatively high level of ‘external’ inoculum entering the system concurrently. Impact of Maize Residue, Variety, and Late Planting on FHB and DON. Residue treatments produced no significant effects on any of the visual disease estimates in any environment (data not shown), however DON was significantly increased by the 80% residue treatment for one environment (2004-PD1). Table 3 presents average DON concentration in grain for all treatments under each environment. There was also a trend toward higher DON in grain with increased maize residue for several environments, though it was not significant. These results mirror closely the average CFU/head data (Table 2). It is hypothesized that inoculum on spikes and final DON in grain may have a strong relationship. This is being investigated further. Impact of Maize Residue Treatments on Spikeborne inoculum. The intermediate objective in plac- The two varieties in the study exhibited different levels ing maize residues in the study area was to establish of disease and DON under all environments and treatthree (or two) distinct levels of local inoculum inten- ments. As expected, ‘Norm’ was significantly higher 110 Session 4: FHB Management in FHB and DON than ‘Alsen’ in nearly all cases. The most noticeable and most significant differences were in DON estimates, where ‘Norm’ accumulated toxin to a much higher level than ‘Alsen’ under comparable environments (Table 3). As overall FHB disease pressure increased (greater inoculum, higher overall disease) across environments, the differences between ‘Norm’ and ‘Alsen’ for all response variables increased, indicating a strong variety by environment interaction, which is also represented in the analysis of variance (Table 4). This suggests that even some level of resistance in the host to FHB was made relatively more valuable as disease pressure increased due to environment. Compared to plots in this study, residue-based inoculum in farm-scale fields would likely be more influential on disease development. Furthermore, a degree of host resistance will be made relatively more valuable under high disease pressure situations, potentially reducing disease and DON compared to more susceptible varieties. With varieties under development and in the first few years of production generally having higher levels of FHB resistance than in earlier decades, overall impact of FHB will likely begin to decline. The risk of severe epidemics such as in the Northern Great Plains in the early 1990’s will be lessened by the widespread adoption of resistant varieties. ACKNOWLEDGEMENT As mentioned, planting dates each year resulted in somewhat unique environments. The effect of late planting can be seen in the higher levels of FHB each year of this study and in the analysis of variance (Table 4). Though the effect of late planting could be inverted under certain environmental conditions such as a dramatic drought or cool period at or just prior to anthesis for a late planted crop, in general, late plantings will experience higher temperatures and greater stress on plants at anthesis. Based on the higher degree of variability in disease and DON across environments than within any one environment, the overall influence of the weather component of the system was probably the largest factor in any of the disease estimates or in toxin accumulation. The environmental variability coupled with the earlier mentioned confounding effects on inoculum load (interplot or ‘external’ inoculum interference) probably masked the relatively subtle effects of the residue treatments. Thus, for this set of experiments, maize residue on the soil surface was not a good predictor of disease development risk. This is contrary to the generally accepted etiological models of FHB which place high risk on corn and small grains residue beneath a susceptible crop (Parry et al. 1995; McMullen et al. 1997; Sutton, 1982; Andersen, 1948; Bai and Shaner 1994; Pererya et al. 2004). The impact of maize residue in this set of studies was much less important than the impact of the environment on disease development. Apparently, non-local inoculum was a significant portion of total inoculum load in this study. This material is based upon work supported by the U.S. Department of Agriculture, under Agreement No. 59-0790-4-107. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. REFERENCES Andersen, A. L. 1948. The development of Gibberella zeae head blight of wheat. Phytopathology 38:595-611. Bai, G-H, and G. Shaner. 1994. Scab of wheat: Prospects for control. Plant disease 78:760-766 Francl, L., G. Bergstrom, J. Gilbert, W. Pedersen, R. Dill-Macky, L. Sweets, B. Corwin, Y. Jin, and D. Gallenberg. 1999. Daily inoculum levels of Gibberella zeae on wheat spikes. Plant disease 83:662-666. Hall, R.G., Rickertson, J, and Kirby, K.K. 2003. Small Grain Variety Recommendations for 2003. South Dakota State University Cooperative Extension Service Publication EC 774. McMullen, M., R. Jones, and D. Gallenberg. 1997. Scab of wheat and barley: a re-emerging disease of devastating impact. Plant disease 81:1340-1348. Osborne, L. E. 2006. Epidemiological research on Fusarium head blight of wheat in South Dakota. Dissertation, Plant Science Dept., South Dakota State University, Brookings, SD. Parry, D.W., P. Jenkinson, and L. McLeod. 1995. Fusarium ear blight (scab) in small grain cereals-a review. Plant pathology 44:207-238. Pereyra, S.A., R. Dill Macky, and A. L. Sims. 2004. Survival and inoculum production of Gibberella zeae in wheat residue. Plant disease 88:724-730. 111 Session 4: FHB Management Stack, R.W., and M.P. McMullin. 1995. A visual scale to estimate severity of Fusarium head blight in wheat. Extension Publication PP-1095, North Dakota State Univ. Extension Service. Stein, J.M, and M.A. Draper. 2005. The Fusarium head blight epidemics of the winter and spring wheat crops in South Dakota for 2005. In Proceedings of the 2005 National Fusarium Head Blight Forum, edited by S. M. Canty, T. Boring, J. Wardwell, S. L. and R. W. Ward. Milwaukee, WI: Michigan State University. Sutton, J. C. 1982. Epidemiology of wheat head blight and maize ear rot caused by Fusarium graminearum. Canadian journal of plant pathology. 4:195. Tacke, B.K., and Casper, H.H. 1996. Determination of deoxynivalenol in wheat, barley, and malt by column and gas chromatography with electron capture detection. J. AOAC Intl. 79:472-475. Table 1. Mean amount (index1) of FHB (%) by variety and year across all residue treatments. Year 2003 2004 2005 2006 Variety ‘Alsen’ ‘Norm’ 1.8 5.2 19.1 48.9 7.0 17.4 0.6 1.5 Average of both varieties 3.5 34.0 12.2 1.1 Disease Category2 Low High Moderate Very Low Differences in disease values were highly significant between varieties each year, and within each variety over years. 1 index=disease incidence*severity; an indicator of disease level within a population. Table 2. Average Daily Inoculum Load on Heads by Residue Treatment. ‘ALSEN’ 0% Residue 30% Residue 80% Residue Inoculum load (cfu) per head per day (average)1 2003 2004 2005 2006 2 PD 1 PD 2 PD 1 PD 2 PD 1 PD 2 PD 1 PD 2 3 a a a a a --388 1408 529 579 29 84a a a --380 1523 ----2133b 475a 662a 39a 43a --493b 2003 ‘NORM’ 0% Residue 30% Residue 80% Residue PD 1 88a 102a 146a Inoculum load (cfu) per head per day (average) 2004 2005 2006 PD 2 PD 1 PD 2 PD 1 PD 2 PD 1 PD 2 a a a a a a 167 472 1654 639 754 34 52a a a a 282 355 1686 ----200a 534a 1990b 693a 781a 38a 39a 1 letters after mean values indicated significant differences among the means (within an environment only) PD=planting date 3 ’Alsen’ was not sampled in 2003, per collaborators protocol. 2 112 Session 4: FHB Management Table 3. Average DON In Grain for each environment ‘ALSEN’ 0% Residue 30% Residue 80% Residue 2 PD 2 0.25 0.25 0.25 DON in grain, ppm (average)1 2004 2005 PD 1 PD 2 PD 1 PD 2 0.53 3.75 0.25 0.66 0.59 4.95 --1.53 7.38 0.51 1.00 PD 1 0.25 -0.25 2006 PD 2 0.25 -0.25 PD 2 0.78 0.87 1.28 DON in grain, ppm (average) 2004 2005 PD 1 PD 2 PD 1 PD 2 4.43 17.18 1.75 2.15 4.63 17.30 --7.93 18.23 2.05 2.43 PD 1 0.25 -0.25 2006 PD 2 0.25 -0.25 2003 PD 1 0.25 0.25 0.25 2003 ‘NORM’ 0% Residue 30% Residue 80% Residue 1 2 PD 1 0.60 0.98 0.40 limit of detection = 0.5ppm, samples below detection limits were assigned the value 0.25ppm for calculations PD=planting date No significant differences were detected within an environment among residue levels for the same variety except for 2004PD1 (80% treatment yielded higher levels of DON in grain for both ‘Alsen’ and ‘Norm’. In all environments except 2006-PD1 and PD2, ‘Norm’ contained significantly higher DON in grain than ‘Alsen’ Table 4. ANOVA for Disease Parameters and Toxin Levels in Grain across environments (2003-2005 only). 1 Analysis of Variance 2 source of variation planting-date(year) variety planting-date(year)*variety 1 2 DON 294.12*** 389.43*** 60.38*** F-values INC SEV 5.24** 20.99*** 36.18*** 109.19*** 1.6 15.06*** PROC Mixed, SAS 9.1, SAS Inc. Cary, NC Prob.>F values indicated by asterisk: *<0.05; **<0.01; ***<0.001 113 INDX 19.78*** 125.91*** 11.91*** Session 4: FHB Management SPORE LOAD, DISEASE, AND DON: AN INOCULUM GRADIENT STUDY USING SISTER WHEAT LINES. Lawrence E. Osborne*, Jeffrey M. Stein, Karl D. Glover and Christopher A. Nelson * Plant Science Dept., South Dakota State University, Brookings, SD 57007 Corresponding Author: PH: (605) 688-5158; Email: Lawrence.Osborne@sdstate.edu ABSTRACT A greenhouse study was conducted utilizing a range of Gibberella zeae inoculum concentrations applied to differentially Fusarium head blight (FHB)-susceptible sister lines of hard red spring wheat. The study was designed to evaluate the relationship between spore concentration on wheat spikes and deoxynivalenol (DON) in grain. Prior observations from several years of field research indicated that high inoculum density on spikes often was associated with high levels of DON in grain, even when disease levels were not well correlated with either DON or inoculum level. The present study utilized aqueous inoculum treatments at seven concentrations from 100 to 100,000 cfu/ml plus a control treatment applied to two sister wheat lines, SD3851 and SD3854. The lines are similar in agronomic characteristics but differ in susceptibility to FHB. Both lines were derived from the same population however line SD3851 possesses resistance conferred by the Fhb1 QTL while SD3854 does not. Inoculated spikes were incubated for 72 hours under 100% RH, then left under ambient GH conditions for 12 additional days. At 15 days post-inoculation, disease assessments were completed and sub-samples of either whole spikes or grain only were ground for mycotoxin analysis. As expected, line SD3851 had less disease and accumulated less DON than SD3854. Both lines tended to accumulate higher levels of DON as inoculum concentration increased, but the susceptible line showed a greater response in both grain and whole-head sub-samples. Whole-head samples contained 4 to 10 times as much DON as grain-only samples, suggesting that chaff tissues might serve as a source of DON which could move to grain under certain environmental conditions. Furthermore, the FHB-susceptible line accumulated about twice as much DON as the FHB-resistant line over the range of inoculum densities comparable to field levels in SD (approximately 125 to 1250 cfu/spike). This further supports the idea that the Fhb1 QTL or any type of host resistance is a crucial part of the USWBSI-stated mission for developing “...control measures that minimize the threat of Fusarium head blight (scab), including the reduction of mycotoxins...”. This study also lends support to the idea that inoculum incident on spikes may be a useful predictor of DON in grain when accompanied by information about host resistance. 114 Session 4: FHB Management A QUANTITATIVE SYNTHESIS OF THE RELATIVE EFFICACY OF TRIAZOLE-BASED FUNGICIDES FOR FHB AND DON CONTROL IN WHEAT. 1* Pierce Paul , Patrick Lipps1, Don Hershman2, Marcia McMullen3, Martin Draper4 and Larry Madden1 The Ohio State University/OARDC, Department of Plant Pathology, Wooster, OH 44691; 2 University of Kentucky, Department of Plant Pathology, Princeton, KY 42445; 3North Dakota State University, Department of Plant Pathology Fargo, ND 58105; and 4 South Dakota State University, Plant Science Department, Brookings, SD 57007 * Corresponding Author: PH: (330) 263-3842; Email: paul.661@osu.edu 1 ABSTRACT Fungicide efficacy against FHB and DON in wheat has been highly inconsistent. Of the classes of fungicides most widely tested, triazoles have been the most effective; however, even among triazoles, the results have been highly variable. A recent quantitative synthesis of the results from over 100 Uniform Fungicide Trials (UFTs), showed that the efficacy of 38.7% tebuconazole (Folicur 3.6F), the longstanding industry standard for FHB and DON control, varied among individual studies and was generally higher in spring wheat than winter wheat. Besides tebuconazole, other triazole-based fungicides have been tested against FHB and DON, with some showing numerically (if not always statistically) superior efficacy relative to tebuconazole. One such fungicide is the recently-registered 41% prothioconazole (Proline 480 SC). In some individual studies, when used as a solo active ingredient or in combination with tebuconazole (either as a premix or a tank mix), this product contributed to significantly greater reduction in FHB and DON than tebuconazole alone. In other studies, however, the differences in efficacy were merely numerical. Similar results were observed in comparisons between metconazole and tebuconazole, suggesting that study- or environment-specific factors were probably influencing the performance of these fungicides. Using data collected from 10 years of UFTs, a multivariate meta-analysis was performed to evaluate the overall and relative efficacy of triazole-based fungicides against FHB and DON, and to determine whether efficacy was consistent across wheat types. Propiconazole (PROP), prothioconazole (PROT), tebuconazole (TEBU), metconazole (METC) and prothioconazole+tebuconazole (PROT+TEBU) fungicides were applied at flowering and disease index and DON concentration were quantified. Based on percent control (C), all fungicides led to a reduction in FHB and DON relative to the untreated check. PROT+TEBU was the most effective product against index, with an overall mean C of 52%, followed by METC (50%), PROT (48%), TEBU (40%), and PROP (32%). For DON, METC was the most effective, with a mean C of 45%; PROT+TEBU and PROT were of equal efficacy, with a C of 42%; whereas TEBU and PROP were the least effective, with mean Cs of 23 and 12%, respectively. In general, fungicide efficacy was higher in spring wheat than in winter wheat studies. When considering the efficacy of PROP+TEBU, METC, PROT and PROP relative to TEBU, that is, using TEBU as the reference for comparison in the meta-analysis instead of the untreated check, all products, with the exception of PROP, were significantly more effective than TEBU against both index and DON. Using the estimated mean efficacy of each fungicide against IND and DON and the estimated between-study variability in efficacy form the meta-analysis, the probability of each fungicide achieving > 50 (p50) percent control in a new (single), randomly-selected study (conducted in a way similar to the UFTs) was estimated. For spring wheat, METC had the highest p50 values for both index (0.64) and DON (0.56), followed closely by TEBU+PROT and PROT. For winter wheat, TEBU+PROT had the highest p50 value for index (0.42), followed by PROT (0.36), 115 Session 4: FHB Management and METC (0.31), whereas for DON, PROT, TEBU+PROT, and METC had comparable p50 values (0.31, 0.27, and 0.26). ACKNOWLEDGEMENT AND DISCLAIMER This material is based upon work supported by the U.S. Department of Agriculture, under Agreement No. 590790-4-112. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture. 116 Session 4: FHB Management AN INTEGRATED APPROACH TO MANAGING FHB AND DON IN WHEAT: UNIFORM TRIALS 2007. P. Paul1*, L. Madden1, M. McMullen2, D. Hershman3, L. Sweets4, S. Wegulo5, W. Bockus6, S. Halley2 and K. Ruden7 1 The Ohio State University/OARDC, Dept. of Plant Pathology, Wooster, OH 44691; 2North Dakota State University, Dept. of Plant Pathology Fargo, ND 58105; 3University of Kentucky, Dept. of Plant Pathology, Princeton, KY 42445; 4University of Missouri, Dept. of Plant Microbiology and Pathology, Columbia, MO 65211 ; 5University of Nebraska-Lincoln, Dept. of Plant Pathology, Lincoln, NE 68583; 6Kansas State University, Dept. of Plant Pathology, Manhattan, KS 66506; and 7South Dakota State University, Plant Science Department, Brookings, SD 57007 * Corresponding Author: PH: (330) 263-3842; Email: paul.661@osu.edu OBJECTIVES 1) Evaluate the integrated effects of multiple strategies for FHB and DON management under a range of environmental conditions; and 2) increase grower adoption of multiple strategies by demonstrating that integrated management is the most effective means of reducing losses due to FHB/DON. are generally most effective at reducing FHB and DON under moderate disease pressure and when applied to moderately resistant varieties than to susceptible varieties. In 2006, members of the then CBCC RAC of the UWBSI met with researcher and established protocols for conducting integrated FHB management trials. In 2007, these trials were implemented for the first time across multiple states and grain classes. The results of these trials are summarized herein. INTRODUCTION MATERIALS AND METHODS Fusarium Head Blight (FHB) and the associated toxin (deoxynevalenol, DON) produced by its causal agent, Fusarium graminearum, continues to be a concern in every sector of the wheat and barley industries. Through years of research funded by the US Wheat and Barley Scab Initiative (USWBSI), several chemical, biological, and cultural management approaches have been evaluated and shown to contribute to FHB and DON reduction. However, when used individually, none of these approaches have been fully effective against FHB and DON. The effects of fungicide application, genetic resistance, and residue management (through crop rotation or tillage) are highly variable and strongly influenced by the environment. Under favorable weather conditions, moderately resistant varieties may become infected and DON contamination may exceed critical threshold levels. In the case of fungicides, efficacy varies from one trial to another, with overall mean percent control between 40 and 60% for index and between 30 and 50% for DON (for the most effective fungicides). Fungicides Field experiments were conducted to investigate the integrated effects of multiple management strategies on FHB and DON accumulation in wheat under natural conditions. The standard experimental design was a split plot with 3 to 6 replicate blocks. Wheat variety and fungicide application served as the whole-plot and sub-plot factors, respectively. In some individual trials, biological control agents and cropping sequence were used as additional treatment factors. Plot dimensions and planting and cropping practices varied somewhat from trial to trial (see individual trial reports for details). In general, between three and six locally adapted and commonly cultivated varieties, with a range of susceptibility to FHB, were planted. There were two adjacent plots of each variety in each block. Sub-plot treatments were established by applying Proline + Folicur (as a tank mix of 3 fl. oz of each) or Prosaro (6.5 fl. oz/A) to one plot of each variety at the flowering date (Feekes’ growth stage 10.5.1) of 117 Session 4: FHB Management the variety and leaving the other plot untreated. A nonionic surfactant was added to the treatment at a rate of 0.125% v/v, and applications were made using CO2-pressurized sprayers, equipped with Twinjet XR8002 nozzles or paired XR8001 nozzles, mounted at an angle (30 or 60o) forward and backward. mean index of 6.7% (Table 2). For DON, Prosarotreated plots had significantly lower mean DON (16.7 ppm) than untreated plots (20.14), averaged across varieties. Among the varieties, mean DON content was highest in Jagalene and lowest in Pioneer 2137, however, this difference was only numerical (Table 2). In each plot, percent FHB incidence (INC), diseasedhead severity (SEV), index (IND; also known as field or plot severity), and Fusarium-damaged kernels (FDK) were measured. Plots were harvested and yield and test weight determined. Milled grain samples from each plot were sent to one of the USWBSI-funded DON Testing Laboratories for DON analysis. Kentucky – Due to dry conditions, FHB intensity and DON contamination were very low in this trial. Mean index and DON ranged from 0.03 to 2% and 0.05 to 1.3 ppm, respectively. For index, the main effects of fungicide and variety were statistically significant, whereas for DON, only the main effect of variety was statistically significant. Analysis of variance (linear mixed model) was used to evaluate the effects of variety, fungicide and their interaction on FHB intensity and DON content at each location. Percent control of FHB and DON was estimated for each treatment and treatment combination by using the level of disease and DON in the untreated plot of the most susceptible variety as the reference. Missouri – Two trials were conducted in Missouri to evaluate fungicide and variety effects on FHB and DON. In the first (MO1), plots were planted no-till into corn residue and in the second (MO2), no-till into soybean residue. In MO1, mean FHB and DON levels ranged from 0.12 to 38% and 0.25 to 5.6 ppm, respectively, whereas in MO2, the corresponding ranges were 0 to 8, and 0.25 to 2, respectively. For index, all main and interaction effects were statistically significant in MO1 and only variety and variety x fungicide effects were significant in MO2. In both trials, the effects of fungicide and variety x fungicide interaction on DON were not statistically significant. In MO1, averaged across varieties, mean index in Proline 3+3treated plots was 7.1% compared to 11.7% in the untreated check. Among the varieties, averaged across fungicide treatments, Elkhart has the highest level of disease (23.8%), followed by Pioneer 25R47 (18.2%), whereas Bess had the lowest mean level of disease (0.32%). Overall, the highest and lowest levels of disease occurred in untreated plots of Elkhart and treated plots of Bess, respectively (Table 2). RESULTS AND DISCUSSION A total of 15 trials were conducted in eight states (Table 1). FHB intensity and DON varied from one location to another, with some trials having zero or nominal disease development and DON contamination. Kansas – Plots in this trial were artificially inoculated with F. graminearum-infested corn kernels and mistirrigated to enhance disease development. As a result, FHB intensity and DON contamination were high, with mean index and DON ranging from 2 to 95% and 7.5 to 30.2 ppm, respectively. The effects of fungicide, variety, and their interaction on FHB index were statistically significant (P < 0.05). For DON, only the main effect of fungicide was statistically significant. Averaged across the three varieties, mean index was 43% in Prosaro-treated plots compared to 70% in the untreated check. For the three varieties evaluated (Harry, Jagalene, and Pioneer 2137) treated plots had significantly lower levels of disease than untreated plots. Untreated plots of Jagalene had the highest level of disease, with a mean index of 87.5%, whereas treated plot of Harry had the lowest level of disease with a Nebraska – Mean FHB index ranged from 2.8 to 47.7% in this trial, with very similar mean levels of disease occurring in fungicide-treated and untreated plots, averaged across varieties. Among the varieties, Pioneer 2137 had the highest mean index (17.5%), followed by Jagalene (16.8%), and Harry (12.8%). The main and interaction effects of variety and fungicide treatment on index were not statistically significant. 118 Session 4: FHB Management North Dakota - Six FHB integrated management trials were conducted in North Dakota - three HRWW, two HRSW and one Durum. Disease and DON levels were not reported for two of the winter wheat trials. For the Durum and one of the HRSW trials, previous crop was used as an additional treatment factor (along with fungicide and variety). For the other spring wheat trial and the third winter wheat trial, 20 different varieties were planted. generally resulted in the highest percent control (Table 2). ACKNOWLEDGEMENT AND DISCLAIMER This material is based upon work supported by the U.S. Department of Agriculture, under Agreement No. 59-0790-4-112. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed For the Durum wheat trial (ND_D), the three-way in this publication are those of the author(s) and do interaction effect of previous crop, variety and fungicide not necessarily reflect the view of the U.S. Department on index was not statistically significant. However, the of Agriculture. main effects of variety and fungicide and the interaction effects of previous crop x variety and fungicide x variety REFERENCES were significant. Mean index (averaged across variety and cropping sequence) in Prosaro-treated plots Champeil, A., and Fourbet, J. F. 2004. Fusarium head blight: epidemiological origin of the effects of cultural practices on (8.54%) was significantly lower than mean index in head blight attacks and the production of mycotoxins by untreated plots (20.04%). Averaged across cropping Fusarium in wheat grains. Plant Sci. 166:1389-1415. sequence and fungicide treatment, Monroe and Divide had the highest and lowest mean levels of disease, Paul, P.A., Lipps, P.E., Hershman, D.E., McMullen, M.P., Draper, M.A., and Madden, L.V. 2007. A quantitative review respectively. Overall, untreated plots of Monroe of tebuconazole effect on Fusarium head blight and planted following HRSW (as the previous crop) had deoxynivalenol content in wheat. Phytopathology 97:211the highest mean index (36.54%). For DON, only the 220. main effects of fungicide and previous crop were Dill Macky, R., and Jones, R. K. 2000. The effect of previous statistically significant. The highest mean level of DON crop residues and tillage on Fusarium head blight of wheat. occurred in untreated plots of Monroe and Lebsock Plant Dis. 84:71-76. (3 ppm) planted after HRSW and the lowest mean McMullen, M., Milus, G., and Prom, L. 1999. 1999 Uniform level occurred in treated plots of Grenora (0.97 ppm) fungicide trials to identify products effective against Fusarium planted after canola. head blight in wheat. Pages 64-68 in: Proc. Of the 1999 National Fusarium Head Blight Forum, Sioux Falls, SD, Dec. 5-7, 1999. Michigan State Univ. CONCLUSIONS Percent control was estimated for a few of the trials to evaluate the efficacy of individual treatments and treatment combinations against index and DON. The results for three trials with the highest levels of disease are presented in Table 2. Trials with nominal levels of disease and DON were not included in the table, because percent control tends to be highly variable at low index and DON levels. In general, moderately resistant variety x fungicide treatment combination resulted in the highest percent control. For the trials with cropping sequence as a treatment factor, nonehost crop + moderately resistant variety + fungicide Nita, M., DeWolf, E., Madden, L., Paul, P., Shaner, G., Adhikari, T., Ali, S., Stein, and Osborne, L. 2006. Effect of corn residue level, fungicide application, and cultivar resistance level on disease incidence and severity of Fusarium head blight and DON concentration. Page 46 in: Proc. 2006 National Fusarium Head Blight Forum, Dec. 10-12, 2006, Research Triangle Park, NC. East Lansing: Michigan State University. Schaafsma, A. W. Tamburic-Ilincic, and Hooker, D.C. 2005. Effect of previous crop, tillage, field size, adjacent crop, and sampling direction on airborne propagules of Gibberella zeae/ Fusarium graminearum, Fusarium head blight severity, and deoxynivalenol accumulation in winter wheat. Can. J. Plant Pathol. 27:217-224. 119 Session 4: FHB Management Table 1. States, principal investigator, institution, wheat class and number of FHB Integrated management trials conducted in 2007. State PI Institution Wheat class No. trials KS Bill Bockus Kansas State Univ. HRWW 1 NE Stephen Wegulo Univ. of Nebraska - Lincoln HRWW 1 MO Laura Sweets Univ. of Missouri SRWW 2 KY Don Hershman Univ. of Kentucky SRWW 1 6 ND Marcia McMullen North Dakota State Univ. HRSW Durum Joel Ransom HRWW Scott Halley Kent McKay Cornell Univ. SRWW 2 NY Gary Bergstroma a South Dakota State Univ. HRSW 1 SD Kay Ruden Ohio State Univ. SRWW 1 OH Pierce Paula a Fusarium head blight did not develop. 120 Table 2. Mean FHB index and DON and estimated percent control for different treatments and treatment combinations. Trial Kansas Missouri MO1 121 Index Previous Crop … … … … … … … … … … … … … … … … DON Variety Jagalene Harry P2137 Elkhart B 25R47 C 25R54 D Roane E Bess A Treatment Untreated Prosaro Untreated Prosaro Untreated Prosaro Untreated Proline 3+3 Untreated Proline 3+3 Untreated Proline 3+3 Untreated Proline 3+3 Untreated Proline 3+3 Mean Index (%) 87.50 67.50 46.00 6.66 77.83 55.00 29.38 18.23 23.68 12.66 2.02 1.92 3.13 2.66 0.42 0.22 Percent Control 0.00 22.86 47.43 92.39 11.05 37.14 0.00 37.95 19.40 56.91 93.12 93.46 89.35 90.95 98.57 99.25 Previous Crop … … … … … … … … … … … … … … … … Variety Jagalene Harry P2137 … … … … … … … … … … Treatment Untreated Prosaro Untreated Prosaro Untreated Prosaro … … … … … … … … … … Mean Index (%) 20.08 18.03 22.53 14.68 17.82 17.60 … … … … … … … … … … Percent Control 0.00 10.21 -12.20 26.89 11.25 12.35 … … … … … … … … … … Session 4: FHB Management Monroe Lebsock Grenora Divide Canola Monroe Lebsock Grenora 122 Divide Untreated Prosaro Untreated Prosaro Untreated Prosaro Untreated Prosaro Untreated Prosaro Untreated Prosaro Untreated Prosaro Untreated Prosaro 36.54 9.79 20.42 8.31 12.41 6.29 14.74 11.89 25.22 4.97 19.4 12.26 23.9 9.37 7.66 5.49 0.00 73.21 44.12 77.26 66.04 82.79 59.66 67.46 30.98 86.40 46.91 66.45 34.59 74.36 79.04 84.98 HRSW Monroe Lebsock Grenora Divide Canola Trials with nominal levels of disease and DON were not included in the table. Monroe Lebsock Grenora Divide Untreated Prosaro Untreated Prosaro Untreated Prosaro Untreated Prosaro Untreated Prosaro Untreated Prosaro Untreated Prosaro Untreated Prosaro 2.98 1.40 3.00 1.98 2.80 2.15 2.32 2.10 2.25 1.08 2.05 1.27 2.00 0.97 1.93 1.15 0.00 53.02 -0.67 33.56 6.04 27.85 22.15 29.53 24.50 63.76 31.21 57.38 32.89 67.45 35.23 61.41 Session 4: FHB Management Table 2. cont. North Dakota HRSW ND_D Session 4: FHB Management FUNGICIDE EFFECTS ON FHB AND DON IN WHEAT ACROSS MULTIPLE LOCATIONS AND WHEAT CLASSES: UNIFORM FUNGICIDE TRIALS 2007. 1* P. Paul , L. Madden1, M. McMullen2, D. Hershman3, D. Brown-Rytlewski4, L. Sweets5, E. Adee6, C. Bradley6, B, Padgett7 and K. Ruden8 The Ohio State University/OARDC, Dept. of Plant Pathology, Wooster, OH 44691; 2North Dakota State University, Dept. of Plant Pathology Fargo, ND 58105; 3University of Kentucky, Dept. of Plant Pathology, Princeton, KY 42445; 4Michigan State University, Dept. of Plant Pathology, East Lansing, MI 48824; 5 University of Missouri, Dept. of Plant Microbiology and Pathology, Columbia, MO 65211 6University of Illinois, Dept. of Crop Sciences, Urbana, IL 61801; 7Louisiana State University, Winnsboro, LA 71295; and 8South Dakota State University, Plant Science Department, Brookings, SD 57007 * Corresponding Author: PH: (330) 263-3842; E-mail: paul.661@osu.edu 1 complete block design, with four replicate blocks (one trial had 3, two had 5, and another had 8 blocks). The Evaluate foliar fungicides for effectiveness against core treatments were: Fusarium head blight (FHB) and deoxynivalenol (DON) accumulation in wheat across multiple trials · Non-treated control; and different wheat classes. · Folicur at 4.0 fl oz/A; · Prosaro at 6.5 fl oz/A; INTRODUCTION · Caramba at 13.5 fl oz/A; · Topguard at 14 fl oz/A; Fusarium head blight (FHB), caused predominantly Proline at 5 fl oz/A; by Fusarium graminearum, continues to impact ev- · Tilt at 4 fl oz/a; ery sector of the wheat and barley industries, causing substantial yield and quality losses. F. graminearum Other treatments evaluated in separate individual triproduces a mycotoxin called deoxynivalenol (DON) als were Proline at 3 fl. oz + at Folicur 3 fl. oz; Caramba (among other toxins) which may accumulate to unac- at 10 fl. oz/A; Caramba at 8.2 fl. oz/A; Punch at 6 fl. ceptable levels in harvested grain. DON levels above oz/A; Proline at 3 fl. oz/A; Topguard at 10 fl. oz/A; 2 ppm may render grain and their by-products unfit Stratego at 10 fl. oz/A; Quadris 8 fl. oz/A; Dithane at for commercialization and consumption. Efforts to mini- 2 fl. oz/A; Quilt at 14 fl. oz/A; Folicur at 2 fl. oz/A + mize the impact of FHB and DON have been based Topguard at 8 fl. oz/A; Headline at 8 fl. oz/A; and on the use of management strategies such as host re- Folicur at 2 fl. oz/A. All treatments were applied at sistance, crop rotation, tillage, and fungicide applica- Feekes 10.5.1. A non-ionic surfactant was added to tion. Through collaborative research involving scien- each treatment at a rate of 0.125% v/v, and applicatists from multiple states, representing various wheat- tions were made using CO2-pressurized sprayers, growing regions, Uniform Fungicide Trials (UFTs) have equipped with Twinjet XR8002 nozzles or paired been conducted annually since 1998 to evaluate fun- XR8001 nozzles, mounted at an angle (30 or 60o) gicide efficacy against FHB and DON. The 2007 re- forward and backward. sults from 23 UFTs across 6 states are presented herein. Planting and crop production practices varied someMATERIALS AND METHODS what from trial to trial. See individual trial reports for details. Most plots were planted with a susceptible In general, each trial consisted of six core fungicide cultivar. To enhance disease development, plots were treatments and an untreated control in a randomized either planted into corn or wheat residue and/or artifiOBJECTIVE 123 Session 4: FHB Management cially inoculated with F. graminearum-infested kernels. Many plots were mist-irrigated as a means of enhancing production of, and infection by fungal inocula. In each plot of each trial, percent FHB incidence (INC), diseased-head severity (SEV), index (IND; also known as plot or field severity), and Fusarium-damaged kernels (FDK) were measured as previously described (McMullen, et al., 1999). DON accumulation was measured at one of the USWBSI-funded DON Testing Laboratories. effective treatment in the Fargo trial (Table 1). The corresponding percent controls (Hershman and Milus, 2003, Paul et al 2007) resulting from these treatments in their respective trials were 97, 91, and 81%. Although the Clarksville trial had the highest level of disease and the greatest difference in mean index between the Topguard treatment (14 fl. oz/A) and the check (17%), this difference was not statistically significant (P = 1.00). This was probably because of the high variability observed in this trial. Similar results were observed for other measures of FHB intensity (SEV, Each trial was analyzed separately using a mixed ef- INC, and FDK). Since index is a direct function of fect model in PROC MIXED of SAS to determine INC and SEV (see Paul et al., 2005), only the results treatment effect on FHB, DON, yield (bu/ac) and test for IND are summarized herein. weight (lb/bu). Linear contrasts were used to make pair-wise comparisons between treatment means or DON content of the grain was reported in 13 of the means across groups of treatments. Studies with zero 23 trials. In nine of these trials, DON levels in the unor nominal levels of disease and DON were not ana- treated check were below 1 ppm. Trials conducted in lyzed. Browntown, IL; Clarksville, MI; East Lansing, MI; and Langdon3, ND were the only trials with DON RESULTS AND DISCUSSION levels in the check close to or above 2 ppm (Table 2). In these trials, Prosaro at 6.5 fl.oz/A, Punch at 6 fl.oz/ Weather conditions in both winter wheat and spring A, Proline at 5 fl. oz/A, and Caramba at 14 fl. oz/A, wheat areas were generally unfavorable (hot and dry respectively, were the most effective treatments. Howduring flowering) for FHB development. In addition, ever, the difference in mean DON between fungicideadverse weather conditions (floods in some areas and treated plots and the untreated check was not significold temperatures in others) caused plots to be lost cantly different from zero in the Clarksville and East and the results to be highly variable in some trials. Lansing trials (Table 2). Punch resulted in a 47% reConsequently, non-irrigated trials and a few irrigated duction in DON relative to the check in the Clarksville trials had nominal disease development. Mean and trial; however, the mean level of DON in Punch-treated maximum FHB index across all replicates of the un- plots still exceeded the critical threshold level of 2 ppm. treated check plots ranged from 0 to 26.28 and 0 to 55.00%, respectively (Table 1). In 15 of the 23 trials, CONCLUSION mean index in the untreated check was less than 1% and less than 2% in 18 of the 23 trials. FHB intensity In summary, in the trials with some level of disease, was highest in the Clarksville and East Lansing trials, fungicide treatments reduced FHB intensity and DON with mean index of 26.3 and 21.9%, respectively. accumulation relative to the untreated check (based mainly on data from four locations). Fungicide effiIn three (Urbana, IL, East Lansing, MI, and Fargo, cacy varied among the trials, with percent control rangND) of the five trials with mean index in the untreated ing from 67 to 97% for index and 41 to 60% for DON. check greater than 2%, fungicide treatment had a sig- However, since the overall levels of disease and DON nificant (P < 0.05) effect on FHB index (Table 1). were very low, these results should be interpreted with Based on pair-wise differences between each treat- caution. Paul et al. (2007a, 2007b) showed that FHB ment and the check, Caramba at 13.6 fl.oz/A was the and DON responses to fungicide treatments were most most effective treatment in the Urbana trial; Proline at variable at low levels of disease (index < 2%) and 3 fl.oz/A was the most effective treatment in the East DON (< 1 ppm) than at intermediate or high levels. In Lansing trial, and Proline at 5 fl.oz/A was the most general, the overall levels of disease and DON in 2007 124 Session 4: FHB Management were too low for us to make broad conclusions re- McMullen, M., Milus, G., and Prom, L. 1999. 1999 Uniform fungicide trials to identify products effective against Fusarium garding the treatments evaluated. ACKNOWLEDGEMENT AND DISCLAIMER This material is based upon work supported by the U.S. Department of Agriculture, under Agreement No. 59-0790-4-112. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture. REFERENCES Hershman, D. E. and Milus, E. A. 2003. Performance of Folicur in Fusarium head blight uniform fungicide trials, 1998-2003. Pages 81-82 in: Proc. of the 2003 National Fusarium Head Blight Forum, Bloomington, MN, Dec 13-15, 2003. Michigan State Univ. head blight in wheat. Pages 64-68 in: Proc. Of the 1999 National Fusarium Head Blight Forum, Sioux Falls, SD, Dec. 5-7, 1999. Michigan State Univ. Paul, P. A., Lipps, P. E., Hershman, D. E., McMullen, M. P., Draper, M. A., and Madden, L. V. 2007. A Quantitative Synthesis of the Relative Efficacy of Triazole-based Fungicides for FHB and DON Control in Wheat. Pages XXX in: Proc. of the 2007 National Fusarium Head Blight Forum, Kansas City, MO, Dec 2-4, 2007. Michigan State Univ. Paul, P. A., Lipps, P. E., Hershman, D. E., McMullen, M. P., Draper, M. A., and Madden, L. V. 2007. A quantitative review of tebuconazole effect on Fusarium head blight and deoxynivalenol content in wheat. Phytopathology 97:211220. Paul, P. A., El-Allaf, S. M., Lipps, P. E., and Madden, L. V. 2005. Relationship between incidence and severity of Fusarium head blight on winter wheat in Ohio. Phytopathology 95:1049-1060. Hershman, D. and Draper, M. 2004. Analysis of 2004 uniform wheat fungicide trials across locations and wheat classes. Pages 318-322 in: Proc. of the second international symposium on Fusarium head blight, Orlando, FL, Dec. 11-15, 2004. Michigan State Univ. 125 Session 4: FHB Management Table 1. Fungicide effect on Fusarium head blight index – 2007 UFT. Most effective Treatmenta Index (%) Check Treat IND Percent P Mean Max State/PI Location (%) Control IL/Adee Browntown W … … … … 0.00 0.00 Monmouth W … … … … 1.23 3.11 IL/Bradley Urbana W Caramba 0.17 97 0.006 5.90 12.73 13.5 fl.oz LA/Padgett Crowley 1 W … … … … 1.20 1.44 Crowley 2 W … … … … 0.34 0.55 Macon Ridge 1 W Prosaro 6.5 0.51 78 0.189 2.37 3.60 fl oz MI/Brown-Rytlewski Clarksville W Topguard 8.73 67 0.100 26.28 55.00 14 floz East Lansing W Proline 3 2.00 91 <0.01 21.90 32.50 fl. oz Saginaw W … … … … 0.00 0.00 Sandusy W … … … … 0.00 0.00 MO/Sweets Columbia 1 W … … … … 1.00 1.60 Columbia 2 W … … … … 0.48 0.80 ND/McMullen Fargo S Proline 5 0.55 81 2.85 3.30 fl. oz <0.01 Langdon 1 S … … … … 0.40 0.50 Langdon 2 S … … … … 0.20 0.50 Langdon 3 S/D … … … … 0.75 1.00 SD/Draper Brookings 1 S … … … … 0.73 1.32 Brookings 2 S … … … … 0.74 1.94 Groton 1 S … … … … 0.00 0.00 Groton 2 S … … … … 0.29 1.00 Watertown 1 S … … … … 0.00 0.00 Watertown 2 S … … … … 0.00 0.00 a Treat = the most effective treatment (s) within each trial based on the pair-wise difference between mean index for each treatment and the check; IND (%) = mean index across plots receiving the most effective treatment; P = level of significance from t test of the difference between mean IND across plots receiving the most effective treatment and the untreated check (P < 0.05 Î significant different). All tests of significance were done using arcsine-transformed IND. … = Trials with zero or nominal levels of disease. Trial Wheat Type 126 Session 4: FHB Management Table 2. Fungicide effect on DON – 2007 UFT. Trial Wheat Type State/PI Location IL/Adee Browntown W Monmouth W IL/Bradley Urbana W MI/Brown-Rytlewski Clarksville W East Lansing W Saginaw Sandusy Fargo W W S Langdon 1 Langdon 2 Langdon 3 S S S/D ND/McMullen Most effective Treatmenta Treat DON % (ppm) Reduction Prosaro 0.76 60 6.5 fl.oz Topguard 0.17 58 14 fl.oz Prosaro 0.21 52 6.5 fl.oz Punch 6 3.60 47 fl.oz Proline 5 1.10 41 fl.oz … … … … … … Proline 5 0.38 58 fl oz … ... … … … … Caramba 1.40 53 13.5 fl.oz a P Index (%) Check Mean Max <0.01 1.91 3.50 0.16 0.40 0.67 0.02 0.43 0.62 0.15 6.80 8.40 0.17 1.85 2.30 … … 0.02 0.00 0.05 0.90 0.00 0.10 1.10 … … 0.01 0.83 0.17 2.97 0.90 0.50 3.80 DON data were not available for some trials or available but equally low (below 1 ppm) for all treatments. Treat = the most effective treatment within each trial based on the pair-wise difference between mean DON for each treatment and the check; DON (ppm = mean DON across plots receiving the most effective treatment; % reduction = percent reduction in DON; P value = level of significance from t test of the difference between mean DON across plots receiving the most effective treatment and the untreated check (P < 0.05 Î significant difference). All tests of significance were done using log-transformed. b … = Trials with zero or nominal levels of DON. 127 Session 4: FHB Management INFLUENCE OF SRWW, HRSW, AND HRWW VARIETIES ON THE RELATIONSHIP BETWEEN FHB AND DON. Pierce A. Paul1*, Larry V. Madden1, Stephen Wegulo2, Tika Adhikari3, Shaukat Ali3 and Erick De Wolf4 1 The Ohio State University/OARDC, Dept. of Plant Pathology, Wooster, OH 44691; 2University of Nebraska-Lincoln, Dept. of Plant Pathology, Lincoln, NE 68583; 3North Dakota State University, Dept. of Plant Pathology, Fargo, ND 58105; and 4Kansas State University, Dept. of Plant Pathology, Manhattan, KS 66506 * Corresponding Author: PH: (330) 263-3842; Email: paul.661@osu.edu ABSTRACT The relationship between visual estimates of Fusarium head blight (FHB) intensity and deoxynivalenol (DON) content of wheat is of interest to both researchers and producers because visual symptoms often are used as an indication of DON contamination of grain. In general, there is a significant positive relationship between FHB and DON, however, this relationship may vary among studies, and in some instances, fairly high levels of DON may accumulate in the absence of visual symptoms of FHB, or conversely, relatively high levels of visual symptoms may be associated with disproportionately low levels of DON contamination. The association between FHB and DON may be influenced by weather conditions, fungicide treatment, pathogen aggressiveness and DON producing ability, and variety resistance to FHB and DON. Field experiments were conducted in Nebraska, North Dakota and Ohio to evaluate the influence of variety resistance on the relationship between FHB and DON. At each location, locally adapted varieties with different levels of resistance to FHB (based on visual symptoms) were planted in a randomized complete block design, with three replicate blocks. The varieties evaluated were HRWW varieties Harry and Pioneer 2137, in Nebraska; HRSW varieties Trooper, Steel-ND, and Glenn, in North Dakota; and SRWW varieties Cooper, Hopewell, and Truman, in Ohio. Plots were inoculated at anthesis, and in each plot of each variety, diseased spikes in different severity categories were tagged. Tagged spikes were hand-harvested, cleaned, and a sample of grain from each disease category was analyzed for DON. DON content varied among varieties in each disease category in the three wheat classes. In all cases, DON generally increased with increase in disease intensity. Of the two HRWW varieties evaluated, Pioneer 2137 had lower mean DON contamination than Harry at all FHB severity levels. Among the SRWW varieties, Hopewell had the highest and Truman the lowest mean levels of DON in all disease categories. In general, Cooper, the susceptible SRWW variety, had DON content comparable to that of Truman, the moderately resistant SRWW variety. Among the spring wheat varieties, Trooper had higher mean DON content than Glenn and Steel-ND at all severity levels. Between Glenn, the moderately resistant HRSW variety, and Steel-ND, the moderately susceptible HRSW variety, the levels of DON contamination were similar in most cases. ACKNOWLEDGEMENT AND DISCLAIMER This material is based upon work supported by the U.S. Department of Agriculture, under Agreement No. 590790-4-112. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture. 128 Session 4: FHB Management DONCAST: SEVEN YEARS OF PREDICTING DON INWHEAT ON A COMMERCIAL SCALE. R. Pitblado1*, D.C. Hooker2, I. Nichols1, R. Danford1 and A.W. Schaafsma3 Weather INnovations Incorporated, Chatham, ON, Canada; 2University of Guelph, Ridgetown Campus, Ridgetown, ON, Canada; and 3Department of Plant Agriculture, University of Guelph, Guelph, ON, Canada * Corresponding Author: PH: (519)352-5334; Email: rpitblado@weatherinnovations.com 1 ABSTRACT Accurate predictions of mycotoxins in harvested grain are useful to help prevent entry of toxins into the food chain. DONcast is an empirical model for predicting mycotoxins in mature wheat grain, mainly as a decision support tool. To our knowledge, DONcast is the only mycotoxin prediction tool that is published and deployed commercially in the world; it was deployed in Ontario (Canada) in 2000, Uruguay (South America) in 2002, and has been undergoing a validation/calibration process in France since 2004. DONcast is attractive to the industry because: 1) prediction accuracies of over 85% have been demonstrated across diverse environments for making decisions on whether or not to apply a fungicide at heading, and 2) of the efficient platform for hosting the model and access to accurate input variables (mainly weather) that result in the following outputs: a) field- or site-specific DON predictions (Site-Specific DONcast – SSD), or b) regional-scaled (map format) outputs, all of which are conveniently managed through Weather INnovations Incorporated or WIN www.weatherinnovations.com (Chatham, ON, Canada). However, after seven years of commercial deployment, users need constant reminders on the limitations of both versions, learn how to interpret predictions toward management decisions, and be warned about unrealistic expectations of model-based predictions especially when they are derived from unrealistic weather or agronomic (or lack thereof) inputs. It has been well documented by others that FHB infection and DON accumulation is highly responsive to weather (mainly around heading), varietal susceptibility, and to the management of crop residue on the soil surface; therefore, these inputs should not be ignored. We will demonstrate that the accuracy of predictions on a regional-scale (i.e., map format) may be less than acceptable because input variables such as weather, wheat variety, crop rotation, and tillage effects tend to be over-generalized; all of these inputs are used in the Site-Specific Calculator. Although prediction maps produced on a regional scale are useful for establishing warnings or trends and are popular amongst growers, the most accurate predictions are derived from input variables that are both accurate and representative of individual fields. Both the input variable database and deployment of these predictions on a field-scale effort are enormous, considering the database is updated daily and the platform must be easily accessible to agribusiness and growers through the internet. The platforms and experiences that have evolved over seven years of commercial use will be presented in more detail. 129 Session 4: FHB Management EFFECTS OF FUNGICIDES ON FHB CONTROL AND YIELD OF WINTER WHEAT CULTIVARS IN NORTH DAKOTA. J.K. Ransom*, M.P. McMullen and S. Meyer * North Dakota State University, Fargo ND Corresponding Author: PH: (701) 293-4067; Email: joel.ransom@ndsu.edu ABSTRACT Research was conducted during three years (2005-2007) to determine the benefits of applying registered fungicides on a range of adapted winter wheat cultivars in North Dakota (ND). Experiments consisted of a factorial combination of fungicides and cultivars laid out in a RCBD with a split plot arrangement. The fungicide treatments served as the main plots and consisted of no fungicide or applying tebuconazole in 2005-06 or tebuconazole and prothioconazole in 2007 at early flowering. Cultivars served as the subplots and consisted of 12-18 cultivars commonly grown in ND or cultivars recently released by breeding programs in the region. Fungicide consistently improved yield and grain quality. Yield increases were associated with the control of leaf spots and especially leaf rust, and in two environments with the control of FHB. Fungicides were profitable when applied to the most disease resistant cultivars when disease pressure was high, but were not beneficial when disease pressure was low. Most of the added value from the use of fungicides resulted from increases in grain yield, but in environments with significant disease pressure, improved test weight also contributed to an increase in the crop’s value. Reductions in DON levels did not improve the grain’s value in the years that it was measured as they were below the threshold where discounts apply. In the least fungicide-responsive environment the return from fungicides did not exceed the cost of the application when applied to the most disease resistant cultivars as they did not produce a yield improvement. In the environment with the greatest disease pressure, however, the most disease resistant types tended to be the ones with the greatest return from fungicides. This indicates the potential value of using disease prediction models if resistant cultivars are used. With the more susceptible cultivars, fungicides should probably always be applied in eastern ND. The value of using resistant varieties, even when planning to use fungicides, was illustrated in the high disease pressure years; the combination of resistance variety and fungicide resulted in the highest yield and generally the greatest return to the fungicide and the highest overall returns. ACKNOWLEDGEMENT AND DISCLAIMER This material is based upon work supported by the U.S. Department of Agriculture under Agreement No. 590790-4-114. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the U.S. Department of Agriculture. 130 Session 4: FHB Management EFFECTS OF FUSARIUM HEAD BLIGHT ON YIELD AND QUALITY PARAMETERS OF WINTER WHEAT. K. Rehorova1*, O. Veskrna1, P. Horcicka2 and T. Sedlacek2 1 Selgen a.s., Stupice 24, 25084 Sibrina, Czech Republic; and 2Research Center SELTON s r.o., Stupice 24, 25084 Sibrina, Czech Republic * Corresponding Author: PH: 00420732237863; Email: rehorova@selgen.cz OBJECTIVES To assess Fusarium Head Blight impact on winter wheat yield reduction, deoxynivalenol (DON) accumulation, sinking of DON content after grading, milling and baking. These parameters are discussed both from the view of susceptible and medium resistant varieties and by application of different fungicide treatment. INTRODUCTION Food safety is nowadays priority for cereal producers and grain-processing industry. Fusarium head blight causes severe yield losses and decreases baking and food quality (Mesterházy, 2003). The most frequent species in Europe are now F. graminearum and F. culmorum (Logrieco et Bottalico, 2001; Mesterházy, 2003), both of which produce mycotoxins (Joffé 1986, Abramson 1998, Chelkowski 1998). The basic toxins are deoxynivalenol (DON), zearalenone and nivalenol (Logrieco et al., 2003). These substances are highly heat and chemically stable. They may be entering the food chain by direct consumption of contaminated foodstuffs, implicitly through feedstuffs and consequently through animal products. (Darwin, Mladka, Sulamit). The project was sown in 3 replications and 4 various fungicide treatments: 1) control – without artificial infection and fungicidal treatment, 2) infection – with artificial Fusarium infection, without fungicide, 3) infection + fungicide, 4) infection + targeted fungicide. Variant infection + fungicide was sprayed with Tango Super (1l/ha, active substances epoxiconazole 84g/ha and fenpropimorph 250g/ha) in growing stage DC 37 – 39. In the variant infection + targeted fungicide was used Tango Super fungicide in DC 37-39 and targeted fungicide Caramba 24 hours before Fusarium infection (1l/ha, active substance metconazole 60g/ha). The experiment was based by small parcel sowing machine type Hege. Final parcel area was 10m2. Inoculum with spore concentrations of 6-7 x 106 spores/ml was prepared and each parcel was infected with 1 liter of inoculum. Infections run up in full flowering period according to each variety term. Symptomatic evaluation was carried in 21st day after the infection. The experiment was harvested by plot harvester. The grain was analyzed; mycotoxins assessment in grain, flour, bread and bran was determined immunochemically using ELISA. RESULTS AND DISCUSSION Most of registered winter wheat varieties are middle or high susceptible to FHB. The results of many researches show us that it is difficult to reach high resistance level and simultaneously high yield and necessary food quality (Mesterházy, 2003). Symptomatic evaluation - The results are average of three years (2005-2007). Head blight symptoms were evaluated on a 1-9 scale (9 - without symptoms, 1 - 100% disease development). Tolerant varieties have with strong infectious pressure significantly lower ocMATERIALS AND METHODS currence of pathogen then susceptible varieties. The difference between infection and non-targeted fungiWe used six winter wheat varieties differed into 2 cide is not significant, while targeted fungicide lead to groups: a) tolerant group (with medium resistant vari- the less presence of symptoms (the evaluation was eties – Sakura, Simila, Petrus), b) susceptible group about 1 point better). 131 Session 4: FHB Management Yield reduction – Targeted treatment was significantly effective in susceptible varieties, which increased their yield about 16% compared to infection variant. Targeted treatment was less significant in tolerant varieties; their yield was higher about 3%. In the susceptible varieties was the lowest yield reduction in targeted treatment (16%), the highest yield reduction was in untreated variant (32%). Yield reduction in tolerant varieties was 12% by targeted treatment, 15% by infection. These results clearly advert to importance of variety tolerence. The active fungicide protection is questionable. Fungicide must be used preventively before symptoms appearance respectively in the right time. ¨ was accurately determined (24 hours after infection), estimation of the application time is doubtful in practice. Non-targeted fungicidal treatment is not explicit. Grading on the 2,2mm sieve causes reduction of the DON content up to 50%. Further manipulation as milling or baking has not so significant influence and major part of DON proceeds to the bread. DON content – In the chart 3 is deoxynivalenol content by 4 fungicide treatments. European Commission devised the limits for DON 1.25 ppm for raw wheat and 0.5 ppm for bread. Tolerant varieties contain about 2/3 less DON than susceptible ones. Targeted fungicide treatment takes positive effect both in tolerant and susceptible varieties and reduces the DON content about more than 50%. Abramson, D. 1998. Mycotoxin formatd environmental factors. In Sinha K.K., Bhatnagar D. (eds), Mycotoxins in Agriculture and Food Safety. Marcel Dekker, New York, pp. 255277 Grain processing and DON content – Figure 4 represent infection variant of 4 varieties, 2 susceptible (Darwin, Sulamit) and 2 medium resistant (Sakura, Simila). Once again is perceived significantly lower DON content in resistant varieties. It is possible to lower DON content just by grading, and that was between 30 – 50%. 70 – 80% of DON proceeds from grain to flour. Due to the high heat stability is the DON occurrence in bread approximately the same as in flour. Development of tolerant varieties is the most effective protection against FHB infection and mycotoxin accumulation. Targeted fungicidal treatment highly influences mycotoxin accumulation and yield in susceptible varieties. However the application date in this work ACKNOWLEDGEMENTS This work was supported by NAZV QG50076 and GAR 521/05/H013. REFERENCES Chelkowski, J. 1998. Distribution of Fusarium species and their mycotoxins in cereal grains. In: In Sinha K.K., Bhatnagar D. (eds), Mycotoxins in Agriculture and Food Safety. Marcel Dekker, New York, pp. 45-64 Joffé, A.Z. 1986. Fusarium Species: Their Biology and Toxicology. John Wiley and Sons, New York. Logrieco, A., Bottalico, A. 2001. Distribution of toxigenic Fusarium species and mycotoxin associated with head blight of wheat in Europe. In: Proceedings of International Conference: Sustainable systems of cereal crop protection against fungal diseases as the way of reduction of toxin occurence in the food webs. 02.-06.07. 2001, Kromeriz, pp. 83-89 Logrieco, A., Bottalico, A., Mul, G., Moretti, A., Perrone, G. 2003. Epidemiology of toxigenic fungi and their associated mycotoxins for some Mediterranean crops. European Journal of Plant Pathology 109:645-667 Mesterházy, A. 2003. Breeding wheat for Fusarium head blight resistance in Europe. In: Leonard, K.J., Bushnell, W.R. (eds), Fusarium Head Blight of Wheat and Barley. The American Phytopathological Society, St. Paul, MN, USA, 312 pp. 132 Session 4: FHB Management 9 8 8.4 SUSCEPTIBLE RESISTANT 7.7 7.3 7.1 6.7 Symptomatic evaluat 7 6 5.4 5 4.4 3.9 4 3 2 1 0 CONTROL TARGETED FUNGICIDE FUNGICIDE INFECTION Fig. 1: Symptomatic Evaluation (2005-2007) 12 SUSCEPTIBLE RESISTANT 100 % 100 % 88 % 84 % 10 87 % 72 % t/ha 8 85 % 68 % 6 4 2 0 CONTROL TARGETED FUNGICIDE FUNGICIDE Fig. 2: Yield in Different Treatment (2005-2007) 133 INFECTION Session 4: FHB Management 18.7 18 SUSCEPTIBLE 16 RESISTANT 16.9 14 12 10 7.7 8 6.0 6 4.5 4 1.9 2 0.2 0.0 0 CONTROL TARGETED FUNGICIDE FUNGICIDE INFECTION Fig. 3: DON Content (2005-2006) 50 45.5 45 DON Content (pp DON Content (pp 20 SUSCEPTIBLE RESISTANT 40 35 30 25 20.9 20 15 10 5 12.9 7.0 3.8 6.9 1.9 1.7 0 BULK GRADING GRADING ABOVE 2.2 mm BELOW 2.2 mm Fig. 4: Grain Processing (2005-2006) 134 BREAD Session 4: FHB Management 2007 UNIFORM FUNGICIDE PERFORMANCE TRIALS FOR THE SUPPRESSION OF FUSARIUM HEAD BLIGHT IN SOUTH DAKOTA. * K.R. Ruden , B.E. Ruden, K.D. Glover and J.L. Kleinjan Plant Science Department, South Dakota State University, Brookings, SD 57007, USA * Corresponding Author: PH: (605) 688-6246; Email: kay.ruden@sdstate.edu ABSTRACT Fusarium head blight (FHB – scab) has been a serious concern for wheat and barley producers in South Dakota for ten years and a serious epidemic impacted the state’s wheat and barley crop in 2005. The objective of this study was to continue to evaluate the efficacy of various fungicides and fungicide combinations for the suppression of Fusarium head blight and other wheat diseases. Two hard red spring wheat cultivars, ‘Briggs’ and ‘Forge’, were planted at three South Dakota locations (Brookings, Groton, and South Shore/ Watertown) and Robust barley was planted at Brookings. Studies at two of these sites were conducted under ambient conditions. At the Brookings site, both the barley and the spring wheat trials received supplemental mist irrigation. Trial treatments from the Uniform Fungicide Trial treatments list for the suppression of FHB included an untreated check, Folicur (tebuconazole) applied at 4.0 fl oz/A, Prosaro (a premix of prothioconazole and tebuconazole) applied at 6.5 fl oz/A, Caramba (metconazole) applied at 13.5 fl oz/A, Topguard (flutriafol) applied at 14 fl oz/A, Proline (prothioconazole) applied at 5 fl oz/A and Tilt (propiconazole) applied at 4 fl oz/ A. All treatments included Induce, a non-ionic surfactant, applied at 0.125% v/v. Spring wheat trials were planted in a factorial randomized complete block design with six replications. The barley trial included four replications. Trial treatments were applied at anthesis (Feekes growth stage 10.51). The spring wheat and barley plots at the Brookings location were inoculated by spreading Fusarium graminearum (isolate Fg4) inoculated corn (Zea mays) grain throughout the field and providing overhead mist irrigation applied from 6:00 pm until 8:00 am each day for two weeks following anthesis. Other sites had natural inoculum from corn stalk residue and natural moisture conditions. Twenty-one days following treatment, plots were evaluated for leaf diseases, FHB incidence, FHB head severity, and FHB field severity. Samples were collected for Fusarium damaged kernels (FDK), deoxynivalenol (DON), grain yield, and test weight. Spring wheat under dryland conditions at South Shore/Watertown and Groton had negligible FHB. No significant differences resulted from the barley trial. On spring wheat in the Brookings trial, Prosaro, Caramba and Proline significantly reduced FHB Incidence, FHB Severity and FHB Index. All products except Folicur significantly increased grain yields with increases ranging from 35-42%. Total leaf disease pressure was very significant, as was leaf rust pressure which occurred late in the season. Control of the leaf diseases likely had a larger effect on yield than FHB control. Data is not yet available for DON. ACKNOWLEDGEMENT AND DISCLAIMER This material is based upon work supported by the U.S. Department of Agriculture, under Agreement No. 590790-4-097. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the U.S. Department of Agriculture. 135 Session 4: FHB Management 2007 UNIFORM TRIALS FOR THE PERFORMANCE OF BIOLOGICAL CONTROL AGENTS IN THE SUPPRESSION OF FUSARIUM HEAD BLIGHT IN SOUTH DAKOTA. K.R. Ruden*, B.H. Bleakley and B.E. Ruden Plant Science Department, South Dakota State University, Brookings, SD 57007, USA * Corresponding Author: PH: (605) 688-6246; Email: kay.ruden@sdstate.edu ABSTRACT Fusarium head blight (FHB – scab) has been a serious concern for wheat and barley producers in South Dakota for ten years and was very severe in parts of SD in 2005. The objective of this study was to continue to evaluate the efficacy of various fungicides and fungicide combinations for the suppression of Fusarium head blight and other wheat diseases under SD conditions. Briggs hard red spring wheat and Robust barley were planted at Brookings, South Dakota. Trial treatments included an untreated check; Prosaro (a premix of prothioconazole and tebuconazole) applied at 6.5 fl oz/A; TrigoCor 1448 (Bacillus sp.) from Cornell University, Ithaca, NY; and TrigoCor 1448 + Prosaro coapplied; 1BA (Bacillus subtilus) from South Dakota State University, Brookings, SD; 1BA + Prosaro coapplied, C3 (Lysobacter enzymogenes) from University of Nebraska, Lincoln, NE; C3 + Prosaro coapplied. The treatments were applied at anthesis. Plots were inoculated by spreading Fusarium graminearum (isolate Fg4) inoculated corn (Zea mays) grain throughout the field and providing overhead mist irrigation applied from 6:00 pm until 8:00 am each day for two weeks following anthesis. Twenty-one days following treatment, plots were evaluated for FHB incidence, FHB head severity, and FHB field severity. Plots were harvested for yield and test weight and samples were collected for Fusarium damaged kernels (FDK) and deoxynivalenol (DON). Even with amending the environment in 2007, significant drought severely limited disease development. In the preliminary analysis, the assessments of FHB Severity and FHB Index disease components indicate a possible treatment effect in the barley study. In the wheat study, all treatments showed a non significant increase in yield as compared to the untreated check. There were no differences among the treatments for the components of FHB. ACKNOWLEDGEMENT AND DISCLAIMER This material is based upon work supported by the U.S. Department of Agriculture, under Agreement No. 590790-4-097. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the U.S. Department of Agriculture. 136 Session 4: FHB Management CHARACTERIZATION OF DON ACCUMULATION IN SRWW CULTIVARS WITH DIFFERENT LEVELS OF TYPE II RESISTANCE TO FHB. Jorge D. Salgado, Gloria Broders, Larry Madden and Pierce Paul* The Ohio State University/OARDC, Department of Plant Pathology, Wooster, OH 44691 * Corresponding Author: PH: (330) 263-3842; Email: paul.661@osu.edu ABSTRACT Under favorable conditions, deoxynivalenol (DON), a mycotoxin produced by Fusarium graminearum, may accumulate to unacceptable levels in harvested grain, making the grain and their by-products unfit for commercialization and consumption. The use of cultivars with resistance to FHB and DON is a widely recommended management approach for reducing the impact of this disease. However, resistance to F. graminearum is complex, with several different types (I, II, III, IV and V) reported, but not completely characterized. While it is clear that there is a positive association between FHB development and DON accumulation, the association between Type II (resistance to disease spread within the spike) and Type III resistance (resistance to DON accumulation) is unclear. It is quite possible for cultivars with similar levels of resistance to FHB to have different levels of resistance to DON accumulation. Some speculate that differential accumulation of DON among cultivars may be the result of differential fungal colonization of grain or the ability of some cultivars to detoxify DON. To quantitatively characterize the associations among FHB, fungal colonization, and DON accumulation in SRWW cultivars with different levels of Type II resistance to FHB, inoculated field trials were conducted at the Ohio Agricultural Research and Development Center, Wooster, during the 2007 growing season. Two experiments (1 and 2) were established, with three wheat cultivars (Cooper, susceptible; Hopewell, moderate susceptible; and Truman, moderately resistant) planted in a randomized complete block design with three replicate blocks. Plots were spray inoculated at early anthesis with a spore suspension (105 spores/ml) containing an equal proportion macroconidia and ascospores of F. graminearum/G. zeae. Approximately 35 days after inoculation, 20 wheat spikes in each of 11 severity categories were tagged. At maturity, spikes in each category were harvested and prepared for DON and PCR analyses. F. graminearum genomic DNA was extracted from each sample and a SYBR green-based real time polymerase chain reaction (RT-PCR) assay used to quantify fungal biomass. DON content was quantified by GC-MS at the USWBSI-sponsored laboratory at the University of Minnesota. Results from an analysis of covariance showed that DON content (ppm) increased with increasing FHB severity in all three cultivars. However, the rate of change in DON with change in severity (the regression slope) was greater for the susceptible cultivars than the resistant cultivar. The magnitude of the difference in DON content at a given level of severity among the cultivars was generally higher at high severity than at low severity. Contrastingly, estimated slopes for relationships between fungal biomass (log-transformed ng/mg) and DON (ppm) were similar for the three cultivars, suggesting similarity in DON accumulation with fungal colonization among the cultivars. However, the heights of the regression lines for the fungal biomass/DON relationships differed among the cultivars, indicating that for a similar level of fungal colonization, DON accumulation differed among the cultivars. Further research is in progress to evaluate these associations under different environmental conditions in an attempt to learn more about possible mechanisms involve in resistance to FHB and DON in wheat. 137 Session 4: FHB Management ACKNOWLEDGEMENT AND DISCLAIMER This material is based upon work supported by the U.S. Department of Agriculture, under Agreement No. 590790-4-112. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture. 138 Session 4: FHB Management CONTRIBUTION OF LOCAL INOCULUM SOURCES TO REGIONAL ATMOSPHERIC POPULATIONS OF GIBBERELLA ZEAE. D.G. Schmale III*, B.R. Dingus, M.D. Keller and A.K. Wood-Jones Virginia Polytechnic Institute and State University, Blacksburg, VA, 24061 * Corresponding Author: PH: (540) 231-6943; Email: dschmale@vt.edu ABSTRACT Decreases in tillage may have contributed to recent epidemics of FHB by increasing the amount of regional atmospheric inoculum available for infection. Where a large, regional source of atmospheric inoculum exists, crop rotation or tillage practices may not effectively reduce the risk of FHB in individual fields. An increased understanding of the contribution of local inoculum sources of Gibberella zeae (Gz) to regional atmospheric populations of the pathogen may aid in developing and/or excluding strategies for managing FHB. In 2007, we conducted 35 sampling flights with unmanned aerial vehicles (UAVs) 100 m above a large clonal inoculum source of Gz established at Virginia Tech’s Kentland Farm. The UAVs were programmed to fly an orbital pattern such that one leg of the sampling path of UAV flew directly over the inoculum source during each of the passes. Our first flight was on 25 March at 11:30 am, and our last flight was on 24 May at 10:30 pm. We collected over 100 isolates of Fusarium spp. during these flights. All of the isolates were single-spored, grown in liquid culture, and suspended in 20% glycerol for cryogenic storage. We have tentatively identified a large portion of these isolates as Gz, and we will be conducting amplified fragment length polymorphisms (AFLPs) on these isolates in the coming months to unambiguously determine the percentage of the clonal isolate of Gz in our atmospheric collections. A series of runs with the atmospheric transport model HYSPLIT suggested that our inoculum source of Gz was transported at least a kilometer away from the ground surface within an hour. Our work continues to (1) elucidate the contribution of a local inoculum sources to atmospheric populations of the pathogen, and (2) develop and test a robust long-distance transport model for FHB forecasting/risk assessment. The ability to predict the regional transport of Gz from local inoculum sources may help refine risk models for FHB. ACKNOWLEDGEMENT AND DISCLAIMER This material is based upon work supported by the U.S. Department of Agriculture, under Agreement No. 590790-7-078. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture. 139 Session 4: FHB Management ENVIRONMENTAL FACTORS INFLUENCING FHB SEVERITY AND DON IN BARLEY. J.M. Stein1*, L.E. Osborne1, S. Neate2 and C. Hollingsworth3 1 Plant Science Department, South Dakota State University, Brookings, SD; 2Department of Plant Pathology, North Dakota State University, Fargo, ND; and 3University of Minnesota, Northwest Research and Outreach Center, Crookston, MN * Corresponding Author: PH: (605) 688-5540; Email: jeff.stein@sdstate.edu ABSTRACT We are investigating the relationship between environmental factors, crop stage, and barley genotype with Fusarium head blight (FHB) and DON accumulation in the grain. This project is associated with the established spring and winter wheat FHB-modeling efforts and aims to produce the information required to either validate one of the current FHB models for use in barley, or generate unique models. Varieties of regionally adapted barley of both 2- and 6-row types were planted at multiple locations in the Northern Great Plains during the 2005-7 growing seasons. At least three varieties were common to each location. Plots were un-irrigated, a minimum of 1.5m x 4.6m in size, and replicated four times in a RCBD. Additional varieties were planted based upon availability and local producer preference. Crop stage was monitored regularly throughout the season and the date at which each plot was at Feekes 10.5 stage was noted. No additional inoculum was introduced into the plots. The incidence and severity of FHB was recorded on a minimum of 50 heads per plot at the soft-dough stage (approximately 21 days after heading). Environmental variables consisting of temperature, relative humidity, and precipitation were recorded by an on-site, or nearby, weather station. Over the past three seasons, we have successfully collected data for 27 of the 38 locations planted. Unsuccessful locations were generally the result of extreme weather-related situations (e.g. floods) that resulted in crop destruction. The remaining locations provide a range in disease intensity, severity, and final deoxynivalenol concentration that we have used to identify weather variables, both simple and complex, that were associated with high FHB/DON situations in barley. For example, the average hourly temperature and relative humidity in the 10 days prior to full head emergence were both significantly correlated with final disease severity, but not DON content. In the available dataset, measurements of humidity after heading (e.g. vapor point depression) were the only factors associated with final DON concentration. From these results, we hypothesize that different environmental factors may be impacting this pathosystem in various ways and the development of a single model for both disease and DON prediction is unlikely. 140 Session 4: FHB Management DIFFERENTIAL SENSITIVITY TO TRIAZOLE-BASED FUNGICIDES AMONG ISOLATES OF FUSARIUM GRAMINEARUM. Matthew Wallhead, Larry Madden and Pierce Paul* The Ohio State University/OARDC, Department of Plant Pathology, Wooster, OH 44691 * Corresponding Author: PH: (330) 263-3842; Email: paul.661@osu.edu ABSTRACT Samples of Fusarium head blight infected wheat spikes were collected from wheat fields across the state of Ohio. All isolates of Fusarium graminearum were identified based on colony and spore morphology. A subset of isolates from different Ohio counties was tested in vitro for sensitivity to Proline (41% prothioconazole) and Folicur (38.7% tebuconazole). Five isolates obtained from conventional wheat fields in Wood, Wayne, Shelby, Van Wert, and Delaware counties and a sixth from an organic wheat field in Wayne County were compared. The fungicides were dissolved in deionized water to achieve stock solutions with the proper concentration of active ingredient and added to PDA. Commercial grade prothioconazole and tebuconazole were evaluated at concentrations of 0.001, 0.01, 0.1 and 1.0 μg/ml and non-amended PDA was used as the control. A 5-mm-diameter plug from the edge of a fully colonized plate was transferred to the center of each plate for each concentration to be evaluated. Colony diameter was measured in three places once every 24 hours for seven consecutive days. The percent growth relative to growth on the control was calculated as the average colony diameter (at each concentration) minus 5 mm (diameter for the PDA plug) divided by the average colony diameter on the non-amended media, multiplied by 100. Sensitivity varied between Proline and Folicur and among isolates of F. graminearum. At concentrations of 0.1 and 1 μg/ml of active ingredient, isolates were generally more sensitive to Proline than Folicur, exhibiting slower and more restricted growth on Prolineamended media than on Folicur-amended media. The sensitivity profile of the isolates was similar for the two fungicides; the same isolates that exhibited the highest and lowest sensitivity to Folicur also exhibited the highest and lowest sensitivity to Proline. With the exception of isolate OHSHE6613, all isolates showed some level of growth on Folicur-amended media at all tested concentration. Conversely, with the exception of OHWAY1619, none of the isolates grew on Proline-amended media at 1.0 μg/ml. Research is in progress to conduct a more comprehensive evaluation of sensitivity of isolates from different wheat-growing regions to Folicur, Proline and other triazole-based fungicides. ACKNOWLEDGEMENT AND DISCLAIMER This material is based upon work supported by the U.S. Department of Agriculture, under Agreement No. 590790-4-112. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture. 141 Session 4: FHB Management A METHOD FOR QUANTIFYING TRICHOTHECENES AND ERGOSTEROL IN SINGLE WHEAT FLORETS USING GAS CHROMATOGRAPHY WITH ELECTRON CAPTURE DETECTION. K.T. Willyerd1, K. Boroczky2 and G.A. Kuldau1* Dept. of Plant Pathology, The Pennsylvania State University, University Park, PA 16802; and 2 Dept. of Entomology, The Pennsylvania State University, University Park, PA 16802 * Corresponding Author: PH (814) 863-7232; Email: kuldau@psu.edu 1 ABSTRACT The relationship between fungal biomass and mycotoxin accumulation during Fusarium Head Blight infections is not completely understood. The purpose of this research is to develop a method to quantify deoxynivalenol and its acetylated derivatives as well as fungal biomass within single wheat florets. Ergosterol, a sterol unique to fungal cell walls, was used to estimate fungal biomass. Gas chromatography with electron capture detection (GC-ECD) and derivatization with hepta-fluorobutyric anhydride (HFBA) were chosen due to their sensitivity. Gas chromatography with mass spectroscopy was subsequently used to confirm these derivatization and detection methods. The extraction solvent used was acetonitrile-water (84:16). Wheat floret extracts were then cleaned through a charcoal alumina column. While previous work has shown GC-ECD to quantify HFBAtrichothecene derivatives, to our knowledge, no studies have used these protocols to also detect ergosterol. This method may be used to study FHB infection patterns within single wheat spikes and the extent of fungal colonization and toxin accumulation within single kernels with varying symptoms. 142 Session 4: FHB Management INFLUENCE OF INFECTION-TIMING ON FUSARIUM HEAD BLIGHT SEVERITY, WHEAT KERNEL DAMAGE AND DEOXYNIVALENOL ACCUMULATION DURING A 2007 FIELD STUDY. K.T. Willyerd1, M. Nita2, E.D. DeWolf2 and G.A. Kuldau1* 1 Dept. of Plant Pathology, The Pennsylvania State University, University Park, PA 16802; and 2Dept. of Plant Pathology, Kansas State University, Manhattan, KS 66506 * Corresponding Author: PH: (814) 863-7232; Email: kuldau@psu.edu ABSTRACT Previous field studies conducted in 2006 have shown that Fusarium graminearum infections during the grain-fill stages of wheat development may lead to kernels with low disease intensity yet significant levels (>2ppm) of deoxynivalenol (DON). Interestingly, infections during both flowering and grain-fill resulted in lower disease severity than when infections occurred during flowering alone. In the most susceptible cultivar a decrease in DON was also observed. The goal of the 2007 study was to gather additional data on infectiontiming patterns. Three winter wheat cultivars were used in this field study: Hopewell (susceptible), Truman (moderately resistant) and Valor (moderately resistant). The experiment was a split-plot design with infectiontiming treatment as the main effect and cultivar as the sub-plot. Four misting treatments were used to facilitate infection: ambient (no supplemental moisture), misting during flowering, misting during grain-fill and misting during flowering and grain-fill. Misting chambers and moveable greenhouses were used to supplement and prevent moisture respectively. All plots were spray inoculated with a mixture of four DON-producing F. graminearum isolates at anthesis and late milk stages. Misting treatments commenced immediately following inoculations and lasted four consecutive nights. Disease incidence and severity were measured in the field during dough stages. Following harvest, yield, kernel damage and DON accumulation were also assessed. Overall, disease intensity and DON levels were low, likely due to dry weather and low humidity during the growing season. In Hopewell, the amount of disease severity and percent kernel damage did not differ between ambient and misting during grain-fill treatments. However, the grain from the misting during grain-fill treatment contained significantly (P d•0.05) higher DON than that grown under ambient conditions. Also in Hopewell, disease severity and DON were significantly (P d•0.05) less under the misting during flowering and grain-fill treatment than in grain grown under the misting during flowering only treatment. This increased moisture – low disease and DON pattern warrants further study. Results from 2007 suggest late infections during grain-fill lead to grain with low disease intensity yet kernels with greater than 1ppm DON. Despite low disease pressure the data gathered in 2007 corroborates with overall infection-timing patterns observed in 2006. 143 Session 4: FHB Management CONTROL OF FUSARIUM INOCULUM PRODUCTION IN CORN RESIDUE BY MECHANICAL, BIOLOGICAL, AND CHEMICAL TREATMENTS. 1* G.Y. Yuen , C.C. Jochum1, J.E. Scott2 and S.Z. Knezevic2 1 Dept. of Plant Pathology, University of Nebraska, Lincoln, NE 68583; and 2 Haskell Research Laboratory, University of Nebraska, Concord, NE, 68728 * Corresponding Author: PH: (402)472-3125; Email: gyuen1@unl.edu OBJECTIVES I. Determine the effects of chopping of field corn residue on the saprophytic growth and sporulation of Gibberella zeae in the residue and the development of Fusarium head blight (FHB) in the following wheat crop. II. Evaluate commercially available fungicides and biological agents as spring applications on the residue to disrupt the sporulation of G. zeae in the residue and reduce the development of FHB in wheat. INTRODUCTION Host resistance and fungicides individually can reduce head infection by G. zeae (=Fusarium graminearum) resulting in partial control reduction of FHB and deoxynivalenol (DON) formation. More complete control, however, will require the integration of these strategies with methods that reduce inoculum production in residue from previous crops. In this study, we examined mechanical, biological and chemical strategies to affect inoculum production. The mechanical strategy involved chopping of residue in the fall. By providing greater surface area for entry of saprophytic organisms and for contact with moisture and soil, chopping could hasten the decomposition of the residue and thus restrict growth of the pathogen. Some residue-colonizing microbes also might be antagonistic to the pathogen and, thus, might be effective in displacing the pathogen from the tissue or preventing its sporulation. The biological and chemical strategies involve application of biocontrol agents and fungicides, respectively, to the residue prior to flowering. Research with fungi, Microsphaeropsis sp. and Trichoderma sp., showed promise in using them to reduce pathogen growth and perithesia production in residue (Bujold and Paulitz, 2001; Fernandez, 1992; Gilbert and Fernando, 2004). Application of fungicides to residue has been investigated on a very limited basis. Tebuconazole impaired decomposition rate and eliminated F. graminearum in residues soaked in the fungicide (Yi et al. 2002), while captan applied to surface residue reduced numbers of fungi (including Fusarium spp.) and slowed residue decomposition (Beare et al., 1993). Thus, it appears that fungicides might directly inhibit the growth of the pathogen in residue but could also have a negative effect on colonization by competitive, decomposing microbes. In the limited studies on the treatment of residue with biological and chemical agents, fall applications were more effective in inhibiting pathogen growth than spring treatments. In this study, we examined whether spring treatments would exert sufficient impact on pathogen spore production in infested residue to reduce FHB and DON. MATERIALS AND METHODS Experiments were conducted in two University of Nebraska experiment station sites, ARDC near Mead and Haskell Agricultural Laboratory near Concord, to test the effects of fall mechanical treatments and spring biological and chemical treatments. Each experiment had a split-plot design with ‘residue type’ (chopped, unchopped, no residue) being the primary factor and ‘spray treatment’ (biocontrol products, fungicides, no treatment) the split factor. At each site, hard red winter wheat ‘Overley’ was block planted in fall 2006 into 4-acre fields having a previous soybean crop. Residue from BT corn in a neighboring field was chopped using a bush hog mower or left intact. The chopped and whole residue was left to decompose in place until early March, 2007. At that time, unchopped 144 Session 4: FHB Management parameters differed between the two experiments. At Concord, there were significant spray treatment effects for FHB incidence and index, with Prosaro being the only treatment to have lower levels than the control (Table 1). Test weight was significantly higher in plots in which residue was sprayed with Headline as compared to the control. When data for each residue type was analyzed across spray treatments, the no-residue control had the lowest DON level and the highest yield measurements, but it also had the highest disease incidence. At Mead, there were significant residue by spray treatment interactions for disease incidence and index, but except for a decrease in disease index in chopped residue by Prosaro, none of the spray treatments reduced disease measurements in any residue types compared to the respective control (Table 2). Instead, Serenade and Headline increased disease incidence and index. Disease incidence, DON level and % FDK, averaged across spray treatments, were significantly higher in the whole residue and chopped Residue samples were collected from plots containing residue plots than in the no-residue plots. In addition, chopped and unchopped residue plots at anthesis presence of residue significantly decreased yield com(Feekes 10.5) for determination of numbers of G. zeae pared to no residue. ascospores. Samples were weighed and then washed in a standard amount of water with Tween. Ascospore CONCLUSIONS concentration in each wash was determined by counting with a hemacytometer. Field data collected were The results with Prosaro suggest there is some promFHB incidence and severity. DON content, yield of ise to the strategy of applying fungicides to residue in kernels, and percentage of Fusarium disease kernels the spring to reduce FHB development in the wheat (FDK) were measured after harvest. All data was sub- crop. The strategy by itself provided on low levels of jected to ANOVA for split-plot design and Fisher’s disease control and DON reduction, so it would need to be integrated with host resistance and/or fungicide LSD was used for means separation. treatments applied to flowering heads. Given that Prosaro might be widely applied to flowering heads, it RESULTS would be not be desirable to use the same product to There was a higher number of ascospores detected treat residue as well because of increased the risks of on chopped residue collected from Mead at anthesis selecting for resistant pathogen populations. Therethan on whole residue (1.95 X 103 per g residue vs. fore, it is necessary to evaluate a larger selection of 5.42 X 103 per g, respectively; P = 0.012). At Con- fungicides with different modes of action than Prosaro cord, there was no significant difference in ascospore specifically for use as residue treatments. In this renumbers between residue types, with fewer than 2 X spect, biocontrol agents theoretically would be good candidates. While the results with the two biological 103 per g being counted. products in this study were not encouraging, there are There was sufficient rain at both sites to cause moder- other commercial agents available for future testing. ate disease incidence, but disease severity was moderate at Mead and low at Concord. The effects of The increased disease development and decreased residue type and spring spray treatments on disease yields observed in the presence of residue as com- corn debris was cut close to the ground with a sickle mower and residue of each type was collected and spread into 10’ X 20’ plots within the wheat field. Approximately 14 Kg (31 lb.) of residue was introduced per plot. There were three blocks containing one strip for each residue type. Each strip had six plots separated by 30’-wide buffer zones of wheat. Each plot within a strip was assigned one of six spray treatments which included three chemicals: Headline, Dithane DF, Prosaro; two biologicals: Serenade (Bacillus subtilis strain QST713) from AgraQuest, T-22 (Trichoderma harzianum strain KRL-AG2) produced by BioWorks; and a distilled water control. Spray treatments were applied to debris on the soil surface once at early stem extension stage (Feekes 68). Each material was applied at manufacturer’s label rate for foliar applications in 20 gal water per acre using a CO2-pressurized backpack sprayer with nozzles configured for spraying herbicides. 145 Session 4: FHB Management pared to no residue were agreement with other reports (Dill-Macky and Jones, 2000). While disease levels in chopped residue tended to be lower than in the whole residue, the differences largely were not significant. This could be related to chopping having only a small influence on sporulation, as suggested by the ascospore counts. Another explanation is that differences could have been masked by substantial aerial inoculum entering the experiment plots, as evidenced by the moderate levels of disease occurring in the noresidue plots. The wet weather experienced in eastern Nebraska during the experiments was atypical. In more typical drier years, the regional inoculum load might be lower and, thus, diminution of residue by chopping might exert a greater effect in reducing inoculum in a given field. ACKNOWLEDGEMENTS The authors wish to thank BioWorks and AgriQuest for providing biological control materials, and Doug Miller, University of Nebraska, for technical assistance. This material is based upon work supported by the U.S. Department of Agriculture, under Agreement No. 59-0790-6-072. reflect the view of the U.S. Department of Agriculture. REFERENCES Beare, M.H., Pohland, B.R., Wright, D.H. and Coleman, D.C. 1993. Residue placement and fungicide effects on fungal communities in conventional and no-tillage soils. J. Soil Sci. Soc Amer. 57:392-399. Bujold, I. and T.C. Paulitz. 2001. Effect of Microsphaeropsis sp. on the production of perithesia and ascospores of Gibberella zeae. Plant Dis. 85:977-984. Fernandez, M.R. 1992. The effect of Trichoderma harzianum on fungal pathogens infesting wheat and black oat straw. Soil Biol. Biochem. 24:1031-1034. Dill-Macky, R. and R.K. Jones. 2000. The effect of previous crop residues and tillage on Fusarium head blight of wheat. Plant Dis. 84:71-76. Gilbert, J. and W.G.D. Fernando. 2004. Epidemiology and biological control of Gibberella zeae. Can. J. Plant Pathol. 26:464472. Yi, C.L., Kaul, H.P., Kubler, E. and Aufhammer, W. 2002. Populations of Fusarium graminearum on crop residues as affected by incorporation depth, nitrogen and fungicide application. Z. Pflanzenkrank. Pflanzenschutz 109:252-263. DISCLAIMER This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily 146 Session 4: FHB Management Table 1. Results from 2007 residue management trial at Concord, Nebraska (Haskell Ag. Lab.) Residue type Spray treatment Incid. (%) Sev. (%) Index (%) DON (ppm) FDK (%) Yield (K) 200seed wt (g) None None Dithane DF Headline Prosaro Serenade T22 None Dithane DF Headline Prosaro Serenade T22 None Dithane DF Headline Prosaro Serenade T22 53 49 46 41 44 44 42 35 41 30 49 49 47 33 37 33 43 36 27 25 22 23 23 25 29 29 24 15 28 27 28 26 28 26 30 21 15 12 10 10 10 11 12 10 10 4 14 13 14 9 10 8 13 8 1.4 1.8 1.8 1.2 2.8 2.4 2.6 2.5 2.8 1.9 3.5 1.9 2.4 1.8 2.3 1.4 2.3 2.2 4.3 3.8 2.7 3.5 4.7 3.8 5.8 3.5 4.7 4.2 4.3 4.3 4.5 4.3 6.3 3.3 5.2 5.7 2.5 3.1 3.0 2.7 3.0 3.1 2.2 2.6 2.5 2.4 1.9 2.4 2.2 2.2 2.9 3.1 2.5 1.6 6.0 6.7 6.5 6.8 6.0 6.3 5.9 6.0 6.4 5.9 5.3 5.7 6.0 6.0 6.6 6.3 6.0 5.3 P residue type X spray trt. NS NS NS NS NS NS NS None Whole Chopped 46 a* 41 ab 38 b 24 25 26 11 11 10 1.9 b 2.5 a 2.1 ab 3.8 4.5 4.9 2.9 a 2.3 b 2.4 b 6.4 a 5.9 b 6.0 b P residue type 0.013 NS NS 0.050 NS 0.014 0.002 Mean across residue types 47 a 39 ab 42 ab 35 b 45 a 43 ab 28 26 25 21 27 24 13 a 10 ab 10 ab 7b 12 a 11 ab 2.1 bc 2.0 bc 2.3 ab 1.5 c 2.9 a 2.2 abc 4.9 3.9 4.6 3.7 4.7 4.6 2.3 2.6 2.8 2.7 2.4 2.4 6.0 bc 6.2 ab 6.5 a 6.3 ab 5.8 c 5.8 c 0.034 NS 0.050 0.018 NS NS 0.001 Whole Chopped Means across spray treatments None Dithane DF Headline Prosaro Serenade T22 P spray treatment * Letters denote means separation at P = 0.05. 147 Session 4: FHB Management Table 2. Results from 2007 residue management trial at Mead, Nebraska (ARDC). Residue type Spray treatment Incid. (%) Sev.( %) Index (%) DON (ppm) FDK (%) Yield (K) 200seed wt (g) 38 d* 50 bcd 42 cd 42 cd 61 abc 53 bcd 45 cd 59 abc 73 a 52 bcd 67 ab 53 bcd 68 ab 53 bcd 49 bcd 51 bcd 58 abc 52 bcd 0.024 29 53 39 37 62 48 35 57 57 44 58 43 59 43 40 33 52 50 NS 11 e 26 abcde 17 cde 16 de 38 abc 25 abcde 16 cde 34 abcd 43 a 25 abcde 40 ab 23 abcde 40 ab 25 abcde 20 bcde 18 cde 30 abcde 27 abcde 0.050 5.3 5.4 4.8 5.2 6.2 6.1 8.4 8.0 10.8 10.0 9.7 12.0 8.9 8.9 7.7 7.0 9.7 7.8 NS 11.0 9.0 10.3 9.8 9.7 11.0 14.5 13.8 17.8 14.0 11.5 14.3 14.2 12.0 14.1 12.5 15.5 12.2 NS 2.7 3.1 3.0 2.4 2.4 3.0 2.4 1.8 2.3 1.6 1.9 2.4 2.3 2.7 2.5 2.6 2.4 2.1 NS 6.3 6.8 6.8 6.8 5.3 6.2 5.9 6.4 6.1 5.9 5.8 5.8 6.0 6.2 6.2 6.4 6.2 6.0 NS 47 b 58 a 55 a 45 49 46 22 30 27 5.5 b 9.8 a 8.4 a 10.2 b 14.3 a 13.4 a 2.8 a 2.1 c 2.4 b 6.4 6.0 6.2 P residue type 0.008 NS 0.087 <0.001 0.006 <0.001 NS Mean across residue types 50 b 54 ab 55 ab 48 b 62 a 52 b 42 51 45 37 57 47 23 28 27 20 36 26 7.5 7.5 7.8 7.4 8.5 8.6 13.2 11.6 14.2 12.1 12.2 12.5 2.5 2.5 2.6 2.2 2.2 2.5 6.0 ab 6.5 a 6.3 a 6.4 a 5.8 b 6.0 ab 0.085 0.06 0.064 NS NS NS 0.039 None None Dithane DF Headline Prosaro Serenade T22 Whole None Dithane DF Headline Prosaro Serenade T22 Chopped None Dithane DF Headline Prosaro Serenade T22 P residue type X spray trt. None Whole Chopped Means across spray treatments None Dithane DF Headline Prosaro Serenade T22 P spray treatment * Letters denote means separation at P = 0.05. 148 Session 4: FHB Management EFFECTS OF SPRAY APPLICATION METHODS ON BIOCONTROL AGENT VIABILITY. G.Y.Yuen1*, C.C.Jochum1, S. Halley2, G. Van Ee3, V. Hoffman4 and B.H. Bleakley5 1 Dept. of Plant Pathology, University of Nebraska, Lincoln, NE 68583; 2Langdon Research Extension Center, North Dakota State University, Langdon, ND 58249; 3Emeritis, Dept. of Agricultural Engineering, Michigan State University, East Lansing, MI 48824; 4Emeritis, Extension Agricultural Engineering, North Dakota State University, Fargo, ND 58105; and 5Biology and Microbiology Dept., South Dakota State University, Brookings, SD 57007 * Corresponding Author: PH: (402) 472-3125; Email: gyuen1@unl.edu OBJECTIVE Determine the effects of commercial ground spray application systems on viability of representative biological control agents. INTRODUCTION All microorganisms used for biological control are sensitive to environmental extremes and thus, environmental conditions occurring during the application process could affect the viability of the agents and, consequently, affect disease control efficacy. Bacteria and yeast have been evaluated as biological agents in field trials for efficacy in controlling Fusarium head blight and reducing deoxynivalenol levels. Field tests conducted thus far utilized CO2 pressurized backpack systems with care taken to avoid subjecting biological materials to temperature extremes. There is no information available as to how biological agents would respond to “real-world” conditions occurring during operation of commercial spray equipment. Under such conditions, accumulation of heat from sunlight or pump motors and sheer forces within the pumps, filters, nozzles and other systems mechanisms could possibly be injurious to biological agents. (Jochum and Yuen, 2006) representing gram-negative, non-spore-forming bacteria; and Cryptococcus aureus OH71-4 (Khan et al., 2004) representing epiphytic yeasts. Spontaneous mutants of the bacterial strains resistant to the drug rifampicin were used so that they could be detected on 10% tryptic soy agar (TSA) medium amended with the drug and a fungicide. The yeast OH71-4 was detected on 10% TSA amended with antibacterial drugs. Quantification of the agents in cell suspensions was done by dilution plating. Cultures of bacterial strains C3 and 1BA in chitin broth and nutrient broth, respectively, and yeast strain OH714 in a proprietary frozen concentrate (provided by D. Schisler, NCAUR) were used in two experiments. One hypothesis tested in the experiments was that heat accumulation in the biocontrol agent suspensions will reduce organism viability. Another hypothesis was that each stage of a spray application (i.e., agitation of cell suspension in tank, pumping of cell suspensions through the spray line, and discharge of cell suspension as droplets through nozzles) also will affect biocontrol agent viability. Experiment 1 was conducted at North Dakota State University research facilities at Langdon using a customized spray system with a 10-gal. tank, shortened lines, and a single nozzle. These changes were made to accommodate small liquid volumes. In addition, MATERIALS AND METHODS ports were added to the line between the pump and the filter and between the filter and nozzle to allow Three microorganisms were tested, each representing collection of liquid samples. Otherwise all parts were a different microorganism group: Bacillus sp. 1BA standard as would be used on conventional spray sys(Draper et al., 2001) representing spore-forming, tems, including a cast iron gear drive centrifugal pump gram-positive bacteria; Lysobacter enzymogenes C3 (HYPRO Model 9006C-O) and XR8002 nozzle. The 149 Session 4: FHB Management bacterial cultures and yeast formulation were diluted with water in the tank to 4 gallons. The pump was operated at standard PTO speed (540 rpm) and manifold pressure was maintained at 40 psi. The contents of the tank were continuously agitated by recirculation (8 gal/min). At 10-minute intervals, temperature within the tank liquid was measured, and samples of liquid were collected from the tank and the two sampling ports. Liquid was then emitted from the nozzle and a sample of the spray collected. Experiment 2 was conducted at the UNL Agronomy Research Farm using an unmodified commercial spray system having a piston pump (Ace model F-1), a 115gal capacity tank, and the same nozzle type as in experiment 1. The biological materials were diluted to 50 gal with tap water. The suspensions were sprayed continuously out three nozzles under 40 psi pressure. As in experiment 1, temperature and tank liquid samples were collected at 10-minute intervals. Spray samples were collected from each of the nozzles as well. RESULTS AND CONCLUSIONS During experiment 1, temperatures in each of the biocontrol suspensions re-circulating in the tank rose rapidly due to transfer of heat from the pump (Fig. 1). Within 30-40 minutes, temperatures exceeded 50oC, which is injurious or lethal to most microorganisms. The heat accumulation was likely the primary factor leading to loss of viability of the biocontrol agents in the liquid (Fig. 2). In this respect, the three organisms exhibited widely different responses to similar rises in temperature. There was little difference in numbers of live cells between samples taken at various points in the spray system and samples collected from the tank (Fig. 3), indicating that passage of the cell suspensions through individual parts (pump, filter, and nozzle) had negligible effects on organism viability and did not account for the drops in population within the tank. When the biocontrol agents were sprayed through commercial equipment in experiment 2, temperatures in the liquid remained stable at favorable levels (Fig. 4). Because ambient temperatures and sunlight conditions were similar between experiments, the lower liquid temperatures recorded in experiment 2 can be attributed to the large liquid volume accumulating heat from the pump at a much slower rate. Consequently, there were no appreciable changes in biocontrol populations within the tank over time (Fig. 5). In addition, biocontrol agent population levels sprayed out of the nozzles were not significantly different from those measured in the tank (Fig. 6), thus confirming that the various mechanisms in a conventional spray system collectively have little effect on biocontrol agent viability. We conclude that when biocontrol agents eventually become available for application to cereals, they will be compatible in the most part with existing equipment used for ground applications of fungicides. Heat accumulation in the liquid may reduce biocontrol agent numbers, but this would most likely occur as tanks are close to being empty. In experiment 1, the high recirculation rate relative to the small liquid volume resulted in foaming of the liquids in the tank. Foaming was particularly a problem with the culture of Bacillus 1BA, resulting in difficulties in maintaining stable manifold pressure. While it is unknown if foaming would affect the viability of the organisms in the liquid, foaming could hasten the degradation of antifungal proteins and antibiotics excreted into broth culture by the bacteria and, thus, could potentially reduce biocontrol effectiveness. In experiment 2, when the biocontrol materials were diluted into much larger water volumes, foaming was not a problem for the bacterium C3 and the yeast. Addition of the an antifoaming agent Biospumex 36K to the suspension of 1BA arrested foaming. Therefore, the use a nontoxic antifoaming agent is recommended as an adjuvant for those biocontrol materials for which foaming may be an issue. ACKNOWLEDGEMENTS The authors wish to thank Dr. David Schisler, NCAUR USDA-ARS, for generously providing preparations of Cryptococcus aureus OH71-4 and Kyle Broderick, University of Nebraska, for technical assistance. This material is based upon work supported by the U.S. Department of Agriculture, under Agreement No. 59-0790-6-072. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. 150 Session 4: FHB Management DISCLAIMER Fusarium Head Blight Forum; 2001 Dec 8-10; Erlanger, KY. East Lansing: Michigan State University. p. 48. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the U.S. Department of Agriculture. Jochum, C.C., L.E. Osborne, and G.Y. Yuen. 2006. Fusarium head blight biological control with Lysobacter enzymogenes strain C3. Biological Control 39:336-344. REFERENCES Khan, N.I, Shisler, D.A, Boehm, M.J., Lipps, P.E., and Slininger, P.J. 2004. Field testing of antagonists of Fusarium head blight incited by Gibberella zeae. Biological Control 29:245-255. Draper, M.A., Bleakley, B.H., Ruden, K.R. and Baye, N. 2001. Greenhouse screening of biological control agents for suppression of Fusarium head blight. In: Canty, S.M., Lewis, J., Siler, L. and Ward, R.W. (Eds.), Proceedings of the National CFU/ml 70 1e+10 60 1e+9 1e+8 50 1e+7 o C 40 1e+6 1e+5 30 1e+4 OH71.4 1BA C3 20 C3 1e+3 OH71.4 1BAC 1e+2 1e+1 10 0 10 20 30 40 50 0 60 10 20 30 40 50 60 Minutes Minutes Fig. 2 Expt 1 - Biocontrol agent populations in suspensions in tank. Fig. 1 Expt 1 - Temperatures in biocontrol agent suspensions in tank. 151 Session 4: FHB Management 70 200 C3 O H 7 1 .4 1BAC % change in population 150 OH71.4 1BA C3 60 50 100 50 o C 40 0 30 -5 0 -1 0 0 20 -1 5 0 B e fo r e A fte r filte r filte r 10 N o z z le 0 Fig. 3 Expt 1 – Changes in biocontrol agent populations at various points in spray system relative to tank populations. Standard deviations shown. 10 20 30 40 50 60 Minutes Fig. 4 Expt 2 - Temperatures in biocontrol agent suspensions in tank. CFU/ml 300 1e+10 1e+9 C3 OH71.4 1BAC 200 % change in population 1e+8 1e+7 1e+6 1e+5 1e+4 C3 1e+3 OH71.4 0 -100 1BAC 1e+2 100 1e+1 0 10 20 30 40 50 -200 60 Minutes Fig. 6 Expt 2 – Changes in biocontrol agent populations at nozzles relative to tank populations. Standard deviations shown. Fig. 5 Expt 2 - Biocontrol agent populations in suspensions in tank. 152 Session 4: FHB Management RESULTS FROM THE 2007 STANDARDIZED EVALUATION OF BIOLOGICAL AGENTS FOR THE CONTROL OF FUSARIUM HEAD BLIGHT ON WHEAT AND BARLEY. G.Y. Yuen1*, C.C. Jochum1, K.R. Ruden2, J. Morgan4, B.H. Bleakley2,3 and L.E. Sweets4 1 Dept. of Plant Pathology, University of Nebraska, Lincoln, NE 68583; 2Plant Science Dept., South Dakota State University, Brookings, SD 57007; 3Biology and Microbiology Dept., South Dakota State University, Brookings, SD 57007; and 4Dept. of Plant Microbiology and Pathology, University of Missouri, Columbia, MO 65211 * Corresponding Author: PH: (402) 472-3125; Email: gyuen1@unl.edu OBJECTIVE To evaluate, using standardized methodology, a set of biological control agents applied alone and in combination with a fungicide for effectiveness in managing Fusarium head blight (FHB) and deoxynivalenol (DON) in wheat and barley across a range of environmental conditions. Uniform fungicide trials in 2006 (Paul et al., 2006) showed increased yield and reduction of DON by a fungicide formulation Prosaro 421 SC that combines tebuconazole and prothioconazole. Therefore, trials in 2007 were designed to test the efficacy of the three biological agents, alone in combination with Prosaro, for the control of FHB and DON. MATERIALS AND METHODS INTRODUCTION Some of the biological agents reported to have potential for controlling FHB are bacterial strains Bacillus subtilis TrigoCor 1448 (Stockwell et al., 2001) and Bacillus sp. 1BA (Draper et al., 2001), and Lysobacter enzymogenes strain C3 (Jochum et al., 2006). Each strain has shown efficacy in some field tests when evaluated separately (Stockwell et al., 2001; Jochum et al., 2006; Yuen and Jochum, 2004). In 2004 through 2006, they were directly compared for efficacy as part of the USWBSI-funded program for standardized evaluation of biological agents. Because combinations of biological control agents and fungicides were reported to be more effective in controlling FHB than the microorganisms or fungicides alone (DaLuz et al., 2003; Khan et al., 2004; Yuen and Jochum, 2004), standardized evaluations in 2005 and 2006, also compared these bacterial strains in combination with the fungicide tebuconazole. In the three years of testing, however, results were inconclusive as to the effectiveness of the treatments across a range of environmental conditions and crop genotypes (Yuen et al, 2004; Yuen et al., 2005; Yuen et al., 2006) due to low disease pressure in most or all test sites. Six trials were conducted across three states on barley and a range of wheat market classes (Table 1). In each trial, three bacterial biological agents (Table 2) were tested alone or in tank mix with the fungicide Prosaro 421 SC (6.5 fl oz/A). There also was a treatment of Prosaro alone and a non-treated control. A broth culture of each organism was provided by the originating laboratory and sent to the researcher in each location. The pre-application population of each agent in the inoculum was determined by the local researcher using dilution plating. All treatment liquids were amended with 0.125% Induce (v/v). One application was made per treatment at early flowering (Feekes 10.51) in 20 gal/acre using CO2-pressurized sprayers (approximately 40 psi) equipped with flat-fan nozzles oriented forward and backward. The size and number of replicate plots varied among trials. Some of the trials were inoculated with Fusarium graminearum spore suspensions and or inoculated corn grain, with mist irrigation systems utilized to stimulate infection. In all trials, FHB incidence (% heads infected per plot), severity (% spikelets infected per diseased head), and index (% plot severity) were determined from at least 40 heads per plot around 3 153 Session 4: FHB Management weeks after anthesis. The incidence of Fusarium-damaged kernels (%FDK), as well as yield of seed and test weight, were determined after harvest. Samples from each plot were sent to the North Dakota State University Veterinary Diagnostic Laboratory, Fargo, ND for analysis of DON content. Analysis of variance was performed on results from each trial separately, with Duncan’s multiple range test used for means separation. Data from Missouri and Nebraska trials were analyzed together using ProcMixed (SAS), with trials being treated as fixed variables and the LSD method used to separate LS means. Results from South Dakota were excluded because of low disease levels. RESULTS AND DISCUSSION Weather conditions in South Dakota were dry, preventing significant disease or DON production. Wet weather in Missouri and Nebraska resulted in higher FHB development. Low temperatures in Missouri occurring during heading, however, caused damage to wheat heads and kernels, particularly in cv. Elkhart, making assessments of FHB severity and FDK difficult. The most effective biological agent applied alone was L. enzymogenes C3, reducing disease severity in two trials and disease index averaged across four trials (Tables 3A and 3B). No combination of a biocontrol agent with Prosaro 421 SC exhibited greater efficacy than the fungicide alone. None of the biocontrol agents alone increased yields over the control (data not shown). Prosaro 421 SC applied alone or in combination with a biological agent was effective in reducing FHB measures and DON levels in multiple trials (Tables 3A and 3B). The fungicide alone and in combinations with some biocontrol agents increase plot yields in the two Nebraska trials and in the Missouri with ‘Roane’ (data not shown). The collective results from this year’s multistate trials indicated L. enzymogenes C3 to the be the most effective biocontrol agent across a range of environments, but treatments with the bacterium are not as effective or as consistent as treatment with Prosaro 421 SC. No benefit was revealed in this study from combining biocontrol agents with Prosaro 421 SC, contrary to past studies with biocontrol agent-tebuconazole combinations. The difference in results is most likely related to the greater effectiveness of Prosaro 421 SC over tebuconazole. Therefore, it may be desirable to explore combinations of biocontrol agents with less efficacious fungicides as a means to broaden the selection of treatments that can be used to protect florets from Fusarium infection. ACKNOWLEDGEMENTS We thank Gary Bergstrom for providing TrigoCor 1448 and valuable suggestions. This material is based upon work supported by the U.S. Department of Agriculture, under Agreement No. 59-0790-6-072. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. DISCLAIMER Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the U.S. Department of Agriculture. REFERENCES Da Luz, W.C., C.A. Stockwell, and G.C. Bergstrom. 2003. Biological Control of Fusarium graminearum. Pages 381-394 in: Fusarium Head Blight of Wheat and Barley. K.J. Leonard and W.R. Bushnell, eds. APS Press. Draper, M.A., B.H. Bleakley, K.R. Ruden, and N Baye. 2001. Greenhouse screening of biological control agents for suppression of Fusarium head blight. Proceedings of the 2001 National Fusarium Head Blight Forum, pg. 48. Jochum, C.C., L.E. Osborne and G.Y. Yuen. 2006 Fusarium head blight biological control with Lysobacter enzymogenes strain C3. Biological Control 39:336-344. Khan, N.I, Shisler, D.A, Boehm, M.J., Lipps, P.E., and Slininger, P.J. 2004. Field testing of antagonists of Fusarium head blight incited by Gibberella zeae. Biological Control 29:245-255. Paul, P., D. Hershman, M. Draper, and L. Madden. 2006. Effect of Fungicides on FHB and DON in Wheat – 2006 Uniform Fungicide Trials. In: Canty, S.M., Clark, A., Van Sanford, D. (Eds.), Proceedings of the National Fusarium Head Blight Forum; 2006 Dec. 10-12; Research Triangle Park, NC. University of Kentucky. pp15-18. 154 Session 4: FHB Management Stockwell, C.A., G.C. Bergstrom, and W.C. da Luz. 2001. Biological control of Fusarium head blight with Bacillus subtilis Trigocor 1448: 2001 field results. Proceedings of the 2001 National Fusarium Head Blight Forum, pp. 91-95. Yuen, G.Y. and C. C. Jochum. 2004. Factors that can affect field efficacy of biological control against Fusarium head blight. Proceedings of the 2nd International Symposium on Fusarium Head Blight; incorporating the 8th European Fusarium Seminar; 2004. p. 379. Yuen, G.Y., B.H. Bleakley, M.A. Draper, C.C. Jochum, E.A. Milus, K.R. Ruden, and L.E. Sweets. 2004. Results From the 2004 Standardized Evaluation of Biological Agents for the Control of Fusarium Head Blight. Proceedings of the 2nd International Symposium on Fusarium Head Blight; incorporating the 8th European Fusarium Seminar; 2004. pp. 380-382. Yuen, G. Y., C.C. Jochum, B.H. Bleakley, M.A. Draper, K.R. Ruden, and L.E. Sweets. 2005. Standardized Evaluation of Biological Agents for the Control of Fusarium Head Blight: 2005 Results. In: Canty, S.M., Boring, T., Wardwell, J., Siler, L., and Ward, R.W. (Eds.), Proceedings of the National Fusarium Head Blight Forum; 2005 Dec. 11-13; Milwaukee, WI. East Lansing: Michigan State University. pp. 237-239. Yuen, G.Y., C.C. Jochum, K.R. Ruden , L.E. Sweets, B.H. Bleakley, M.A. Draper. 2006. 2006 Results From the Standardized Evaluation of Biological Agents for the Control of Fusarium Head Blight on Wheat and Barley. In: Canty, S.M., Clark, A., Van Sanford, D. (Eds.), Proceedings of the National Fusarium Head Blight Forum; 2006 Dec. 10-12; Research Triangle Park, NC. University of Kentucky. pp. 27-30. Table 1. 2007 uniform biological control trial locations, crop cultivars, and researchers State (location) Crop market class and cultivar Researcher and Institution MO Soft red winter wheat ‘Roane’ L. Sweets, University of Missouri MO Soft red winter wheat ‘Elkhart’ L. Sweets, University of Missouri NE -1 Hard red winter wheat ‘2137’ C. Jochum & G. Yuen, University of Nebraska (Mead) NE - 2 Hard red winter wheat ‘2137’ C. Jochum & G. Yuen, University of Nebraska (Lincoln) SD Hard red spring wheat ‘Briggs’ K. Ruden & B. Bleakley, South Dakota St. Univ. SD Six-rowed barley ‘Robust’ K. Ruden & B. Bleakley, South Dakota St. Univ. Table 2. Biological control agents tested in 2007 uniform trials. Organism Supplier Bacillus sp.1BA Bruce Bleakley, South Dakota State University Bacillus subtilis TrigoCor 1448 Gary Bergstrom, Cornell University Lysobacter enzymogenes C3 Gary Yuen, University of Nebraska 155 Session 4: FHB Management Table 3A. 2007 results across six uniform biocontrol trials denoted by state and crop MO MO NE-1 NE-2 SD SD Treatment ‘Roane’ ‘Elkhart’ 2137 2137 Briggs Barley INCIDENCE (% heads infected) Control 12 a* 15 c 95 a 68 a 2 57 Prosaro 7 cd 22 abc 58 b 47 c 1 42 1BA 9 bc 18 bc 87 a 70 a 3 62 1BA + Prosaro 6d 24 abc 65 b 52 bc 2 65 TrigoCor 1448 10 ab 22 abc 92 a 69 a 1 90 TrigoCor 1448 + 7 cd 30 a 62 b 47 c 1 50 Prosaro C3 12 a 26 ab 84 a 62 ab 1 79 C3 + Prosaro 7 cd 16 c 70 b 58 abc 1 84 P <0.001 0.045 <0.001 <0.001 0.758 0.174 SEVERITY (% spikelets infected) Control 16 a 32 Prosaro 6c 32 1BA 12 ab 23 1BA + Prosaro 7 bc 36 TrigoCor 1448 11 ab 26 TrigoCor 1448 + 9 bc 34 Prosaro C3 10 bc 28 C3 + Prosaro 9 bc 19 P 0.017 0.175 38.0 a 15.6 e 33.0 ab 20.0 de 35.8 a LS mean 59 a 40 b 58 a 44 b 60 a 43 b 56 a 46 b <0.0001 29 b 28 b 28 b 28 b 28 b 45 17 13 12 38 5 5 5 5 7 31 a 21 cd 27 bc 23 bcd 28 ab 22.1 cde 36 a 22 5 26 abc 28.1 bc 24.7 cd <0.001 27 5 0.755 28 b 26 b 0.039 6 10 .0545 *Means separation (P=0.05) shown only when treatment effect was significant. 156 25 abcd 20 d <0.0001 Session 4: FHB Management Table 3B. 2007 results across six uniform biocontrol trials denoted by state and crop. MO MO NE - 1 NE - 2 SD SD Treatment ‘Roane’ ‘Elkhart' 2137 2137 Wheat Barley INDEX (plot severity) Control 1.9 a 4 36 a 20 a 1.1 3b Prosaro 0.4 c 8 9e 13 c 0.7 2b 1BA 1.1 b 4 29 ab 20 a 0.6 3b 1BA + Prosaro 0.4 c 9 14 de 14 bc 0.3 3b TrigoCor 1448 1.2 b 7 33 a 19 ab 0.8 6 ab TrigoCor 1448 + 0.6 bc 10 15 de 17 abc 0.3 2b Prosaro C3 1.1 b 8 24 bc 17 abc 0.5 5 ab C3 + Prosaro 0.7 bc 4 18 cd 15 bc 0.2 8a P 0.001 0.133 <0.001 0.039 0.894 0.029 FDK (%) Control Prosaro 1BA 1BA + Prosaro TrigoCor 1448 TrigoCor 1448 + Prosaro C3 C3 + Prosaro DON (ppm) Control Prosaro 1BA 1BA + Prosaro TrigoCor 1448 TrigoCor 1448 + Prosaro C3 C3 + Prosaro LS Mean 20 a 9d 18 ab 11 d 19 ab 12 cd 15 bc 11 d <0.0001 10 7 11 9 12 16 20 22 18 19 18 10 16 13 12 15 9 15 12 12 1.2 0.5 0.8 0.5 1.0 ND# ND ND ND ND 10 7 11 9 10 11 23 11 11 0.8 ND 10 19 17 0.232 15 14 0.150 13 14 0.130 0.8 0.8 0.517 ND ND --- 10 9 0.121 <0.5 <0.5 <0.5 <0.5 <0.5 0.8 c 1.4 ab 1.1 bc 1.2 abc 1.0 bc 5.4 a 2.6 c 4.9 a 2.9 c 4.6 a 3.6 a 2.3 b 3.4 a 2.7 ab 3.3 a <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 3.7 a† 2.2 b 3.5 a 2.3 b 3.4 a <0.5 1.6 a 2.6 c 2.1 b <0.5 <0.5 2.2 b 12 5 P 0.164 <0.5 1.3 ab 4.2 ab 3.4 a <0.5 <0.5 <0.5 1.0 bc 2.9 bc 2.3 b <0.5 <0.5 P --0.027 <0.001 0.003 ----*Means separation (P=0.05) shown only when treatment effect was significant. #ND=no data. †Data from MO ‘Elkhart’ and NE trials were used to calculate LS means for DON. 157 3.3 a 2.2 b <0.0001 SESSION 5: VARIETY DEVELOPMENT AND HOST RESISTANCE Chairpersons: Gina Brown-Guedira and Mohamed Mergoum Session 5: Variety Development and Host Resistance AIR SEPARATION AND DIGITAL PHOTO ANALYSIS AS NOVEL METHODS TO MEASURE THE PERCENTAGE OF FUSARIUM DAMAGED KERNELS. Andres Agostinelli, Anthony Clark and D. Van Sanford* * Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY Corresponding Author: PH (859) 257-5020 ext. 80770; Email: dvs@email.uky.edu ABSTRACT One of the greatest problems in breeding for Fusarium head blight (FHB) resistance lies in the difficulty of assessing the disease. At the present time, researchers generally measure disease incidence and severity in the field, deoxynivalenol (DON) content and percentage Fusarium damaged kernels (FDK). FDK is presently measured in two ways: i) visual comparison of samples with reference samples and ii) manual separation of diseased and healthy kernels. Visual comparison of samples is a quick way of assessing FDK but is arguably too subjective. On the other hand, manual separation could be less subjective but is highly time consuming. Furthermore in manual separation, due to the amount of work that it takes, only small samples (e.g. 100 kernels) can be evaluated. This may not be an adequately representative sample size. To improve the efficiency of FDK measurement we should look for a method that: (i) reduces subjectivity, (ii) reduces the amount of work and time required, and (iii) allows increased sample size. To achieve this, two new methods are being proposed: air separation and digital photo analysis. Air separation methods have long been used in the seed industry for seed conditioning purposes and in seed labs to measure the proportion of different components of seed samples. An air separation machine was specifically developed from a Precision Machine head thresher and a Shop-Vac vacuum to separate scabby kernels from healthy ones. Once a sample is loaded into the machine air-driven elevation of the lighter portion of wheat (i.e. scabby seeds) occurs until it reaches the top of the column where is collected in a receptacle. The heavier portion of wheat (i.e. asymptomatic seeds) is suspended midair and does not reach the top of the column. Once the air is turned off, the asymptomatic seeds fall and are collected in the bottom of the column. Finally, both portions of the sample are weighed separately and FDK is calculated. Approximate time per sample is 90 seconds. In the digital photo analysis method, samples are evaluated based in their color composition. Color histograms are generated from the digital photos of the samples by image editing software. Mean blue value appears to have a consistent correlation with the FHB damage of kernels. The air separation and the photo analysis methods emerge as two prospective techniques to measure FDK in an efficient and objective way and, ultimately, appear as promising tools in the difficult endeavor of assessing FHB in scab breeding programs. 161 Session 5: Variety Development and Host Resistance ACKNOWLEDGEMENT AND DISCLAIMER This material is based upon work supported by the U.S. Department of Agriculture, under Agreement No. 590790-6-056. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture. 162 Session 5: Variety Development and Host Resistance USE OF MAS FOR FHB RESISTANCE: IS IT WORKING FOR WHEAT BREEDERS? James A. Anderson1*, Sixin Liu1 and Shiaoman Chao2 Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, MN; and 2USDA-ARS, Biosciences Research Lab, Fargo, ND * Corresponding Author: PH: (612) 625-9763; Email: ander319@umn.edu 1 ABSTRACT Marker-assisted selection (MAS) is most appropriate and efficient when 1) the gene/QTL under selection has a large contribution to the phenotype; and 2) diagnostic markers are available. MAS is especially attractive to wheat breeders needing to improve FHB resistance because of the difficulties posed by phenotypic selection, including multi-genic inheritance and the need to establish inoculated, misted nurseries. Although not a substitute for established phenotypic screening methods, MAS, when used in early generations (F2-F4) can effectively increase the frequency of resistant genotypes. The Fhb1 QTL on chromosome 3BS is the best known and by far the most heavily selected QTL for FHB resistance. The presence of this QTL results in 20% or more reduction of disease symptoms, making it the most potent FHB QTL mapped to date. The SSR markers Xbarc133 and Xgwm493 are in a 5 cM interval that flank Fhb1. Xbarc133 is less than 2 cM from Fhb1 and can be effectively used as a stand-alone marker, but is not diagnostic in all genetic backgrounds. New STS markers based on the sequences of candidate genes should be more efficient than marker Xbarc133. The QTL on chromosome 5AS, Qfhs.ifa-5A, provides only Type I (initial infection) resistance, but is complementary to Fhb1. However, this QTL is in a centromeric region of 5AS, making it less accessible for fine mapping and development of diagnostic markers. We have had success using Xbarc180 to track this QTL in our germplasm. From our surveys of germplasm in the U.S. spring wheat region and some soft red winter cultivars, we discovered numerous cases of highly resistant material not having Fhb1, so the presence of this gene cannot be inferred based on pedigree and high levels of resistance. Robust phenotypic screening is still essential to identify resistant cultivars. Survey results regarding the use of these and other markers for FHB resistance will be presented. ACKNOWLEDGEMENT AND DISCLAIMER This material is based upon work supported by the U.S. Department of Agriculture, under Agreement No. 590790-4-091. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the U.S. Department of Agriculture. 163 Session 5: Variety Development and Host Resistance MARKER-ASSISTED TRANSFER OF 3BS QTL FOR FHB RESISTANCE INTO HARD WINTER WHEAT. G.-H. Bai1*, P. St. Amand1, D.-D. Zhang2, A. Ibrahim3, S. Baenziger4, B. Bockus5 and A. Fritz2 USDA-ARS, Plant Science and Entomology Research Unit and 2Dept. of Agronomy, Kansas State University, Manhattan, KS; 3Plant Science Dept., South Dakota State University, Brookings, SD; 4 Dept. of Agronomy and Horticulture, University of Nebraska, Lincoln, NE; and 5 Dept. of Plant Pathology, Kansas State University, Manhattan, KS * Corresponding Author: PH: (785)-532-1124; Email: guihua.bai@ars.usda.gov 1 ABSTRACT Epidemics of Fusarium head blight (FHB) can significantly reduce wheat grain yield and quality in the central and northern Great Plains of the U.S.A. Use of resistant cultivars is the most effective measure to control the disease. However, most hard winter wheat (HWW) cultivars currently grown in this area are highly susceptible to FHB. In addition, the disease screening procedure is laborious, time consuming, and costly, the progress in breeding for resistant HWW cultivars has been relatively slow with conventional methods. We used highthroughput marker-assisted backcross method to successfully transfer the major quantitative trait locus (QTL) from Sumai 3 and its derivatives into locally adapted HWW with minor FHB-resistance QTL to develop marketable FHB resistant HWW cultivars and useful germplasm lines. Three crosses were made between Sumai 3 derived soft red wheat lines and three locally adapted hard winter wheat cultivars (Harding, Wesley and Trego). Harding and Wesley are red wheat cultivars from South Dakota and Nebraska, respectively, and Trego is a white wheat cultivar from Kansas. Using marker-assisted backcross, about 80 Bc2F2 plants homozygous for the 3BS QTL were selected from each backcross population based on closely linked markers to the 3BS QTL. All selected Bc2F3 lines were evaluated in the greenhouse for Type II resistance in the USDA Genotyping Center in the fall of 2006. The result indicated that most selected lines were either highly resistant or moderately resistant. These materials have also been planted in mist-irrigated fields for further selection of FHB resistance, winter hardiness, hard-textured grains, and other traits. Some lines with good FHB resistance and other desirable traits will be released as new germplasm or cultivars in the hard winter wheat growning region after further yield trials. ACKNOWLEDGEMENTS AND DISCLAIMER This material is based upon work supported by the U.S. Department of Agriculture. This is a cooperative project with the U.S.Wheat & Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture. 164 Session 5: Variety Development and Host Resistance GENETIC LINKAGE MAPPING WITH DART MARKERS TO DETECT SCAB RESISTANCE QTLS IN A ‘SUMAI-3’ DERIVED WHEAT POPULATION. Bhoja. R. Basnet1, Yang Yen1, Shiaoman Chao2 and Karl D. Glover1* 1 Plant Science Department, South Dakota State University, Brookings, SD 57007; and 2USDA-ARS Biosciences Research Laboratory, Fargo, ND 58105 * Corresponding Author: PH: (605) 688-4769; Email: Karl.Glover@sdstate.edu ABSTRACT Much effort has been invested in identification of molecular markers linked to quantitative trait loci (QTL) that confer Fusarium Head Blight (FHB) resistance, though levels significantly higher than those of ‘Sumai-3’ remain elusive. Additional resistance QTLs may exist which typically are overshadowed by partial resistance of susceptible parents used in development of mapping populations. The objectives of this research were: (1) to create a genetic linkage map using Diversity Array Technology (DArT) and Simple Sequence Repeat (SSR) markers and (2) to associate FHB resistance phenotypes with the markers. Our population was created by crossing Sumai-3 with the very susceptible ‘Y1193-6’ (a Tibetan accession with unknown pedigree). An F2:6 recombinant inbred mapping population was developed. A framework map consisting of 65 polymorphic SSR markers has been used to place most of 352 DArT markers. Our report will include associations between markers and FHB resistance QTLs for disease incidence, severity, index, and FDK percentage values. 165 Session 5: Variety Development and Host Resistance USING THE AFFYMETRIX ARRAY TO DISCOVER SINGLE NUCLEOTIDE POLYMORPHISMS IN WHEAT. A.N. Bernardo1, S.-W. Hu1, P.J. Bradbury2, R.L. Bowden3, E.S. Buckler2 and G-H. Bai3* 1 Dept. of Plant Pathology, Kansas State University, Manhattan KS; 2USDA-ARS, Maize Genetic Diversity Laboratory, Ithaca NY; and 3USDA-ARS Plant Science and Entomology Unit, Manhattan, KS * Corresponding Author: PH: (785) 532-1124; Email: guihua.bai@ars.usda.gov ABSTRACT Gene expression arrays have been used to discover single nucleotide polymorphisms (SNPs) in several crop species. This study was designed to explore the possibility of using the Affymetrix Wheat Genome Array for the discovery of SNPs in wheat. Complementary DNAs synthesized from the mRNA isolated from the seedlings of six wheat cultivars of diverse origins (Ning 7840, Clark, Jagger, Encruzilhada, Chinese Spring and Opata 85) were hybridized to Affymetrix Wheat Genome Arrays. Cluster analysis of array data selected a total of 396 genes/probe sets with a signal intensity of at least 200, p-value of <1e-10 and overall R2 ratio >0.8 for SNP confirmation through DNA sequencing. Sequencing results confirmed that 87 probe sets had at least one SNP within the probe sequences. In addition, SNPs were also identified in 21 additional genes, but they were detected outside the probe sequences. A total of 387 SNPs were discovered from the 108 genes. One SNP was selected from each gene to design primers for SNP analysis in a mapping population using SNaPshot kit and only 62 primers were successfully designed for SNaPshot analysis. Forty-two SNP markers were further analyzed in 96 F8-12 recombinant inbred lines from the cross of Ning 7840/Clark and 25 markers were integrated into the existing SSR map of the population. The result shows that Affymetrix arrays can be used to discover SNP markers in wheat. ACKNOWLEDGEMENT AND DISCLAIMER This material is based upon work supported by the U.S. Department of Agriculture. This is a cooperative project with the U.S.Wheat & Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture. 166 Session 5: Variety Development and Host Resistance ENHANCING HOST RESISTANCE TO FUSARIUM HEAD BLIGHT: PYRAMIDING GENES IN SPRING WHEAT. W.A. Berzonsky1*, E.L. Gamotin1, G.D. Leach1 and T. Adhikari2 Department of Plant Sciences, and 2Department of Plant Pathology, North Dakota State University, Fargo, ND 58105 * Corresponding Author: PH: 701-231-8156; Email: bill.berzonsky@ndsu.edu 1 ABSTRACT Spring wheat (Triticum aestivum L.) production in the Northern Great Plains is severely affected by Fusarium head blight (FHB). Damage due to FHB is characterized by bleached spikes with white shriveled kernels, which ultimately reduces grain yield, lowers test weight, and results in an accumulation of deoxynivalenol (DON) in the kernels. Type II resistance has been associated with genes on chromosome 3B of Sumai 3 and chromosome 3A of Triticum turgidum L. var. dicoccoides. The objective of this study was to evaluate and compare the FHB resistance of two lines. One line, designated Line 1 has the Sumai 3 source only and another line, designated Line 2 has both the Sumai 3 and T. dicoccoides sources. These lines were developed by crossing a synthetic hexaploid wheat having the T. dicoccoides resistance to Alsen, a hard red spring wheat with the Sumai 3 source of resistance. The F1 hybrid was backcrossed twice to Alsen and then pollinated with maize to produce doubled-haploid lines. These Alsen backcross-derived doubled-haploid (BC2F1DH) lines were initially screened with the SSR markers Xgwm533 and Xgwm2 for detection of the 3B QTL of Sumai 3 and the 3A QTL of T. dicoccoides, respectively. Additional STS markers were later used to verify the presence of these QTL. Phenotypic evaluation of these lines was done in three greenhouse seasons. A 10 μl inoculum with 50,000 spores ml-1 was injected into a single floret in the middle of the spike at anthesis. FHB resistance was assessed by measuring disease severity at 7, 14, and 21 d after inoculation (dai), percent Fusarium-damaged kernels (FDK), and DON content of the grain. A combined ANOVA indicated a significant genotype by dai interaction for disease severity. When means among genotypes were compared at the same dai, no significant differences were observed at 7 dai. However, a significant increase in disease severity was noted at 14 and 21 dai. The disease severity of Line 1 and Line 2 was not significantly different from Alsen, but both lines exhibited significantly lower disease severity than the synthetic parent at 14 dai. At 21 dai, Line 1 exhibited significantly higher disease severity than either Line 2 or Alsen. When comparisons were made within the same genotype across different dai, Line 1 exhibited a significant increase in disease severity, progressing from 5% severity at 7 dai to 17% severity at 21 dai. However, Line 2 exhibited no significant change, progressing from 5% severity at 7 dai to 8% severity at 21 dai. Alsen and the synthetic wheat exhibited a significant change in disease severity, progressing from 5% (7 dai) to 13% (14 dai) and 5% (7 dai) to 33% (14 dai) severity, respectively. The percent FDK of Line 1 and Line 2 was not significantly different from Alsen, but the FDK of both lines was significantly lower than the synthetic wheat. There were no significant differences in DON content for either Line 1, Line 2, or Alsen across greenhouse seasons, but the synthetic wheat had a significantly higher DON content in one of the three greenhouse seasons. Line 1 and Line 2 exhibited Type II resistance in all evaluations, and although differences between them were not always significant, in some instances, the combined effect of the 3A and 3B QTL in Line 2 may have contributed to a lower expression of FHB severity, percent FDK and DON content. 167 Session 5: Variety Development and Host Resistance QTL ASSOCIATED WITH REDUCED KERNEL DAMAGE AND RESISTANCE TO FUSARIUM HEAD BLIGHT IN WHEAT. C.M. Bonin*, F.L. Kolb and E.A. Brucker University of Illinois, Department of Crop Sciences, 1102 S. Goodwin Ave. Urbana, IL 61801 * Corresponding Author: PH (217) 244-2568; Email: cbonin2@uiuc.edu ABSTRACT Resistance to Fusarium head blight (FHB) is controlled by a number of genes, suggesting that the identification and accumulation of multiple resistance genes in a variety would result in increased resistance. Our objective in this study was to identify QTL associated with resistance to FHB, especially QTL associated with a decrease in percent Fusarium damaged kernels (FDK). A population of 269 recombinant inbred lines (RILs) was developed from a cross between Patton and IL94-1653, and evaluated for FHB resistance in a mistirrigated FHB nursery in Urbana, IL in 2006 and 2007. The parent lines, Patton and IL94-1653, are moderately susceptible to FHB, and IL94-1653 appears to exhibit resistance to kernel damage. The RIL population was genotyped using SSR markers and QTL analysis was performed using MapQTL 4.0. Preliminary results identified a QTL on chromosome 4B associated with reduced percent FDK in both years. The markers associated with this region are gwm513, gwm495, and wmc47. Chromosome 4B was also associated with DON content in 2007 but not in 2006. In 2006, a QTL on 2B was associated with reduced percent FDK, severity, FHB index, and ISK index. This QTL was not significant in 2007. Further genotyping and QTL analysis is in progress to identify additional resistance QTL in this population. 168 Session 5: Variety Development and Host Resistance RESISTANCE TO KERNEL DAMAGE CAUSED BY FUSARIUM HEAD BLIGHT IN AN RIL POPULATION. C.M. Bonin* and F.L. Kolb University of Illinois, Department of Crop Sciences, 1102 S. Goodwin Ave., Urbana, IL 61801 * Corresponding Author: PH: (217) 244-2568; Email: cbonin2@uiuc.edu ABSTRACT Fusarium head blight (FHB) infection of wheat results in Fusarium damaged kernels (FDK) that contain the mycotoxin deoxynivalenol (DON), reducing the value of the grain. By developing lines with resistance to kernel damage resulting in a low percent FDK, breeders may be able to reduce DON content in grain. A population of 269 recombinant inbred lines (RILs) was developed from a cross between two soft red winter wheat lines, IL94-1653 and Patton, with IL94-1653 thought to exhibit resistance to kernel damage. Both parents are moderately susceptible to FHB. The RIL population was evaluated for FHB resistance in the greenhouse in 2005 and 2006, and in field in 2006 and 2007. A wide range of FHB symptoms and DON content were observed in the RIL population. The RIL population also exhibited transgressive segregation for all measures of FHB resistance, including disease severity, percent FDK, and DON content. In the field, the correlation between percent FDK and DON content was positive, and varied by year from moderately low to medium (r = 0.38 in 2006; r = 0.53 in 2007). The ISK index, a combination of severity, incidence, and percent FDK measurements, gave a better correlation with DON content for both years (r = 0.47 in 2006 and r = 0.64 in 2007), with all correlation values significant at p<0.0001. However, identifying RILs with consistent resistance to kernel damage was difficult and seems to be influenced by environmental variation as well as by resistance to initial infection and resistance to spread of infection. 169 Session 5: Variety Development and Host Resistance BARLEY CHROMOSOME 2(2H) BIN 10 FUSARIUM HEAD BLIGHT RESISTANCE QTL: MAPPING AND DEVELOPMENT OF ISOLINES. Christine N. Boyd1, Richard Horsley3 and Andris Kleinhofs1,2* Dept. of Crop and Soil Sciences, 2School of Molecular Biosciences, Washington State University, Pullman, WA 99164-4660, USA; and 3Dept. of Plant Sciences, North Dakota State University, Fargo, ND 58105-5051, USA * Corresponding Author: PH: 509-335-4389; E-mail: andyk@wsu.edu 1 has been difficult to saturate, as it has been for others who have published maps for this region (Marcel et Development of commercially acceptable cultivars with al., 2006; Stein et al., 2007). Nevertheless, there are Fusarium Head Blight (FHB) resistance and good nine loci with 20 markers and 125 BAC clones identiagronomic qualities is the goal of the barley SCAB fied in our laboratory (Fig. 1). There are a total of 295 project. One of the best FHB resistance quantitative BAC clones identified for this region if one includes trait loci (QTL) resides in the chromosome 2(2H) bin those in the Tim Close barley physical map database. 10 region. Our contributions are focused on genetic Some of those BAC clones likely belong to different and physical mapping of this region with the long-term loci, but nevertheless the physical map is fairly satugoal of cloning the genes responsible for FHB resis- rated. Based on just the BAC clones identified in our tance. To facilitate this, we have isolated recombi- laboratory (because we are more confident that these nant lines with introgressed small chromosome 2(2H) really belong in this region), we have identified nine bin 10 genomic segments in a susceptible genomic BAC clone contigs. We can reasonably exclude the background. We have also developed 6-rowed re- two BAC contigs associated with the MWG503 locombinants in the resistant CI4196 genomic back- cus because that region is far from the FHB resistance ground. To further facilitate development of agro- QTL (Horsley et al., 2006). The most likely FHB nomically acceptable barley cultivars with FHB resis- resistance peak resides around marker BF265762A tance, we have undertaken to modify the resistant line just below the Vrs1 locus (Fig. 1). This region is covCI4196 by mutagenesis. Mutants with desirable traits ered by a completed BAC contig that also includes such as semi-dwarf, early and 6-rowed are easily se- the next downstream marker BI955972. The area lected. These provide improved FHB resistant par- between Vrs1 and BF265762A has been fine-mapped ent material that can be rapidly incorporated in breed- by Pourkheirandish et al. (2007), providing further probes for picking BAC clones to build a complete ing programs. contig. This region should be sequenced as soon as possible. RESULTS AND DISCUSSION INTRODUCTION Genetic and physical mapping Isoline development We have developed an extensive genetic map for the bins 8-10 region (markers ABC306-MWG882). This map includes 43 loci, 111 markers, and 423 bacterial artificial chromosome (BAC) clones identified in our laboratory (the Tim Close barley physical map database increases this number to over 800). The details of this map will be published elsewhere. Here we focus on the bin 10 region from MWG699 to MWG503. This region has relatively few markers and We have been developing small introgressed CI4196 FHB resistant line chromosome 2(2H) bin 10 region fragments into a susceptible cv. Morex background in order to more accurately define the region responsible for FHB resistance (Fig. 2). We now have identified three homozygous recombinant lines from the A171 x Morex cross that integrate either the region directly below Vrs1 (07-83-11) or a slightly larger region (0776 and 07-84). An additional five homozygous lines 170 Session 5: Variety Development and Host Resistance were developed from the cross A80 x Morex (0785-1; 07-87; 07-90; 07-91; 07-97). These lines contain the BG369629 to BG416977 CI4196 region, which we do not think is important in FHB resistance. They also contain various small fragments introgressed from the Vrs1 to BG343659 region that appears to be critical to FHB resistance. All of these lines are being phenotyped in China winter ’07-’08. A number of less advanced recombinant lines have already been tested for FHB resistance in China and North Dakota. Among them, line 06-310-18 stands out as being reproducibly FHB resistant and about the same height as Foster. This line is six-rowed, but the spike is not very robust, probably due to the presence of the two-rowed Int-c allele. This allele will be replaced by the mutant int-c allele that we have isolated from CI4196 (see below). Mutant selection and analysis All of the agronomically important CI4196 mutants that were identified in the field 2006 and 2007 were confirmed as CI4196 by PCR (Boyd et al., 2006). The mutants isolated in 2006 were phenotyped in China winter ’06-’07 (Table 1). The male sterile mutants show good FHB resistance. These should be useful to facilitate backcrossing and breeding efforts. Early mutants (desirable for breeding) were identified and confirmed, but only two (G07-83 and G07-105) showed FHB resistance levels comparable to CI4196. The higher FHB susceptibility may be due to longer exposure of the early-emerging spike to the disease (Nduulu, 2007), or it may be the product of other unintended mutations. Of the two semi-dwarf mutants tested, only G07-66 is about the same height as Foster with FHB resistance comparable to CI4196. This line may be suitable as an improved CI4196 parent for FHB resistance breeding. Another line of potential interest is lax spike (G07-52). This line maintains CI4196 FHB resistance and is not excessively tall. The lax spike trait may be useful in reducing the opportunities for FHB infection. The intermedium line isolated in 2006 is not very promising and may be just a distorted head mutant. Two much more promising apparently intermedium type mutants were isolated in 2007 (see below). New mutants identified in the field 2007 from a gamma irradiated population and sent for FHB phenotyping in China winter ’07-’08 include intermedium, early, premature ripening, semi-dwarf, dwarf, erectoides, anthocyanin-less, and glossy head. The two intermedium types are particularly interesting. They have reasonably good 6-rowed heads and are probably of the int-c type (based on phenotype). The Vrs1 gene was sequenced from these mutants and appears to be identical to the CI4196 Vrs1 gene. Thus, unless the mutation is in a regulatory region, they are not Vrs1 mutants. However, they will be very useful for crossing with the 06-310-18 line described above to recover a true 6-rowed head in a mostly CI4196 line. We are also very much interested in the FHB response of the new semi-dwarf and early mutants. Since these come from a gamma irradiated population, we expect that the genome may not be as highly rearranged as in the mutants selected from fast neutron irradiated material in 2006. ACKNOWLEDGEMENTS This work was supported by the U.S. Department of Agriculture, under Agreement No. 59-0790-4-110. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. DISCLAIMER Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the U.S. Department of Agriculture. REFERENCES Boyd, C., Maier, C., Sushailo, S., Horsley, R., Kleinhofs, A. 2006. Genetic and Physical Mapping of the Barley Chromosome 2(2H) vrs1 Region Fusarium Head Blight Resistance QTLs. 2006 NFHB Forum Proceedings p. 87-90. Horsley, R.D., Schmierer, D., Maier, C., Kudrna, D., Urrea, C.A., Steffenson, B.J., Schwartz, P.B., Franckowiak, J.D., Green, M.J., Zhang, B., Kleinhofs, A. 2006. Identification of QTL associated with Fusarium Head Blight resistance in barley accession CIho 4196. Crop Science 46: 145-156. 171 Session 5: Variety Development and Host Resistance Marcel, T.C., Varshney, R.K., Barbieri, M., Jafary, H., de Kock, M.J.D., Graner, A., Niks, R.E. 2006. A high-density consensus map of barley to compare the distribution of QTLs for partial resistance to Puccinia hordei and of defence gene homologues. Theor. Appl. Genet. 114:487-500. Pourkheirandish, M., Wicker, T., Stein, N., Fujimura, T., Komatsuda, T. 2007. Analysis of the barley chromosome 2 region containing the six-rowed spike gene vrs1 reveals a breakdown of rice-barley micro collinearity by a transposition. Theor. Appl. Genet. 114:1357-1365. Nduulu, L.M., Mesfin, A., Muehlbauer, G.J., Smith, K.P. 2007. Analysis of the chromosome 2(2H) region of barley associated with the correlated traits Fusarium head blight resistance and heading date. Theor. Appl. Genet. 115:561-570. Stein, N., Prasad, M., Scholz, U., Thiel, T., Zhang, H., Wolf, M., Kota, R., Varshney, R.K., Perovic, D., Grosse, I., Graner, A. 2007. A 1,000-loci transcript map of the barley genome: new anchoring points for integrative grass genomics. Theor. Appl. Genet. 114:823-839. 172 Session 5: Variety Development and Host Resistance HAPLOTYPING OF KNOWN FHB RESISTANCE QTL IN PACIFIC NORTHWEST WHEAT GENOTYPES. Jianli Chen1*, Juliet Windes1, Robert Zemetra1 and Carl Griffey2 University of Idaho, Aberdeen, ID 83210; and 2Virginia Polytechnic Institute and State University, Blacksburg, VA 24061 * Corresponding Author: PH: 540-231-9540; Email: jchen@uidaho.edu 1 ABSTRACT This study was conducted to discern the genetic variation of Fusarium head blight resistance in PNW wheat genotypes via haplotyping of fifteen SSR and STS markers associated with six QTL for FHB resistance previously identified in known resistance sources. A total of 15 markers on six chromosome regions (2D, 3A, 3BS, 5AS, and 6B) are being used in 94 PNW wheat genotypes which have no Sumai 3 related backgrounds. Based on the preliminary results derived from haplotyping of six markers, we identified some of the known major FHB QTL present in the adapted PNW cultivars/lines. The frequency of the known QTL on 3BS and 6BS were higher than expected. The known target alleles for marker STS3B-256 on 3BS was present in 30 lines and the one for WMC 152 on 6BS was present in 45 lines out of the 94 genotypes studied. Evaluation of field FHB resistance of these genotypes is needed and they will be done in the 2007-08 growing season. This study has the potential to identify novel and adapted sources of resistance through allele size comparisons of known SSR loci associated with QTL identified in known resistance sources. Identified cultivars/lines having good field FHB resistance and/or known FHB resistance QTL can then be grown in PNW region and used as adapted resistance sources in the PNW and Great Plains breeding programs. 173 Session 5: Variety Development and Host Resistance VALIDATION OF SIX QTLS ASSOCIATED WITH FUSARIUM HEAD BLIGHT RESISTANCE IN ADAPTED SOFT RED WINTER WHEAT. Jianli Chen1*, Carl Griffey2, Shiaoman Chao3 and Gina Brown-Guedira4 1 University of Idaho, Aberdeen, ID 83210; 2Virginia Polytechnic Institute and State University, Blacksburg, VA 24061;3USDA-ARS, Biosciences Research Lab, Fargo, ND 58105; and 4USDA-ARS, Plant Science Research Unit, Raleigh, NC 27695 * Corresponding Author: PH: 540-231-9540; Email: jchen@uidaho.edu ABSTRACT This study was conducted to validate molecular markers linked to six FHB resistance QTL previously identified in different bi-parental populations using elite breeding lines incorporated FHB resistance to initial infection, spread, and DON accumulation in different genetic backgrounds. A total of 129 SSRs were characterized in the 145 breeding lines. Forty-four unrelated SSRs (4 SSRs per chromosome) were used in background selection and the remaining 85 SSRs were used in validation of target QTL. The 145 wheat lines were also evaluated in yield performance trials at two locations, Blacksburg and Warsaw, VA, and for type I, type II, and DON resistance in a scab nursery at Blacksburg, VA in 2005 and 2006. Molecular markers linked to scab resistance genes located on wheat chromosomes 2BS, 2DS, 3AS, 3BS, 5AS, and 6BS were confirmed and allelic effects of associated marker loci were analyzed. Adapted resistant lines with novel alleles different from known exotic sources were characterized. Renwood 3260 and its derived lines have good overall resistance and high yield potential. These lines have unique resistance with alleles differing from those of known resistance sources W14 and Sumai 3 at marker loci Gwm429, Gwm120, Gwm261, Barc133, and Gwm186 in the chromosome 2BS, 2DS, 3BS, and 5AS QTL regions. Ernie and its derived lines also have good overall resistance but didn’t produce promising grain yields in Virginia. These lines have unique resistance comprised of the same resistant alleles as Renwood 3260 at loci Gwm429, Gwm120, and Gwm261 in 2BS and 2DS QTL regions. Both the Ernie and Renwood 3260 derivatives contain the same resistant alleles as donor parent W14 at loci Wmc264, Barc133, and Barc117 in 3AS, 3BS, and 5AS QTL regions. In addition, these lines have unique resistant alleles in their background at Gwm493 and Wmc152 in 3BS and 6BS QTL regions.This is the first study validating six FHB QTL in elite breeding lines. QTL-markers validated in the current study have been used widely in parental selection, gene pyramiding, and in postulating and selection of FHB resistance of progeny derived from such newly developed FHB resistant lines. This is also the first study evaluating the effects of allelic differences and genetic backgrounds on FHB resistance. Newly developed FHB resistant lines with unique QTL/allele combinations have been used as parental lines in most of eastern wheat breeding program. Some of these lines will be released as varieties and/or adapted germplasm. The newly developed FHB resistant lines and unique QTL/marker allele profiles identified in this study will set the stage for using MAS not only for FHB resistance but also in combining FHB resistance with other important agronomic traits. 174 Session 5: Variety Development and Host Resistance DEVELOPMENT OF SCAB RESISTANT SOFT RED WINTER WHEAT GERMPLASM USING MARKER-ASSISTED SELECTION. Jose M. Costa1*, Leila Al-Tukhaim1, Raquel Brown1, Neely Gal-Edd1, Alice Ku1, Erin Wenger1, David Van Sanford2 and Gina Brown-Guedira3 1 University of Maryland, PSLA Dept. 2102 Plant Sciences Bldg., College Park, MD 20742-4452; 2 University of Kentucky, Dept. of Plant Sciences, Lexington, KY 40546; and 3 USDA-ARS, Plant Science Research Unit, Raleigh, NC 27695 * Corresponding Author: PH: 301-314-9308; Email: costaj@umd.edu ABSTRACT Scab of wheat, caused by Fusarium graminearum, is a disease that periodically strikes the US mid-Atlantic region. Breeding for resistance is an effective measure of disease control. The objective of this study was to develop scab resistant soft red winter wheat germplasm adapted to the US mid-Atlantic region using markerassisted selection. McCormick, a genotype adapted to the Mid-Atlantic region, was used in a backcross program with the Chinese variety Ning7840. An accelerated backcross scheme was developed to incorporate scab resistance QTLs found on chromosomes 3BS, 5A and 2DL in the Chinese variety Ning7840. Two rounds of backcrossing were completed using McCormick as the female parent. Progenies from the first round of backcrossing were selected for the presence of the Ning7840 scab resistance alleles at 3BS, 5A, and 2DL, and then for a high background of McCormick alleles. Two backcross progenies had over 60% McCormick background. Using these two selected BC1F1s, 400 BC2F1s were produced in a second round of backcrossing. Additionally, the two selected BC1F1s were crossed with a wheat line with stripe rust resistance (GA96229-3A41). 800 BC2F1 seeds were screened with molecular markers to identify those with Ning7840 alleles (on 3BS, 5A and 2DL) and most McCormick background. A single BC2F2s population derived from a selected BC2F1 plant was screened with 3BS, 5AS, and 2DL markers to select those homozygous for the resistant alleles. Additionally, we derived near-isogenic lines from this F2 population to identify the effect of each QTL on scab resistance, agronomic and quality traits. We plan to test some of the BC2F3s for scab resistance in the spring of 2008. We anticipate having a small amount of seed of selected BC2F4s, containing the Ning7840 alleles in the McCormick background, available for distribution to other soft red winter wheat breeders for crossing in the fall of 2008. 175 Session 5: Variety Development and Host Resistance APPLYING SINGLE KERNEL SORTING TECHNOLOGY TO DEVELOPING SCAB RESISTANT LINES. F.E. Dowell* and E.B. Maghirang USDA-ARS, Grain Marketing and Production Research Center, Engineering Research Unit, Manhattan, KS * Corresponding Author: PH: (785) 776-2753; Email: floyd.dowell@ars.usda.gov ABSTRACT We are using automated single-kernel near-infrared (SKNIR) spectroscopy instrumentation to sort Fusarium head blight (FHB) infected kernels from healthy kernels, and to sort segregating populations by hardness to enhance the development of scab resistant hard and soft wheat varieties. We sorted 3 replicates of 192 samples into a damaged fraction yielding an average of 61.3 ppm DON, and a healthy fraction yielding an average of 0.73 ppm DON. This collaborative work with Dr. Gene Milus and Peter Horevaj investigated the resistance of soft red winter wheat lines to DON and NIV chemotypes of Fusarium graminearum. In another study, we also sorted the soft portion of a hard x soft cross into FHB infected and healthy fractions, and likewise sorted the hard portion into FHB infected and healthy fractions. The hard x soft crosses were separated into the hard and soft portions in 2006 where the respective portions were inoculated and planted. The 2007 scabby and healthy fractions of the hard and soft lines will be planted this fall to determine if our sorting will result in populations with FHB resistance. This work is in cooperation with Dr. Anne McKendry and Dr. Stephen Baenziger. Another project that was done in cooperation with Dr. Stephen Wegulo, Julie Breathnach and Dr. Stephen Baenziger used the automated SKNIR system to rapidly assess lines for FHB resistance by running multiple samples and obtaining a count of infected and healthy kernels. We have done this for about 300 lines and the information is being used to select resistant lines for further developement. 176 Session 5: Variety Development and Host Resistance TUNISIAN DURUM WHEAT AS NEW SOURCES OF RESISTANCE TO FUSARIUM HEAD BLIGHT. Farhad Ghavami, Melissa Huhn, Elias Elias and Shahryar Kianian* Department of Plant Sciences, North Dakota State University, Fargo, ND 58105 * Corresponding Author: PH: 701-231-7574; Email: s.kianian@ndsu.edu ABSTRACT There are limited sources of resistance to FHB which mostly restricted to Chinese hexaploid genotypes like Sumai3 and Wangshuibai. Therefore it is necessary to use other sources of resistance to expand the number of genes that may be used in the gene pyramiding programs. The North Dakota Durum Wheat breeding program has identified four tetraploid wheat sources of resistance from Tunisia, which were selected among a large number of lines evaluated over 55 repeated FHB trials. Since their identification, these lines have been extensively used in the breeding program to derive resistant breeding lines. We used a collection of backcross derived advanced resistant lines, susceptible sibs, and parental lines to identify markers that are associated with these novel sources of resistance. In this study we used 184 BC1F6 and 189 BC1F7 lines derived from crossing of Tun7, Tun18, Tun34, Tun36 with durum cultivars ‘Ben’, ‘Maier’, ‘Lebsock’ and ‘Mountrail’ for association studies. As Tunisian lines pedigree shows no relation to Chinese genotypes, they probably carry different genes or alleles for resistance to FHB. We checked all the parents and RILs in the greenhouse in two seasons for type II resistance to FHB by single floret injection inoculation method. The data showed that the Tunisian lines have different amount of resistance varying from 18% to 10% infection rate through the spikes. The data also showed Maier may have some minor resistance genes because it showed a moderate resistance in our greenhouse study and the crosses between Maier and different Tunisians had more transgressive resistant progenies compared with the other crosses. To accelerate the identification of markers associated to FHB resistance, we initially screened the parents. The amounts of recombination in wheat chromosome arms are low so we picked 10-14 SSR markers per chromosome which were roughly 10cm apart and cover the whole genome. Among the 179 SSR markers that we applied on the parents about 45% showed polymorphism for at least two parents and about 8 % showed polymorphism between the whole set of Tunisian lines and susceptible cultivars. The most polymorphism was found on chromosomes 5A and 3B and the least on chromosome 6A. About 22 SSR markers that had been mentioned in different articles to be linked to FHB resistant were also applied to the parents. Among them barc117 and gwm129 from chromosome 5A showed the same pattern in Tunisian lines but not the susceptible lines. We also did the Diversity array (DArT) marker analysis to have a more complete coverage of the whole genome and to find closer markers to the genes of interest. DArT analysis used 2300 markers which showed 25% polymorphism between the parents. About 8% of the polymorphic markers were present in all the Tunisian lines but not the susceptible cultivars. The cluster analysis of the polymorphic markers revealed three distinct groups. Tun7 was in a separate group far from the other two and all the other Tunisian lines fell in a separate group from susceptible cultivars. Our data shows Tun7 and Tun18 are potential candidates for new sources of resistance which will be discussed in detail in our presentation. 177 Session 5: Variety Development and Host Resistance ACKNOWLEDGEMENTS AND DISCLAIMER We greatly appreciate the technical assistance of Justin Hegstad, Stan Stancyk and Sarah R. Underdahl from NDSU. This material is based upon work supported by the U.S. Department of Agriculture, under Agreement No59-0790-4-109 to Shahryar F. Kianian. This is a cooperative project with the U.S. Wheat and Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the U.S. Department of Agriculture. 178 Session 5: Variety Development and Host Resistance RESPONDING TO FUSARIUM HEAD BLIGHT FOR THE NORTHERN ROCKY MOUNTAINS AND WESTERN GREAT PLAINS. W. Grey*, A. Dyer and L. Talbert Department of Plant Sciences and Plant Pathology, Montana State University, Bozeman, MT 59717-3150 * Corresponding Author: PH: (406) 994-5687; Email: wgrey@montana.edu OBJECTIVES To improve the efficiency of individual breeding programs’ development of FHB resistance and minimize the impacts of associated diseases in irrigated systems. INTRODUCTION Fusarium head blight is a perennial disease problem for irrigated acreage in northern Rocky Mountains and western Great Plains. Montana, alone, has 150,000 acres of irrigated spring wheat with an annual production of 9.8 million bushels amounting to $50 million annually. All of this acreage is potentially impacted by this disease. Neighboring states in the northern intermountain region (Idaho, Washington, Oregon and Wyoming) have an additional 390,000 irrigated acres, all of which may be affected by FHB. FHB tolerant varieties have been utilized by producers in the past years but there are significant deficiencies which discouraged their continued use. In particular, varieties had problems with lodging, ergot and black chaff. In addition, current FHB tolerant varieties lack sawfly resistance, which is important to the western Great Plains region. The purpose of this proposal is to develop new lines specifically adapted for these areas and to determine the suitability advance FHB tolerant materials from other states for high-yielding, irrigated production. MATERIALS AND METHODS FHB Nursery - Wheat scab conditions were optimized with sprinkler center pivot irrigation, continuous cropping of wheat, and wheat residue serving as inoculum of F. pseudograminearum and F. graminearum. Residue management involved a fall cultivation with irrigation to germinate volunteer seed with residue remaining on the soil surface. Spring cultivation was a minimum tillage following by the planting with a no-till drill. Nursery was planted with a no-till small plot drill and seed was treated with RaxilMD. Individual plots were 3.5 x 20 ft, 4 replications, planted May 9, 2006 and 3.0 x 12 ft, 3 replications planted May 8, 2007. Pre-plant, top dress and fertigation at soft dough was applied for potential yield of 90-100 bu. MCPA and Discover broadleaf herbicide and Quilt fungicide for leafspots during seedling stage. Best management practice to minimize FHB consisted of a Folicur fungicide (14 oz/acre) applied at anthesis (Feekes 10.5) and irrigation discontinued for nine days, July 7 to July 16. Normal sprinkler irrigation was on a daily cycle, 0.3 in/day, with 4.5 in up to flowering and then resumed on alternate days with a 4.5 in through grain development. Harvest completed with a small plot combine and chaff air volume was minimized to avoid loss of light, scabby grain. Spring wheat entries, 15 in total, were selected based on commercially available variety or advanced lines. Hank was entered twice as a susceptible check and for evalution of the uniformity of FHB distribution in the nursery. Disease Evaluation - Scabs heads were visually determined by the pre-mature head blight and dark tan of the peduncle at ripening and hard dough stage. DON mycotoxin in the grain was processed according to standard protocols and evaluated by the NDSU Veterinary Diagnostic Lab. Germination blotter test for viability of seed and ergot quantity on percentage weight was conducted by the MSU Seed Analysis Lab. Lodging of the variety was determined as a visual assessment for the plot area at harvest. 179 Session 5: Variety Development and Host Resistance RESULTS AND CONCLUSIONS FHB Effects & Agronomic performance - The overall grain deoxynivalenol (DON) concentration was 2.2 ppm for 2006 and 2007 MSU FHB nursery. “Hank” is highly susceptible to FHB with a grain DON of 8.8 ppm, yield loss of 37%, test weight of 54 lb/bu and a head scab incidence of 58%. Other varieties without Sumai3 gene had 0.6 to 2.0 ppm grain DON, including; Vida, Howard, Granite, Explorer (HWW), Choteau and Expresso. Tolerant varieties had less than 0.5 ppm grain DON, including; Kuntz, Volt, Freyr, Granite, Knudson, Alsen, Glenn, Kelby, and an experimental line MT0550 (Choteau/ND709-9). Tolerant varieties had a 7% incidence of symptomatic scab heads as compared to 33% among varieties lacking the Sumai3 gene. Overall, varieties with the Sumai3 gene yielded 67 bu/ac or 18% higher than varieties lacking tolerance to FHB. Grain test weights were 62.3 lb/bu in the tolerant varieties and 57.8 lb/bu in the susceptible varieties. Seed germination by a blotter test was below an acceptable “92% for foundation class” on those varieties lacking tolerance to FHB. Several of the short statured varieties are adapted to high production under irrigation, but susceptible to FHB, whereas the FHB tolerant varieties will lodge under these conditions. Ergot and bacterial black chaff susceptibility of varieties are a concern for irrigated wheat production in these regions. Breeding Lines -We have used molecular markers to backcross the Sumai3 QTL into Choteau, a solidstem variety with resistance to the wheat stem sawfly, and MT0249, a variety with long green leaf duration and short stature. Molecular marker GWM 533 was utilized for selection in Choteau lines, such as MT0550, and this line is expressing tolerance to FHB. A similar marker selection line, MT0551, was removed from consideration following poor FHB tolerance in the 2006 nursery BARC 133 was used when MT0249 was a recurrent parent and there are several shorter statured lines that are under evaluation for FHB tolerance. We will have sufficient seed of for single screening and seed increase rows in the MSU FHB nursery in 2008. An FHB resistant line similar to Choteau will find utility in the western Great Plains and the northern Rocky Mountains. ACKNOWLEDGEMENT This material is based upon work supported by the U.S. Department of Agriculture, under Agreement No. 59-0790-6-059. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. REFERENCES Anderson, J. A., R. W. Stack, S. Liu, B. L. Waldron, A. D. Fjeld, et al. 2001. DNA markers for Fusarium head blight resistance in QTL in two wheat populations. Theor. Appl. Genet. 102:1164-1168. Lanning, S. K., G. R. Carlson, D. Nash, D. M. Wichman, K. D. Kephart, R. N. Stougaard, G. D. Kushnak, J. L. Eckhoff, W. E. Grey and L. E. Talbert. 2004. Registration of Choteau wheat. Crop Sci. 44:2264-2265. Menzies, J.G., 2004. The reactions of Canadian spring wheat genotypes to inoculation with Claviceps purpurea, the causal agent of ergot. , Canadian Journal. Plt. Sci.: 84:625-629. Waldron, B. L., B. Morena-Sevilla, J. A. Anderson, R. W. Stack, and R. C. Frohberg. 1999. RFLP mapping of QTL for Fusarium head blight in wheat. Crop Sci. 39:805-811. 180 Session 5: Variety Development and Host Resistance Table 1. Effects of Fusarium head blight on performance of spring wheat varieties under sprinkler irrigation in Montana during 2006 and 2007. VARIETY FHB reaction Grain Yield 2 yr aver Bu/ac EXPLORER 53.2 HANK 56.7 HANK 59.3 HOWARD 65.6 VIDA 69.8 GLENN Sumai3 70.7 ALSEN Sumai3 70.7 GRANITE Tolerant 75.0 EXPRESSO 76.7 MT0550 Sumai3 77.8 CHOTEAU* 77.9 KELBY Sumai3 80.8 KNUDSON Sumai3 83.8 KUNTZ Sumai3 85.9 VOLT Tolerant 87.6 FREYR Sumai3 87.6 Lsd P<0.05 C.V.% *Choteau 2007 Grain DON 2 yr aver ppm 2.2 8.8 8.4 1.1 1.4 0.2 0.2 0.4 3.6 0.3 2.8 0.5 0.4 0.4 0.5 0.3 Test Wt 2 yr aver lb/bu 55.4 53.5 54.2 60.2 57.7 63.9 62.2 62.3 59.9 61.9 60.8 61.8 60.3 61.3 62.4 61.5 181 Scab Heads 2 yr aver % 13.6 53.9 48.9 21.6 18.1 3.1 5.9 16.2 25.8 4.8 21.5 16.6 5.6 11.9 5.6 6.3 Germ Blot Test % 83.6 57.3 57.1 86.6 83.4 96.6 95.6 93.9 81.1 94.4 na 92.6 92.6 94.9 95.6 91.3 3.5 2.8 Lodge Ergot 1 yr 2006 % 37 0 0 64 50 31 10 5 5 49 na 24 63 16 8 78 22 49 1 yr 2006 % wt 0.00 0.00 0.00 0.04 0.00 0.01 0.14 0.07 0.00 0.02 na 0.02 0.02 0.01 0.02 0.03 Ns Session 5: Variety Development and Host Resistance RESISTANT GERMPLASM FROM SUSCEPTIBLE PARENTS: AN EVOLUTIONARY APPROACH. Steve Haber* and Jeannie Gilbert Agriculture and Agri-Food Canada, Cereal Research Centre, 195 Dafoe Rd., Winnipeg MB R3T 2M9, Canada * Corresponding Author: PH: (204) 983-1467; Email: shaber@agr.gc.ca ABSTRACT The Chinese wheat line Sumai 3 is widely considered the standard for resistance to Fusarium Head Blight (FHB). Much research effort aims to introgress its FHB resistance into germplasm with better quality, agronomic traits, geographic adaptation and resistance to other diseases. As several, or perhaps many, genes are involved in expressing the trait, it has proved difficult to combine high FHB resistance with desirable agronomic and quality traits while advancing through cycles of crosses with elite, FHB-susceptible parents. Instead of aiming to transfer the complement of genes that condition FHB resistance in Sumai 3, we pursued an alternative approach of emulating in our desired germplasm the evolution of resistance that had occurred in Sumai 3 itself as it derived from moderately susceptible parents Evolution within a population can be speeded by enhancing variation, selection and generation of fertile progeny among its individual members, and is more readily followed using small populations that can be carefully examined and advanced through as many as 4 generations per year (3 indoors and 1 field trial). After initial crosses with lines resistant to wheat streak mosaic virus (WSMV), we identified and selected in each generation of back-crossing regimes those individuals that appeared to combine vigorous growth under pressure from virus infection with the best resemblance to the recurrent cultivar parent. After the BC2 generation, lines that consistently performed well under pressure from WSMV infection were spray-inoculated with macroconidial suspensions of Fusarium graminearum and the most promising individuals selected for additional backcrossing and selection. We had chosen a Canadian amber durum spring wheat cultivar, Strongfield, to apply this novel approach of ‘speeded evolution’, as the germplasm in this wheat class is highly susceptible to FHB and there are no wellcharacterized tetraploid wheat sources of resistance that might readily be introgressed. To date, we have generated lines equivalent to BC3F4-6, and with repeated backcrosses and selection, the lines have increasingly come to resemble Strongfield in all desirable agronomic traits. In 2007, BC3F4 and BC3F5 lines were evaluated in FHB nurseries. Families of lines were observed with excellent FHB reactions (similar to the most resistant hexaploids) that were consistent with results seen in predecessor generations selected in indoor tests. The ‘speeded evolution’ approach allows only small quantities of seed to advance from each generation of highly selected germplasm, precluding testing for quality until disease reactions and agronomic traits are consistently good. Initial assessments, however, of gluten index of selected lines of BC3F5 and BC3F6 seed harvested from 2007 FHB nursery plots indicate several of these FHB-resistant lines have acceptable quality. 182 Session 5: Variety Development and Host Resistance RESISTANCE OF WINTER WHEAT LINES TO DEOXYNIVALENOL AND NIVALENOL CHEMOTYPES OF FUSARIUM GRAMINEARUM. P. Horevaj1, E.A. Milus1*, L.R. Gale2 and H.C. Kistler2 1 Dept. of Plant Pathology, University of Arkansas, Fayetteville, AR, 72701; and 2USDA-ARS, Cereal Disease Laboratory, St. Paul, MN 55108 * Corresponding Author: PH: (479) 575-2676; Email: gmilus@uark.edu OBJECTIVES The objectives of this research were to determine if wheat lines selected for resistance to Fusarium head blight (FHB) caused by deoxynivalenol (DON) chemotypes also have resistance to the nivalenol (NIV) chemotypes and to determine which lines appear to have resistance to mycotoxin accumulation in the grain. INTRODUCTION Even after several decades of intensive research on FHB of wheat caused by Fusarium graminearum, mycotoxins produced by this pathogen still cause serious concerns for food and feed safety. The most prevalent mycotoxins produced by F. graminearum are DON and NIV. Strains that produce mainly DON (DON chemotypes) predominate in the U.S. (Chandler et al. 2003). However, strains that produce mainly NIV (NIV chemotypes) were found recently in Louisiana and Arkansas (Gale et al. 2005 and 2007), and NIV is ten times more toxic to humans than DON (Ueno and Ishii 1985). Developing resistant wheat cultivars is perceived to be the most effective means for managing FHB and reducing levels of mycotoxins in grain. The presence of both DON and NIV chemotypes in the Midsouth necessitates having resistance to both chemotypes in wheat cultivars adapted to this region. MATERIALS AND METHODS A susceptible check and 15 resistant winter wheat lines (Table 1) were grown in the greenhouse. Heads were inoculated at flowering with two DON and two NIV chemotypes of F. graminearum. Inoculum (20μl at 5.104 cfu/μl) was injected into one floret of each head, and plants were misted for 72 hours to pro- mote infection. The number of infected florets per head was counted 21 days after inoculation, and the percentage of infected florets (%IF) was calculated. Heads were harvested at maturity and threshed by hand to retain all of the rachis tissue and grain. Grain and rachis tissue from heads within a pot were bulked, and each bulked grain sample was separated into “healthy” and “scabby” fractions by the USDAARS Grain Marketing and Production Research Center at Manhattan, KS. Healthy and scabby grain and rachis tissue were ground and sent to the University of Minnesota for mycotoxin analyses using GC/MS. The statistical model was a full factorial with 16 lines, two DON chemotypes, two NIV chemotypes, and three replications (pots). Analysis of %IF was based on three experiments, and analyses of toxin concentrations were based on two experiments because there was little variation for toxin concentration in the third experiment. Data were analyzed using JMP version 7.0. Data were transformed before analyses to achieve homogeneity of variances using the most appropriate transformation suggested by the software. Means were back-transformed for presentation of results. RESULTS AND DISCUSSION Percentage of infected florets (%IF). The two isolates within each chemotype caused similar levels of disease, and data were pooled by chemotype for analysis. The line ´ chemotype interaction was not significant (P=0.0713), indicating that lines ranked similarly for both chemotypes. The two DON isolates averaged 16.9% IF and caused significantly more disease (P<0.0001) than the two NIV isolates that averaged 11.9% IF. All resistant lines had significantly lower %IF than the susceptible check for both chemotypes, and lines Fg 368, ARGE97-1033-10183 Session 5: Variety Development and Host Resistance will be evaluated to determine which are the most appropriate, and data for healthy and scabby grain fractions will be analyzed to determine if these data are useful for characterizing resistance to FHB and mycotoxin accumulation. 2 and VA04W-433 had the lowest %IF (Table 2). Wheat lines with resistance to isolates of the DON chemotype were even more resistant to isolates of the NIV chemotype, and therefore selecting lines for resistance to the DON chemotype should also select for resistance to the NIV chemotype. ACKNOWLEDGEMENTS Toxin concentration in grain and rachis tissue. Averaged across 16 wheat lines in experiments 1 and 2, the two isolates of the NIV chemotype primarily produced NIV and little to no DON, 3-ADON, or 15ADON, and the two isolates of the DON chemotype primarily produced DON and little to no NIV, 3ADON, or 15-ADON in both grain and rachis tissue (Table 3). Therefore, all comparisons of toxins only considered NIV for the NIV chemotype and DON for the DON chemotype. The two isolates within each chemotype produced similar levels of mycotoxins (Table 3), and data were pooled by chemotype for analysis. Averaged across 16 wheat lines in experiments 1 and 2, toxin concentrations for the two NIV and two DON isolates were 0.25 ppm NIV and 0.73 ppm DON, respectively, in the healthy grain fraction and 25.71 ppm NIV and 61.30 ppm DON, respectively, in the scabby grain fraction. These results indicate that the grain sorting procedure effectively sorted grain into healthy and scabby fractions. There were significant wheat line ´ chemotype interactions for toxin concentration in grain (P<0.0001) and in rachis tissue (P=0.02). For each line, however, the NIV concentration was always less than the DON concentration (Table 2), indicating that the interactions were due only to the magnitude of the differences between DON and NIV concentrations. Lines ARGE97-1033-10-2 and VA04W-433 had the lowest concentrations of toxin in both grain and rachis tissue and were among the most resistant lines as measured by the percentage of infected florets (Table 2). Both lines have the cultivar ‘Freedom’ in their pedigree, and Freedom may have contributed genes for resistance. We thank Peter Rohman and Jody Hedge for technical assistance, Floyd Dowell and Elizabeth Maghirand for separating grain fractions, Yanhong Dong for toxin analyses, and Andy Mauromoustakos for statistical advice. This material is based upon work supported by the U.S. Department of Agriculture, under Agreement No. 59-0790-9-054. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. DISCLAIMER Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the U.S. Department of Agriculture. REFERENCES Chandler, E. A., Simpson, D. R., Thomsett, M. A., and Nicholson, P. 2003. Development of PCR assays to Tri7 and Tri13 trichothecene biosynthetic genes, and characterization of chemotypes of Fusarium graminearum, Fusarium culmorum and Fusarium cerealis. Physiol. Mol. Plant Pathol. 62:355-367. Gale, L.R., Ward, T.J., O’Donnell, K., Harrison, S.A., and Kistler, H.C. 2005. Fusarium head blight of wheat in Louisiana is caused largely by nivalenol producers of Fusarium graminearum and Fusarium asiaticum. Page 159 in Proceedings of the 2005 National Fusarium Head Blight Forum, Milwaukee, Wisconsin. Gale, L.R., Harrison, S.A., Milus, E.A., Ochocki, J.E., O’Donnell, K., Ward, T.J., and Kistler, H.C. 2007. Diversity of Fusarium graminearum from the United States: an update. Proceedings of the 2007 National Fusarium Head Blight Forum (in press). Ueno, Y., Ishii, K. 1985. Pages 307-316 in: Trichothecenes and Other Mycotoxins. Lacey, J. editor. Willey & Sons, Chichester, England. This report is based on a preliminary analysis of the data. Additional statistical models and transformations 184 Session 5: Variety Development and Host Resistance Table 1. Winter wheat lines used in this study, their pedigree and contributors. Line Pedigree Contributor ARGE97-1033-10-2 ARGE97-1042-4-5 ARGE97-1047-4-2 ARGE97-1048-3-6 ARGE97-1064-13-5 VA04W-433 VA04W-628 ROANE AR97002-2-1 BESS NC03-11465 COKER 9835 SZ 13 SZ 14 Fg 365 Fg 368 FREEDOM/CATBIRD MASON / CATBIRD P2643 / 3 NING 7840 // PARULA / VEERY # 6 MASON // SHA 3 / CATBIRD MASON//FREEDOM/SUPER ZLATNO NING 7840/PION2684//96-54-244 (CK9803/FREEDOM) ERNIE//NING7840/ERNIE VA71-54-147(CI17449)/Coker68-15//IN65309C1-18-2-3-2 AR396-4-2/NING 8026 MO 11769/MADISON NING 7804/P2643//NC95-22426 Susceptible check Ringo Star / Nobeoka Bozu Ringo Star / Nobeoka Bozu Sgv / Nb / MM / Sum3 Zu / Re / Nobeoka Bozu Milus Milus Milus Milus Milus Griffey Griffey Griffey Bacon McKendry Murphy Check Mesterházy Mesterházy Mesterházy Mesterházy 185 Toxin concentrations (mg/kg) 1 grain2 186 Line ARGE97-1033-10-2 VA04W-433 VA04W-628 AR97002-2-1 ROANE BESS Fg 368 SZ 14 Fg 365 NC03-11465 SZ 13 ARGE97-1064-13-5 ARGE97-1047-4-2 ARGE97-1042-4-5 ARGE97-1048-3-6 COKER 9835 1 Infected florets (%) 1 rachis3 DON for DON chemotype NIV for NIV chemotype DON for DON chemotype NIV for NIV chemotype 0.54 0.65 1.39 1.72 1.76 1.97 2.88 7.93 8.57 11.35 11.81 13.19 27.41 31.53 40.90 178.05 0.37 0.29 0.58 0.95 0.25 0.72 0.82 0.60 4.69 0.95 0.66 1.41 3.80 9.96 1.40 12.18 75.61 73.84 119.33 136.91 165.66 138.61 175.37 226.49 238.08 192.73 229.72 223.83 201.90 217.24 201.11 438.73 13.97 21.44 26.94 30.69 24.54 28.24 28.03 37.86 56.29 36.01 45.82 30.82 40.91 53.69 23.53 172.01 fg fg fg defg efg cdef cdef bcde bcd bcd bcde bc b b b a cd cd bcd bcd d bcd cd cd ab bcd cd bcd abc a bcd a d d cd bcd bcd bcd bc bc b bc bc bc bc bc bc a c bc bc bc bc bc bc bc b bc bc bc bc b bc a 6.20 6.42 9.43 10.00 9.52 8.31 5.74 10.60 13.24 15.57 9.45 16.90 17.65 20.46 15.44 48.13 e e de de de de de cde bcd bc bcd bc b b b a Values within a chemotype followed by the same letter are not significantly different by a Tukey’s HSD test at P=0.05. Data were transformed for statistical analyses by the formula: Log( :Tox.Con. in grain) * 1.10101230397926 ; however, values represent actual back-transformed LS means for each line and variable. 3 Data were transformed for statistical analyses by the formula: ( :Name("TOX_Rachis (ppm)_F") ^ 0.4 - 1) / 0.0350989281248155 ; however, values represent actual back-transformed LS means for each line and variable. 2 Session 5: Variety Development and Host Resistance Table 2. Toxin concentrations in grain and rachis and percentage of infected florets for each winter wheat line. Table 3. Toxin concentrations in grain and rachis for each isolate of Fusarium graminearum DON and NIV chemotypes (averaged across 16 wheat lines and two experiments). Toxin concentrations in harvested grain1, 2 Chemotype NIV DON Isolate DON NIV 3-ADON 15-ADON Toxin concentrations in rachis1, 3 DON NIV 3-ADON 15-ADON 03-29 0.06a 1.93a 0.00a mg/kg 0.00a 1.74a 03-112 0.08a 3.03a 0.00a 0.00a 0.24a 43.75a 0.01a 0.00a 03-57 17.69a 0.07a 0.07a 0.00a 199.78a 0.51a 13.93a 0.59a 03-113 25.01a 0.10a 0.11a 0.00a 182.11a 0.40a 13.67a 0.46a 40.10a 0.10a 0.00a 1 Values within a chemotype followed by the same letter are not significantly different by an Student’s t – test at P=0.05. Data were transformed for statistical analyses by the formula: Log( :Tox.Con. in grain) * 1.10101230397926 ; however, values represent actual back-transformed LS means for each isolate and variable. 3 Data were transformed for statistical analyses by the formula: ( :Name("TOX_Rachis (ppm)_F") ^ 0.4 - 1) / 0.0350989281248155 ; however, values represent actual back-transformed LS means for each isolate and variable. 2 Session 5: Variety Development and Host Resistance 187 Session 5: Variety Development and Host Resistance CURRENT STRATEGIES FOR BREEDING FUSARIUM HEAD BLIGHT RESISTANT WHEAT IN CANADA. G. Humphreys1*, D. Somers1, S. Fox1, D. Brown1, H. Voldeng2, A. Brule-Babel3, F. Eudes4 and A. Comeau5 Cereal Research Centre, Agriculture and Agri-Food Canada, 195 Dafoe Road, Winnipeg, Manitoba, Canada R3T 2M9; 2Eastern Corn and Oilseeds Research Centre, Agriculture and Agri-Food Canada, Central Experimental Farm, Ottawa, Ontario K1A 0C6; 3Dept. of Plant Science, 66 Dafoe Road, University of Manitoba, Winnipeg, Manitoba, R3T 2N2; 4Lethbridge Research Centre, Agriculture and Agri-Food Canada, 5403 1st Avenue South, Lethbridge, Alberta, Canada T1J 4B1; and 5Station de Recherche, Agriculture and Agri-Food Canada, 2560 Boulevard Hochelaga, Ste-Foy, Quebec, Canada G1V 2J3 * Corresponding Author: PH: (204) 984-0123; Email: ghumphreys@agr.gc.ca 1 ABSTRACT Fusarium head blight (FHB) is a destructive fungal disease of wheat that annually results in yield and grade losses for producers as well as reduced feed and end-use quality for the wheat industry. Efforts to develop FHB resistant wheat cultivars can be roughly divided into three research areas: (1)Germplasm development; (2)Molecular breeding; and (3)Line development and evaluation. Non-Canadian sources of FHB resistance, from Brazil, China, CIMMYT, Germany, Japan and the USA, are being used to develop FHB resistant material suited to Canadian growing conditions and registration requirements. FHB screening in inoculated nurseries has been established across Canada permitting evaluation of germplasm and breeding materials in multiple environments. Novel germplasm is being developed through in vitro selection of wheat microspores for tricothecene resistance. Molecular breeding strategies have been used to develop new breeding materials that combine resistance genes from multiple sources in improved backgrounds. Fine mapping of genes fhb1 and fhb2 has facilitated screening for FHB resistance in parental, in vitro selected, backcrossed, and doubled haploid lines. High throughput screening technologies such as DNA extraction robotics and multi-channel capillary electrophoresis permit the screening of multiple markers. In our wheat breeding program, lines are regularly screened for FHB resistance loci on chromosomes 3BS, 5A, 6BS, and 2D. Haplotype analyses of breeding materials at these loci permit selection of FHB resistant lines for advancement and crossing. Future breeding efforts will focus on traits which reduce deoxynivalenol content, and the mapping and deployment of non-Asian FHB resistance. 188 Session 5: Variety Development and Host Resistance EFFECTS OF AGRONOMIC AND MORPHOLOGICAL CHARACTERS ON FHB SEVERITY, DEOXYNIVALENOL AND ERGOSTEROL CONCENTRATIONS IN NEAR-ISOGENIC LINE PAIRS OF BARLEY. Haiyan Jia1, Brian J. Steffenson2* and Gary J. Muehlbauer1 Department of Agronomy and Plant Genetics, and 2Department of Plant Pathology, University of Minnesota, St. Paul, MN 55108 * Corresponding Author: PH: 612-625-4735; Email: bsteffen@umn.edu 1 ABSTRACT Fusarium head blight (FHB) is a devastating disease on barley. Several agronomic traits have been shown to be associated with lower FHB severity in barley. To evaluate the relationship between different agronomic and morphological traits and FHB levels, we examined FHB severity, deoxynivalenol (DON) and ergosterol concentrations in 20 pairs of near-isogenic lines (NILs) carrying the traits two-rowed/six-rowed, lax/dense, erect/ normal, club/normal spike, hulled/hulless caryopsis, fertile/infertile lateral florets, and early maturity/late maturity. These lines were planted in FHB disease nurseries at Langdon, ND, Fargo, ND, St. Paul, MN, and Crookston, MN in 1995, 2000, 2005, and 2007. Inoculations were applied either by spreading Fusarium graminearum infected barley kernels over the nursery for consecutive weeks starting 10 days prior to spike emergence or by spraying macroconidia inoculum once after head emergence. The FHB severity (number of FHB infected kernels out of total number of kernels per spike) was evaluated on 10 or 20 randomly selected spikes in each replicate at the mid-dough stage of development. After harvest, the concentrations of DON and ergosterol were assessed in random 3 gram grain samples of each replicate. Differences between the means of NILs for levels of FHB severity, DON and ergosterol concentrations were analyzed for statistical significance using the paired t-test. The results will be presented in the poster. Overall, few statistically significant and consistent differences were observed for the scored disease parameters (FHB severity, DON and ergosterol concentrations) on the NIL pairs. However, in general loci controlling six-rowed and six-rowed like spike phenotypes exhibited more disease symptoms. 189 Session 5: Variety Development and Host Resistance FUSARIUM HEAD BLIGHT (FHB) RESISTANCE INTO SOFT RED WINTER WHEAT AGS2000. Jerry Johnson1*, Zhenbang Chen1, James Buck2 and Mingli Wang3 Department of Crop and Soil Science, Griffin-Campus, University of GA, Griffin, Georgia; 2 Department of Plant Pathology, Griffin-Campus, University of Georgia, Griffin, GA; and 3USDA-ARS, PGRCU, Griffin, GA * Corresponding Author: PH: (770) 228-7345; Email: jjohnson@griffin.uga.edu 1 ABSTRACT Fusarium head blight (FHB) is a potential devastating disease in the southeast region in the United States where low temperature and misted weather occurs frequently during soft red winter wheat flowering. Releasing new cultivars resistant to FHB is the most effective option to minimize the chance of FHB incidence and reducing DON contamination. Crosses were made since 2001 between AGS2000 or its derivatives and FHB resistant donor VA01-461 to introduce the exotic resistant genes into our widely local adaptive genetic background. Twelve advanced lines, 941523-E21, 991109-6E8, 991109-6A7, 991371-6E12, 991371-6E13, 031454DH7, 031454-DH31, 031307-DH6, 031307-DH14, 031354-DH30, 981621-5E34, 951306-2E13, derived from VA01W-461, which is a derivate of Sumai3, were evaluated in scab nursery and field in 2006 and 2007 for FHB resistance and agronomy performances with Ernie and Coker 9835 as resistant and susceptible control respectively under misted conditions in Griffin-Campus, Georgia. DNA markers, XGWM533, BARC133, XGWM493, STS3B-256 for QTL on 3BS; BARC117, XGWM156, BARC186, BARC56, for QTL on 5AS; BARC18, and BARC91 for QTL on 2BS were employed to genotype 12 new lines with the donor parent of VA01W-461. Here, we reported the results of DNA genotyping and performances of our elite lines. The scab resistance and QTLs in VA01-461 are discussed in this study. 190 Session 5: Variety Development and Host Resistance CHARACTERIZATION OF RESISTANCE TO DEOXYNIVALENOL (DON) ACCUMULATION IN DIFFERENT WHEAT LINES. L. Kong1, Y. Dong 2 and H.W. Ohm1* 1 Department of Agronomy, Purdue University, 915 W. State Street, West Lafayette, IN 47907; and 2Department of Plant Pathology, University of Minnesota, St Paul, MN 55108 * Corresponding Author: PH: 765-494-8072; Email: hohm@purdue.edu ABSTRACT ANOVA results suggested highly significant differences among wheat genotypes for DON accumulation (F= 22.72, P< 0.0001). As expected, disease and genotype × disease interaction for resistance to DON accumulation were also significant: F=329.66 and 15.98, respectively, P<0.0001. In the combined analysis for DON accumulation: replicate, collection date, rep × genotype, collection date × genotype, and genotype × collection date × disease interaction effects were not significant: F= 0.166-0.382, P= 0.202-0.841. Significant variation for DON content was observed between ‘healthy + diseased’ seeds and ‘healthy’ seeds. But, no significant difference was observed in the healthy seeds between resistant and susceptible genotypes. However, for the DON content in the ‘healthy + diseased’ samples, the FHB-resistant or moderately resistant genotypes including 0128A1, INW0411, INW0412, Bess, Freedom, and Truman, exhibited lower DON accumulation than FHB-susceptible cultivars Patterson and Pioneer2545. Patterson and Pioneer2545 both are susceptible to FHB, but, on average, the DON content for Patterson (12.4 ppm) was only approximately half of that of Pioneer2545 (21.2 ppm). 191 Session 5: Variety Development and Host Resistance DEVELOPMENT OF CIMMYT’S 11TH SCAB RESISTANCE SCREENING NURSERY. J.M. Lewis, T. Ban, R.Ward and E. Duveiller* CIMMYT (International Maize and Wheat Improvement Center), Km 45, Carretera Mex-Veracruz, El Batan, Texcoco, Mexico CP 56130, Mexico * Corresponding Author: PH: 52 (595) 9521900; Email: e.duveiller@cgiar.org ABSTRACT CIMMYT has regularly developed and distributed a Scab Resistant Screening Nursery (SRSN) over the past decade. These nurseries have consisted of the best scab resistant material identified through CIMMYT’s FHB screening trials and have been distributed to interested programs around the world upon request. The most recent nursery distributed was the 10th SRSN, which was made available in 2006. Since that time CIMMYT’s method for screening FHB has been modified for more effective identification of FHB resistant germplasm. These changes have included modifications in the location of the screening nursery, isolates used for inoculation, inoculation technique and misting technology. After two years of screening a range of materials using the modified methodologies, entries for the 11th SRSN have been identified. This nursery primarily includes the best FHB resistant advanced lines developed by the CIMMYT wheat breeding programs. The 11th SRSN will be available for distribution in 2008. 192 Session 5: Variety Development and Host Resistance PRELIMINARY EXAMINATION OF THE INFLUENCE OF GRAIN COLOR IN FHB RESISTANCE. J.M. Lewis*, R. Trethowan, T. Ban, E. Duveiller and R. Ward CIMMYT (International Maize and Wheat Improvement Center), Km 45, Carretera Mex-Veracruz, El Batan, Texcoco, Mexico CP 56130, Mexico * Corresponding Author: PH: 517-355-0271 x 1185; Email: lewisja6@msu.edu 1 ABSTRACT Breeding programs around the world have seen noteworthy improvements in FHB resistance in recent years. However, there is the perception that white wheat lags behind red wheat in the development of resistant varieties. In our study, we asked the question whether or not grain color itself is influencing the amount of disease development. Thirty-six genotypes from fourteen sibling groups originating from different breadwheat crosses were examined. Each sibling group was comprised of at least one red and one white sibling pair. In 2006 and 2007, genotypes were screened for FHB resistance in a four replication incomplete block design at CIMMYT headquarters, Mexico. Plots were spray inoculated at anthesis and three days following, and were rated for % severity and % incidence at thirty-one days post inoculation. Post harvest, DON levels of the 2006 samples were evaluated via ELISA. Preliminary results of this study will be shared. 193 Session 5: Variety Development and Host Resistance FHB RESISTANCE AND DON CONTAMINATION IN VIRGINIA BARLEY. S. Liu, D. G. Schmale III, W.S. Brooks, P.A. Khatibi and C.A. Griffey* Virginia Polytechnic Institute and State Univeristy, Blacksburg, VA, 24061 * Corresponding Author: PH: (540) 231-9789; Email: cgriffey@vt.edu ABSTRACT Knowledge of FHB resistance and DON contamination in Virginia barley is essential for providing growers and producers with new and improved commercial cultivars. In 2006 and 2007, we screened 12 hulless (HLS) and 19 hulled (HLD) lines of barley in inoculated, mist-irrigated field plots at Blacksburg, VA for FHB incidence, FHB severity, and DON contamination. In 2006 and 2007, FHB incidence ranged from 40% to 80% for HLS lines and from 30% to 90% for HLD lines, and FHB severity ranged from 4.0% to 12.5% for HLS lines and from 6.1% to 33.5% for HLD lines. In 2006, DON concentrations ranged from 0.1 to 2.0 ppm for HLS lines and from 0.5 to 11.5 ppm for HLD lines; in 2007, DON concentrations ranged from 0.2 to 2.4 ppm for HLS lines and from 0.1 to 3.3 ppm for HLD lines. In 2006, FHB incidence was correlated with DON for HLS lines (r = 0.92, P < 0.001) and HLD lines (r = 0.48, P < 0.05); in 2007, FHB incidence was not significantly correlated with DON for HLS lines (r = 0.14, P = 0.6) or HLD lines (r =-0.16, P = 0.5), but FHB development was generally low in 2007 in VA. DON concentrations in 100 g kernel lots were correlated among DON testing labs in MN (Dong), ND (Schwarz), and VA (Schmale) (range of r from 0.81 to 0.89, P < 0.001). HLS line VA01H-125 had the lowest level of DON contamination in both years (0.2 ppm in 2006 and 2007), and HLD line VA92-42-46 had relatively low levels of DON contamination in both years (1.0 ppm in 2006, 0.36 ppm in 2007). We are continuing to develop and test new cultivars of barley in VA for FHB resistance and reduced DON potential. ACKNOWLEDGEMENT AND DISCLAIMER This material is based upon work supported by the U.S. Department of Agriculture, under Agreement No. 590790-4-102. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture. 194 Session 5: Variety Development and Host Resistance META-ANALYSES OF QTL ASSOCIATED WITH FUSARIUM HEAD BLIGHT RESISTANCE. Shuyu Liu1, Carl A. Griffey1*, Anne L. McKendry2 and Marla D. Hall1 1 Crop Soil Environmental Science, Virginia Polytechnic and State University, Blacksburg, VA 24061; and 2Division of Plant Science, University of Missouri-Columbia, Columbia, MO 65211 * Corresponding Author: PH: 540-231-9789; Email: cgriffey@vt.edu map. This permits the QTL position to be compared to determine if they overlap. This may provide some The objective of this study is to estimate the CI’s of distinguishable common flanking markers linked to dif63 QTL associated with different types of FHB resis- ferent QTL that can be used more effectively in MAS tance using meta-analyses and align them onto the breeding. consensus ITMI maps to determine if different QTL on the same chromosomes from different studies over- MATERIALS AND METHODS lap. Estimate CI of QTL - The following formulas were used to estimate the 95% CI of each QTL: INTRODUCTION OBJECTIVES Many QTL have been mapped on to chromosomes of resistant sources from China, Japan, Brazil, USA and various European countries. This paper reviewed 63 QTL from 23 studies including four QTL identified for type I FHB resistance (resistance to initial infection), 50 QTL for type II resistance (resistance to spread), six QTL for type III resistance (resistance to DON accumulation) and three QTL for type IV resistance (resistance to kernel damage). Among these studies, 10 chromosomes were identified that have more than two FHB QTL regions. Each QTL explained more than 10% of the phenotypic variation in the corresponding experiment was included in the analyses. Some QTL identified from the same resistance source mapped onto the same chromosome but were not linked to the same marker loci. Furthermore, some QTL from different sources mapped in proximity to each other on the same chromosome. Differences in marker orders across studies make it very difficult to select markers linked to respective QTL from individual studies. CI = 530 /(N*R2) for backcross [1] CI = 530 /(N*R2) for F2 intercross [2] CI = 163 /(N*R2) for RILs [3] CI = 132 /(N*R2) for DH [4] Where N is the number of lines in the mapping population and R2 is the percentage of phenotypic variation explained by the identified QTL. Formulas [1] and [2] were deduced by Darvasi and Soller (1997) through simulation. The CI formulas [3] and [4] for RIL and DH, respectively, were derived via further detailed characterization of genetic parameters (Weller and Soller, 2004; Weller, 2007, personal communication). Classification of FHB resistance QTL - Criteria similar to those reported by Guo et al. (2006) were used in the current study to classify QTL into three classes: (i) suggestive QTL if LOD < 4.0 (or p value > 0.0001), (ii) significant QTL if LOD e” 4.0 (or p value d” 0.0001) and, (iii) confirmed QTL if the QTL was identified in two or more separate studies (Lander and Kruglyak, 1995) and was significant in at least one Meta-analyses have been used to estimate the confi- study. dence intervals (CI) of identified QTL in plants and animals (Wise et al., 1999; Guo et al., 2006). It com- Meta-analyses of marker-QTL associations bines the information for each individual study to esti- Where the estimated CI of QTL regions overlapped, mate QTL CI which can be aligned on the consensus those QTL were grouped into one cluster. QTL alleles 195 Session 5: Variety Development and Host Resistance within the same cluster were assumed to be the same (Guo et al., 2006). QTL were classified into different clusters if none of the estimated CI regions overlapped and were more than 20 cM apart. RESULTS AND DISCUSSION Significant and confirmed type II resistance QTL in the same cluster - Significant QTL from different sources but in the same cluster were distributed on chromosomes 3AS, 5A, 7AL, 3BS, 4B, 6B, and 2DS (Fig. 1). Separate mapping studies of several derivatives of Sumai3 (W14, CM82036, DH181, and Ning7840) provide evidence of a confirmed type II FHB resistance QTL located on chromosome 3BS (Fig. 1). Another confirmed type II FHB resistance QTL also located on chromosome 3BS is derived from Wangshuibai. The third confirmed QTL is on 5A of CM82036 and W14, two Sumai 3 derivatives (Fig. 1). These are confirmed and significant type II FHB resistance QTL, present in different sources and located in the same position along the respective chromosomes Markers flanking the most common regions of these QTL’s CI can be applied in MAS to increase the efficiency. Significant type II resistance QTL in different clusters - The type II resistance QTL of Renan is on chromosome 5AL, which is different from another cluster located around the centromere of chromosome 5A in other sources, such as CM82036, Ernie, Frontana and W14 (Fig. 1). Other type II resistance QTL have been located on chromosome 1BS of Fundulea 201R and 1BL of Wangshuibai and Arina (Fig. 1). A second type II resistance QTL in Renan was mapped near the centromere of chromosome 2B while the QTL in Dream is located on distal region of the same chromosome (Fig. 1). A QTL close to centromere of chromosome 3B in Ernie is in a separate cluster compared to the primary QTL located in the distal region of chromosome 3BS of Sumai 3 and its derivatives. The type II resistance QTL on chromosome 5BS of Wangshuibai does not overlap with the QTL of Arina on chromosome 5BL. These QTL belong to different clusters and, therefore, should provide different FHB resistance alleles for breeding. The flanking markers identified in the original studies should be validated to confirm their effectiveness in MAS prior to using them for pyramiding these QTL. The application of respective tightly linked markers to pyramid these QTL should be effective to breed durable FHB resistances. Different Types of FHB Resistance QTL in the Same Sources and Clusters - Types I and II resistance QTL were found on chromosomes 3AS in Frontana, 5A in W14, and 4B in Wangshuibai. Types II, III, and IV resistance QTL were identified on chromosomes 3BSc and 5A in Ernie while types I, II, III, and IV resistance QTL were discovered on 3BS in W14, a Sumai 3 derivative (Fig. 1). QTL conferring different types of FHB resistance were identified and located in the same clusters suggesting a pleiotropic effect or association among them. FHB resistance types other than type II have been evaluated only in a limited number of resistant sources and environments with most of them being greenhouse studies. Therefore, the common QTL reported for these different types of resistance (Chen et al., 2006; Abate et al., 2007; Liu et al., 2007) need to be proved with more evaluation in additional sources and genetic backgrounds, and in studies specifically designed to assess and distinguish resistance types I, III and IV. Such studies are needed to elucidate whether these QTL have pleiotropic effects or if their interrelatedness is simply a function of the highly correlated effects that FHB assessment methods, particularly single floret point inoculation, has on multiple types of FHB resistance. ACKNOWLEDGEMENTS This material is based upon work supported by the U.S. Department of Agriculture, under Agreement No. 59-0790-4-102, and also Virginia Small Grain Board. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. DISCLAIMER Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the 196 Session 5: Variety Development and Host Resistance author(s) and do not necessarily reflect the view of the Liu, S., Zhang, X., Pumphrey, M.O., Stack, R.W., Gill, B.S., and Anderson, J.A. 2006. Complex microcolinearity among U.S. Department of Agriculture. wheat, rice and barley revealed by fine mapping of the genomic region harboring a major QTL for resistance to Fusarium head blight in wheat. Funct. Integr. Genomics 6:83–89. REFERENCES Abate, Z., Liu, S., McKendry, A.L. 2007. Quantitative trait loci associated with deoxynivalenol content and kernel quality in the soft red winter wheat Ernie. Crops Sci. In press. Buerstmayr, H., Lemmens, M., Hartl, L., Doldi, L., Steiner, B., Stierschneider, M., and Ruckenbauer, P. 2002. Molecular mapping of QTLs for Fusarium head blight resistance in spring wheat. I. Resistance to fungal spread Type II resistance. Theor Appl Genet 104:84–91. Chen, J., Griffey, C.A., Maroof, G.A.S., Stromberg, E.L., Biyashev, R.M., Zhao, W., Chappell, M.R., Pridgen, T.H., Dong, Y., and Zeng, Z. 2006. Validation of two major quantitative trait loci for Fusarium head blight resistance in Chinese wheat line W14. Plant Breed. 125:99–101. Chen, X.F., Faris, J.D., Hu, J., Stack, R.W., Adhikari, T., Elias, E.M., Kianian, S.F., Cai, X. 2007. Saturation and comparative mapping of a major Fusarium head blight resistance QTL in tetraploid wheat. Molecular breed 19:113–124. Cuthbert, P.A., Somers, D.J., Brulé-Babel, A. 2007. Mapping of Fhb2 on chromosome 6BS: a gene controlling Fusarium head blight Weld resistance in bread wheat Triticum aestivum L. Theor Appl Genet 114:429–437. Gervais, L., Dedryver, F., Morlais. J-Y., Bodusseau, V., Negre, S., Bilous, M., Groos, C., Trottet, M. 2003. Mapping of quantitative trait loci for Weld resistance to Fusarium head blight in a European winter wheat. Theor Appl Genet 106:961–970. Guo, B., Sleper, D.A., Lu, P., Shannon, J.G., Nguyen, H.T., and Arelli, P.R. 2006. QTLs Associated with Resistance to Soybean Cyst Nematode in Soybean: Meta-Analysis of QTL Locations. Crop Sci. 46:595–602. Lin, F., Kong, Z.X., Zhu, H.L., Xue, S.L., Wu, J.Z., Tian, D.G., Wei, J.B., Zhang, C.Q., Ma, Z.Q. 2004. Mapping QTL associated with resistance to Fusarium head blight in the Nanda2419 × Wangshuibai population. I. Type II resistance. Theor Appl Genet 109, 1504–11. Liu, S., Abate, Z.A., Lu, H., Musket, T., Davis, G.L., McKendry, A.L. 2007. QTL associated with Fusarium head blight resistance in the soft red winter wheat Ernie. Theor Appl Genet 115: 417–427. Otto, C.D., Kianian, S.F., Elias, E.M., Stack, R.W., and Joppa, L.R. 2002. Genetic dissection of a major Fusarium head blight QTL tetraploid wheat. Plant Mol. Biol. 48:625–632. Paillard, S., Schnurbusch, T., Tiwari, R., Messmer, M., Winseler, M., Keller, B., Schachermayr, G. 2004. QTL analysis of resistance to Fusarium head blight in Swiss winter wheat. Theor Appl Genet 109:323–332. Shen, X., Zhou, M., Lu, W., Ohm, H. 2003a. Detection of Fusarium head blight resistance QTL in a wheat population using bulked segregant analysis. Theor Appl Genet 106:1041– 1047. Somers, D.J., Fedak, G., Savard, M. 2003. Molecular mapping of novel genes controlling Fusarium head blight resistance and deoxynivalenol accumulation in spring wheat. Genome 46:555–564. Steiner, B., Lemmens, M., Griesser, M., Scholz, U., Schondelmaier, J., Buerstmayr, H. 2004. Molecular mapping of resistance to Fusarium head blight in the spring wheat cultivar Frontana. Theor Appl Genet 109:215–224. Weller, J.I., and Soller, M. 2004. An analytical formula to estimate confidence interval of QTL location with a saturated genetic map as a function of experiment design. Theor. Appl. Genet. 109:1224–1229. Zhou, W.C., Kolb, F.L., Bai, G.H., Shaner, G., and Domier, L.L. 2002. Genetic analysis of scab resistance QTL in wheat with microsatellite and AFLP markers. Genome 45: 719–727. 197 Session 5: Variety Development and Host Resistance Figure 1a. 3A 7A 5A 1B Fig. 1. The 95% confidence intervals of QTL associated with different types of Fusarium head blight (FHB) resistance. Significant QTL are represented by solid black bars (LOD > 4 or p <0.0001) above the respective chromosome region; suggestive QTL (LOD < 4 or p > 0.0001) are represented by open bars and; confirmed QTL are shown as solid black bars with bold and italic font-type. The QTL name is the source followed by an underscore and a Roman number which indicated the type of FHB resistance identified. The frame of ITMI genetic chromosome maps from Song et al. (2005) was used with the consensus map from Somers et al. (2004) as reference. 198 Session 5: Variety Development and Host Resistance Figure 1b. 2B 4B 5B 6B 3B 2D 199 Session 5: Variety Development and Host Resistance PYRAMIDING FHB RESISTANCE QTL USING MARKERASSISTED SELECTION IN WHEAT. S.Y. Liu1, C.A. Griffey1*, J. Chen2 and G. Brown-Guedira3 1 Crop and Soil Environmental Sciences, Virginia Tech, Blacksburg VA; 2Aberdeen Research & Extension Center, University of Idaho, Aberdeen ID; and 3Eastern Regional Small Grains Genotyping Lab, USDA-ARS, NCSU-Crop Science, Raleigh NC * Corresponding Author: PH: (540) 231-9789; Email: cgriffey@vt.edu ABSTRACT This study was conducted to pyramid FHB resistance genes from the Chinese source Futai8944 and adapted sources Ernie and Tribute into adapted backgrounds using marker assisted selection. A three-way cross VA02W-713/Tribute//VA07W-120 was made. VA07W-120 is a backcross-derived line with the donor parent Futai8944 and the recurrent parent Ernie. This line contains QTL from both Futai8944 and Ernie and has exhibited a high level FHB resistance in both greenhouse and field tests. Marker assisted selection was applied in F1 and F2 generations in spring 2007 and will be applied in F2:3 generation during winter 2007. Nineteen F1 plants derived from the 3-way cross were selected for advancement on the basis of the presence of target alleles for 12 markers on chromosomes 2DS, 3A, 3BS, 5AS, and 6B. About 900 F2 plants were characterized with 19 markers on 2BS, 2DS, 3A, 3BS, 5AS, and 6B. More than 200 F2 plants having different combinations of target marker alleles for the five QTL regions were selected for advancement. All of these F2:3 lines were planted as head rows this fall in a field scab nursery. Among the 210 F2:3 lines, 31 having target marker alleles for two or more QTL fixed in a homozygous state also will be further evaluated and selected in greenhouse tests for plant phenotype, marker haplotypes, and Type II FHB resistance. Among 19 marker loci, these 31 progeny lines have only one to three loci in the heterozygous state and, thus, the primary goal is to identify desirable progeny having fixed target alleles in all five QTL regions. Based on the number of heterozygous marker loci in the progeny, 10 to 30 plants of each F2:3 family will be further screened and selected using target markers. This study will assess the efficiency of marker-assisted selection for pyramiding different FHB resistance QTL. ACKNOWLEDGEMENTS AND DISCLAIMER The authors thank the technical help from Marla D. Hall, Patricia G. Gundrum, Wynse S. Brooks and Bryan C. Will. This material is based upon work supported by the U.S. Department of Agriculture, under Agreement No. 59-0790-4-102, and also theVirginia Small Grain Board. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture 200 Session 5: Variety Development and Host Resistance WINTER AND SPRING WHEAT PARENTAL DIALLEL ANALYSIS FOR SCAB RESISTANCE. S. Malla1*, A.M.H. Ibrahim2 and K. Glover1 1 Plant Science Department, South Dakota State University, Brookings, SD 57007; and 2 Dep. of Soil and Crop Sciences, Texas A&M University, College Station, TX 77843 *Corresponding Author: PH: (605) 688-4951; Email: mallas@sdstate.edu ABSTRACT Fusarium head blight (FHB), caused by Fusarium graminearum, is an important disease of wheat in South Dakota. The study was conducted to determine combining ability and gene effects in populations derived from mating among spring, winter and facultative wheat genotypes. Six genotypes consisting of susceptible winter wheat ‘Nekota’ and ‘2137’, moderately susceptible winter wheat ‘Harding’, moderately resistant spring wheat ‘ND2710’ and ‘BacUp’ and resistant facultative wheat ‘Ning7840’ were crossed in a partial diallel mating design. F4:5 lines were hand transplanted in May 2006 and 2007 and screened under mistirrigated field conditions. Artificial inoculation consisted of corn spawn spread at jointing and inoculum suspension spray at flowering stages. Disease index percentage (incidence percentage * severity percentage/ 100) of the crosses was analyzed using Griffing’s method 4 and model 1. General and specific combining abilities were highly significant (P < 0.01) for both years. The result showed that both additive and nonadditive gene effects were involved in the inheritance of FHB resistance. The ratio of combining ability variation components [2σ2GCA/(2σ2GCA+ σ2SCA)] was 0.85 and 0.81 in 2006 and 2007, respectively. The homogeneity of the data over two years was tested. The calculated F-value for the ratio of error variances (F = larger error MS/smaller error MS) for two years was 1.09 (P = 0.10, Dfnum = 846, Dfden = 867). The test of homogeneity indicated that the two years data could be pooled. The pooled analysis showed that general combining ability was significant (P < 0.01) but not the specific combining ability (P = 0.17). Both the individual and pooled analysis showed that additive gene effects were more important than non-additive gene effects. Thus, progress in developing resistance in wheat can be made by selection. ACKNOWLEDGEMENT AND DISCLAIMER This material is based upon work supported by the U.S. Department of Agriculture, under Agreement No. 59-0790-4-130. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture. 201 Session 5: Variety Development and Host Resistance ROLE OF A PLASMA MEMBRANE CA2+-ATPASE IN THE RESISTANCE OF POTATO CELLS TO FUSARIUM SOLANI. A.M. Manadilova*, G.T. Tuleeva and R.M. Kunaeva * Institute of Molecular Biology and Biochemistry, Almaty, Kazakhstan Corresponding Author: PH: 7 3272 937091; Email: Man_Alija@mail.ru ABSTRACT In natural conditions, plants are always under attack of numerous pathogenic micro organisms and remain, at that, resistant to most of them. To a large extent, the resistance is due to the main enzyme of plasma membrane – Ca2+-ATPase, which plays an important role in the metabolism of the plant cells both in normal conditions and under pathogenesis. This research was aimed at studying the activity and some physical-chemical characteristics Ca2+- ATPase of the potato varieties of different resistance level both in normal conditions and under infection of the fungus Fusarium solani. The research targets were the tubers and cells culture of the potato varieties Tamasha and Santa, which differ in their resistance to Fusarium solani. Membrane preparations were obtained by differential sucrose density-gradient centrifugation. The activity of Ca2+-ATPase was evaluated by the number of Pi, obtained as a result of ATP hydrolysis. The fungus Fusarium solani was grown on the modified Chapek medium. The use of differential centrifugation method resulted in the isolation of pure preparation of plasma membrane, rich in ions Ca2+. Maximal enzyme activity was identified at the ion concentration Ca2+- 2,25mM, and pH – 7,0. The change of Ca2+-ATPase activity under potation infection with the conidia of Fusarium solani was also studied. The increase of Ca2+-ATPase transport activity is revealed in the first hours of infection with the fungus in the resistant potato varieties, where as no change of activity is observed with non-resistant potato varieties. In 24 hours, the increase of enzyme activity is observed in both resistant and non-resistant varieties. Activation of Ca2+-ATPase during the fungus pathogens leads to the increased inflow of Ca2+ ions through plasmalemma, which results in the improvement of protective mechanisms. The studying of ATPase activity kinetics showed that for plasma membrane fractions, isolated from the resistant variety Tamasha, the maximum speed of hydrolysis from incubation time, was 1.5 higher than in the non-resistant variety Santa. The infected cells of potato, resistant to Fusarium solani, show the increase of Km. Infection leads to the change of physical-chemical enzyme parameters and, respectively, to the increased Ca2+ ion flow through membrane. 202 Session 5: Variety Development and Host Resistance PROSPECTS FOR IDENTIFYING FUSARIUM HEAD BLIGHT RESISTANCE QTL BY ASSOCIATION MAPPING USING BREEDING GERMPLASM. Jon Massman and Kevin Smith* Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, MN 55108, USA * Corresponding Author: PH: 612-624-1211; Email: smith376@umn.edu ABSTRACT In barley, there have been numerous quantitative trait loci (QTL) identified through bi-parental mapping, but very few have been utilized in marker assisted selection (MAS). A new tool, association mapping, may overcome some of the problems that accompany bi-parental mapping by: identifying QTL which are segregating in relevant germplasm, as well as give appropriate estimates of allelic effects. Use of association mapping may shorten the time to implementation of MAS. Two important considerations arise when using association mapping, the extent of linkage disequilibrium and the amount of phenotypic variation present in the mapping population. The objective of this study was to assess phenotypic variation, for FHB and DON, among representative breeding germplasm from four barley breeding programs in the upper Midwest. To accomplish this goal 768 lines were evaluated in seven environments over 2006 and 2007 with each line being assessed in at least four environments. Artificial inoculation, overhead spray and grain spawn, and mist irrigation were used to encourage disease development. Significant variation was found among lines for both traits, with heritabilities, calculated on an environment basis, of 0.45 to 0.52 (FHB) and 0.64 to 0.70 (DON). Histograms showed a range of phenotypic variation that is comparable to bi-parental mapping and therefore should be useful for mapping. Lines will be genotyped using 3000 SNP markers, which have been developed through the USDA Barley CAP. Mapping will be conducted using a mixed model approach to find significant markers. 203 Session 5: Variety Development and Host Resistance BREEDING FOR FHB RESISTANCE IN WINTER WHEAT: WHAT’S AHEAD? Anne L. McKendry Division of Plant Sciences, University of Missouri, Columbia MO 65211 Corresponding Author: PH (573) 882-7708; Email: mckendrya@missouri.edu ABSTRACT Significant yield losses caused by Fusarium graminearum Schwabe (teleomorph Gibberella zeae (Schwein.), the pathogen known to cause Fusarium head blight (FHB), have occurred in Missouri for more than 70 years but have become more frequent in the last 15-20 years due to increased corn acreage, reduced tillage practices aimed at soil conservation, and the lack of effective cultural and/or fungicide control. Severe state-wide outbreaks in both 1990 and 1991 resulted in losses that were estimated at more than $250 million. In addition to the direct yield losses, test weights were reduced and associated deoxynivalenol (DON) accumulation in the grain prevented harvested grain from being marketed. In 1993, FHB resistance was included as a major breeding objective in the Missouri wheat breeding program and systemic screening of both Missouri breeding lines and germplasm that had been introduced through collaborations with CIMMYT became a routine part of the breeding effort. In 1998, germplasm screening efforts were augmented through U.S. Wheat and Barley Scab Initiative funding. Despite evaluating more than 10,000 genotypes from targeted regions globally between 1993 and 2005 we discovered that some of the best sources of resistance were in our own program in genetic backgrounds that were adapted in much of the soft red winter wheat region. It was clear that this ‘native’ resistance would lead to the most rapid release of resistant cultivars. Since 1994, three FHB resistant cultivars have been released from the Missouri breeding program including: ‘Ernie’ released in 1994; Truman, released in 2003; and Bess, an early maturing full-sib of Truman, released in 2005. All have been widely accepted in Missouri and Bess and Truman, which are more widely adapted than Ernie, are being grown on significant acreage outside of the state. The identification of native sources of resistance within the Missouri program has enabled us to have a productive pipeline of FHB resistant germplasm in adapted backgrounds and has led to our focus on this source of resistance. Truman and its early maturing full-sib Bess, have good to excellent levels of types I and II resistances coupled with low DON and good kernel quality retention. They are unique in that this high level of resistance is in an agronomic background that couples excellent yield and test weight with broad geographic adaptation. Haplotype data using known FHB resistance markers suggests that resistance alleles in Truman and Bess probably differ from those in other widely-used sources of resistance. Coupled with its potentially unique resistance alleles, Truman has excellent combining ability (both general and specific) for FHB resistance, producing progeny populations with a high percentage of agronomically desirable, FHB-resistant offspring. This paper will explore opportunities for further enhancing FHB resistance in winter wheat cultivars and accelerating their release by building on the broad-based native resistance available in the winter wheat region. It will focus on the use Truman and its early maturing, full sib, Bess. ACKNOWLEDGEMENT AND DISCLAIMER This material is based upon work supported by the U.S. Department of Agriculture under Agreement No. 590790-4-113. This is a cooperative project with the U.S. Wheat and Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author and do not necessarily reflect the view of the U.S. Department of Agriculture. 204 Session 5: Variety Development and Host Resistance SCAB EPIDEMIC IN NEBRASKA. Neway Mengistu1, P. Stephen Baenziger1*, Stephen Wegulo2, Julie Breathnach2 and Janelle Cousell2 Dept. of Agronomy and Horticulture; and 2Dept. of Plant Pathology, University of Nebraska, Lincoln, NE, 68583-0915 * Corresponding Author: PH: 402-472-1538; Email: pbaenziger1@unl.edu 1 ABSTRACT Fusarium head blight (FHB), caused by Fusarium graminearum, is an important disease of wheat. Natural epidemics of the disease may result in severe yield losses, reduction in quality, and contamination of the harvested grain by mycotoxins. Deoxynivalenol (DON) is the most important mycotoxin that affects all sectors of the wheat industry and it has serious food safety implications in marketing, exporting, processing, and feeding scabby grain. FHB is an episodic disease in the hard winter wheat region of the Great Plains that is known for its diverse and highly variable climate. In eastern Nebraska, the predominant rotation is corn-soybeans, but wheat acreage is increasing as wheat price increases, and wheat continues to be an important winter annual rotational crop. In the 2007 cropping season, a severe epidemic of FHB occurred in the eastern, southeastern, south central, and southwestern parts of Nebraska starting from Omaha to Ogallala (>435 km; >320, 000 ha of wheat). To gain an understanding of the impact of the disease, a sample of grain from each of sixty elite hard winter wheat experimental lines grown in four different locations (Lincoln, Mead, Clay Center, and North Platte) were tested for DON content. The overall mean DON level at each location ranged from <0.5 ppm to 2.3 ppm, the average across locations being 0.8 ppm. The level of DON was highest at Clay Center (3.9 ppm) followed by Lincoln (2.5 ppm), Mead (2.2 ppm), and North Platte (1.2 ppm). Of the sixty experimental lines NE05568, NE05418, and Overland had consistently low DON levels (a mean of <0.5 ppm) at all four locations and also in our mist-irrigated nursery at Mead. Two of the elite lines (NE04653 and Harry) which had the highest DON levels (a mean of >2 ppm) at all locations also showed elevated levels of DON in the mist-irrigated field nursery. This year’s scab epidemic impacted many wheat growers in Nebraska. The ongoing FHB research will help in developing adapted FHB-resistant/tolerant cultivars. ACKNOWLEDGEMENT AND DISCLAIMER This material is based upon work supported by the U.S. Department of Agriculture, under Agreement No. 590790-4-092. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture. 205 Session 5: Variety Development and Host Resistance ‘FALLER’: A NEW HARD RED SPRING WHEAT CULTIVAR WITH HIGH YIELD AND QUALITY ADDED TO COMBAT FUSARIUM HEAD BLIGHT DISEASE. Mohamed Mergoum1*, Richard C. Frohberg1 and Robert W. Stack2 1 Dept. of Plant Sciences, and 2Dept. of Plant Pathology, North Dakota State University, Fargo, ND, 58105 * Corresponding Author: PH: (701) 231-8478; Email: Mohamed.mergoum@ndsu.edu OBJECTIVES To develop a new improved hard red spring wheat (HRSW) cultivar which combines resistance to Fusarium Head Blight (FHB) disease and superior grain yield and bread-making quality. than 20% of total HRSW grown in ND (N.D. Agricultural Statistics Service, USDA. 2006; 2007). The rapid increase in acreage planted to both Alsen and Glenn indicates the desire of ND wheat growers to produce such HRSW cultivars. MATERIAL AND METHODS INTRODUCTION Scab or FHB has been a serious threat to wheat production throughout the world (Schroeder and Christenson, 1963; Bai and Shaner, 1994; McMullen et al., 1997; Stack, 2003). In North America, FHB is caused mainly by Fusarium graminearum Schwabe [telomorph Gibberella zeae (Schwein.)] (Bai and Shaner, 1994; McMullen et al., 1997). In the spring wheat region, FHB has been a major disease for HRSW produced in North Dakota and neighboring states since 1993. The most recent economic report (Nganje et al., 2004) estimate combined direct and secondary economic losses caused by FHB for all crops were at $7.7 billion. ND and MN account for about 68% ($5.2 billion) of the total dollar losses. Direct losses from 1993 trough 2001 for wheat only were estimated to $2.492 billion (Nganje et al., 2004). The use of genetically resistant cultivars is believed to be the most efficient and economical method of controlling this FHB in wheat. This has been demonstrated in 2002, 2003, and 2004 when ‘Alsen’, a moderate FHB resistance cultivar derived from the Chinese source ‘Sumai 3’, released in 2000 by NDSU (with the support of the scab initiative funds) was planted on more than 2.1, 2.4, and 1.9 million acres representing 30.8, 37.4, and 28.9% of ND wheat acreages, respectively (N.D. Agricultural Statistics Service, USDA. 2002; 2003; 2004). Similar scenario was repeated in 2007 when ‘Glenn’, the 2005 NDSU release jumped from about 2% to more Faller was developed using a modified bulk breeding procedure. It was selected from the “ND2857/ ND2814” cross made at NDSU in the fall of 1997. ND2857 (ND2709/ND688) is a hard red spring experimental lines that has good resistance to FHB originating from ND2709 line derived from the cross involving ‘Sumai3’ (PI 481542). Sumai3, a spring wheat from China, is arguably the most used source of resistance to FHB in the world. Both ND 2709 and ND688 are HRSW experimental lines developed by the NDSU breeding program. ND2814 (‘KITT’ (PI 518818)/‘AMIDON’ (PI 527682)//‘GRANDIN’ (PI 531005) /’STOA S’ (PI 520297)) is a HRSW line developed by NDSU HRSW breeding program. Kitt is HRSW cultivar released in1975 by the Minnesota Agricultural Experiment Station and the USDA-ARS while Amidon, Grandin and Stoa are HRSW cultivars released by NDAES in 1988, 1989, 1984, respectively. Faller was selected from a bulk of one purified F5 row-plot selected in 2001 at Christchurch, NZ. Faller was initially put in PYT in the summer of 2001. Subsequently, Faller was tested in the advanced yield trials (AYT) and elite yield trials (YET) at four locations in ND in 2002 and 2003, respectively. Faller was tested as ND 805 at 21 location-years in the North Dakota Variety Trials (NDVT) from 2004 to 2006 and in the HRSW Uniform Regional Nursery (URN) (18 locations) in 2005. The URN is conducted in the 206 Session 5: Variety Development and Host Resistance states of North Dakota, Minnesota, South Dakota, Nebraska, Montana, Wyoming, Washington, and Manitoba, Canada. The first seed increase of Faller was grown in Prosper, ND in the summer of 2004. Faller was tested for its reaction to different races of tan spot, leaf and stem rusts, SNB, STB, and FHB in the greenhouse and in the field during the period of 2001- 2006. The SNB, STB and tan spot are the major components of the leaf spotting disease complex of wheat in North America. A complex of these diseases occurs in nature. Hence managing leaf spots is difficult; however, resistant cultivars are the most effective and economical means of controlling leaf spot. RESULTS Faller was tested under experimental line ND 805 and was released because it combines very high yield (Table 1), resistance to FHB and leaf diseases (Table 2), and very good end-use quality (Table 3). The name of Faller was chosen as recognition to late James Faller, a former technician in the HRSW breeding program for almost three decades. Based on 27 site-years of testing in the NDVT and AYT, grain yield of Faller (4467 kg ha-1) was significantly (p<0.05) higher than all previously NDSU released cultivars including Alsen (3763 kg ha-1), Glenn (3743 kg ha-1), ‘Parshall’ (3607 kg ha-1), ‘SteeleND’ (4052 kg ha-1), ‘Reeder’ (3625 kg ha-1), and ‘Howard’ (3943 kg ha-1) (Table 1). In 19 site-years of testing in the URN trials conducted in 2006, Faller yielded 4055 kg ha-1 compared to 3631, 4095, and 2952 kg ha-1 for ‘Keene’, ‘Verde’, and ‘Chris’, respectively. Other agronomic traits including kernel weight, heading date, plant height and straw strength of Faller and other HRSW cultivars are reported in Table 1. Quality parameter including Falling number, Flour extraction, dough and baking parameters for Faller and major grown NDSU HRSW cultivars are reported in Table 2. Mean grain volume weight of Faller (757 kg m-3) over 26 site-years in NDVT was similar to Reeder (753 kg m-3) and ‘Dapps’ (756 kg m-3), but significantly (p<0.05) lower than Glenn (797 kg m-3) and Howard (778 kg m-3) (Table 1). Similarly, grain protein of Faller (150 g kg-1) was comparable to Reeder (155 g kg-1) and Parshall (156 g kg-1), but lower (p<0.05) than Alsen (157 g kg-1) and Dapps (165 g kg-1) (Table 1). The seedling and adult plant screening tests conducted under greenhouse conditions from 2003-2006 showed that Faller posses high level of resistance to pathotype THBL, the predominant race of leaf rust (caused by Puccinia triticina Eriks.) in the region (Table 3). Faller was also evaluated for resistance to stem rust (caused by Puccinia graminis Per.:Pers. f. sp. tritici Eriks. & E. Henn) and was found to be highly resistant to pathotypes Pgt-QCCJ, -QTHJ, -RTQQ, -TMLK, -TPMK, and –HPHJ (Table 3). Faller was screened in the greenhouse for Septoria nodorum [caused by Stagonospora nodorum (Berk.) Castellani & E.G. Germano] and tan spot [caused by Pyrenophora tritici-repentis (Died.) Drechs]. On a scale of 1 to 5 where 1 is resistant and 5 susceptible, Faller had average scores of 1.7, 2.1, 3.7, 2.7, and 3.1 in reaction to tan spot race, 1, 2, 5, Septoria tritici, and Spetoria nodorum, respectively (Table 3). AKNOWLEDGEMENTS This material is based upon work supported by the U.S. Department of Agriculture, under Agreement No. 59-0790-4-100. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. The authors thank T. Olson (Dep. of Plant Sciences, NDSU, Fargo), for quality analysis; Dr J. B. Rasmussen (Dep. of Plant Pathology, NDSU, Fargo), for leaf rust evaluation; Dr T. L. Friesen (USDA-ARS, Northern Crop Science Laboratory, Fargo, ND), for stem evaluation; and Dr S. Ali (Dep. of Plant Pathology, NDSU, Fargo), for tan spot and septoria evaluations. REFERENCES Anderson, J.A., R.W. Stack, S. Liu, B.L. Waldron, A.D. Fjeld, C. Coyne, B. Moreno-Sevilla, J.M. Fetch, Q.J. Song, P.B. Cregan, and R.C. Frohberg. 2001. DNA markers for Fusarium head blight resistance QTL in two wheat populations. Theor. Appl. Genet. 102:1164-1168. 207 Session 5: Variety Development and Host Resistance Bai, G.H., and G. Shaner. 1994. Scab of wheat: Prospects for control. Plant Disease. 78: 760-766. N.D. Agricultural Statistics Service, USDA. 2004. 2007 North Dakota wheat varieties. McMullen, M. P., R. Jones, and D. Gallenberg. 1997. Scab of wheat and barley: A re-emerging disease of devastating impact. Plant Disease 81:1340-1348. Nganje W. E., S. Kaitibie, W. W. Wilson, F. L. Leistritz, and D. A. Bangsund. 2004. Economic Impacts of Fusarium Head Blight in Wheat and Barley: 1993-2001. Agribusiness and Applied Economics Report No 538. N.D. Agricultural Statistics Service, USDA. 2002. 2002 North Dakota wheat varieties. N.D. Agricultural Statistics Service, USDA. 2003. 2003 North Dakota wheat varieties. N.D. Agricultural Statistics Service, USDA. 2004. 2004 North Dakota wheat varieties. N.D. Agricultural Statistics Service, USDA. 2003. 2006 North Dakota wheat varieties. Schroeder, H. W., and J. J. Christensen. 1963. Factors affecting resistance of wheat to scab caused by Gibberella zeae. Phytopathology 53: 831-838. Stack, R. W. 2003. History of Fusarium head blight with emphasis on North America. P. 1-34. In K. J. Leonard and W. R. Bushnell (ed.) Fusarium head blight of wheat and barley. APS Press, St Paul, MN. 208 Table 2. Quality parameters for Faller hard red spring wheat (HRSW) and check cultivars tested in the ND HRSW Variety Trials (2003-2006). Falling Flour Mixing Mixing Loaf Water Cultivar number Extraction time tolerance volume absorption -1 g kg min % sec min 71.1 15.8 1042 64.3 Faller 423 8.1 69.7 12.2 1007 64.5 Howard 427 8.2 67.6 20.6 1102 64.9 Glenn 401 9.3 70.1 13.5 1011 Steele-ND 425 8.5 68.6 16.2 1057 64.8 Alsen 412 9.0 69.2 14.9 1081 64.7 Parshall 415 8.3 67.8 12.0 1002 63.8 Reeder 431 7.0 Observations 21 21 21 21 21 21 Session 5: Variety Development and Host Resistance 209 Table 1. Summary of agronomic data for Faller hard red spring wheat (HRSW) and check cultivars tested in the ND HRSW Variety Trials (2003-2006). Grain Thousand Grain volume Cultivar Grain yield protein kernel weight weight Heading date Height Straw strength -1 -3 kg ha % g kg m days from 06/1 cm score† Faller 4467c† 15.0a 29.8cd 757a 29 85 0.6 Howard 3943b 15.3ab 29.0cd 778b 28 (<0.05)‡ 85(<0.10) 0.5 (1.00) Glenn 3743a 15.7b 30.2d 797c 28 (<0.05) 83 (<0.50) 0.5 (1.00) Steele-ND 4052b 15.3ab 28.4bc 773b 28 (<0.05) 85 (<0.10) 2.0 (<0.01) Dapps 3646a 16.5c 29.5c 756a 26 (<0.01) 93 (<0.01) 0.5 (1.00) Alsen 3763ab 15.7b 26.9ab 777b 27 (<0.05) 80 (<0.05) 0.3 (0.50) Parshall 3607a 15.6b 26.5a 770b 27 (<0.05) 86 (1.00) 0.2 (0.50) Reeder 3625a 15.5ab 26.7a 753a 29 (1.00) 80(<0.05) 1.0 (0.50) Observations 27 26 26 26 26 26 7 † Lodging score: 1=completely erect to 9=completely flat at harvest. ‡ P values (in parentheses) represent the significance of the comparison between Faller and the respective check cultivar based on a Student’s paired t-test procedure (SAS-JMP version 6.0.3, SAS Institute Inc., Cary, NC). Session 5: Variety Development and Host Resistance 210 Table 3. Diseases reactions of Faller hard red spring wheat (HRSW) and check cultivars tested in the ND HRSW Variety Trials (2003-2006). Septoria Septoria Cultivar FHB† Leaf rust Stem rust Tan spot tritici nodorum Greenhouse‡ Field Greenhouse§ Field Race 1 Race 2 Race 5 1-5# 1-5 1-5 % ¶ Faller 27 R R R tR 1.7 2.1 3.7 2.7 3.1 Alsen 22 R MR/MS MR/R 5R 2.7 4.4 Traverse R MR/MS R R 2.9 2.6 Knudson R R R 2.2 1.6 Reeder 55 R S MR/R 5R 2.9 2.2 Baart S S 50MS Tatcher S Glenlea 4.3 2.0 1.9 2.4 3.7 Salamouni 1.4 1.4 1.3 1.7 1.7 6B662 1.7 1.7 4.0 1.9 1.4 6B365 1.7 4.1 1.9 2.1 1.7 Observations 9 5 4 9 6 6 6 4 4 † FHB (Fusarium Head blight) severity as described by (Stack and Frohberg, 2000). ‡ Greenhouse reactions for leaf rust races MCDL and THBJ. § Greenhouse reactions for stem rust races Pgt TPMK, TMLK, RTQQ, QFCQ, QTHJ, THTS, and TCMJ. ¶ R=resistant, MR=Moderate resistant, MS=Moderate susceptible, S=Susceptible, tR= trace/Resistant. # The 1-5 scale developed by Lamari and Bernier (1989) was used to score the genotype Session 5: Variety Development and Host Resistance PUTATIVE FHB RESISTANCE COMPONENTS RESISTANCE TO KERNEL INFECTION AND TOLERANCE IN THE SSRWW NURSERY, 2005-2007. Á. Mesterházy1*, A. Szabo-Hever1, B. Toth1, G. Kaszonyi2 and Sz. Lehoczki-Krsjak2 Cereal Research Non.profit Company; and 2Department of Biotechnology and Resistance Research, Szeged, Hungary * Corresponding Author: PH: (36) 30 915430; Email: akos.mesterhazy@gabonakutato.hu ABSTRACT The Southern Soft Red Winter Wheat Nursery has been tested in Hungary since 2003 with four isolates of F. graminearum and F. culmorum separately in three replicates, so four epidemic situations could be studied at the same time. Three bagged groups of heads served as controls. From 2005 we applied one inoculation date as the flowering differences were not larger than 4-5 days, and so an important source of mistake could be neutralized and the data become more suitable to estimate resistance components. After spraying the heads of 15-20 in a group with Fusarium suspension, polyethylene bags over 48 hrs secured the high humidity to initiate infection. As the members of the nursery changed from year to year, a repeatability of the Szeged results could not be tested over years, however, we provide a way to estimate the presence of the resistance to kernel infection and tolerance, which can be applied on the multilocated data set of the nursery. The statistical evaluation is as follows: A linear regression slope will be counted between FHB and FDK data. By using the function, the data points of FDK data for all FHB data points will be developed. From the original FDK data the counted FDK values will be extracted, so for each genotype we receive a difference. When this difference is larger than the LSD 5 % from the ANOVA of the FDK values, we will have two sorts of deviations. When the value will be negative, e. g. the predicted FDK is larger than the original data, we speak about the presence of the kernel infection resistance component, e. g. the kernel infection is lower than would be calculated from the function. On the opposite, for positive number the predicted value is smaller than the original data, we speak about an extra sensitivity. Both are important and this is one reason why FHB alone does not sufficiently describe resistance behavior of the given genotype. In 2007 for example, six genotypes of 45 provided resistance and seven revealed extra susceptibility. The estimation of tolerance has the same pattern. We think that such additional analyses of the data will help to evaluate additional resistance components and understand better behavior of the genotypes under different epidemic conditions. ACKNOWLEDGEMENTS The authors express their thanks to NKTH-KPI projects signed as OMFB 01286/2004 and OMFB 00313/ 2006 for financial support. 211 Session 5: Variety Development and Host Resistance EVALUATION OF FHB PROFILES OF ADVANCED WHEAT BREEDING LINES TREATED WITH A FUNGICIDE. N. Mundell1, D. Hershman2, Chad Lee1 and D. Van Sanford1* 1 Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY; and 2Department of Plant Pathology, University of Kentucky Research and Education Center, Princeton, KY * Corresponding Author: PH (859) 257-5020 ext. 80770; Email: dvs@email.uky.edu ABSTRACT In evaluating FHB resistance of wheat breeding lines, breeders strive to estimate, as accurately as possible, the genetic component of resistance. The possible benefits of management tools, e.g., fungicides, are often ignored by the breeder. The purpose of this study was to evaluate the FHB profile of a set of advanced breeding lines in the presence and absence of the fungicide Prosaro. The study was conducted at Princeton and Lexington, KY. The Princeton location was non-irrigated and inoculated with a single application of scabby corn at Feeke’s growth stage 9 and two conidial sprays (1 X 106 spores ml-1) at flowering and one week later. The Lexington location was irrigated and inoculated with scabby corn at Feeke’s growth stage 9. Rainfall across Kentucky was inadequate for FHB development, but measurable levels of disease were achieved in both nurseries. Diseases other than FHB were present in Lexington, but at very low levels. A factorial design with 3 replications was used at each location. At Princeton the experimental unit was a conventional 6 row yield plot, 15 ft. long; at Lexington the experimental unit was a 4 row plot, 4 ft long, planted with a headrow planter. Plots at each location were treated at flowering with a tank mix of Prosaro fungicide (6.5 fl. oz. acre-1) with Induce (0.125% w/v). Three replicates were left untreated for comparison. FHB symptoms were evaluated 21 days after flowering using a 5 point visual rating scale that encompasses both severity and incidence. After harvest, percentage Fusarium damaged kernels (FDK), deoxynivalenol concentration (DON), yield and test weight were measured. In Princeton, where rain was a limiting factor, there was no significant difference between fungicide-treated and control plots for rating, FDK, DON, and test weight. There was a significant difference in yield, with the average yield of the control plots 11.5 % less than the average yield of the treated plots. In the irrigated Lexington nursery, FHB rating, FDK and DON were all significantly lower in treated than in controls. Yield and test weight were significantly higher for the treated plots than for the control plots. Particularly interesting was the DON level reduction in Lexington. Fungicide x genotype interaction was apparent. For instance, in the control plots, KY99C-1205-06-1 had the lowest DON with 15.4 ppm, but it was third lowest in the treated plots with 13.2 ppm, a 2.2 ppm reduction. In KY98C-1324-01-3 the reduction was 21.4 ppm. The study suggests that advanced breeding lines should routinely be screened with a fungicide as part of the candidate variety evaluation process. ACKNOWLEDGEMENT AND DISCLAIMER This material is based upon work supported by the U.S. Department of Agriculture, under Agreement No. 590790-6-056. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture. 212 Session 5: Variety Development and Host Resistance THE 2006-07 SOUTHERN UNIFORM WINTER WHEAT SCAB NURSERY. J.P. Murphy* and R.A. Navarro Department of Crop Science, Box 7629, North Carolina State University, Raleigh, NC 27695 * Corresponding Author: PH: (919) 513-0000; Email: paul_murphy@ncsu.edu ABSTRACT Most components of Fusarium Head Blight (FHB) resistance are greatly influenced by genotype by environment interaction which limits the heritability of resistance estimated by a single program in any given year. The Southern Uniform Winter Wheat Scab Nursery provides breeders in the public and private sectors with an opportunity for multi-environment, independent evaluations of FHB resistance in advanced generation breeding lines compared with the current most resistant check varieties Ernie and Bess. In addition, the nursery facilitates the sharing of the best resistant materials throughout the breeding community. The 2006-07 nursery comprised 42 advanced generation breeding lines and three check cultivars, ‘Ernie’ and ‘Bess’ (partially resistant) and ‘Coker 9835’ (susceptible). Six U.S. public programs (Univ. of Arkansas, Univ. of Georgia, Louisiana State University, Univ. of Maryland, N.C. State Univ. and VA Tech.), and one private company, Agripro-Coker, submitted entries. The nursery was distributed to 11 U.S., one Hungarian, and one Romanian cooperator for field and / or greenhouse evaluations. In addition three USDA-ARS laboratories conducted evaluations for Hessian Fly resistance, milling and baking quality and haplotypes based on established SSR markers. Several nights of uncharacteristic freezing temperatures during the April 6th to April 9th, 2007 period severely damaged the wheat crop throughout the southern US. As a result, no data were obtained by our AgriproCoker and University of Georgia cooperators. Partial data were provided by the Univ. of Illinois and N.C. State Univ. Copies of the full report will be available at the 2007 National Fusarium Head Blight Forum and subsequently on line at the USWBSI web site: http://www.scabusa.org/. ACKNOWLEDGEMENT AND DISCLAIMER This material is based upon work supported by the U.S. Department of Agriculture, under Agreement No. 590790-4-117. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the U.S. Department of Agriculture. 213 Session 5: Variety Development and Host Resistance HAPLOTYPE STRUCTURE AND GENETIC DIVERSITY AT FUSARIUM HEAD BLIGHT RESISTANCE QTLS IN SOFT WINTER WHEAT GERMPLASM. Leandro Perugini1, Clay Sneller2, Fred Kolb3, David VanSanford4, Carl Griffey5, Herb Ohm6 and Gina Brown-Guedira1* USDA-ARS Plant Sciences Research, Dept. of Crop Science, North Carolina State University, Raleigh, NC 27695; 2Horticulture & Crop Science Department, Ohio State University, Wooster, OH, USA; 3Dept. of Crop Science, University of Illinois, Urbana, IL, USA; 4Dept. of Agronomy, University of Kentucky, Lexington, KY, USA; 5Crop & Soil Environmental Sciences Dept., Virginia Tech, Blacksburg VA, USA; and 6Dept. of Agronomy, Purdue University, West Lafayette, IN, USA * Corresponding Author: PH: (919) 532-0696; Email: Gina_Brown-Guedira@ncsu.edu 1 INTRODUCTION AND OBJECTIVES MATERIALS AND METHODS Several quantitative trait loci (QTLs) for resistance to Fusarium head blight (FHB) have been mapped in wheat. Among these, three were mapped in the Chinese cultivar Sumai 3 and its derivatives on chromosomes 3BS, 5AS, and 6BS (Anderson et al. 2001; Buerstmayr et al. 2002; McCartney et al. 2004; Yang et al. 2003; Zhou et al. 2002). The 3BS FHB resistance QTL (designated Fhb1) has been by far the most deployed by breeding programs worldwide (McCartney et al. 2004). Other FHB resistance QTLs have been mapped in Wuhan 1 on chromosomes 2DL and 4BL, (Somers et al 2003) and in the soft red winter wheat cultivar Ernie on chromosomes 4BL, 5A and 3BSc near the centromere (Liu et al. 2007). Haplotyping strategies make use of previous QTL mapping and molecular marker information. In our study we selected markers reported to be near FHB resistance QTL mapped in Sumai 3, Wuhan 1 and Ernie to haplotype a large set of eastern SW wheat lines submitted by breeders . Two hundred forty-five SW wheat lines with moderately low to strong FHB resistance from native and/or exotic sources, including susceptible and resistant checks, were grown in screening nurseries at Wooster, OH; Urbana, IL; Lexington, KY; and Blacksburg, VA in 2005 to test for type I and type II resistance to FHB. Some exotic FHB resistant accessions (Sumai 3, Ning 7840, Futai 8944, W14, Wuhan 1, F201R, and VR95B717) were planted only for the purpose of obtaining DNA for the molecular marker analysis because they were in the pedigrees of some of the entries screened for FHB and they served as reference for alleles sizes for markers linked to FHB resistance. Forty-seven SSR markers and one STS marker that map near or at FHB-resistance QTLs on chromosome 2DL and 4BL based on Wuhan 1; 3BS, 5AS, and 6BS based on Sumai 3; and 3BS and 5A based on Ernie were selected. The objectives of this research were to (1) determine the genetic relationship among soft winter (SW) wheat lines with native and exotic sources of resistance using SSR markers data, (2) compare the SSR marker haplotypes of SW wheat lines with those of Sumai 3, Wuhan 1, and Ernie at known FHB resistance QTLs, and (3) identify lines with novel sources of FHB resistance. Statistical analysis of phenotypic data was performed using the SAS software package (SAS Institute Inc., NC, USA), and wheat accessions were classified as resistant (R), moderately resistant (MR), moderate (M), moderately susceptible (MS), and susceptible (S). Scoring of polymorphic DNA fragments generated by SSR markers at each locus was conducted by using GeneMarker v1.5 (SoftGenetics LLC, State College, PA). The freeware package PowerMarker version 3.25 (Liu and Muse 2005) was used to perform the phylogenetic analysis and to obtain genetic 214 Session 5: Variety Development and Host Resistance les observed for SSR markers ranged from two (Xgwm508) to 22 (Xgwm601) with a mean number of 10.42 alleles per locus. The 3BS QTL region had the lowest mean number of alleles detected by SSR markers (6.3), which resulted in the lowest mean PIC value of 0.493. In constrast, the 3BSc QTL interval had the highest mean number of alleles detected by SSR markers (12.1) and the highest mean PIC value of 0.7. A total of 251 haplotypes were detected by the 48 SSR markers, indicating that although there were RESULTS AND DISCUSSION several full-sib lines in included in the study, no entries The table with haplotypes and the dendrogram had the exact same alleles at all loci. of all the wheat lines/cultivars included in this study were too large to be included in these pro- Cluster Analysis ceedings; this information can be found at http://www.cropsci.ncsu.edu/sggenotyping/index.htm. Entries were grouped into 16 clusters that were generally based on breeding program or geographic origin of lines. The Chinese wheat cultivars having the Phenotypic Evaluation Sumai 3 haplotype at Xbarc75, Xgwm533, Reaction of the SW entries evaluated was skewed Xgwm133, Xsts3B-256, Xbarc147, and Xgwm493; toward resistance, with 59 lines classified as resistant, and therefore the Fhb1 resistance gene, were grouped 116 moderately resistant, and 28 intermediate. Only separately from all other entries. One exception was 12 and 18 lines were considered moderately suscep- inclusion of entry VA01W-476 (W14 x Roane) in this tible and susceptible, respectively. Of the resistant lines, cluster. VA01W-476 had the highest level of resistance 24 have exotic sources of resistance in the pedigree of all entries evaluated in the study. and the remaining resistant lines had only SW germplasm in their pedigrees. The SW wheat culti- Soft white winter wheat accessions were grouped tovars NC-Neuse and Truman were resistant to FHB. gether and with the resistant Chinese cultivar Wuhan Of the seventy-three entries in the experiment having 1, and the susceptible cultivars Madison and Pioneer exotic sources of FHB resistance in their pedigrees, 2545. In general, entries from breeding programs in index scores ranged from resistant (VA 01W-476, the Corn Belt (IN, OH, MO and IL) were in different IND = 4.1) to susceptible (VA41W-495, IND = clusters than entries from the Southeast. The French 41.9). The exotic sources of resistance in pedigrees line VR95B717 that was used as a resistance source of lines in this study included Sumai 3, Ning 7840, by the Virginia Tech. breeding program was included Ning 8026, Ermai 9, Futai 8944, ZM10782, W14, in a cluster of resistant to moderately resistant Corn Catbird, F201R, and VR95B717. The most com- Belt entries. mon SW wheat cultivars in pedigrees with moderate resistance were Roane, Freedom, Patton, and Ernie. Comparison of haplotypes of SW wheat with Sumai 3, Wuhan 1 and Ernie Marker Diversity Markers linked to the Fhb1 locus had the lowest geThe forty-eight SSR markers evaluated had PIC val- netic diversity and linkage disequilibrium was observed ues that ranged from 0.175 (Xgwm113) to 0.922 between markers across the interval. Markers (Xcfd233) with a mean value of 0.639. Only ten SSR Xgwm533 and Xsts3B-256 proved to be the best markers had PIC values of less than 0.50. Two alleles available for identifying wheat lines with the Fhb1 gene were observed for STS marker Xsts3B-256 that is derived from Chinese sources. The Fhb1 resistance closely linked to the Fhb1 gene. The number of alle- gene was not present in all entries derived from crosses diversity estimates; polymorphism information content (PIC) values; and a total number of allele at each SSR marker locus. Haplotype numbers were calculated (1) on the basis of shared alleles at all loci to detect putative novel QTLs in germplasm lines, and (2) on the basis of the allelic distribution of SSR markers linked to 2DL and 4BL QTLs in Wuhan 1; 3BS, 5AS, and 6BS in Sumai 3; and 3BSc and 5A in Ernie. 215 Session 5: Variety Development and Host Resistance with donor Chinese lines. However, all eight entries in nine resistant SW wheat lines, 11 moderately resistant this study that have the Fhb1 resistance gene based and 2 susceptible lines were identical to Ernie. on haplotype data were resistant in the field evaluation. Wuhan 1 was not in the pedigree of any of the lines in this experiment. None of the SW wheat entries shared At the 6BS interval, no entries matched the Sumai 3 the complete haplotype of Wuhan 1 for markers in the haplotype. The most common haplotype on this chro- 2DL or 4BL QTL intervals. The highest degree of simimosome region was Xgwm518 (224 bp), Xgwm508 larity in the 2DL region was observed in soft white (Null), Xwmc398 (168 bp), Xgwm133 (131 bp), winter wheat lines from Michigan and New York. The Xwmc397 (178 bp), Xwmc152 (Null), and most similar accession to Wuhan 1 was the suscepXgwm219 (205 bp), which was shared by a group of tible soft white winter wheat cultivar Geneva. Wuhan moderately resistant to susceptible lines of diverse ori- 1 alleles for proximal SSR markers Xwmc245, gin. The two moderately resistant SRW wheat culti- Xwmc144, and Xwmc601 were common among the vars Patton and Goldfield, while different from Sumai soft white wheat lines. These haplotypes were not 3, had the most closely related haplotypes in this in- observed among the soft red winter wheat entries. terval, with matching alleles at six of the seven SSR marker loci assayed. Resistance QTL were located on chromosome 4B in cultivars Ernie and Wuhan 1. The Qfhs.umc-4BL QTL Resistance QTLs located on chromosome 5A have peak in Ernie was located near marker Xgwm495. been identified in a number of lines, including Sumai 3, Only three backcross-derived lines had the Ernie hapNing 7840 and Ernie. Chinese lines Futai 8944, Shaan lotype for SSR markers spanning this interval 85-2, and Ning 7840 had the Sumai 3 haplotype across (Xwmc238, Xgwm165 and Xgwm495). Eighteen the ten loci at the 5AS interval analyzed. No SW wheat resistant to moderately resistant and 2 susceptible lines line had the complete Sumai 3 haplotype at all ten had the Ernie haplotype for markers Xgwm165 and markers. Sumai 3 alleles at SSR marker loci Xbarc117, Xgwm495. Released cultivars Pat, Superior, FreeXgwm304, Xbarc186 and Xgwm415 reported near dom, and Truman, as well as the French line the Qfhs.ifa-5A QTL peak were common in the SW VR95B717, shared this haplotype. entries evaluated, with frequencies of 0.42, 0.39, 0.31 and 0.26, respectively. However, when these four Wheat cultivars Freedom and Patton, and the SW marker alleles were considered to form a Sumai 3 hap- wheat line VA04W-608 had the Ernie haplotype at all lotype, only controls Ning 7840, W14, Shaan 85-2 eight loci in the 3BSc QTL interval, and they showed and Futai 8944, and four resistant and moderately re- moderately resistance reactions to FHB. Thirty-six sistant SW lines derived from crosses with ZM10782 moderately resistant to resistant lines had a partial Ernie (OH904 and OH902), and with Ning 8026 haplotype for SSR markers Xwmc625, Xgwm285, (AR9700-2-1 and AR9700-2-2) had the donor par- Xwmc307, and Xwmc418, including the French line ent haplotype. VR95B717, the soft red winter wheat cultivars Freedom, Roane, Patton, and their backcross-derived lines. No lines had the complete Ernie haplotype at all ten marker loci on 5A. Liu et al. (2007) reported the lo- CONCLUSIONS cation of the Qfhs.umc-5A FHB resistance QTL between markers Xbarc56 and Xbarc40. The alleles The Xsts3B-256 and Xgwm533 markers can be observed in Ernie at SSR marker loci Xbarc56, clearly used to identify lines with the Fhb1 resistance Xbarc165, Xbarc40, and Xgwm156 were common gene. However, there is a need for fine mapping other in the SW germplasm in this study having allele fre- regions in which FHB resistance QTLs have been loquencies of 0.53, 0.41, 0.34, and 0.40, respectively. cated. This seems to be particularly important for reWhen these alleles were considered as a haplotype, sistance from Ernie. Allele sizes of Ernie at 5A and 216 Session 5: Variety Development and Host Resistance 4BL QTL intervals are common among Eastern soft wheat germplasm. The haplotypes at these loci, along with the 3BSc region, suggest that SW wheat cultivars such as Patton, Freedom, and Roane, that have been considered important sources of native FHB resistance, may share resistance QTL with Ernie. However, fine mapping and marker enrichment of these intervals could provide closer and better molecular markers to be used in germplasm characterization as well as marker-assisted selection programs. There were 59 wheat entries with high levels of FHB resistance in this study. Many of the most resistant lines had the Fhb1 QTL combined with native resistance. However, more than half of the resistant lines had no exotic parentage, including the cultivars NCNeuse and Truman. Neither of these cultivars had the complete haplotype of the donor sources for any of the interval examined in this research. A number of other SW wheat breeding lines did not share any haplotype at known QTLs evaluated in this study. These lines likely carry novel sources of FHB resistance. In contrast, similarity of marker alleles in the 3BSc and 4BL regions suggests that resistance in the line VR95B717 may be due to the QTL identified in Ernie. These data are useful in prioritizing germplasm for QTL mapping and identifying diverse sources of resistance that can be combined to further increase the level of FHB resistance. DISCLAIMER Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture. REFERENCES Anderson JA, Stack RW, Liu S, Waldron BL, Fjeld AD, Coyne C, et al. (2001) DNA markers for Fusarium head blight-resistance QTLs in two wheat populations. Theor Appl Genet 102:1164-1168 Buerstmayr H, Lemmens M, Hartl L, Doldi L, Steiner B, Stierschneider M, Ruckenbauer P (2002) Molecular mapping of QTLs for Fusarium head blight resistance in spring wheat. I. Resistance to fungal spread (type II resistance). Theor Appl Genet 104:84-91 Liu S, Abate ZA, Lu H, Musket T, Davis GL, McKendry AL (2007) QTL associated with Fusarium head blight resistance in the soft red winter wheat Ernie. Theor Appl Genet 115:417427 Liu, K, Muse SV (2005) PowerMarker: Integrate system environment for genetic marker data. Bioinformatics 21(9): 21282129 McCartney CA, Somers DJ, Fedak G, Cao W (2004) Haplotype diversity at Fusarium head blight resistance QTLs in wheat. Theor Appl Genet 109:261-271. SAS Institute (2003) The SAS system for windows, version 9.1. SAS Inst. Cary, NC. Somers DJ, Fedak G, Savard M (2003) Molecular mapping of novel genes controlling Fusarium head blight resistance and deoxynivalenol accumulation in spring wheat. Genome 46:555564 Yang ZP; Gilbert J, Somers DJ, Fedak G, Procunier JD, McKenzie IH (2003) Marker assisted selection of Fusarium head blight resistance genes in two doubled haploid populations of wheat. Molecular Breeding 12:309-317 Zhou,WC, Kolb FL, Bai GH, Shaner GE, Domier LL (2002) Genetic analysis of scab resistance QTL in wheat with microsatellite and AFLP markers. Genome 45:719-727 217 Session 5: Variety Development and Host Resistance SOLVING THE FHB PROBLEM: GROWERS, EXPORT MARKET AND WHEAT COMMODITY PERSPECTIVES. Jim Peterson North Dakota Wheat Commission, 4023 State Street, Bismark, ND 58503 Corresponding Author: PH: 701-328-5111; E-mail: jpeterso@ndwheat.com ABSTRACT The Fusarium Head Blight (FHB) disease of cereal crops has become a very important disease in recent years. The favorable environmental conditions including wet weather during flowering and grain filling and major changes in cropping system (introduction of Maize and minimum tillage) has favored disease development. All classes of wheat produced in the United States, as well as barley, have been impacted by this disease, some regions and classes more than others with the most pronounced impacts in regions that tend to have higher precipitation during the growing season or a greater concentration of Maize in the crop rotation such the US Northern Plains. In the Northern Plains region, FHB had a devastating impact on the wheat and barley production in 1993, and has continued to cause minor to major annual impacts on cereal crops since. The impact of FHB has not only caused significant economic loss to producers in the region but has also impacted domestic and export customers by threatening the quality reputation; particularly the accumulation of mycotoxins such as DON; and reliability of the Northern Plains in its ability to supply their demands. The Northern Plains has a strong history of being a quality source for hard red spring wheat (HRSW) and durum wheat, and malting and feed barley. Customers in the United States and in numerous export markets have come to rely on the regions wheat and barley production for making specialty breads, premium pasta and couscous and well known brands of beer. Solving the FHB problem is a top priority for wheat, durum and barley research programs in the region, including developing varieties with higher levels of tolerance, research on optimal crop rotation and management practices that incorporate fungicide applications, and studying ways to help millers and processors handle the inherent quality problems that FHB has on the raw wheat and barley and their products. While notable gains have been made in wheat, durum and barley varieties during the past 15 years, further advancements in varietal tolerance and other tangent production research is needed. The significant financial contributions from the US Wheat and Barley Scab Initiative (USWBSI) have been complemented with State dollars and direct contributions from producers themselves through wheat and barley check-off programs, to help producers in the region and the customers they serve reduce the impacts from FHB. The successful incorporation of resistance genes to FHB from the Chinese source ‘Sumai 3’ wheat and other sources such as wheat wild relative Triticum dicoccoides into hard red spring and other market classes wheat varieties like Alsen, Glenn, Steele-ND, Freyr, Truman, Neuse, Tribute, McCormick, and others has given producers some attractive options to ward off FHB pressures. An added bonus of many of these varieties is that they also maintain the end-use quality standards demanded by our domestic and international customers. For example, Glenn, released by NDSU and the mostly grown cultivar in the spring wheat region (1.3 millions in 2007) is currently the HRSW quality standard accepted by the US wh eat quality industry and for the regional breeding programs. Worldwide, Glenn has received numerous top quality ratings from key customers participating in the U.S. Wheat Associates Overseas Variety Analysis (OVA) program. In addition, 218 Session 5: Variety Development and Host Resistance major improvements in forecast models, available fungicides and application methods have allowed producers to more effectively manage the disease in the field. Development of tolerant varieties has been more of a challenge with barley and durum due to smaller germplasm pools. However, successful incorporation of tolerance has accelerated in recent years due to new breeding techniques. Advanced lines of both are showing more promise, and it now looks possible to have varieties available for commercial production within the next two to three years that could have the same level of tolerance as is found in some classes of red wheat. Producers and end-users have already received significant dividends from the enhancement to breeding programs through the USWBSI and other complementary funding. Additional improvements are still needed however, and solving the FHB threat remains a top priority and will likely be for the foreseeable future for small grain producers and researchers in the Northern Plains and the entire US. Domestic and international customers need the additional research to ensure they are able to draw from quality cereal crop production to continue making premium priced, safe and wholesome food products. The producers in the United States and around the world need the additional research to help them maintain viable cereal crop options in their farming operations. 219 Session 5: Variety Development and Host Resistance ASSOCIATION MAPPING OF COMPLEX TRAITS IN A DIVERSE DURUM WHEAT POPULATION. C.J. Pozniak1*, S. Reimer1, D.J. Somers2, F.R. Clarke3, J.M. Clarke3, R.E. Knox3, A.K. Singh3 and T. Fetch2 1 Crop Development Centre, University of Saskatchewan, Saskatoon, Saskatchewan, Canada, S7N 5A8; 2 Agriculture and Agri-Food Canada, Cereal Research Centre, Winnipeg, Manitoba, Canada, R3T-2M9; and 3Agriculture and Agri-Food Canada, Semiarid Prairie Agriculture Research Centre, Swift Current, Saskatchewan, Canada, S9H 3X2 * Corresponding Author: PH: 306-966-2361; Email: curtis.pozniak@usask.ca OBJECTIVE To assess the potential of association mapping to complement existing mapping efforts in durum wheat. INTRODUCTION In plants, the traditional approach to quantitative trait loci (QTL) mapping is to develop a set of random lines derived from crossing two inbred lines that vary in phenotypic values for a particular trait of interest. Although this method has proven effective, there are a number of inherent limitations. First, QTL resolution is generally poor due to the low number of recombination events sampled (Nordborg et al. 2002). This limits potential for marker assisted selection and prevents identification of candidate genes coincident with the QTL. Second, allele variation is restricted to the alleles present in the two parents and QTL localization is restricted to loci segregating between the two parents. This prevents identification of novel alleles and QTL outside of the mapping population (Buckler and Thornsberry, 2002). Association mapping (AM) mapping is employed in medical studies, and is expected to be a complementary strategy for describing associations between genotype and phenotype in crop plants. The most notable attributes of AM mapping are an improved level of molecular polymorphism as multiple alleles are detected at each locus. Second, accessions or breeding lines have been derived over many generations of meiotic events and this increases the number of crossover events in a defined chromosome interval which is expected to improve the resolution of trait/genotype associations. In plants, two strategies have been used for AM mapping: a) genome scans where the entire genome can be analyzed with molecular markers of sufficient density to localize the QTL (Kraakman et al. 2004; Rafalski 2002) or b) where a candidate gene has been identified, an association of polymorphic markers within the gene and a trait are examined (Thornsberry et al. 2001). An attractive feature of AM is the ability to perform marker trait associations in well phenotyped breeding populations and locally adapted varieties. However, population structure, due to selection and high levels of co-ancestry, is expected in pedigree-based breeding programs, resulting in a high probability of identifying of spurious associations (Pritchard et al. 2000). However, computational methods have been developed to account for population structure and relatedness to reduce identification of spurious associations (Yu et al. 2005). Studies on association mapping have been conducted in a number of crop species including barley (Kraakman et al. 2004) and wheat (Tommasini et al. 2007). However, most studies in wheat have focused on specific chromosomes where major QTL have previously been reported and further research to assess the potential of genome-wide association mapping for complex traits in wheat is needed. In this study, we performed genome-wide AM and report on a) the agreement of identified associations with previously published QTL b) and identification of novel QTL yet to be reported in the literature. 220 Session 5: Variety Development and Host Resistance MATERIALS AND METHODS AM Population and Molecular Analysis – Ninetysix (96) diverse durum wheat cultivars and breeding lines collected from breeding programs in Canada (25), Argentina (5), Australia (9), France (3), Italy (18), Germany (2), Mexico (3), Morrocco (3), United States (12), New Zealand (1), Russia (1), Iran (4), Spain (9), as well as one line of unknown origin formed the AM population. Genotyping was performed on an ABI 3100 capillary electrophoresis using M13labeled microsatellites. A total of 241 microsatellite (SSR) markers were used to amplify 245 loci. Trait Analysis and AM Mapping – Two traits were selected for genome wide AM study. Grain yellow pigment content (YP; mg kg-1) was assessed on a plot basis using AACC approved method 14-50 on samples collected from replicated trials grown at Saskatoon and Swift Current, Saskatchewan, Canada in 2005 and 2006. Resistance to stem rust race TTKS (UG99) was also assessed in 2007 at an endemic nursery in Kenya. Prior to AM analysis, population structure was assessed using a selection of 147 SSRs were selected at 2 cM intervals within the program STRUCUTRE v.2 (Pritchard et al, 2000). Structure parameter settings were: linkage model, allele frequencies correlated, burn-in length 10,000, and 10,000 repetitions. The highest likelihood was observed for K (no. sub-populations)=5, but little difference was observed between K=3 and K=5. Therefore, the Q matrix was estimated as the average of five runs for K=3. Marker-trait associations were determined using a general linear model in TASSEL version 2.0.1 with the Q-matrix as covariates. Pair-wise linkage disequilibrium (LD) of each SSR with allele specific CAPS markers for phytoene synthase genes Psy1A1 and Psy1-B1 was estimated as the squared allele frequency correlation (r2) within TASSEL. In cases of multiple alleles, a weighted average of r2 between loci was calculated. RESULTS AND DISCUSSION We first examined YP as a validation trait for AM as the genetics of this trait are well understood and QTL for have been well documented in the literature (Pozniak et al. 2007). Variation for YP was large, ranging from less than 5 mg kg-1 to greater than 12 mg ha-1, regardless of testing environments. Heritability estimates were high, ranging from 0.95 to 0.99. Using the sub-population Q-matrix as covariates in a general linear model, marker associations for YP were identified on five chromosomes (Fig. 1) and were statistically significant (P<0.01) in all four environments. These associations were coincident with previously published QTL (Fig. 1). Additional markers on chromosomes 2B, 3A and 3B were identified, but these were not consistent among testing environments (data not shown). Phytoene synthase (Psy) is the first critical enzyme in the biosynthesis of lutein, the major xanthophyll responsible for yellow pigment. Genes coding for Psy1 have been mapped in durum, and Psy1-B1 co-segregates with a QTL for YP on chromosome 7B (Pozniak et al. 2007). In genetic mapping studies, Psy1-B1 mapped approx. 4 cM from gwm146 and Pair-wise LD analysis revealed a CAPs marker for Psy1-B1 was in strong LD with that SSR (Fig. 2). AM confirmed that this gene was also associated with variation in YP (Fig. 1). Psy1-A1 was in disequilibrium with cfa2257 on 7A and genetic studies have confirmed that this gene is linked to cfa2257. Using AM, Psy1-A1 was associated with YP in the durum population (Fig. 1), and localizes to a region on 7A previously associated with YP (Elouafi et al., 2001). These results suggest that LD analysis can be used to correctly position genes in the durum wheat genome, and to determine their association with phenotypic variation. Interestingly, both genes were in LD with a region on 1A where a putative QTL for YP has been identified. Given the apparent success of genome wide AM mapping for YP, similar analyses were performed for resistance to stem rust race TTKS. Nearly half of the durum wheat accessions evaluated in the 2007 Kenya nursery were scored as moderately to high resistant. The remaining lines possessed intermediate resistance (n=25), or were scored as being moderately susceptible (n=21) or susceptible (n=10). Marker associations for TTKS using numerical severity ratings were 221 Session 5: Variety Development and Host Resistance identified on chromosomes 1B, 4B, and the group 5 disease resistance traits, including Fusarium head blight and 7 chromosomes (Fig. 3). (FHB) resistance. Only a few FHB resistance QTL have been reported in durum wheat, but given the reOf these, only two regions identified are known to sults of this study, AM could be used for identification house mapped Sr resistance genes. Two regions were of novel chromosome regions and alleles that confer identified on chromosome 7A, one distal to the cen- some degree of resistance. Although FHB resistance tromere, and a second at gwm276 and cfa2257 (Fig. has yet to be evaluated in balanced field trials in this 3). Sr22 is linked to gwm276, and that gene is effec- population, sufficient variation for resistance is known tive against TTKS (Jin et al., 2007). Linked markers to exist in and efforts to perform AM will be pursued. on 4B, including the lipoxygenase gene Lpx-B1.1, were Inclusion of additional FHB resistant breeding lines significantly associated with variation in disease resis- known to carry similar resistance (i.e. progeny of tance. Lipoxygenase is known to play a role in dis- known lines with improved tolerance) is being considease resistance and enzyme activity has been reported ered to improve statistical power for detecting relevant to increase in wheat treated with a rust fungal elicitor marker associations. (Bohland et al. 1997). Sr gene Tmp from winter wheat cultivar ‘Triumph 64’ is effective against TTKS (Jin et REFERENCES al., 2007) and that gene is believed to reside on 4B. Marker associations were identified for gwm291 and Bohland,C. et al. (1997) Differential Induction of lipoxygenase isoforms in wheat upon treatment with Rust Fungus Elicitor, wmc727 near the distal end of 5A and linked markers Chitin Oligosaccharides, Chitosan, and Methyl Jasmonate. wmc537 and Cdu1 on 5B, were also significant. Al- Plant Physiol. 114, 679-685. though the association on 5B is in a region were Sr genes have yet to be identified, this region has recently Buckler,E.S. and Thornsberry,J.M. (2002) Plant molecular diversity and applications to genomics. Curr. Opin. Plant Biol been associated with stem rust resistance in hexaploid 5, 107-111. wheat using AM (Crossa et al., 2007). Interestingly Psy1-B1 was associated with TTKS resistance (Fig. Crossa,J. et al. (2007) Association analysis of historical wheat germplasm using additive genetic covariance of relatives and 3). Although linkage with leaf rust resistance with YP population structure. Genetics, doi:10.1534/genethas been reported on 7A, linkage with stem rust has ics.107.078659 not been reported on 7B Elouafi,I. et al. (2001) Identification of a microsatellite on chromosome 7B showing a strong linkage with yellow pigment in durum wheat. Hereditas 135, 255-261. CONCLUSIONS Our results experimentally validate the potential of genome-wide AM to detect marker associations for complex traits in durum wheat. In the case of YP, marker associations were in agreement with previously identified QTL, suggesting that AM for that trait was effective in this population. For TTKS resistance, novel regions not associated with previously mapped Sr resistance genes were identified, and further validation of these marker-trait associations is a high priority. In particular, the association with Lpx-B1.1 will need to be confirmed, as high enzyme activity results in undesirable colour loss in pasta products. Given the apparent success of AM in this population, we are currently in the process of performing additional genotyping and more detailed phenotyping for mapping of other important agronomic, end-use quality and Jin,Y. et al. (2007) Characterization of Seedling Infection Types and Adult Plant Infection Responses of Monogenic Sr Gene Lines to Race TTKS of Puccinia graminis f. sp. tritici. Plant Disease 91, 1096-1099. Kraakman, AT. et al. (2004) Linkage disequilibrium mapping of yield and yield stability in modern spring barley cultivars. Genetics 168, 435-446. Nordborg,M. et al. (2002) The extent of linkage disequilibrium in Arabidopsis. Nat. Genet. 30, 190-193. Pozniak,C.J. et al. (2007) Identification of QTL and association of a phytoene synthase gene with endosperm colour in durum wheat. TAG. Pritchard,J.K. et al. (2000) Inference of population structure using multilocus genotype data. Genetics 155, 945-959. Rafalski,A. (2002) Applications of single nucleotide polymorphisms in crop genetics. Curr. Opin. Plant Biol 5, 94-100. 222 Session 5: Variety Development and Host Resistance Thornsberry,J.M. et al. (2001) Dwarf8 polymorphisms associate with variation in flowering time. Nat. Genet. 28, 286-289. Yu,J. et al. (2005) A unified mixed-model method for association mapping that accounts for multiple levels of relatedness. Nat. Genet. Tommasini,L. et al. (2007) Association mapping of Stagonospora nodorum blotch resistance in modern European winter wheat varieties. TAG 115, 697-708. 2A 4B 6B gwm192 wmc238 wmc89 wmc48 gwm368 barc20 wmc710 wmc177 wmc522 wmc296 gwm95 gwm558 gwm372 barc5 7A cfd13 gwm518 wmc494 gwm193 gwm311 7B wmc790 cfa2019 gwm554 Psy1-A1 wmc311 cfa2040 cfa2040 wmc10 Psy1-B1 gwm146 wmc809 barc24 Figure 1. Marker associations for YP in the durum wheat association mapping population. Bars to the right of the linkage group regions where significant associations (p<0.001) were identified at all four testing environments. Highlighted regions in the centre of the linkage groups are QTL regions previously identified using bi-parental mapping populations. Only relevant regions of the linkage groups are shown. 0.5 1A 1B 2A 2B 3A 3B Chromosome 4A 4B 5A 5B 6A 6B 7A 0.4 7B gwm146 0.3 R2 cfa2257 0.2 0.1 0 0 200 400 600 800 1000 1200 1400 1600 Cum ulative dis tance (cM) Figure 2. Pairwise LD (r2) with Psy1-B1 (__) and Psy1-A1 (__). The dashed line represents where the cumulative frequency of genome wide r2 reached 95%. 223 Session 5: Variety Development and Host Resistance 1B 21 25 26 30 31 32 34 35 37 38 4B gwm264 barc8 gwm413 wmc128 gwm498 wmc419 cfd65 cfd65 barc137 wmc626 wmc216 wmc694 barc181 19 20 25 28 30 34 36 37 38 48 5A wmc349 Lpx-B1.1 gwm251 gwm149 gwm192 wmc238 wmc89 wmc48 gwm368 barc20 wmc710 149 151 154 163 5B 80 82 84 89 gwm595 wmc524 wmc727 gwm291 7A wmc415 Cdu1 wmc537 gwm554 7B 40 47 48 55 wmc283 barc127 cfa2028 wmc83 65 71 72 73 76 83 92 barc174 barc108 wmc9 wmc603 barc121 gwm276 cfa2257 cfd20 145 146 150 wmc10 Psy1-B1 gwm146 Figure 3. Marker associations for TTKS in the durum wheat association mapping population. Bars to the right of the chromosomes represent regions where significant associations (p<0.001) were identified. Only relevant regions are presented. 224 Session 5: Variety Development and Host Resistance MOLECULAR CHARACTERIZATION OF A WHEAT-LEYMUS COMPENSATING TRANSLOCATION LINE CONFERRING RESISTANCE TO FUSARIUM HEAD BLIGHT. 1 L.L. Qi , M.O. Pumphrey2, B. Friebe1, P.D. Chen3 and B.S. Gill1* 1 Wheat Genetic and Genomic Resources Center, Department of Plant Pathology, Kansas State University, Manhattan KS; 2ARS-USDA, Plant Science and Entomology Research Unit, Manhattan KS; and 3The National Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing Jiangsu * Corresponding Author: PH (785) 532-1391; Email: bsg@ksu.edu ABSTRACT Fusarium head blight (FHB) resistance was identified in the alien species Leymus racemosus (syn Elymus giganteus). Several wheat-Leymus translocation lines with FHB resistance were developed using radiation treatment or gametocidal gene action. However, all of them are noncompensating, preventing their use in cultivar improvement. We have further screened 58 wheat-Leymus introgression lines from 14 siblings for their resistance to spread of infection within spikes. Of 24 lines with high levels of resistance to FHB, we determined that three lines (T01, T09, and T14) were lacking Sumai 3-type alleles at marker loci linked to Fhb1, indicating that the FHB resistance in these lines was most likely derived from L. racemosus. Previous cytogenetic data revealed that line T01 has a translocation, T4BS·4BL-7Lr#1S, and line T14 has a translocation, T6BL·6BS5Lr#1L. Line T09 has an unknown wheat-Leymus translocation chromosome. A total of 33 RFLP markers selected from seven homoeologous groups of wheat were used to screen T09. Only short arm markers of group-7 detected Leymus specific fragments in line T09. The 7AS-specific RFLP fragments were missing in T09, indicating that this line has a compensating Robertsonian translocation involving the long arm of wheat chromosome 7A and the short arm of Leymus chromosome 7Lr#1. C-banding and genomic in situ hybridization (GISH) analyses using Leymus genomic DNA as probe confirmed the RFLP results. RFLP analysis was further conducted in lines T01 and T14, as well as the wheat-Leymus disomic addition lines of DA5Lr#1 and DA7Lr#1, with 11 group-5 markers (five on the short arm and six on the long arm) and eight short arm markers of group-7 chromosomes. The results revealed that both lines T01 and T14 were complex translocations involving the short arms of Leymus chromosomes 5Lr#1 and 7Lr#1. These two lines have similar segments from 5Lr#1S. However, the length of 7Lr#1S segment in T01 and T14 is different. Line T01 contains an almost complete short arm of 7Lr#1, whereas about 50% of the distal portion of 7Lr#1S is transferred to the translocation chromosome in the line T14. Three translocation lines and the disomic addition 7Lr#1 were consistently resistant to FHB, whereas the disomic addition 5Lr#1 was susceptible. Because three translocation lines share a common distal segment of 7Lr#1S, a novel scab resistance gene from Leymus most likely resides in the distal region of the short arm of chromosome 7Lr#1. Three PCR-based markers, BE586744STS, BE404728-STS, and BE586111-STS, were developed to accommodate marker-assisted selection in breeding programs. Development of wheat-Leymus compensating recombinant lines with smaller alien segments that retain FHB resistance is underway by ph1b-induced homoeologous recombination. 225 Session 5: Variety Development and Host Resistance FHB RESISTANCE OF WHEAT LINES NEAR-ISOGENIC FOR FIVE DIFFERENT FHB RESISTANCE QTLS. Pilar Rojas-Barros1, Zachary J. Blankenheim2, Karen J. Wennberg1, Amar M. Elakkad1, Ruth Dill-Macky1 and David F. Garvin2* Dept. of Plant Pathology, University of Minnesota, St. Paul, MN; and 2USDA-ARS Plant Science Research Unit, St. Paul, MN * Corresponding Author: PH: (612) 625-1975; Email: garvi007@umn.edu 1 ABSTRACT To continue improving FHB resistance in hard red spring wheat (HRSW), it is imperative that new FHB resistance genes from wheat and its relatives be introduced into the HRSW germplasm base. In 2001 we initiated a program to use marker-assisted backcrossing to individually introgress five confirmed or postulated FHB resistance QTLs from diverse germplasm sources into three FHB-susceptible HRSW backgrounds (Norm, Wheaton, Apogee). The QTLs include two from Sumai 3 (Fhb1 and Qfhs.ifa-5AS) to serve as reference QTLs, one from the soft red winter wheat Freedom reportedly on chromosome arm 2AS, one from the Brazilian wheat Frontana on chromosome arm 3AL, and one from chromosome 3A of wild emmer (Qfhs.ndsu-3A). This individual QTL introgression permits evaluation of the effect of each QTL while simultaneously performing prebreeding introgression into HRSW germplasm. The development of the BC4-derived QTL near-isogenic lines (QTL-NILs) is now complete. For each genetic background/QTL combination, 4 to 5 independent resistant and susceptible NIL pairs were developed, except for the QTL Qfhs.ndsu-3A, for which genetic barriers prevented introgression into Norm and Apogee. These lines have been subjected to comparative FHB resistance evaluations both in the field and greenhouse. Results of these evaluations reveal the effect of these QTLs on FHB resistance improvement in the different HRSW genetic backgrounds. Evidence from greenhouse and/or field evaluations has accrued to indicate enhancement of FHB resistance by each of the QTLs introgressed, with both genetic background and the efficacy of the molecular markers contributing to the outcomes. The QTL-NIL series we have developed represent new germplasm for HRSW FHB resistance breeding efforts, and they are a useful resource for additional research on FHB resistance. For instance, we currently are using the lines to examine epistatic interactions between these QTLs to determine which combinations most effectively reduce FHB symptoms. Additional scientific uses include exploring the molecular basis of the wheat-F. graminearum interaction as well as the biological basis both of type I and type II resistance. ACKNOWLEDEMENT AND DISCLAIMER This material is based upon work supported by the U.S. Department of Agriculture, under Agreement No. 590790-4-096. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the U.S. Department of Agriculture. 226 Session 5: Variety Development and Host Resistance FAMILY BASED MAPPING OF FHB RESISTANCE QTLS IN HEXAPLOID WHEAT. Rosyara, R.U., Maxson-Stein, K.L., Glover, K.D., Stein, J.M and *Gonzalez-Hernandez, J.L. Dept. of Plant Sciences. South Dakota State University. SNP247. Brookings, SD 57007 * Corresponding Author: PH: 605-688-6907; Email: jose.gonzalez@sdstate.edu ABSTRACT Traditionally quantitative trait loci (QTL) mapping and marker aided selection programs have been two different ventures. For application in breeding programs, novel QTLs must be mapped, though mapping populations are generally not highly useful to breeders because the bi-parental cross is often composed of an unadapted parent. In contrast, breeding programs obtain the most success by creating a large number of breeding populations, or families, using adapted parental germplasm. We combined data from many breeding populations to map QTLs initially, and subsequently used markers of interest for selection. This single step approach is quick, simple, and employs pedigree information and variance component based linkage analysis to place QTLs with substantial effects. Experiments were conducted to validate the approach by mapping Fhb1. As part of an ongoing spring wheat breeding program, forty-five susceptible spring wheat genotypes were crossed in different combinations with at least one founder (i.e., a parent containing Fhb1). Eighty-three unique families were generated and 793 individuals were screened for resistance in the greenhouse using a point inoculation method. Genotyping was done using mapped simple sequence repeat markers. The QTL was placed in the same position as previous studies with a high probability value. These results demonstrate the usefulness of this approach to quickly map QTLs with relatively large effects, and should allow for marker aided selection as generations are advanced. 227 Session 5: Variety Development and Host Resistance FACING THE FHB CHALLENGES TO MALTING BARLEY AND BREWING THROUGH BARLEY BREEDING. L.G. Skoglund Busch Agricultural Resources, Inc., Fort Collins, CO 80524 Corresponding Author: PH: (970) 472-2332; Email: linnea.skoglund@anheuser-busch.com INTRODUCTION SOURCES OF RESISTANCE Fusarium head blight has posed the biggest challenge to the malting and brewing industries, possibly since Prohibition. While disease has lead to reduced yield and quality problems for farmers, DON has caused scarcity of high quality malting barley and relocation of production areas farther from the bricks and mortar (malt houses and breweries) of the industry. In response, BARI Seed Research integrated breeding for resistance into its domestic 6 rowed spring malting barley program with an emphasis on resistance to DON accumulation. Sources of resistance have been identified from the National Small Grains Collection (NSGC), Composite lines, ICARDA/CIMMYT breeding program, Swiss landraces, the Vavilov Collection in Russia and others. Screening of the NSGC was done by Skoglund and Menert at BARI (Skoglund and Menert, 2002) from 1998-2001 and Steffenson and Scholz at NDSU (Steffenson and Scholz, 2001) from 1999-2001. Over 8100 6 rowed spring barley accessions were screened by one or both groups. Screening was carried out in the field and in the greenhouse. The BARI group identified 15 accessions for possible breeding and the NDSU group identified 10. Only one accession, CIho 6613 (Seed Stocks 1148-1) was identified by both groups. THE CHALLENGES All malting barley cultivars grown in the 1990s were susceptible to FHB. Breeding programs had little or no resistance in their up-and-coming lines. The Challenge - Where to get resistance? Even under high disease pressure, it was risky to rely on natural infection in the field. Field trials are expensive and require many location years. Greenhouse inoculations, while giving high levels of disease and DON, overwhelmed the low levels of resistance available. The Challenge - How to insure good screening? FHB is difficult to evaluate and DON is costly to determine. The Challenge - How to measure DON in thousands of breeding lines? Meeting and overcoming these challenges has required a collaborative effort involving the entire global barley scientific community. What is reported here includes results from that global effort. BARI began collaboration with the ICARDA/ CIMMYT barley breeding program in 1999. Since then there has been 1) exchange of germplasm, 2) crossing of elite malting germplasm to resistant sources and 3) screening of various germplasm in nurseries run by BARI and collaborators in North Dakota and Minnesota. Some of the best parents from ICARDA/ CIMMYT are listed in Table 1. Dr. Brian Steffenson (University of Minnesota) has been instrumental in identifying and distributing sources of resistance from other, less accessible sources. These include Composite Cross XXX, Swiss landraces, the Vavilov Collection in Russia and Nordic Gene Bank (Steffenson, 2003; Steffenson and Dahl, 2003: Steffenson, Dahl and Luskutov, 2005). Table 2 has a list of the accessions used by BARI. 228 Session 5: Variety Development and Host Resistance FHB NURSERIES AND COLLABORATIVE SCREENING A number of misted, inoculated nurseries have been established around the world (Table 3). Data collected usually include FHB severity and, in some cases, DON concentration. BARI has collaborated with these institutions to have breeding lines tested. These nurseries have proven invaluable as insurance against low DON years in our yield trials. There are a number of collaborative screening trials in which BARI participates, some intended for FHB only and others that are primarily for yield and agronomic characteristics. The North American Barley Scab Evaluation Nursery (NABSEN) is an international screening nursery that includes six breeding programs and is planted in 8-10 locations, both dryland and irrigated. Data collected includes FHB incidence and severity, DON and any other (heading date, etc.). The Mississippi Valley Barley Nursery and the Midwest Coop are examples of trials that are grown by multiple collaborators in multiple locations. Where feasible, these are evaluated for resistance to FHB and DON. genes available within the BARI germplasm as well as UM and NDSU germplasm. Overall, this approach has been a matter of integrating multiple genes with minor effects into an elite line that has acceptable malt quality – not an easy task. Many additional sources of resistance to FHB/DON are in use at BARI. Projects are currently underway at various universities to determine their genetic diversity. Meanwhile, the most advanced of these were planted as F6 headrows in summer 2007. Selections from these were planted in the nursery in China in Fall 2007. Some of these should advance to first year yield trial status in 2008. These include crosses to the following resistant parents: • • • • • • • • COMP 351 & 355 HV 746, 779 and 531 B2027/5/Ataco/Bermejo//Higo/3/CLN/ Gloria/Copal/4/Chevron Legacy/Chamico Merit//Canela/Zhedar#2 Merit/MSEL Merit/4/Gob/Humai10//Canela/3/Aleli Arupo/K8755//Mora/3/Gob/ Humai10/4/ Shyri DON TESTING For the past 8-10 years, we have placed high priority on screening for DON accumulation in the BARI breeding program. This has been facilitated by the establishment and/or expansion of a number of facilities (Table 4). We primarily rely on the NDSU DON Testing Lab. Also, BARI Seed Research has collaborated with Dr. Nick Hill, Agrinostics Inc, in testing an ELISA-based technique for quantifying Fusarium graminearum mycelium in grain. Seed Stocks 1148-1 (CIho 6613) was crossed with Legacy in 2000. Though several selections did well in field trials, they failed to advance through our selection process. HV 527, VIR 16537, 28797 and 28807 and NGB9443 are in the current crossing block. THE FINAL CHALLENGE Most elite lines never make it through AMBA testing and Anheuser-Busch acceptance. The Final Challenge - How to get resistant cultivars into the beer? WHERE ARE WE NOW? Progress has been painfully slow due to lack of major genes with large effects in a background close to that needed for brewing. Legacy (accepted in 2001) has reduced DON accumulation to about 30% of that typically found in Robust. Lines in the program now reduce DON by 30-50% of Robust. This progress is the result of incremental integration of BARI continues to submit our best resistant lines to AMBA for testing, as do NDSU and UM. At this time, two lines have made it through AMBA and will be tested in brewing trials with the 2008 crop. These are the UM line M122 and the NDSU line ND20448. 229 Session 5: Variety Development and Host Resistance CONCLUSION Steffenson, B.J. and U. Scholz. 2001. Evaluation of Hordeum accessions for resistance to Fusarium head blight. Pages 208211 in: Proc 2001 National Fusarium Head Blight Forum. Dec. 8-10, 2001. Erlanger, KY. Malting barley breeding programs are making progress and meeting the challenges faced by industry. There have been incremental improvements in DON accumulation in advanced lines using a variety of genetic resources. These lines are slowly progressing through the testing and acceptance procedures on their way to farmers’ fields and the brew house. Steffenson, B.J. 2003. A population approach for identifying Fusarium head blight resistance in barley. Page 233 in: Proc 2003 National Fusarium Head Blight Forum. Dec. 13-15, 2003. Bloomington, MN. Steffenson, B.J. and S.K. Dahl. 2003. Evaluation of Swiss barley landraces for resistance to Fusarium head blight. Page 234 in: Proc 2003 National Fusarium Head Blight Forum. Dec. 13-15, 2003. Bloomington, MN. REFERENCES Skoglund, L.G. and J.L. Menert. 2002. Evaluation of the National Small Grains Collection of barley for resistance to Fusarium head blight and deoxynivalenol accumulation. Pages 213-215 in: Proc 2002 National Fusarium Head Blight Forum. Dec. 7-9, 2002. Erlanger, KY. Steffenson, B.J., S.K. Dahl and I. Loskutov. 2005. Fusarium head blight resistance in barley accessions from the N.I. Vavilov Institute. Page92 in: Proc 2005 National Fusarium Head Blight Forum. Dec. 11-13, 2005. Milwaukee, WI. Table 1. Two rowed and six rowed spring barley lines developed by ICARDA/CIMMYT with resistance to Fusarium head blight and other diseases. LINE SVANHALS-BAR/MSEL//AZAF/GOB24DH GOB/HUMAI10/3/ATAH92/ALELI TOCTE//GOB/HUMAI10/3/ATAH92/ALELI CANELA/ZHEDAR#2 ATACO/BERMEJO//HIGO/3/CLN/GLORIA/COPAL/4/CHEVRON CHAMICO MADRE SELVA PENCO/CHEVRON TYPE 2 rowed 2 rowed 2 rowed 2 rowed 2 rowed 6 rowed 2 rowed 6 rowed Table 2. New sources of resistance to Fusarium head blight utilized in the Busch Agricultural Resources, Inc. barley breeding program. ACCESSIONS COMP 351 COMP 355 HV 746 HV 779 HV 527 HV 531 VIR 20738 VIR 20733 VIR 16537 VIR 28797 VIR 28807 NGB 9443 TYPE 6 rowed 6 rowed 6 rowed 6 rowed 2 rowed 2 rowed 6 rowed 2 rowed 2 rowed 6 rowed 2 rowed 6 rowed YEAR 2003 2003 2003 2003 2003 2003 2004 2004 2007 2007 2007 2007 SOURCE Composite Cross Composite Cross Swiss Landrace s Swiss Landrace Swiss Landrace Swiss Landrace Vavilov Collection Vavilov Collection Vavilov Collection Vavilov Collection Vavilov Collection Nordic Gene Bank 230 STATUS Active Active Active Active Active Dropped Dropped Dropped Active Active Active Active Session 5: Variety Development and Host Resistance Table 3. Misted and inoculated Fusarium head blight nurseries available to barley breeding programs. INSTITUTION NDSU UM LOCATIONS Osnabrock, ND St. Paul, Morris and Crookston, MN BARI Fort Collins, CO AAFC Brandon, MB ZHEJIANG UNIVERSITY Hangzhou, China CIMMYT El Batan, Mexico # BARLEY ENTRIES 21000 hills, 1200 rows, 90 plots 12000 rows 350 rows 18000 rows 10000 half rows 3000-3500 rows Table 4. DON testing facilities available to barley breeders. USWBSI FUNDING Yes COMMENT Up from 4000 samples in 2000 INSTITUTION NDSU # BARLEY SAMPLES 18000 in 2006 12000 in 2007 (est) UM 5000 in 2006/2007 Yes AAFC 7000-9000 samples/yr No Private lab - 3000 samples CIMMYT unknown No Est. by Dr. Lucy Gilchrest 231 Session 5: Variety Development and Host Resistance DEVELOPMENT OF BARLEY VARIETY CANDIDATE M122WITH ENHANCED RESISTANCE TO FHB. Kevin P. Smith*, Ed Schiefelbein and Guillermo Velasquez Dept. of Agronomy and Plant Genetics, University of Minnesota, St. Paul, MN 55108 * Corresponding Author: PH: 612-624-1211; Email: smith376@umn.edu ABSTRACT M122 is a barley variety candidate with enhanced FHB resistance, good malting quality and yield performance similar to currently grown varieties in the Midwest. This line was developed from the sources of resistance Chevron and Zhedar 1 over three breeding cycles. Based on 27 field experiments conducted between 2002 and 2007, M122 has FHB severity and DON levels that are 47% and 52% of the common variety check Robust, respectively. M122 was developed using field-based screening for FHB resistance and low DON in harvested grain. Simultaneous selection was imposed for agronomic performance and malting quality. M122 does not appear to contain resistance source alleles at known and validated QTL for FHB or DON. Development M122 is an experimental breeding line, with enhanced FHB resistance, from the University of Minnesota that has potential as a new six-rowed malting variety for the Upper Midwest barley production area. M122 is an F5 derived selection from the cross FEG18-20 / M110 (Figure 1). FEG18-20 was a line selected for its FHB (Fusarium Head Blight) tolerance over several years of disease screening from the cross MNBrite / SI4-29. SI4-29 was also a line selected for its FHB tolerance from the cross Zhedar 1 / Stander // Foster, which was given to us by Rich Horsley as F2 seed in 1994. M122 was advanced by single seed descent from the F1 thru F4 generations. It was selected from an FHB tolerant population (FEG65) in 2002 as an F4.5 line, then seed from a single F5 plant was increased in New Zealand for preliminary yield testing and malt quality evaluations in 2003. M122 was included in the Mississippi Valley Nursery in 2005 (limited locations), 2006 and 2007. M122 was entered into AMBA Pilot Malt evaluations with the 2005 and 2006 crops. M122 is scheduled to be evaluated in plant-scale brewing evaluations with the 2008 crop. From 2002 – 2007, M122 was evaluated for FHB resistance and DON level in numerous FHB nurseries. FHB Screening and Performance Screening for resistance that lead to the development of M122 was conducted entirely in inoculated and mist-irrigated field nurseries in Minnesota or in an offseason nursery in China. Disease evaluation in the barley improvement project is a large collaborative effort that involves personnel from the barley breeding project, Ruth Dill-Macky and Char Hollingsworth’s pathology programs, and staff at the UM Research and Outreach Centers at Morris and Crookston. Off-season screening in Hangzhou China was done in collaboration with Dr. Bingxin Zhang at Zhejiang University in Hangzhou. Disease screening of M122 began 2002 in replicated F4:5 plots. M122 has been evaluated for resistance to FHB and DON in 25 field experiments conducted between 2002 and 2007. M122 has FHB severity and DON levels that are 47% and 52% of the common variety check Robust, respectively. Agronomic Performance In Minnesota State Variety Trials (2004-2007) based on the 12 location mean, M122 was the top yielding variety (Table 1). In on-farm trials conducted in Minnesota, M122 was among the higher yielding lines although had slightly more lodging (Table 2). In North Dakota trials in 2007, M122 yielded better than Ro- 232 Session 5: Variety Development and Host Resistance bust but not as high as Lacey, Tradition and Legacy (Table 3). 59-0790-4-120. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. Graphical Genotype REFERENCES We assessed M122 and all of the parents in the pedigree (Figure 1) with DArT markers (Wenzl et al., 2006). A total of 884 markers provided a good signal in the assay and 165 of those were polymorphic between the parents of M122. We examined chromosome 2H and 6H since these chromosomes have been implicated in FHB resistance in mapping studies with Chevron, Zheddar 2 and other sources of resistance(de la Peña et al., 1999; Dahleen et al., 2003; Canci et al., 2004). In the two QTL regions that have been mapped and validated in Chevron, M122 appears to carry the susceptible parent allele. We were also able to confirm that M122 does not carry the Zhedar 1 allele in these regions. This indicates that we should be able to improve resistance in M122 via MAS for the Chevron or Zhedar 1 alleles at these QTL. Canci, P. C., L.M. Nduulu, R. Dill-Macky, G.J. Muehlbauer, D.C. Rasmusson, and K.P. Smith. 2004. Validation of Quantitative Trait Loci for Fusarium Head Blight and Kernel Discoloration Resistance in Barley. Mol. Breeding 14:91-104. ACKNOWLEDGEMENT This material is based upon work supported by the U.S. Department of Agriculture, under Agreement No. Zhedar 1 Stander Foster ZS 1 Dahleen, L.S., H.A. Agrama R.D. Horsley, B.J. Steffenson, P.B. Schwarz, A. Mesfin, J.D. Franckowiak. 2003. Identification of QTLs associated with Fusarium head blight resistance in Zhedar 2 barley. Theor Appl Genet (2003) 108:95– 104. de la Peña, R. C., Smith, K. P., Capettini, F., Muehlbauer, G. J, Gallo-Meagher, M., Dill-Macky, R., Somers, D. A., and Rasmusson D. C. 1999. Quantitative trait loci associated with resistance to Fusarium head blight and kernel discoloration in barley. Theor. App. Genet. 99:561-569. Wenzl, Peter, Haobing Li, Jason Carling, Meixue Zhou, Harsh Raman, Edie Edie, Phillippa Hearnden, Christina Maier, Ling Xia, Vanessa Caig, Jaroslava Ovesna, Mehmet Cakir, David Poulsen, Junping Wang, Rosy Raman, Kevin P Smith, Gary J Muehlbauer, Ken J Chalmers, Andris Kleinhofs, Eric Huttner and Andrzej Kilian. 2006. A high-density consensus map of barley linking DArT markers to SSR, RFLP and STS loci and agricultural traits. BMC Genomics 2006, 7:206 Figure 1. M122 was developed over three breeding cycles and has two resistant sources in its pedigree (Zhedar 1 and MNBrite) MNBrite derives its resistance from Chevron. Lines or varieties in boxes are susceptible to FHB and those in ovals have enhanced resistance to FHB. SI4-29 MNBrite Feg18-20 M110 M122 233 Session 5: Variety Development and Host Resistance 120% M122 MNBrite DON (% of Robust) 100% 80% 60% 40% 20% 8. 8 10 .0 10 .1 10 .6 10 .8 11 .0 11 .3 12 .0 12 .2 16 .3 29 .6 48 .3 8. 0 4. 7 4. 4 4. 5 4. 3 4. 0 3. 9 3. 7 3. 3 3. 4 2. 9 1. 7 1. 6 0% Mean DON level in nursery (Robust, Stander, MNBrite, M122) Figure 2. DON levels as a percent of Robust for M122 and MNBrite in 25 trials representing a wide range of disease pressure. Table 1. Yield comparisons of M122 compared to check varieties 2005-2007 in Minnesota State Variety Trials (mean of 12 trials). Crookston Morris Stephen St.Paul Roseau 12 3 yr. 3 yr. 2 yr. 2 yr. 2 yr. Sta.Yr. Variety Ave. Ave. Ave. Ave. Ave. Ave. Robust Stander MNBrite Lacey Drummond Stellar ND Legacy Tradition Conlon 91 94 92 94 93 96 95 96 97 71 77 70 79 72 66 79 80 69 84 86 86 99 92 94 103 101 96 100 98 93 96 106 90 106 95 81 86 92 74 76 84 84 85 88 73 85 88 83 88 88 85 92 91 83 M122 103 77 96 113 81 93 234 Session 5: Variety Development and Host Resistance Table 2. Yield comparisons of M122 compared to check varieties 2006-2007 in Minnesota On-Farm trials (mean of 5 locations). Yield Test Plant Variety/Line (Bu/A) Weight Protein Plump Height Lodging* Drummond 107.5 43.9 13.2 72.5 32.6 1.0 Lacey 105.5 45.6 13.6 73.9 31.4 1.0 Legacy 99.1 41.5 13.0 61.8 31.2 1.3 Robust 100.1 44.6 13.8 69.4 32.4 1.8 Stellar 107.2 44.2 12.7 77.1 31.9 1.7 Tradition 101.7 44.5 13.2 74.7 31.7 1.0 M122 105.5 43.4 13.3 Data provided courtesy Jochum Wiersma *data from 2007 only 68.7 32.6 3.2 Table 3. Yield comparisons of M122 compared to check varieties 2007 in North Dakota State Variety Trials (mean of 5 trials). Days to Plant Heading Height Lodging Stem break Yield Variety / Line (June) (inches) (%) (1-5) (Bu/A) Robust 25.0 33 33 2.5 75.7 Lacey 24.7 32 30 2.5 84.0 Drummond 24.4 32 25 1.9 78.1 Stellar-ND 24.3 32 26 2.6 76.7 Legacy 26.5 32 34 2.2 82.8 Tradition 25.2 32 31 1.5 80.5 M122 24.9 31 31 2.3 79.0 Data provided courtesy Richard Horsley 235 Session 5: Variety Development and Host Resistance 6H 2H 0 0 20 20 40 40 60 60 80 80 100 100 120 120 140 140 160 160 Figure 3. Graphical genotype of DArT markers for M122 on chromosomes 2H and 6H. Numbers are centimorgans. Dark squares indicate that M122 carries the resistant parent (Feg18-20) allele at the marker locus and grey squares indicate M122 carries the susceptible parent allele (M110). Ovals indicate position of QTL for FHB. 236 Session 5: Variety Development and Host Resistance REPORT ON THE 2006-07 NORTHERN UNIFORM WINTER WHEAT SCAB NURSERIES (NUWWSN AND PNUWWSN). C. Sneller1*, P. Paul2, L. Herald1, B. Sugerman1 and A. Johnston2 Dept. of Horticulture and Crop Science, and 2Dept. Plant Pathology, The Ohio State University, Wooster, Ohio 44691 * Corresponding Author: PH: (330) 263-3944; Email: sneller.5@osu.edu 1 from 13 programs while the PNUWWSN entries came from nine programs (Table 2). OBJECTIVES This is a summary of the report on the 2006-2007 Northern Uniform Winter Wheat Scab Nursery (NUWWSN) and the Preliminary Northern Uniform Winter Wheat Scab Nursery (PNUWWSN). A full report will be available on the USWBSI web site prior to the 2007 forum. The objective of these tests is to screen winter wheat genotype adapted to the northern portion of the eastern US for scab resistance. RESULTS There are eight FHB traits for each trail. Entries with means that were not significantly different than the lowest mean for six or more FHB traits are shown in Tables 3 and 4 (eg entries with at least 6 “l”s). Only three entries had DON < 2 ppm (entries 5, 22, and 26 in the PNUWWSN, see Tables 4 and 5). MATERIAL AND METHODS The traits assessed and locations that reported data are listed in Table 1. Entries for the NUWWSN came Table 1. Traits assessed in the 2006-07 PNUWWSN and NUWWSN tests. Code Trait SEV Disease severity from field tests % of infected spikelets in an infected head. IL,KY,MI,MO,ON,VA IL,KY,MD,MI,MO,NE,NY,OH,ON, VA INC Disease incidence % of heads with at least one infected spikelets IL,KY,MI,MO,ON,VA IL,KY,MD,MI,MO,NE,NY,OH,ON, VA IND Disease index IND = (SEVxINC)/100 KR Kernel rating A visual assessment of the percent infected kernels IL,IN,,KY,MI,MO,OH,ON, VA IL IL,IN,KS,KY,MD,MI,MO,NE,NY, OH,ON,RO,VA IL,KS PSS Percent scabby seed Composite of head and kernel traits KY,MO KY,MD,MO,NE,RO IL,KY,MO IL,KY,MD,MO,NE ISK DON GH DON (vomitoxin) Description Percent of scabby seed by weight ISK Index = .3 (Severity) + .3 (Incidence)+.4 (% FDK or PSS) PPM of vomitoxin in grain Greenhouse Same as SEV except from severity greenhouse * ON and RO indicate Ontario Canada, and Romania, respectively PNUWWSN Locations* NUWWSN Locations* IL,KY,VA IL,KS,KY,MD,NE,NY,VA IL IL,MO 237 Session 5: Variety Development and Host Resistance Table 2. Entries in the 2006-07 PNUWWSN and NUWWSN. NAME ERNIE TRUMAN FREEDOM PIONEER 2545 P.981129A1--17 P.99751RA1--94 PNUWWSN PEDIGREE Moderate Resistant Check Mod Resistant/Resistant Check Moderate Resistant Check Susceptible Check 92829A1/Patton 92212/961331/5/92212/4/F201R/3/9547// Patterson/Ernie 981129A1/981312A1 NAME KS04HW47-3 KS04HW101-3 P.011035A1-71 P.011036A1-14 P.02444A1-23-6 P.03647A1-1 NUWWSN PEDIGREE (CONTINUED) X921012-A-7-1/TGO 98HW423/98HW170 981128A1/981477A1//92145E8 981128A1/97462A1//92145E8 981129A1/99793RE2//INW0301/92145E8 981477A1/INW0315//981517A1/97462A1 P.04287A1-10 INW0315*2/5/INW0304/4/9346/CS5A// 91202/3/INW0301/INW0315 INW0315/9895C1/3/INW0301/INW0304// 981542A1 99751RA1/INW0315//981358C1/97462 P25R57/SE1694-12 NE01643 NE04653 NE03490 MD01W233-06-11 M03-3002 M03-3104 M03-3616 M03*3877 M03*3861 RCUOG19/21 RCUOGF110202D/4 RCUOGF111202A/3 AC Ron/WEK0609H3xACRon SD97060 x Ringo Star Freedom x Harding RCUOGDHACF1109O2D RCUOGNS984-1 IL00-8530 IL01-11445 IL01-11934 SD97060 x Freedom Not available IL89-1687 // IL90-6364 / IL93-2489 IL87-2834-1 / IL95-678 IL90-6364 / IL94-1909 IL02-19463 IL02-23168 KY97C-0540-01-03 KY97C-0554-03-06 KY97C-0554-04-05 KY97C-0508-01-01A-1 KY97C-0554-03-02 MO 040165 MO 050101 MO 050143 MO 050197 Patton / Cardinal // IL96-2550 IL94-1909 / Pioneer25R26 // IL95-4162 COKER 9803/L910097//2552 VA94-54-549/Roane//Kristy VA94-54-549/Roane//Kristy FFR 555W/VA94-52-25//2568 VA94-54-549/Roane//Kristy Bess RS, earlier Bess RS, same Bess RS, shorter MO 12278/Pioneer 2552 VA06W-608 VA06W-627 TAISHANG1/GR863//CARDINAL T814/L880119 KM2186-92/M94*1649//Patton Pio26R61/Patton VA94-54-479/Pio2628 Madison/Roane M94*1586-1/Roane N/A ACRONxSVP/R/FR.#1 2737W x EX9806/TF13 SVPx ACRON/TF18 ACRON x R/FR. #1 IL97-3574 / IL95-4162 IL96-2526 / IL97-3574 Ernie/ IL95-4162 IL84-2191 / IL87-2834 // IL90-6364 / IL9624851 IL94-6727 / IL96-6472 KY 89C-804-11/KY 89C-225-5//2540 2552/2684//2540 NC96 BGT 6/2552//25R26 Kingraze/Bess ‘S’ 950016/3/950016//90X54-1-1/MO 91-1009 Truman ‘S’/MO 960815 Ernie/Truman ‘S’ P89118RC1-X-9-3-3-1/TRIBUTE//M94-1069 IL 94-1549/AGS 2000,F8 P88288C1-6-1-2-8/VAN98W-346//RC STRAT. FREEDOM/NC96-13374//RC STRATEG, F7 IL 94-1549/VA97W-375//COKER 9025, F7 NE94482 (=ARA/ABILENE//NE86488)/ND8974 NE90614 /NE87612 NE96644//PAVON/*3SCOUT66/3/NE9465 3 N97S084//W96-500W/N95L158 WI90-540W/NE93554 MCCORMICK/CHOPTANK Winter/Winter FHB Bulk Hopewell / M94-1107 Hopewell / Patton T8141 / D93-6093 Pio2552 / M94-1407 OH03-183-32 OH03-235-2 OH03-41-45 15497 /897A OH552 /HOPEWELL IL91-14167 /OH599 MSU Line E3023 MSU Line E5015 MSU Line E6001 MSU Line E6002 MSU Line E6003 VA06W-600 OH03-97-6 P88288C1-6-1-2 /OH536 OH03-75-58 HOPEWELL /OH655 CALEDONIA/NY85020-395 CALEDONIA/PIONEER_25W33 PIONEER_25W60/CJ9306 VA96W-403WS /CJ9403 VA96W-403WS /W14 P89118RC1-X-9-3-3-1/TRIBUTE// M94-1069,F7 P89118RC1-X-9-3-3-1/TRIBUTE// M94-1069, F7 ROANE//OH 552/AGS 2000, F7 P88288C1-6-1-2-8/VAN98W-346//RC STRATEGY Roane / Ernie//McCORMICK,F8 P.0128A1-44-1-7 P.03528A1-10 P.03630A1-18 SE981089-34 SE91 1492-4 SE94-1012-25 M04-4843 M04-4788 M04*5109 M04-4258 M04-4393 RCUOGGold.Val RCUOGL15 RCUOGL4 RCUOGL17 RCUOG10/18 IL03-18438 IL03-15452 IL03-453 IL01-34159 IL79-002T-B-B KY99C-1298-08-1 KY99C-1051-03-1 KY99C-1176-02-1 MO 050600 MO 050699 MO 050917 MO 050921 VA06W-598 VA06W-557 VA06W-595 HARRY NI04421 VA06W-602 VA06W-587 VA06W-594 NAME ERNIE TRUMAN FREEDOM PIONEER 2545 NY88046-8138 NUWWSN PEDIGREE Moderate Resistant Check Mod Resistant/Resistant Check Moderate Resistant Check Susceptible Check Susquehanna/Harus NY93285SP-7343 NY93285-7110 NY91028SP-9082 NY93306-7091 SuMei Comp: 92002 SuMei Comp: 92002 Harus/4/CS/A.Curvif//Glenn/3/Ald/Pvn(M-30) 18cc-59/Pio2548 VA06W-585 OH02-15978 OH02-12678 OH02-12686 OH02-13567 238 PATTERSON/HOPEWELL FOSTER/HOPEWELL//OH581/OH569 FOSTER/HOPEWELL//OH581/OH569 OH581/IN83127E1-24-5-2//5088B-D-321/OH601 Session 5: Variety Development and Host Resistance Table 3. Best entries (top) and worst (bottom) from the 2006-07 NUWWSN. Summary statistics are for all 60 entries. 2 51 43 45 44 50 59 46 31 49 29 58 NAME TRUMAN MSU Line E6003 MO 040165 MO 050143 MO 050101 MSU Line E6002 OH02-12686 MO 050197 RCUOGDHACF 1109O2D MSU Line E6001 RCUOGF110202D/4 OH02-12678 SEV 11.2 14.0 20.2 19.5 21.4 17.5 20.2 17.7 18.3 19.1 22.9 19.4 l l l l l l l l l l l INC 26.7 18.3 34.7 38.1 40.4 27.1 35.5 33.7 21.4 41.5 25.7 39.5 l l l l l IND 6.1 6.1 12.2 12.2 12.7 12.9 14.6 11.6 12.1 14.2 14.7 17.0 l l l l l l l l l l l KR 17.5 24.4 16.2 20.6 26.2 35.6 25.0 17.9 47.1 33.1 32.1 15.3 l l l l l l l l 5 32 24 4 10 PSS 12.6 17.4 15.4 7.1 7.3 13.9 17.1 8.6 22.7 l l l l l l l l h ISK 16.6 12.6 25.6 26.7 26.3 26.7 26.0 21.3 26.7 l l l l l l l l l DON 3.9 4.8 3.1 3.9 3.9 4.6 2.1 3.7 3.9 l l l l l l l l l GHSEV 3.4 8.4 4.6 6.2 4.5 25.5 24.0 39.1 25.4 14.4 11.1 11.2 l l l 25.0 23.5 25.2 l l l 4.6 1.7 4.2 l l l h h h h h 7.8 8.2 8.4 11.6 16.6 5.7 3.9 0.8 60.9 NY88046-8138 38.6 h 54.1 h 24.8 h 44.6 28.0 h 48.5 RCUOGNS984-1 38.0 h 61.1 h 28.3 h 45.6 26.6 h 42.6 M03-3104 31.4 h 64.3 h 25.5 h 50.9 h 22.6 h 49.5 PIONEER 2545 39.0 h 62.3 h 30.0 h 49.8 h 18.5 lh 50.3 KS04HW47-3 41.5 h 62.7 h 34.1 h 65.0 h 21.2 h 46.1 AVERAGE 24.1 40.4 17.0 33.3 16.2 30.5 LSD 11.2 14.3 10.0 17.6 14.8 14.1 R2 0.53 0.73 0.52 0.79 0.48 0.52 CV 46.7 34.7 66.2 27.6 64.9 32.6 n 10 10 13 2 5 5 l,h indicate a mean that is not significantly different than the lowest (l) or highest (h) mean in that column 239 h l #l 8 8 7 7 7 7 7 6 6 #h 0 0 0 0 0 0 0 0 1 4.9 10.7 13.3 l l l 6 6 6 0 0 0 46.6 46.9 59.1 50.5 70.6 26.1 28.8 0.71 57.8 2 h h h h h 0 0 0 1 0 4.8 l l l l l l l 6 6 7 7 8 2.1 Session 5: Variety Development and Host Resistance Table 4. Best entries (top) and worst (bottom) from the 2006-07 PNUWWSN. Summary statistics are for all 44 entries. 2 26 31 34 27 40 23 5 6 37 8 43 24 9 32 22 1 44 25 33 16 13 NAME TRUMAN IL01-34159 MO 050600 MO 050921 IL79-002T-B-B OH03-183-32 IL03-18438 P.981129A1--17 P.99751RA1--94 VA06W-595 P.03528A1-10 OH03-97-6 IL03-15452 P.03630A1-18 MO 050699 RCUOG10/18 ERNIE OH03-75-58 IL03-453 MO 050917 M04-4258 M04-4843 SEV 16.9 18.9 21.7 19.9 19.4 19.5 23.1 23.7 23.3 25.0 16.9 18.8 22.0 21.0 22.6 31.0 22.8 23.6 24.3 24.3 29.4 32.3 l l l l l l l l l l l l l l l l l l l l INC 38.4 40.1 39.1 42.7 40.3 46.1 54.7 47.0 47.9 49.9 43.2 47.7 40.0 50.8 50.8 48.0 44.5 50.0 57.5 55.7 56.8 42.5 l l l l l l l l l l l l l l l l l l l l IND 7.3 8.0 10.0 10.4 11.1 12.2 13.3 13.4 15.2 16.7 9.6 10.0 11.0 11.2 12.6 16.8 12.9 13.3 15.8 16.0 17.9 20.4 l l l l l l l l l l l l l l l l l l l l l KR 13.0 8.0 20.0 15.0 11.0 15.0 18.0 23.0 20.0 25.0 30.0 33.0 12.0 27.0 25.0 11.0 33.0 60.0 25.0 37.0 22.0 15.0 l l l l l l l l l l l l l l l l 4 12 10 18 PSS 9.4 5.5 9.8 6.4 4.0 7.7 5.1 19.2 15.6 13.7 5.6 18.6 7.6 10.5 10.6 6.8 12.7 12.5 3.0 6.9 9.1 10.8 l l l l l l l l l l l l l l l l l l l l l l ISK 17.8 14.9 18.9 17.2 11.8 19.7 20.2 22.4 23.8 22.3 22.2 24.1 15.1 24.0 22.5 16.9 23.6 32.5 23.5 31.0 24.9 20.0 PIONEER 2545 38.1 69.0 h 30.9 h 47.0 28.6 h 39.8 SE94-1012-25 49.6 h 60.0 33.5 h 63.0 h 32.0 h 43.5 SE981089-34 47.8 h 64.4 h 36.4 h 80.0 h 42.8 h 48.0 RCUOGGoldenValue 53.3 h 80.4 h 38.9 h 40.1 h 46.7 Average 26.5 50.3 16.3 31.7 13.6 24.9 LSD 13.9 17.5 11.6 17.8 19.6 12.8 R2 0.6 0.6 0.6 0.8 0.7 0.8 CV 42.5 42.5 65.5 33.4 65.3 29.4 # Locations 6 6 8 1 2 3 l,h indicate a mean that is not significantly different than the lowest (l) or highest (h) mean in that column 240 l l l l l l l l l l l l l l l l l l l h h h h DON 2.2 0.3 1.8 2.7 3.4 2.0 4.1 0.4 3.9 3.4 3.6 4.0 3.7 2.6 4.9 1.3 4.9 3.5 3.7 4.4 4.1 2.6 6.5 6.7 9.9 8.3 3.8 4.3 0.9 55.3 3 l l l l l l l l l l l l l l l l l l l l h h h h GHSEV 12.3 3.2 18.0 4.0 21.1 32.9 12.5 3.8 4.1 37.5 28.8 37.8 58.2 3.4 6.0 17.4 28.6 40.8 59.3 27.5 4.8 36.1 62.1 38.8 85.4 36.7 38.6 0.4 90.1 1 #l l l l l l l l l l l l l l l l l l l l l h l h 8 8 8 8 8 8 8 8 8 8 7 7 7 7 7 7 6 6 6 6 6 6 #h 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 4.8 5 7 7 7 1.2 Session 5: Variety Development and Host Resistance Table 5. Summary of results of the 2006-076 PNUWWSN. NAME SEV INC IND KR PSS ISK ERNIE 22.8 l 44.5 l 12.9 l 33.0 12.7 l 23.6 TRUMAN 16.9 l 38.4 l 7.3 l 13.0 l 9.4 l 17.8 FREEDOM 28.3 l 47.5 l 15.6 l 37.0 12.5 l 26.2 PIONEER 2545 38.1 69.0 h 30.9 h 47.0 28.6 h 39.8 P.981129A1--17 23.7 l 47.0 l 13.4 l 23.0 l 19.2 l 22.4 P.99751RA1--94 23.3 l 47.9 l 15.2 l 20.0 l 15.6 l 23.8 P.0128A1-44-1-7 27.6 l 56.9 17.1 l 40.0 17.1 l 30.3 P.03528A1-10 16.9 l 43.2 l 9.6 l 30.0 5.6 l 22.2 P.03630A1-18 21.0 l 50.8 l 11.2 l 27.0 10.5 l 24.0 SE981089-34 47.8 h 64.4 h 36.4 h 80.0 h 42.8 h 48.0 SE91 1492-4 31.7 55.0 l 21.5 32.0 13.0 l 28.7 SE94-1012-25 49.6 h 60.0 33.5 h 63.0 h 32.0 h 43.5 M04-4843 32.3 42.5 l 20.4 15.0 l 10.8 l 20.0 M04-4788 38.4 42.1 l 16.4 l 37.0 20.1 l 29.2 M04*5109 25.0 l 53.7 l 16.0 l 50.0 4.6 l 25.6 M04-4258 29.4 l 56.8 17.9 l 22.0 l 9.1 l 24.9 M04-4393 24.7 l 59.7 14.7 l 35.0 14.0 l 29.1 RCUOGGoldenValue 53.3 h 80.4 h 38.9 h 40.1 h 46.7 RCUOGL15 29.8 l 52.1 l 17.9 l 43.0 23.1 35.6 RCUOGL4 37.8 53.8 l 22.6 47.0 13.9 l 31.1 RCUOGL17 31.6 51.8 l 17.7 l 40.0 11.2 l 25.6 RCUOG10/18 31.0 48.0 l 16.8 l 11.0 l 6.8 l 16.9 IL03-18438 23.1 l 54.7 l 13.3 l 18.0 l 5.1 l 20.2 IL03-15452 22.0 l 40.0 l 11.0 l 12.0 l 7.6 l 15.1 IL03-453 24.3 l 57.5 15.8 l 25.0 l 3.0 l 23.5 IL01-34159 18.9 l 40.1 l 8.0 l 8.0 l 5.5 l 14.9 IL79-002T-B-B 19.4 l 40.3 l 11.1 l 11.0 l 4.0 l 11.8 KY99C-1298-08-1 30.0 l 50.4 l 19.9 35.0 13.7 l 24.3 KY99C-1051-03-1 33.3 67.8 h 25.0 43.0 14.8 l 33.6 KY99C-1176-02-1 31.7 63.3 h 19.7 37.0 28.8 h 34.4 MO 050600 21.7 l 39.1 l 10.0 l 20.0 l 9.8 l 18.9 MO 050699 22.6 l 50.8 l 12.6 l 25.0 l 10.6 l 22.5 MO 050917 24.3 l 55.7 l 16.0 l 37.0 6.9 l 31.0 MO 050921 19.9 l 42.7 l 10.4 l 15.0 l 6.4 l 17.2 VA06W-598 28.9 l 51.4 l 22.0 30.0 11.3 l 25.7 VA06W-557 35.5 62.4 27.1 50.0 26.3 h 30.5 VA06W-595 25.0 l 49.9 l 16.7 l 25.0 l 13.7 l 22.3 VA06W-608 30.9 62.6 17.8 l 35.0 16.0 l 30.8 VA06W-627 35.1 62.7 27.8 h 38.0 19.6 l 34.4 OH03-183-32 19.5 l 46.1 l 12.2 l 15.0 l 7.7 l 19.7 OH03-235-2 28.7 l 61.4 19.6 38.0 27.3 h 30.8 OH03-41-45 34.3 56.6 21.6 30.0 14.5 l 29.4 OH03-97-6 18.8 l 47.7 l 10.0 l 33.0 18.6 l 24.1 OH03-75-58 23.6 l 50.0 l 13.3 l 60.0 12.5 l 32.5 Average 28.5 52.7 17.8 31.7 14.9 26.9 LSD 13.9 17.5 11.6 17.8 19.6 12.8 R2 0.6 0.6 0.6 0.8 0.7 0.8 CV 42.5 42.5 65.5 33.4 65.3 29.4 # Locations 6 6 8 1 2 3 l,h indicate a mean that is not significantly different than the lowest (l) or highest (h) mean in that column 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 241 l l h l l l l h h l h h l l l l l l l l l l l l l DON 4.9 2.2 6.0 6.5 0.4 3.9 3.0 3.6 2.6 9.9 4.9 6.7 2.6 2.8 5.8 4.1 5.6 8.3 4.6 6.9 4.5 1.3 4.1 3.7 3.7 0.3 3.4 7.8 9.1 7.5 1.8 4.9 4.4 2.7 4.0 8.4 3.4 5.6 7.2 2.0 6.3 4.7 4.0 3.5 4.6 4.3 0.9 55.3 3 l h h l l l l l h h l l h l h h l h l l l l l l l h h h l l l l h l h h l h l l GHSEV 28.6 12.3 5.0 3.8 4.1 4.9 28.8 3.4 38.8 44.5 62.1 36.1 100.0 21.5 4.8 65.4 85.4 76.2 40.3 66.7 17.4 12.5 58.2 59.3 3.2 21.1 40.9 52.1 62.2 18.0 6.0 27.5 4.0 60.7 42.4 37.5 32.5 66.0 32.9 61.5 52.7 37.8 40.8 36.7 38.6 0.4 90.1 1 #l l l l l l l l l l h l h l l h h h l h l l l l l h l l l l l l h l h l l 6 8 5 0 8 8 5 7 7 1 2 0 6 4 5 6 3 0 4 3 4 7 8 7 6 8 8 5 1 0 8 7 6 8 4 0 8 3 1 8 1 1 7 6 4.8 #h 0 0 1 5 0 0 0 0 0 7 0 7 0 1 1 0 2 7 2 1 1 0 0 0 0 0 0 1 2 4 0 0 0 0 0 2 0 1 3 0 3 0 0 0 1.2 Session 5: Variety Development and Host Resistance Table 6. Summary of results of the 2006-07 NUWWSN (l,h indicate a mean that is not significantly different than the lowest (l) or highest (h) mean in the col.). 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 NAME ERNIE TRUMAN FREEDOM PIONEER 2545 NY88046-8138 NY93285SP-7343 NY93285-7110 NY91028SP-9082 NY93306-7091 KS04HW47-3 KS04HW101-3 P.011035A1-71 P.011036A1-14 P.02444A1-23-6 P.03647A1-1 P.04287A1-10 NE01643 HARRY NI04421 NE04653 NE03490 MD01W233-06-11 M03-3002 M03-3104 M03-3616 M03*3877 M03*3861 RCUOG19/21 RCUOGF110202D/4 RCUOGF111202A/3 RCUOGDHACF1109O2D RCUOGNS984-1 IL00-8530 IL01-11445 IL01-11934 IL02-19463 IL02-23168 KY97C-0540-01-03 KY97C-0554-03-06 KY97C-0554-04-05 KY97C-0508-01-01A-1 KY97C-0554-03-02 MO 040165 MO 050101 MO 050143 MO 050197 MSU Line E3023 MSU Line E5015 MSU Line E6001 MSU Line E6002 MSU Line E6003 VA06W-600 VA06W-602 VA06W-587 VA06W-594 VA06W-585 OH02-15978 OH02-12678 OH02-12686 OH02-13567 AVERAGE LSD R2 SEV 27.1 11.2 24.0 39.0 38.6 25.6 34.1 29.9 24.2 41.5 33.8 29.7 32.6 24.7 23.6 30.4 22.5 23.2 28.5 27.2 29.1 27.2 33.1 31.4 28.2 27.5 27.6 28.6 22.9 35.4 18.3 38.0 30.3 32.5 24.0 34.7 28.8 35.4 23.7 24.0 23.8 20.9 20.2 21.4 19.5 17.7 35.4 27.2 19.1 17.5 14.0 30.1 32.9 33.7 25.9 21.2 25.7 19.4 20.2 18.8 26.9 11.2 0.53 l h h h h h h h h h h l h h h h h l l l l l h l l l h h l l l l INC 45.7 26.7 47.2 62.3 54.1 33.3 32.7 58.7 52.2 62.7 49.2 53.9 46.9 46.2 38.7 51.2 44.1 51.1 56.8 51.5 49.8 48.3 50.8 64.3 45.2 61.8 61.6 44.9 25.7 58.5 21.4 61.1 48.4 39.2 40.2 37.4 45.3 57.9 55.7 48.6 42.8 50.7 34.7 40.4 38.1 33.7 50.0 55.3 41.5 27.1 18.3 45.1 49.6 55.4 52.3 47.4 41.5 39.5 35.5 43.8 46.2 14.3 0.73 l h h h h h h h h h h h h h h l h l h h h h h h l l h h IND 16.8 6.1 15.2 30.0 24.8 18.4 15.8 20.4 17.1 34.1 24.5 23.4 23.0 17.1 13.8 23.8 19.2 17.6 20.3 20.6 21.8 19.1 26.0 25.5 20.5 23.3 21.9 15.8 14.7 21.9 12.1 28.3 20.0 22.3 18.0 22.5 20.5 25.6 16.0 16.8 17.9 16.4 12.2 12.7 12.2 11.6 22.0 20.8 14.2 12.9 6.1 27.3 27.7 23.6 18.3 18.6 17.5 17.0 14.6 12.8 19.2 10.0 0.52 l l h h l h h l h h l l l h h l l l l l l l l h h l l KR 40.6 17.5 38.4 49.8 44.6 24.4 26.3 36.9 45.6 65.0 41.3 31.5 32.7 17.1 22.9 39.4 41.6 57.1 46.6 45.6 41.0 25.2 36.5 50.9 26.5 37.9 44.1 34.0 32.1 32.5 47.1 45.6 18.4 15.9 15.0 21.0 24.1 30.0 38.8 23.5 45.9 22.9 16.2 26.2 20.6 17.9 28.1 29.1 33.1 35.6 24.4 29.4 19.0 20.9 24.4 9.4 37.9 15.3 25.0 28.5 31.9 17.6 0.79 242 l h l l h l l h l h l l l l l l l l l l l l l l l l l l l PSS 4.3 12.6 7.1 18.5 28.0 33.2 24.4 25.1 31.4 21.2 31.8 21.4 18.6 16.1 17.8 13.2 10.9 16.4 25.7 20.6 16.8 24.4 12.7 22.6 27.0 20.2 11.7 14.2 11.1 22.4 22.7 26.6 19.9 7.0 11.9 9.7 19.1 14.5 22.5 30.4 27.7 15.5 15.4 7.3 7.1 8.6 12.7 23.6 14.4 13.9 17.4 22.5 18.5 23.4 13.8 15.1 14.1 11.2 17.1 15.8 18.0 14.8 0.48 l l l lh h h h h h h h h lh l l l l l h h l h l h h h l l l h h h h l l l lh l h h h l l l l l l h l l l h lh h l l l l l l ISK 30.7 16.6 34.9 50.3 48.5 32.6 29.5 42.2 37.9 46.1 35.4 43.4 39.0 39.8 27.6 34.7 30.3 35.9 39.2 31.1 37.5 31.1 43.7 49.5 36.5 37.8 34.1 29.5 23.5 42.5 26.7 42.6 30.9 34.0 26.3 34.8 32.4 40.3 42.9 42.3 31.9 29.2 25.6 26.3 26.7 21.3 38.7 33.9 25.0 26.7 12.6 42.2 38.1 43.1 35.8 34.2 31.3 25.2 26.0 29.6 34.1 14.1 0.52 l h h h h h h h h h h h h h h l h l h l h h h l l l l h l l l h h h l l DON 6.2 3.9 5.8 11.6 7.8 4.4 4.2 11.6 10.1 16.6 8.8 4.2 4.9 3.9 3.4 6.6 6.7 11.2 10.3 8.8 7.2 3.7 6.1 8.4 4.9 8.5 8.7 7.8 1.7 5.9 3.9 8.2 3 3.5 3.1 4 4 7.1 4.4 4.1 6.4 3.5 3.1 3.9 3.9 3.7 7.3 10.7 4.6 4.6 4.8 4.1 4.1 4 4.8 3.5 5.7 4.2 2.1 5.2 5.9 3.9 0.8 l l l h l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l GHS 18.0 3.4 20.7 50.5 46.6 22.3 32.8 42.6 42.6 70.6 40.5 22.1 10.6 19.8 10.2 21.8 21.4 14.1 19.5 19.7 25.9 25.2 35.6 59.1 21.7 26.7 20.3 31.2 10.7 25.5 25.4 46.9 14.7 11.1 11.6 25.3 31.8 39.6 12.1 9.8 28.5 8.8 4.6 4.5 6.2 39.1 50.7 17.1 4.9 25.5 8.4 39.5 26.8 34.4 18.8 19.1 50.2 13.3 24.0 12.3 24.9 28.8 0.71 l l l h h l h h h l l l l l l l l l l l h l l l l l l l h l l l l l l l l l l l l h l l l l l l l h l l l #l 2 8 3 1 0 3 3 0 0 0 0 2 3 4 5 2 2 2 1 1 2 3 1 0 3 1 2 3 6 1 6 0 3 4 5 4 4 1 3 3 1 5 7 7 7 6 1 1 6 7 8 1 4 2 4 5 1 6 7 5 3.1 #h 0 0 0 7 6 1 2 4 4 8 3 3 3 1 0 2 0 2 3 2 1 1 4 7 2 3 1 0 0 4 1 6 2 1 0 1 1 4 3 2 1 1 0 0 0 0 4 2 0 0 0 3 4 4 1 0 1 0 0 0 1.9 Session 5: Variety Development and Host Resistance DEOXYNIVALENOL ACCUMULATION AND FUSARIUM HEAD BLIGHT SEVERITY IN WINTER WHEAT AFTER SPRAYINOCULATION WITH MIXTURE OR SINGLE ISOLATES OF FUSARIUM GRAMINEARUM. L. Tamburic-Ilincic* and A.W. Schaafsma. Ridgetown Campus, University of Guelph, Ridgetown, Ontario, N0P 2C0, Canada Corresponding Author: PH: (519) 674-1557; Email: ltamburi@ridgetownc.uoguelph.ca * OBJECTIVES as r=0.18, to as high as r=0.70, depending on the year and level of FHB resistance in cultivars/lines tested in F. graminearum inoculated nurseries (unpublished data). Resistance to FHB infection and DON content may be controlled by independent loci or genes (Somers et al., 2003; Tamburic-Ilincic et al., 2002). 1) to compare aggressiveness of four F. graminearum isolates and their mixture based on FHB severity and DON accumulation in grain, after spray-inoculation of winter wheat cultivars with known FHB resistance and 2) to test the influence of isolates, wheat cultivar, year and their interactions on level of FHB symptoms and MATERIALS AND METHODS DON accumulation to ensure that there was no isolate-specific resistance from different wheat cultivars Winter wheat cultivars were planted in mid October in 2003, 2004 and 2005 at Ridgetown, Ontario, Canada with respect to DON accumulation. in single rows, 4 m long, spaced 17.8 cm apart, containing 270 seeds each. The experiments were arINTRODUCTION ranged in a 5 x 4 factorial randomized complete block Fusarium head blight (FHB), caused by Fusarium design with four replications. There were four cultigraminearum (Schwabe), is an important wheat dis- vars (FHB susceptible (S) ‘AC Ron’ and three FHB ease. In Canada FHB is caused primarily by Fusarium moderately resistant (MR): ‘Wisdom’, ‘Vienna’ and graminearum (Schwabe) [teleomorph: Gibberella ‘AC Morley’) and four isolates of F. graminearum zeae Schw. (Petch)]. Apart from yield and quality (DAOM178148, DAOM234041, DAOM234042 losses, the most serious concern associated with FHB and DAOM234043) and a mixture of the four. infection is the contamination of the harvested crop with mycotoxins. Deoxynivalenol (DON) is the myc- Spray-inoculations of each row with a suspension of otoxin most commonly recovered from wheat grain in 50-ml of macroconidia (50,000 spores/ml) of each of Canada. In Ontario, losses in winter wheat produc- four F. graminearum isolates (1-4) and their mixture tion from yield and quality reductions, were more than (5) were done, when primary wheat heads were at 50% anthesis for each cultivar (Zadoks Growth Stage, $ 100 million CAD in 1996 (Schaafsma , 2002). ZGS 65) (Zadoks et al., 1974) using a back-sprayer. Host specific strains have not been demonstrated and The suspension was produced in liquid shake culture resistance to FHB in wheat is generally considered using Bilay’s medium. F. graminearum isolate #1 horizontal (race-nonspecific) (Van Euwijk et al., 1995). (DAOM178148) was obtained from Agriculture Canada (ECORC, Ottawa, Ontario, Canada) and was When breeding for FHB and DON resistance, a isolated from wheat. Isolates #2 (DAOM234041), 3 breeder often has to rely on FHB symptoms rather (DAOM234042), and 4 (DAOM234043), were isothan to perform costly DON analysis. However, FHB lated from spring barley varieties ‘Chapais’, ‘AC severity and DON content are not always well corre- Stephen’, and ‘C231-0141’, respectively in 2000 at lated; we have experienced correlations, from as low Elora Research Station, Guelph, Ontario, Canada by 243 Session 5: Variety Development and Host Resistance Tamburic-Ilincic. After isolation the isolates were kept in liquid nitrogen and they have been submitted to the Canadian collection of fungal cultures in Ottawa and are available as DAOM234041, DAOM234042 and DAOM234043. All the plots were misted daily beginning after the first inoculations with about 7.5 mm of water each day. The mist system was engaged until three days after the last cultivar was inoculated. In each year, cultivars were assessed for visual symptoms when the early dough stage (ZGS 83) was reached. Disease levels were estimated as Fusarium head blight severity on a scale of 1-9 where 1 was disease free and 9 was total. The entire grain sample for each cultivar was harvested in mid July in 2004, 2005 and 2006 and finely ground through a ROMER mill (Model 2A, Romer Labs, Inc. Union, MO). Deoxynivalenol (DON) content was estimated from the three replications with highest mean FHB severity using a quantitative fluorometric test-FluoroQuan (Romer® Labs, Inc, Union MO). PROC UNIVARIATE (SAS Institute Inc., 2003) was used to test ANOVA assumptions to determine if transformations were needed. DON content was transformed by log (x+0.5). The data was analyzed using PROC GLM (SAS Institute Inc., 2003) where year, cultivar, isolate and their interactions were sources of variation. RESULTS AND DISCUSSION FHB severity and DON accumulation in this experiment depended on year, isolate and cultivar and the interaction between year and isolate and year and cultivar, but there was no significant interaction between isolates and cultivars (Table 1), suggesting that variation in resistance in these winter wheat cultivars was independent of the variation in aggressiveness of the F. graminearum isolates tested. The results from the present study are also in agreement with Mesterhazy (1997) who also reported that DON level in wheat depends on both cultivar and isolate. FHB severity and DON content in winter wheat cultivars after inoculation with four isolates and their mixture during three years are shown in Fig. 1 and Fig. 2. Aggressiveness of F. graminearum isolates High isolate x year interaction was reported in the present study (Table 1). Average FHB severity after spray-inoculation with isolates #3 (DAOM234042), #4 (DAOM234043) and the mixture of all (5) four across the cultivars was higher in 2004 than in 2005 or 2006 (Fig. 1a). Average DON accumulation across the cultivars was also higher in 2004 than in 2005 or 2006 (Fig. 1b). In 2004, after spray-inoculation with isolate #4 (DAOM234043) the DON level across the cultivars was significantly higher than after inoculation with the three other isolates or the mixture of all four (Fig. 1b). The summer of 2005 and 2006 was hot and dry and was not favorable to DON accumulation in winter wheat in Ontario. DON content measured in a survey of winter wheat in Ontario in 2005 and 2006 ranged from 0.1 ppm to 1.0 ppm and from 0.1 ppm to 0.3 ppm, respectively, which was much lower than found in a similar survey in 2004, where the highest level of DON was 4.9 ppm (Tamburic-Ilincic et al., 2006). FHB susceptibility and DON accumulation in winter wheat cultivars In 2004 and 2005, the average FHB severity was the highest for the FHB-susceptible cv. ‘AC Ron’ and significantly higher than three FHB MR cultivars (Fig. 2a). In 2006, cv. ‘AC Ron’ had significantly higher FHB severity than ‘AC Morley’ and ‘Wisdom’ (Fig. 2a). ‘AC Ron’ had the highest average DON level after inoculation in all three years; while the relative ranking of the three MR cultivars was slightly different in each year (Fig. 2b). In 2004, ‘Wisdom’ had the lowest DON level (Fig. 2b); ‘Wisdom’ and ‘Vienna’ accumulated the least DON in 2005 (Fig. 2b). There was no significant difference in DON accumulation among MR cultivars in 2006 (Fig. 2b). In conclusion, it is cautiously suggest that perhaps the selection of isolates may be less important when 244 Session 5: Variety Development and Host Resistance screening for FHB symptoms than when screening for the propensity to accumulate DON. Relatively similar FHB ratings between years resulted in very different DON levels between years. Although inconclusive the data suggest that a mixture of isolates might contribute some stability in results over years. This possibility would need further study with more cultivars under more environments and years. It seems from the results that high DON-producing isolates would allow for differentiation among cultivars which differ in their propensity to produce DON, and are recommended for screening wheat cultivars for simultaneous FHB resistance and DON accumulation. A highly pathogenic isolates of F. graminearum, that simultaneously produce a high level of FHB symptoms and DON, or a mixture of isolates is recommended for breeding programs targeting FHB resistance and reduced DON production in winter wheat. ACKNOWLEDGEMENTS SAS Institute Inc. 2003. 8th ed. SAS Institute Inc. Cary N.C. Schaafsma, A. W, 2002. Economic changes imposed by mycotoxins in food grains: case study of deoxynivalenol in winter wheat. In DeVries, J. W. et al. (ed.), Mycotoxins and Food Safety. 271-276. Kluwer Academic/Plenum Publishers. Somers, D. J., Fedak, G., and Savard, M. 2003. Molecular mapping of novel genes controlling Fusarium head blight resistance and deoxynivalenol accumulation in spring wheat. Genome. 46:555-564. Tamburic-Ilincic, L., Fedak, G., and Schaafsma, A. W. 2002. Study on deoxynivalenol (DON) and Fusarium Head Blight (FHB) resistance in a F2 winter wheat population. J. Appl. Genet. 43A: 333-340. Tamburic-Ilincic, L., Schaafsma, A. W., and Falk. D. 2006. 2005 survey for Fusarium head blight of winter wheat in S.W. Ontario. Can. Plant Dis. Surv. 86: 87 (www.cps-scp.ca/ cpds.htm) Van Euwijk, F. A., Mesterhazy, A., Kling, C. I., Ruckenbauer, P., Saur, L., Burstmayr, H., Lemmens, M., Keizer, L.C.P., Maurin N., and Snijders, C. H. A. 1995. Assessing non-specificity of resistance in wheat to head blight caused by inoculation with European strains of Fusarium culmorum, F. graminearum and F. nivale using a multiplicative model for interaction. Theor. Appl. Genet. 90: 221-228. This project was funded by partnership between the Canadian Adaptation Council (CanAdapt) the Ontario Wheat Producers Marketing Board and the Ontario Zadoks, J. C., Chang, T. T., and Konzak, C. F. 1974: A decimal Ministry of Agriculture and Food and Rural Affairs code for the growth stages of cereals. Weed Research. 14:415through the University of Guelph Contract. Technical 421. assistance by Diane Paul and Todd Phibbs is gratefully acknowledged. REFERENCES Mesterhazy, A., 1997. Breeding for resistance to Fusarium head blight of wheat. In H.J. Dubin, L. Gilchrist, J. Reeves, and A. McNab (eds), Fusarium Head Blight: Global Status and Future Prospects. CIMMYT, Mexico, 79-85. 245 Session 5: Variety Development and Host Resistance Table 1. Analysis of variance for the effect of year, Fusarium graminearum isolate, winter wheat cultivar, and interactions on: a) FHB severity (1-9) and b) transformed (log (x+0.5)) deoxynivalenol (DON) content (ppm) in winter wheat. Ridgetown, ON, 2003-2004, 2004-2005 and 2005-2006. a) FHB severity (1-9) Source Year Isolate Cultivar Year*Isolate Year*Cultivar Isolate*Cultivar Year*Isolate*Cultivar df 2 4 3 8 6 12 24 Mean square 6.254 4.235 1.249 4.192 3.249 0.474 0.547 F 19.16 12.98 3.83 12.84 9.95 1.45 1.68 P>F <.0001 <.0001 0.0098 <.0001 <.0001 0.1459 0.0310 b) DON accumulation Source Year Isolate Cultivar Year*Isolate Year*Cultivar Isolate*Cultivar Year*Isolate*Cultivar df 2 4 3 8 6 12 24 Mean square 90.034 1.584 5.027 1.710 2.125 0.202 0.195 F 427.24 7.52 23.86 8.11 10.09 0.96 0.93 P>F <.0001 <.0001 <.0001 <.0001 <.0001 0.4912 0.5671 246 Session 5: Variety Development and Host Resistance a 4.5 a FHB severity (1-9) 4 b 3.5 3 2.5 a ab c a ab ab a d a ab b b 2004 2005 2 2006 1.5 1 0.5 0 1 2 3 4 5 Isolates Figure 1 a 12 a DON (ppm) 10 8 b b b 2004 2005 6 4 2006 c a 2 ab b b ab ab ab 1 2 3 a a ab 0 4 5 Isolates Figure 1 b Figure. 1. The effect of Fusarium graminearum isolates (1-4) and their mixture (5) ( + SE) on: a) FHB severity (1-9) and b) deoxynivalenol (DON) content (ppm) across winter wheat cultivars. Ridgetown, ON. Means within years followed by the same letter are not different according to Fisher’s protected least significant difference test (P= 0.05). 247 Session 5: Variety Development and Host Resistance 4 a a 3.5 b a b FHB severity (1-9) 3 b b ab b c c 2.5 b 2004 2 2005 1.5 2006 1 0.5 0 AC RON AC MORLEY VIENNA WISDOM Winter wheat cultivars Figure 2 a a 10 9 8 DON (ppm) 7 bc 6 5 2004 b 2005 c 4 3 2 2006 a a b b c b c b 1 0 AC RON AC MORLEY VIENNA WISDOM Winter wheat cultivars Figure 2 b Figure. 2. The effect of winter wheat cultivars (‘AC RON’, ‘AC Morley’, ‘Vienna’ and ‘Wisdom’) on: a) FHB severity (1-9) and b) deoxynivalenol (DON) content (ppm) ( + SE) after spray-inoculation across Fusarium graminearum isolates. Ridgetown, ON. Means within years followed by the same letter are not different according to Fisher’s protected least significant difference test (P= 0.05). 248 Session 5: Variety Development and Host Resistance THE EFFECT OF FUSARIUM SOLANI METABOLITES ON PEROXIDASE ACTIVITY IN POTATO. A.Sh.Utarbayeva*, O.A.Sapko and R.M.Kunaeva M.A.Aitkhozhin Institute of Molecular Biology and Biochemistry, Almaty, Kazakstan * Corresponding Author: PH: 7 (3272) 93-70-91; Email: a_utarbayeva@yahoo.com ABSTRACT The fungus Fusarium solani cause the dry rot of potato tubers. The disease advances in tubers during storage. It leads to great losses of harvest. The reveal of qualities of host- pathogen interaction and natural biochemical mechanisms of tolerance is an actual scientific problem. It is known that so-called “oxidative burst” is one from early defense reaction of plant cells to attack of pathogens. The formation reactive oxygen species such as hydrogen peroxide, superoxide radicals etc. to result from “oxidative burst”. The many enzymes involved into cascade of oxidative reaction. The peroxidase (POD, EC.1.11.1.7) is one antioxidant enzymes which play important role in defense reaction. POD to oxidize substances with use hydrogen peroxide or molecular oxygen and participate in biosynthesis of toxic compounds for pathogen and processes of lignification or suberinization for forming barriers against pathogens. The various POD activate on different stages of protective mechanism. The role of soluble and cell-wall bound forms of POD in protective reactions of host-pathogen system “Solanum tuberosum – Fusarium solani” was studied. The changes of enzyme activity in tubers and in vitro cultivated cells of potato by additional different fractions of fungal metabolites were investigated. Changes showed response reactions of cells depended from plant’s sensitivity, localization of enzyme and chemical composition of fungal isolates. The effect of proteins and non-proteins isolates of cultural filtrate and mycelium on the disks of tubers was studied. The proteins of cultural filtrate stimulated rapid induction of soluble (in 2-2,5 time) and especially cell-wall bound (in 5,5-8 time) forms of POD as in tolerance as in sensitivity sorts. The non-proteins isolates also insignificantly induced soluble PODs (in 1,8-2 time). Induction of cell-wall bound PODs depend from resistible of plant. In tolerance sort was more significant increase (in 46 time) of enzyme activity than in sensitivity (in 1,2-1,5 time). The influence of metabolites of cultural filtrate and mycelium on POD activity in suspension cells was studied. The metabolites of cultural filtrate induced activity only of soluble forms POD. The magnitude of induction depended from filtrate concentration. The low concentrations (dilution 1:50) increased POD in sensitivity, but high (dilution 1:5) – in resistible cells. Metabolites of mycelium along with cytotoxic effect to suspension cells caused exchanges as soluble as cell-wall bound PODs. The metabolites of ethanol fraction of mycelium induced rapid increase of soluble forms in 2 time, but metabolites of acetone and water-soluble fractions increased activities only cell-wall bound PODs in 2-3 and 3-5 time accordingly and independently from initial sensitivity of cells to fungus. 249 Session 5: Variety Development and Host Resistance SEARCHING FOR NEW SOURCES OF FHB RESISTANCE IN THE RELATIVES OF WHEAT. S.S. Xu1*, R.E. Oliver2, X. Cai3, T.L. Friesen1, S. Halley4 and E.M. Elias3 1 USDA-ARS Cereal Crops Unit, Northern Crop Science Laboratory, Fargo, ND 58105; 2USDA-ARS National Small Grains Germplasm Research Facility, Aberdeen, ID 83210; 3Department of Plant Sciences, North Dakota State University, Fargo, ND 58105; and 4Langdon Research Extension Center, North Dakota State University, Langdon, ND 58249 * Corresponding Author: PH: (701) 239-1327; E-mail: steven.xu@ars.usda.gov ABSTRACT Epidemics of Fusarium head blight (FHB), caused mainly by Fusarium graminearum Schwabe, have threatened the production of bread wheat (Triticum aestivum L.) and durum wheat (T. turgidum L., subsp. durum) in North America in recent years. Deployment of FHB-resistant cultivars has been considered the most efficient and cost-effective strategy to combat this disease. However, only limited sources of FHB resistance are currently available, especially in durum, which makes the development of FHB-resistant varieties difficult. In an effort to identify novel sources of FHB resistance, we have screened about 900 accessions of wheat relative species and their derived lines for Type II resistance in greenhouse and in field nurseries (Fargo and Langdon, ND) during the past four years. A number of accessions and derived lines of the relative species have exhibited resistance or moderate resistance to FHB in these screening experiments. Resistant lines include 16 T. carthlicum and 20 T. dicoccum accessions, two synthetic hexaploid wheat lines, one T. timopheevi-derived hexaploid line, one ‘Fukuhokomuji’-Elymus rectisetus disomic addition line, and two ‘Chinese Spring’- Thinopyrum junceum disomic addition lines. These materials likely represent new sources of FHB resistance for durum and bread wheat. The resistant tetraploid wheat accessions are currently utilized for developing durum wheat germplasm resistant to FHB. Introgression of FHB resistance from the derived lines of wild species is currently in progress. ACKNOWLEDGEMENT AND DISCLAIMER This material is based upon work supported by the U.S. Department of Agriculture. This is a cooperative project with the U. S. Wheat & Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the U.S. Department of Agriculture. 250 Session 5: Variety Development and Host Resistance COMPARISON OF BARLEY SEED PROTEOMIC PROFILES ASSOCIATED WITH FUSARIUM HEAD BLIGHT REACTION. J. Zantinge1, K. Kumar1*, K. Xi1, A. Murray2, M. Johns2, J.H. Helm3 and P. Juskiw3 1 Field Crop Development Centre, Alberta Agriculture Food, 6000 C & E Trail, Lacombe, AB, T4L 1W1, Canada; 2Lacombe Research Centre, Agriculture and Agri-Food Canada, 6000 C & E Trail, Lacombe, AB, T4L 1W1 Canada; and 3Field Crop Development Centre, Alberta Agriculture Food, 5030 50 St., Lacombe, AB, T4L 1W8, Canada * Corresponding Author: PH: 403-782-8880; Email: krishan.kumar@gov.ab.ca ABSTRACT Plants have evolved a complex array of chemical and enzymatic defenses expressed both constitutive and inducible, that influence pathogenesis and disease resistance. To better understand the constitutive molecular mechanisms associated with the differential reactions to FHB, mature seed of barley genotypes and sister lines differing in FHB reactions were subject to proteome analysis using two dimensional gel electrophoresis (2DE). A total of 38 protein spots were correlated with FHB resistance and susceptibility. Several of these proteins were previously identified as important for disease resistance or as pathogen related proteins (PR-protein). Aldehyde dehydrogenase (BIS1) upregulated in resistant lines has been previously identified as a PR-protein upregulated in barley during stem infection. Protein spot #155 also upregulated in resistant sister lines, was identified as aconitate hydratase. Aconitate hydratase is important for Pto-mediated plant defense response. Putative NADP Malic enzyme found elevated in resistant barley lines, has been previously identified in EST libraries prepared from the barley lemma, palea and FHB infected spikes. Alpha amylase inhibitor (BDAI-1) also upregulated in resistant lines is also known to have antifungal activities. Several proteins were identified to ESTs or proteins, for which their functions were unknown. Selecting for constitutively expressed proteins or enzyme activities that correlate with enhanced FHB resistance may allow the development of new in vitro tissue test that could be used to select for improved FHB resistance in new barley lines. 251 Session 5: Variety Development and Host Resistance NOVEL FUSARIUM HEAD BLIGHT RESISTANCE IN TRITICUM AESTIVUM REVEALED BY HAPLOTYPING WITH DNA MARKERS ASSOCIATED WITH A KNOWN RESISTANCE QTL. C. Zila, J.R. Recker, X. Shen, L. Kong and H.W. Ohm* Department of Agronomy, Purdue University, 915 W. State Street, West Lafayette, IN 47907 * Corresponding Author: PH: 765-494-8072; Email: hohm@purdue.edu ABSTRACT Fusarium head blight (FHB) is a devastating disease of wheat (Tritcium aestivum L.) that causes reduced grain yield, and the fungus, Fusarium graminearum, produces a mycotoxin, deoxynivalenol (DON), that renders infected wheat grain to be unfit for food or feed. Several sources of resistance in wheat and certain related grass species have been identified. However, research to date indicates that several resistance genes need to be combined to result in highly effective resistance. Thus, there is need to identify additional novel sources of resistance. Xing 117, a wheat line obtained from China that has FHB resistance, the seven other wheat lines Ning 7840, Frontana, Wangshuibai, Arina, Renan, F201R, and Chokwang, all previously identified as having partial FHB resistance, as well as P9762 and P9774 that are susceptible to FHB, were haplotyped at marker loci that are associated with FHB resistance of the seven partially resistant wheat lines. Xing 117 was polymorphic at all previously identified marker loci of the seven wheat lines with partial FHB resistance, as well as these marker loci of P9762 and P9774, indicating that the FHB resistance of Xing 117 is likely novel compared to the resistances of the seven wheat lines in this study. 252

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Fusarium Head Blight Outbreak Frequency Under a Changing Climate Environment

Fusarium Head Blight Outbreak Frequency Under a Changing Climate Environment

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