2002 National
Fusarium Head Blight
Forum Proceedings
Holiday Inn Cincinnati-Airport
Erlanger, KY
December 7-9, 2002
2002 National Fusarium Head
Blight Forum Proceedings
Holiday Inn Cincinnati-Airport
Erlanger, KY
December 7-9, 2002
Organized by:
U.S. Wheat & Barley
Scab Initiative
Proceedings compiled by: Susan M. Canty, Janet Lewis, Lee Siler, and Richard W. Ward
Michigan State University
©Copyright 2002 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 Kinko’s, Okemos, MI 48864.
Copies of this publication can be viewed at http://www.scabusa.org.
We would like to acknowledge
the following companies and organizations
for their sponsorship of the
2002 National Fusarium Head Blight Forum
American Malting Barley Association, Inc.
Table of Contents
BIOTECHNOLOGY
Fusarium Virulence and Plant Resistance Mechanisms: a Project within the Austrian
Genome Programme GEN-AU
G. Adam and J. Glössl ...................................................................................................................... 1
QTL Analysis of Fusarium Head Blight in Barley Using the Chinese Line Zhedar 2
H.A. Agrama, L.S. Dahleen, R.D. Horsley, B.J. Steffenson, P.B. Schwarz,
A. Mesfin, and J.D. Franckowiak .................................................................................................... 2
Isolation and Characterization of Tri16 from Fusarium sporotrichioides
N.J. Alexander, S.P. McCormick, T.M. Larson, and J. E. Jurgenson .................................................. 3
A Systematic Approach for Identifying Antifungal Proteins with Enhanced
Resistance to Scab
Ajith Anand, Harold N. Trick, Bikram S. Gill, and S. Muthukrishnan ................................................. 4
Verification of Molecular Markers Linked to Fusarium Head Blight Resistance
QTLs in Wheat
N. Angerer, D. Lengauer, B. Steiner, and H. Buerstmayr ................................................................... 9
Wheat Transformation for Enhanced Fusarium Head Blight Tolerance
P. Stephen Baenziger, A. Mitra, M.Dickman, T. Clemente, S. Sato, S. Mitra,
J. Schimelfenig, and J. Watkins ........................................................................................................ 10
Molecular Characterization of Scab Resistance QTL in Wheat
G-H. Bai, A. Bernardo, P-G. Guo, K. Xiao, M. Das, X-Y. Xu, and S. R. Gaddam ......................... 14
Genetic Diversity of New Fusarium Head Blight Resistant Barley Sources
K.M. Belina, W.J. Wingbermuehle, and K.P. Smith ......................................................................... 16
Mapping Fusarium Head Blight Resistance QTL in the Chinese Wheat Line Fujian 5114
D.E. Bowen, S. Liu, R. Dill-Macky, C.K. Evans, and J.A. Anderson ............................................... 21
Molecular Mapping of QTLs for Fusarium Head Blight Resistance in Spring Wheat
H. Buerstmayr, B. Steiner, L. Hartl, M. Griesser, N. Angerer
D. Lengauer, and M. Lemmens ....................................................................................................... 22
QTL Mapping and SSR Genotyping of Fusarium Head Blight Resistance in Virginia
Tech Wheat Breeding Program
J. Chen, C, A, Griffey, M. A. Saghai Maroof, W. Zhao, J. Wilson, and D. Nabati ............................ 26
Insight in the Differentially Expressed Genes in Response to Fusarium Mycotoxins in
FHB Resistance Wheat Nobeokabouzu-Komug
I. Elouafi and T. Ban ....................................................................................................................... 27
2002 National Fusarium Head Blight Forum Proceedings
Control of Scab with Puroindoline-Containing Transgenic Wheat
S.A. Gerhardt, C. Balconi, and J.E. Sherwood ................................................................................ 28
Genetic Analysis of Type II Fusarium Head Blight (FHB) Resistance in the Hexaploid
Wheat Cultivar ‘Wangshubai’
Jose L. Gonzalez-Hernandez, A. del Blanco, B. Berzonsky, and S.F. Kianian .................................. 29
Identification of Scab Resistance Gene Expression in Wheat Following Inoculation
with Fusarium
L. Kong, J.M. Anderson, and H.W.Ohm ........................................................................................ 30
Mapping Genes Conferring Fusarium Head Blight Resistance in C93-3230-24
K.E. Lamb, M.J. Green, R.D. Horsley, and Zhang Bingxing ............................................................. 31
Targeted Saturation Mapping of Qfhs.ndsu-3BS Using Wheat ESTs and Synteny with
the Rice Genome
S. Liu and J. A. Anderson ............................................................................................................... 32
Identification of QTL Associated with Scab Resistance in Ernie
Shuyu Liu, Theresa Musket, Anne L. McKendry, and Georgia L. Davis ........................................... 33
Over-Expression of Anti-fungal Protein Genes Increases Resistance of Transgenic Wheat
to Fusarium Head Blight
C.A. Mackintosh, S.J. Heinen, L.A. Smith, M.N. Wyckoff, R.J. Zeyen, G.D. Baldridge,
and G.J. Muehlbauer ...................................................................................................................... 34
Effect of Chevron Alleles At Two Fusarium Head Blight Resistance QTL Determined
Using Near-Isogenic Lines
L. M. Nduulu, A. Mesfin, G.J. Muehlbauer, and K.P. Smith ............................................................. 35
Saturation Genetic and Physical Mapping of Chromosome 3 Fusarium Head Blight
QTL Region
Deric Schmierer, Kara Johnson, Thomas Drader, and Andris Kleinhofs ............................................ 39
Microsatellite Genetic Map in Wheat
J.R. Shi, Q. J. Song, S-Singh, R.W. Ward, P.B. Cregan, and B.S. Gill ............................................. 40
Strategies for Combating Fusarium in Barley Through Gene Expression Targeting,
Metabolic Profiling and Signaling Analysis
R.W. Skadsen, T. Abebe, M.L. Federico, J. Fu, C. Henson, and H.F. Kaeppler ............................ 41
Transgene Expression in Spring Wheat (Triticum aestivum L.) Transformed with
Candidate Anti-Fusarium Genes
M. Somleva, P. Okubara, and A. Blechl .......................................................................................... 42
Molecular Mapping of Resistance to Fusarium Head Blight in the Spring Wheat
Cultivar Frontana
B. Steiner, M. Griesser, M. Lemmens, and H. Buerstmayr ............................................................... 46
Examination of Molecular Variability of Fusarium culmorum Isolates
B. Tóth, Á. Mesterházy, J. Téren, and J. Varga ................................................................................ 47
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2002 National Fusarium Head Blight Forum Proceedings
A Non-Coding Wheat RNA May Play an Important Role in Wheat Resistance to Fusarium
Head Blight
D.H. Xing, Y. Yen, and Y. Jin .......................................................................................................... 49
A Putative Acyl-CoA-Binding-Protein of Fusarium graminearum May Play an Important
Role in the FHB Pathogenesis in Wheat
D.H. Xing, Y. Yen, and Y. Jin ......................................................................................................... 50
Identification of Chromosome Regions Associated with Fusarium Head Blight Resistance
in Bread Wheat Cultivar Sumai 3 with its Susceptible NILs by Using DNA Markers
D.H. Xu, M. Nohda, H.G. Chen, and T. Ban .................................................................................. 51
Transposon-mediated Generation of Marker-free Barley Plants Expressing Putative
Antifungal Proteins
X-H. Yu, P. Bregitzer, M-J. Cho, M.L. Chung, and P.G. Lemaux ..................................................... 52
CHEMICAL AND BIOLOGICAL CONTROL
Effect of Bacterial Growth Medium Composition on Antifungal Activity of Bacillus sp.
Strains Used in Biological Control of Fusarium Head Blight
Nichole Baye, Bruce H. Bleakley, Martin A. Draper, and Kay R. Ruden .......................................... 54
Taxonomic Affiliation of Bacterial Strains Used in the Biological Control of Fusarium Head
Blight Suggests Possible Role of Lipopeptide Antibiotic in Fungal Antagonism
Nichole Baye and Bruce H. Bleakley .............................................................................................. 55
JAU 6476 for the Control of Fusarium graminearum and Other Diseases in Cereals
J. R. Bloomberg, D.E. Rasmussen, and T. K. Kroll ......................................................................... 56
Effect of Fungicide Treatments on Fusarium Head Blight and Leaf Disease Incidence in
Winter Wheat
A.L. Brûlé-Babel and D. Fernando ................................................................................................. 57
Population Dynamics in the Field of a Biocontrol Agent for Fusarium Head Blight of Wheat
A.B. Core, D.A. Schisler, T.E. Hicks, P.E. Lipps, and M.J. Boehm ................................................. 61
Variations in Fungicide Application Techniques to Control Fusarium Head Blight
Martha Diaz de Ackermann, Mohan Kohli, and Vilfredo Ibañez ....................................................... 62
Aerial Spray Coverage Trials in South Dakota – 2002
M.A. Draper, J.A. Wilson, B.E. Ruden, D.S. Humburg, K.R. Ruden,
and S.M. Schilling ........................................................................................................................... 63
Uniform Trials for Biological Control Agent Performance in the Suppression of Fusarium
Head Blight in South Dakota – 2002
M.A. Draper, B.H. Bleakley, K.R. Ruden, N.L. Baye, A.L. LeBouc,
and S.M. Schilling ........................................................................................................................... 65
Uniform Fungicide Performance Trials in South Dakota – 2002
M.A. Draper, K.D. Glover, K.R. Ruden, A.L. LeBouc, S.M. Schilling,
and G. Lammers ............................................................................................................................. 67
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Fusarium Head Blight: Epidemics and Control
Samia M. El-Allaf, P.E. Lipps, and L.V. Madden ............................................................................. 69
Effect of Three Bacillus Sp. from Wheat on FHB Reduction
W.G.D. Fernando, Y. Chen, and P. Parks ....................................................................................... 73
An Extension Agronomist’s Experiences with Fungicide Application Techniques to
Improve Control of FHB
T.D. Gregoire ................................................................................................................................. 76
Barley Cultivar Response to Fungicide Application for the Control of Fusarium Head
Blight and Leaf Disease
S. Halley ........................................................................................................................................ 77
Analysis of the 2002 Uniform Wheat Fungicide and Biocontrol Trials Across Locations
D.E. Hershman and E.A. Milus ....................................................................................................... 82
Management of Fusarium Head Blight in Wheat Using Selected Biological Control Agents
and Foliar Fungicides, 2002
D.E. Hershman, P.R. Bachi, D.M. TeKrony, and D.A. VanSanford .................................................. 88
Multiple Infection Events and Split Timing of Folicur Fungicide Applications for Control
of FHB in Hard Red Spring Wheat, Durum Wheat, and Spring Barley, 2002
J. Jordahl, S. Meyer, and M. McMullen .......................................................................................... 91
Evaluation of Foliar Fungicides and Bioprotectants for Control of Fusarium Head Blight
of Winter Wheat in New York in 2002
S.O. Kawamoto, C.A. Stockwell, D.J. Otis, W.J. Cox, M.E. Sorrells,
and G.C. Bergstrom ....................................................................................................................... 92
History and Accomplishments of the USWBSI Uniform Fungicide and Biological Control
Trials, 1998-2002
M. McMullen, and E. Milus ............................................................................................................ 96
ND Uniform Wheat Fungicide and Biological Agent Trials, 2002
M. McMullen, J. Lukach, K. McKay, and B. Schatz ....................................................................... 97
New and Effective Fungicides Against the FHB in Wheat
Á. Mesterházy, T. Bartók, and G. Kászonyi .................................................................................. 100
Uniform Barley Fungicide and Biological Agent Trials, Fargo, ND, 2002
S. Meyer, J. Jordahl, and M. McMullen ........................................................................................ 104
Efficacy of Fungicides and Biocontrols Against FHB on Wheat in Arkansas in 2002
Eugene A. Milus, Peter Rohman, and Samuel Markell ................................................................... 106
Practical Aspects of Ground Application of Foliar Fungicides
Philip Needham ............................................................................................................................ 109
Efficacy of Fungicides in Controlling Barley Fusarim Head Blight in Lines With
Partial Resistance
J.D. Pederson, R.D. Horsley, M. McMullen, and K. McKay ......................................................... 110
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2002 National Fusarium Head Blight Forum Proceedings
Automated Control of a Watering System for Fusarium Head Blight Research
T. Scherer, D. Kirkpatrick, and M. McMullen ................................................................................ 111
USDA-ARS, Ohio State University Cooperative Research on Biologically Controlling
Fusarium Head Blight 1: Discovery and Scale-up of a Freeze-drying Protocol for
Biomass of Antagonist Cryptococcus nodaensis OH 182.9 (NRRL Y-30216)
D.A. Schisler, J.E. VanCauwenberge, and M.J. Boehm ................................................................. 115
USDA-ARS, Ohio State University Cooperative Research on Biologically Controlling
Fusarium Head Blight 2: 2002 Field Tests of Antagonist and Antagonist/
Fungicide Mixtures
D.A. Schisler, M.J. Boehm, T.E. Hicks, and P.E. Lipps ................................................................. 119
Evaluation of Fungicides for the Control of Fusarium Head Blight and Leaf Diseases on
‘Elkhart’ and ‘Pioneer variety 2540’ Winter Wheat in Missouri
L.E. Sweets .................................................................................................................................. 123
Report on Induced Resistance and Field Biological Control of Fusarium Head Blight by
Lysobacter enzymogenes Strain C3
Gary Yuen and C.C. Jochum ......................................................................................................... 127
EPIDEMIOLOGY AND DISEASE MANAGEMENT
Influence of Crop Rotation and Cover Crop on Fusarium Head Blight of Wheat
H.U. Ahmed, J. Gilbert, W.G. D. Fernando, A. Brûlé-Babel, A. Schoofs, and M. Entz .................. 128
Determination of Wetness Duration Using Radar-Derived Precipitation Estimates
J.A. Andresen, T.M. Aichele, and A.Pollyea ................................................................................. 132
A Second Genetic Map of Gibberella zeae
R.L. Bowden, J.E. Jurgenson, J.K. Lee, Y.-W. Lee, S-H. Yun, K. Zeller, and J.F. Leslie ............... 133
What Part Does Programmed Cell Death Play In Fusarium Head Blight?
W.R. Bushnell and T.M. Seeland ................................................................................................... 134
Influence of Irrigation Following Disease Assessment on Deoxynivalenol Accumulation in
Fusarium-Infected Wheat
M.D. Culler and R. Dill-Macky ..................................................................................................... 135
Spatial Patterns of Fusarium Head Blight in New York Wheat Fields in 2002
E.M. Del Ponte, D.A. Shah, and G.C. Bergstrom .......................................................................... 136
Influence of Corn Residue and Cultivar Susceptibility on the Accuracy of Fusarium Head
Blight Risk Assessment Models
E. De Wolf, P. Lipps, L. Madden, and L. Francl ............................................................................ 137
Effect of Cereal Residue Burning on the Incidence and Stratified Distribution of Fusarium
graminearum and Cochliobolus sativus in Wheat and Barley Plants
R. Dill-Macky and B. Salas .......................................................................................................... 140
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Identification of Environmental Variables That Affect Perithecial Development of
Gibberella zeae
N. Dufault, E. De Wolf, P. Lipps, and L. Madden .......................................................................... 141
Relationship of Temperature and Moisture to Gibberella zeae Perithecial Development
in a Controlled Environment
N. Dufault, E. De Wolf, P. Lipps, and L. Madden .......................................................................... 142
Incidence-Severity Relationships for Fusarium Head Blight on Wheat
S. M. El-Allaf, L. V. Madden, and P. E. Lipps ............................................................................... 145
Spatial Aspects of Fusarium Head Blight Epidemics on Wheat in Ohio
S. M. El-Allaf, L. V. Madden, and P. E. Lipps ............................................................................... 146
Effect of Wheat Floral Structure Extracts and Endogenous Compounds on the Growth
of Fusarium graminearum
Jessica S. Engle, Patrick E. Lipps, Terry L. Graham, and Michael J. Boehm ................................... 151
A Phenology-Based Predictive Model for Fusarium Head Blight of Wheat
J.M.C. Fernandes and W. Pavan .................................................................................................. 154
AFLP-Assisted Genetic Characterization of Fusarium graminearum Isolates from Canada
W.G.D. Fernando, R. Ramarathnam, J. Gilbert, and R. Clear ........................................................ 159
Assessment of the Differential Ability of Fusarium Strains to Spread on Wheat and Rice
Rubella S. Goswami and H. Corby Kistler .................................................................................... 163
Development of Gibberella zeae on Wheat Tissue
John Guenther and Frances Trail ................................................................................................... 164
The DONcast Model: Using Weather Variables Pre- and Post-Heading to Predict
Deoxynivalenol Content in Winter Wheat
David C. Hooker, Arthur W. Schaafsma, and Lily Tamburic-Ilincic ................................................ 165
Fusarium Head Scab Risk Forecasting for Ohio, 2002
Patrick Lipps, Dennis Mills, Erick DeWolf, and Larry Madden ...................................................... 166
Practical Application of Fusarium Head Blight Risk Predictions
Patrick Lipps, Erick De Wolf, Dennis Mills, and Larry Madden ..................................................... 167
Epidemiological Studies on Fusarium Head Blight of Wheat in South Dakota for 2002
L. Osborne and Y. Jin ................................................................................................................... 171
FHB Inoculum Distribution on Wheat Plants Within the Canopy
L. Osborne, Y. Jin, F. Rosolen, and M.J. Hannoun ........................................................................ 175
South Dakota Fusarium Head Blight Risk Advisory for 2002
L. Osborne and Y. Jin ................................................................................................................... 176
Incidence of Fusarium graminearum and Cochliobolus sativus in Wheat and Barley
Cultivars at Three Locations in Minnesota
B. Salas, R. Dill-Macky, and J.J. Wiersma .................................................................................... 177
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2002 National Fusarium Head Blight Forum Proceedings
Airborne Populations of Gibberella zeae: Spatial and Temporal Dynamics of Spore
Deposition in a Localized Fusarium Head Blight Epidemic
David G. Schmale III, Elson J. Shields, and Gary C. Bergstrom ..................................................... 178
Development of Fusarium Head Blight in Indiana, 2002
G. Shaner and G. Buechley ........................................................................................................... 179
Comparison of Spray, Point Inoculation Methods, and FDK to Facilitate Early Generation
Selection for Fusarium Head Blight Resistance in Winter Wheat
L. Tamburic-Ilincic, G. Fedak, and A.W.Schaafsma ...................................................................... 180
REMI Mutagenesis in the Wheat Scab Fungus Fusarium graminearum
Miles Tracy, Zhanming Hou, H. Corby Kistler, and Jin-Rong Xu .................................................... 185
The Fusarium graminearum Genomics Project
Frances Trail, Jin-Rong Xu and H. Corby Kistler ........................................................................... 186
Comparative Virulence of Isolates of Fusarium Species Causing Head Blight in Wheat
A.G. Xue, K.C. Armstrong, H.D. Voldeng, G. Fedak, Y. Chen, and F. Sabo ................................. 187
Population Genetic Differentiation and Lineage Composition Among Gibberella zeae
(Fusarium graminearum) in North and South America
K.A. Zeller, J.I. Vargas, G. Valdovinos-Ponce, J.F. Leslie, and R.L. Bowden ................................. 188
FOOD SAFETY, TOXICOLOGY AND UTILIZATION
Metabolism of Trichothecenes by Wheat
L.-F. Chen, H.-Y. Yao, G. Yu, W.-P. Xie, and H. C. Kistler .......................................................... 189
Yeast Strains Allowing Phenotypic Detection of Estrogenic Activity: Development of a
Sensitive and Inexpensive Yeast Bioassay for Zearalenone
R. Mitterbauer, H. Weindorfer, N. Safaie, H. Bachmann, and G. Adam ......................................... 190
Diagnostic Vomitoxin (DON) Services in 2002/2003 Samples
M.S. Mostrom, P. Schwarz, Y. Dong, and P. Hart ......................................................................... 191
Human Susceptibility toTrichothecene Mycotoxins
James J. Pestka, Kristen Penner, and Jennifer Gray ....................................................................... 195
Using Near Infrared Transmittance as a Screening Tool for Don in Barley
H. Pettersson, L. Aberg, J.A. Persson, H. Andren, and M. Matteson ............................................ 197
Storage of Scabby Wheat: Fusarium Goes Away, DON Doesn’t
Robert W. Stack, Howard H. Casper, and Dennis J. Tobias .......................................................... 198
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GERMPLASM INTRODUCTION AND ENHANCEMENT
Variation for Resistance to Fusarium Head Blight in Triticum dicoccoides
H. Buerstmayr, M. Stierschneider, B. Steiner, M. Lemmens, M. Griesser,
E. Nevo, and T. Fahima ................................................................................................................ 199
Designating Types Of Scab Resistance: A Discussion
W. R. Bushnell .............................................................................................................................. 200
Inheritance of Fusarium Head Blight Resistance (Type II) in New Wheat Germplasm
CJ 9306 and CJ 9403
Guo-Liang Jiang and Richard W. Ward ......................................................................................... 201
Screening Winter and Facultative Wheats for Fusarium Head Blight Infection
Mohan Kohli, Martin Quincke, and Martha Diaz de Ackermann .................................................... 202
Types I, II and Field Resistance to Fusarium Head Blight in Winter and Spring
Wheat Germplasm
Anne L. McKendry, Kara S. Bestgen, and David N. Tague ........................................................... 204
Resistance in Hexaploid Wheat to Fusarium Head Blight
Gregory Shaner ............................................................................................................................ 208
Novel Source of Type II Resistance to Fusarium Head Blight
Xiaorong Shen, Lingrang Kong and Herbert Ohm ......................................................................... 212
Evaluation of the National Small Grains Collection of Barley for Resistance to Fusarium
Head Blight and Deoxynivalenol Accumulation
L.G. Skoglund and J.L. Menert ..................................................................................................... 213
Fusarium Head Blight Type II Resistance of a Spring Wheat Population Derived from a
Hungarian Winter Wheat
R.W. Stack, R.C. Frohberg, and M. Mergoum ............................................................................. 216
Proposed Chromosomal Location of FHB Resistance Genes in Additional Sets of Durum
Disomic Substitution Lines Derived from Different T. dicoccoides Accessions
R.W. Stack, J.D. Miller, and L.R. Joppa ....................................................................................... 217
Wild Emmer, Triticum dicoccoides, as a Source of FHB Resistance for Tetraploid and
Hexaploid Wheats
Robert W. Stack and James D. Miller ........................................................................................... 218
Efficiency and Efficacy of Marker Assisted Selection over Phenotypic Selection for FHB
Resistance in Durum Wheat
B. Suresh, E.M. Elias, J.L. González-Hernández, and S.F. Kianian ................................................ 219
Putative Sources of Fusarium Head Blight Resistance in Spring Wheat Identified from
the USDA Small Grains Collection
X. Zhang and Y. Jin ...................................................................................................................... 220
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VARIETY DEVELOPMENT AND UNIFORM NURSERIES
The Development of Scab (Fusarium graminearum) Resistant Varieties of Wheat
P.S. Baenziger, J. Schimelfenig, and J.E. Watkins .......................................................................... 223
Number of Location-Years Needed to Determine the Reaction of Winter Wheat Cultivars
to Fusarium Head Blight
William W. Bockus, Mark A. Davis, and Karen A. Garrett ............................................................ 228
Identification of DNA Markers for Fusarium Head Blight Resistance of Wheat Line
Huapei 57-2
William Bourdoncle and Herbert W. Ohm ..................................................................................... 229
Coordinated Fusarium Head Blight Screening Nursery for Wheat Breeding Programs in
Western Canada
A.L. Brûlé-Babel, D. Fernando, P. Hucl, G. Hughes, S. Fox, R. DePauw, M. Fernandez,
J. Clarke, R. Knox, J. Gilbert, G. Humphreys, and D. Brown ........................................................ 230
Timing of Inoculations of Dryland Wheat Plots and the Effect on Fusarium Head
Blight (FHB) Severity and Mycotoxinaccumulation Due to Fusarium graminearum
Infection
C. K. Evans and R. Dill-Macky .................................................................................................... 231
Variety Development and Uniform Nurseries: FHB Resistance in Barley
J.D. Franckowiak ......................................................................................................................... 232
A Fusarium Resistance Gene and an Awn Promotor are Associated on Chromosome 5A
of Spring Wheat
Richard C. Frohberg, Robert W. Stack, and S.S. Maan ................................................................ 234
A Historical Analysis of the Uniform Regional Scab Nursery for Spring Wheat Parents
D.F. Garvin and J.A. Anderson ..................................................................................................... 235
Genes with Major Effects on FHB Resistance Promise Easy Marker Application
L. Gilchrist, M. van Ginkel, R. Trethowan, and E. Hernandez ......................................................... 239
Sources of Combined Resistance to Fusarium Head Blight, Stripe Rust, and BYD
in Triticale
L. Gilchrist, A. Hede, R. Gonzalez, and R.M. Lopez ..................................................................... 242
Progress in Breeding Fusarium Head Blight Resistance in Soft Red Winter Wheat
C.A. Griffey, J. Wilson, D. Nabati, J. Chen, and T. Pridgen ........................................................... 246
Comparison of FHB Development on Hard Winter Wheat Using Different
Planting Schemes
D.M. Gustafson, A.M.H. Ibrahim, and L. Peterson ....................................................................... 247
Stability of Type II Resistance and DON Levels Across Isolate and Soft Red Winter
Wheat Genotype
Anne L. McKendry, Kara S. Bestgen, David N. Tague, and Zewdie Abate ................................... 248
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Developing FHB-resistant Cultivars and Germplasm for the Mid South
E.A. Milus, R.K. Bacon, S.A. Harrison, P. Rohman, S. Markell, and J. Kelly ................................ 249
Uniform Southern Soft Red Winter Wheat Fusarium Head Blight Screening Nursery
J.P. Murphy, R.A. Navarro, and D.A. Van Sanford ....................................................................... 253
Developed Evaluation Method of Fusarium Head Blight (FHB) Resistance in Wheat by
Continuous Simulated Rainfall and Diversity of FHB Resistance in Domestic Wheat
Zenta Nishio, Kanenori Takata, Tadashi Tabiki, and Tomohiro Ban ................................................ 254
Phenotypic Effects of Qfhs.ndsu-3BS on Fusarium Head Blight Resistance in Near-Isogenic
Wheat Lines
M.O. Pumphrey and J.A. Anderson .............................................................................................. 255
SSR Mapping of Fusarium Head Blight Resistance in Wheat
Xiaorong Shen and Herbert Ohm .................................................................................................. 260
Summary Report on the 2002 Northern Uniform Winter Wheat Scab Nursery (NUWWSN)
Clay Sneller, Patrick Lipps, and Larry Herald ................................................................................ 261
Fusarium Head Blight in Hexaploid Wheat Populations Derived from Lines with
Type I Resistance
R.W. Stack, R.C. Frohberg, and M. Mergoum ............................................................................. 265
Scab Screening Using Frozen Spikes
A.J. Stewart, B. Kennedy, and D. A. Van Sanford ........................................................................ 266
Fusarium graminearum and DON in Single Seeds Following Greenhouse Point Inoculation
Dennis M. TeKrony, David VanSanford, Marcy Rucker, Cheryl Edge,
and Yanhong Dong ....................................................................................................................... 267
How to Make Intelligent Crosses to Accumulate Fusarium Head Blight Resistance
Genes Based on Knowledge of the Underlying Resistance Mechanisms
M. van Ginkel and L. Gilchrist ....................................................................................................... 268
Apparent and Actual Seed Quality in Soft Red Winter Wheat Infected with Fusarium
graminearum
V.L.Verges, B. Kennedy, A.J.Stewart, D. TeKrony, and D.A. Van Sanford .................................... 273
Effect of Sumai 3 Chromosomes on Type II and Type V Scab Resistance in Wheat
Wenchun Zhou, Frederic L. Kolb, Larry K. Boze, Norman J. Smith, Guihua Bai,
Leslie L. Domier, and Jingbao Yao ................................................................................................ 274
OTHER REPORTS
Estimating the Economic Impact of a Crop Disease: The Case of Fusarium Head Blight in
U.S. Wheat and Barley
William E. Nganje, Dean A. Bangsund, F. Larry Leistritz, William W. Wilson,
and Napoleon M. Tiapo ............................................................................................................... 275
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FUSARIUM VIRULENCE AND PLANT RESISTANCE MECHANISMS: A
PROJECT WITHIN THE AUSTRIAN GENOME PROGRAMME GEN-AU
G. Adam* and J. Glössl
Center of Applied Genetics, University of Agricultural Sciences, Vienna, Austria,
*Corresponding Author: PH: 43-1-36006-6380; E-mail: adam@edv2.boku.ac.at
ABSTRACT
In 2002 the Austrian Federal Ministry for Education, Science and Culture has established
the national genome programme GEN-AU (GENome Research in AUstria: http://www.genau.at/). The first call has brought together a broad spectrum of Austrian scientists (coordinator GA) focussing on the Fusarium problem. We proposed (as one part) that Austria should
contribute 25% of the costs of sequencing the F. graminearum genome, but the panel felt
that our proposal was too much dependent on the (in April 2002) not yet submitted proposal
by US partners (Birren, Kistler, Xu, Trail). In the meantime a dramatically downscaled pilotproject (with only 5 of the initially 12 partner institutions remaining) has been funded (about
795.000 US dollar).
In the next two years researchers from the Center of Applied Genetics (CAG) of the University of Agricultural Sciences, Vienna, the Technical University (TU) of Vienna, the Institute for
Agrobiotechnology in Tulln (IFA), the Austrian Research Center Seibersdorf (ARCS), and
from the wheat breeding company Saatzucht Donau will collaborate on several aspects.
The following principal investigators are involved: Josef Straub, Gerhard Adam (CAG) and
Robert Mach (TU) will collaborate on the development of efficient gene disruption methods
for F. graminearum. Mutants will be tested for altered virulence at the IFA Tulln (Marc
Lemmens) and for altered metabolite production by LC-MS-MS (Rudolf Krska, IFA Center
for Analytical Chemistry). Also analytical techniques and reference materials for “masked
mycotoxins” will be developed. In the group of Gerhard Adam Arabidopsis thaliana genes
encoding mycotoxin inactivating enzymes will be characterized, and Marie-Theres Hauser
will explore the role of zearalenone in plants (CAG). The group from ARCS will establish
wheat suspension cultures and work on the identification of differentially expressed genes
in wheat and the development of DNA arrays. The genetic basis of so far uncharacterized
highly Fusarium resistant wheat genetic resources will be elucidated by Hermann
Büerstmayr (IFA), the knowledge gained will be utilized by the commercial partner (Julia
Lafferty, Saatzucht Donau) in a marker assisted backcross breeding program (QTL
pryamiding).
The aim of the GEN-AU program is “to secure and expand Austria’s competitiveness and
ability to cooperate on an international level”. It may be of interest for US researchers, that
the EU 6th framework programme is open to the participation of entities from nonmember
countries on the project level on the basis of mutual benefit.
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2002 National Fusarium Head Blight Forum Proceedings
QTL ANALYSIS OF FUSARIUM HEAD BLIGHT IN BARLEY
USING THE CHINESE LINE ZHEDAR 2
H.A. Agrama1*, L.S. Dahleen2, R.D. Horsley3, B.J. Steffenson4, P.B. Schwarz5,
A. Mesfin3, and J.D. Franckowiak3
Dept of Plant Pathology, North Dakota State University, Fargo, ND; 2ARS-USDA, NCSL, Fargo, ND; 3Dept
of Plant Sciences, North Dakota State University, Fargo, ND; 4Dept. of Plant Pathology, University of
Minnesota, St. Paul, MN; and 5Dept. of Cereal Sciences, North Dakota State University, Fargo, ND
*Corresponding Author: PH (701) 239-1345; E-mail: agramah@fargo.ars.usda.gov
1
ABSTRACT
Fusarium head blight (FHB) in barley and wheat caused by Fusarium graminearum is a
continual problem worldwide. Primarily, FHB reduces yield and quality and produces the
toxin deoxynivalenol (DON), which can affect food safety. Locating QTLs for FHB severity,
DON level and related traits heading date (HD) and plant height (HT) with consistent effects
across a set of environments would increase the efficiency of selection for resistance. A
population of seventy-five double haploid lines, developed from the three-way cross Zhedar
2/ND9712//Foster, was used for genome mapping and FHB evaluation. Phenotypic data
were collected in replicated field trails from five environments in two growing seasons. A
linkage map of 214 RFLP, SSR and AFLP markers was constructed. The data were analyzed using MQTL software to detect QTL x environment interaction. Because of the presence of QTL x E, the MQM in MAPQTL was applied to identify QTLs in single environments.
MQM mapping identified nine QTLs for FHB severity and five for low DON. Only three of
these QTLs were consistent across environments. Five QTLs were associated with HD and
two with HT. Regions that appear to be promising candidates for MAS and further genetic
analysis including the two FHB QTLs on chromosome 2H and one on 6H which also were
associated with low DON and later heading date in multiple environments. This study
provides a starting point for manipulating Zhedar 2-derived resistance by MAS in barley to
develop varieties that will show effective resistance under disease pressure.
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2002 National Fusarium Head Blight Forum Proceedings
ISOLATION AND CHARACTERIZATION OF TRI16 FROM
FUSARIUM SPOROTRICHIOIDES
N.J. Alexander1*, S.P. McCormick1, T.M. Larson1,2 and J. E. Jurgenson3
1
Mycotoxin Research Unit, USDA/ARS/NCAUR, Peoria, IL; 2Dept. of Biol., Bradley University, Peoria, IL;
and 3Dept. of Biol., University of Northern Iowa, Cedar Falls, IA
*Corresponding Author: PH: (309) 681-6295; E-mail: alexannj@ncaur.usda.gov
ABSTRACT
Many of the genes involved in the trichothecene biosynthetic pathway in Fusarium have
now been identified within a 29 kb section of DNA. Within this cluster are 3 genes (Tri4,
Tri11, and Tri13) encoding P450 oxygenases, a gene (Tri5) encoding sesquiterpene cyclase, a gene encoding an esterase (Tri8), two acetyltransferase genes (Tri3 and Tri7), a
transport pump gene (Tri12), and two regulatory genes (Tri6 and Tri10). One gene encoding
an acetyltransferase, Tri101, is not located within the cluster. However, not all of these genes
are functional in every Fusarium species. The Fusarium toxins can be divided into two
groups based on the substitution of the A ring. Fusarium sporotrichioides produces A-type
trichothecenes, such as T-2 toxin or 4,15-diacetoxyscirpenol, while F. graminearum produces B-type trichothecenes, such as deoxynivalenol (DON), that have a carbonyl at C-8.
These differences in side groups are due, at least, to the non-functional Tri7 and Tri13 in Btype trichothecene producers. In the search for the remaining trichothecene genes, the use
of an EST library from a toxin over-producing strain carrying an altered Tri10 has identified
Tri16, a gene believed to be involved with trichothecene biosynthesis. We isolated and
cloned this gene from F. sporotrichioides, then formed disruption vectors through insertional
disruption and truncated disruption. Insertional disruption vectors produced only single
cross-over events when the vector was transformed into the host protoplasts thus producing
a transformant with both a disrupted as well as an intact copy of the gene. Transformants
carrying the truncated disruption vector were also tested by PCR and Southern hybridization for disruption events and analyzed for toxin production. Disruption of Tri16 does not
affect toxin production. Northern analyses suggest that Tri16 is regulated like a secondary
metabolite as it is turned on in later cultures like several of the other toxin biosynthetic
genes. Tri16 is physically located on linkage group 2 whereas the main trichothecene
cluster is on linkage group 1. Even though Tri16 is found in the EST library, these studies
show that Tri16 is not necessary for toxin production.
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2002 National Fusarium Head Blight Forum Proceedings
A SYSTEMATIC APPROACH FOR IDENTIFYING ANTIFUNGAL
PROTEINS WITH ENHANCED RESISTANCE TO SCAB
Ajith Anand1*, Harold N. Trick2, Bikram S. Gill2 and S. Muthukrishnan1
Department of Biochemistry and 2Department of Plant Pathology,
Kansas State University, Manhattan, KS 66506
*Corresponding Author: PH: (785) 532-6939; E-mail: ajith@ksu.edu
1
OBJECTIVES
1) Identification and purification of antifungal proteins from apoplastic fluids of wheat plants.
2) Expression of recombinant antifungal proteins and test the effectiveness of the purified
proteins singly or in different combinations against scab using in vitro assays. 3) Incorporate
the desired antifungal genes into elite germplasm of wheat using the transgenic approach.
INTRODUCTION
One of the strategies to enhance disease resistance in plants is to make effective use of
their natural defenses such as pathogenesis-related (PR-) proteins. Many genes for PRproteins were shown to be induced upon scab infection of wheat indicating their importance
in plant defense (Li et al., 2001; Pritsch et al., 2001). In the past, we have utilized antifungal
genes with different cellular targets in wheat transformation studies without experimental
evidence that these proteins are actually effective against Fusarium graminearum (Chen et
al., 1999; Anand et al., 2001). Even though some of these transgenic plants were moderately resistant under greenhouse trials, field evaluation did not show any significantly improved resistance to scab suggesting that these lines could not withstand continuous pathogen pressure encountered in the field where both the type I and type II resistance is required
for survival (Anand et al., 2002 manuscript communicated). In order to speed up the effort to
obtain transgenic plants with enhanced scab resistance and to improve the chance of
success within a limited period of time, it will be useful to identify the genes encoding proteins that are effective against scab in preliminary in vitro assays. Thus a multi-pronged
approach relying on identification of genes that are likely to have antifungal activity, isolating
these genes from appropriate sources, and introducing them into wheat or barley plants has
been developed.
MATERIALS AND METHODS
Field testing of the transgenic wheat plants- Field testing of transgenic and control lines
were carried out in spring 2002 at the Plant Pathology Experimental Farm located near
Manhattan, KS, USA. A randomized complete block design was used with 20 replicates for
each treatment. Corn kernels (93 gm-2) colonized by F. graminearum were applied to the
soil.
Extraction of the Apoplastic fluid from wheat leaves- Ten grams of fresh leaves from
mature plants were vacuum infiltrated with 100 mM sodium phosphate buffer (pH 6.8). The
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2002 National Fusarium Head Blight Forum Proceedings
infiltrated leaves were dried on filter paper sheets and centrifuged at 5000 rpm for 30’. The
supernatant representing the apopalstic fraction was collected and stored at -70°C.
Recombinant expression of antifungal genes- The wheat cDNA clones isolated from the
fungus-infected plants of wheat (Li et al., 2001) or rice were used for expression. The
coding region fragments (minus signal peptide) were moved into the E. coli expression
vectors, pQE60 or pTOPO under the control of the lac promoter and induced with isopropyl
thiogalactoside (IPTG) for different time intervals to optimize maximum expression.
RESULTS AND DISCUSSION
Greenhouse and field testing of transgenic plants- Greenhouse testing was carried out
in spring 2001 and fall 2002 with 4 independent homozygous transgenic (see Table 1). The
line over-expressing the 383 chitinase/ 638 glucanase transgenes (#32A) showed a delay
in the development of disease symptoms, and was scored as moderately resistant, while
three other transgenic lines did not show any elevated levels of resistance reaction to scab
(Table 1). We suspect that there may be a requirement for a threshold level of PR-proteins
in order to be effective against scab. The results of the field evaluation are presented in
Table 2. The transgenic lines did not have any enhanced resistance against scab, suggesting that these lines could not withstand continuous pathogen pressure encountered in the
field where both the type I and type II resistance is required for survival.
Table 1. Greenhouse trials with the homozygous progenies of different transgenic wheat
lines.
E n try
# 3 2A
# 3 2C
M N 9 91 1 2
B o b w h ite
# 3 2A
# 3 2C
M N 9 91 1 2
B o b w h ite
#76
#78
# 82
M N 9 91 1 2
B o b w h ite
#76
#78
# 82
M N 9 91 1 2
B o b w h ite
T o tal no . p lan ts
in ocu lated
25
31
24
42
25
31
24
42
44
51
70
42
64
44
51
69
42
62
D ays after
in o cu lation
10
10
10
10
14
14
14
14
10
10
10
10
10
14
14
14
14
14
5
M ean in fected sp ik elets /
h ead
3 .7 b
6 .6 a
2 .0 c
6 .8 a
7 .4 b
1 2 .5 a
4 .2 c
1 3 .8 a
6 .5 b c
6 .1 c
7 .1 a
2 .05 d
6 .8 b a
1 1 .8 d
1 4 .0 b a
1 4 .6 a
3 .1 e
1 3 .0 c
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2002 National Fusarium Head Blight Forum Proceedings
Homozygous plants of #32A (383 chitinase and 638 glucanase), #32C (silenced line) used
as an epigenetic control, Line # 76 and # 78 (289 glucanase and 383 chitinase) and line
#82 (638 glucanase) were tested along with MN99112 (resistant check) and non-transformed ‘Bobwhite’ plants (susceptible check) respectively.
Table 2. Field evaluation of transgenic plants and control plants in spring 2002
E ntry
B T -14 -18
32 A
32C
B o bw h ite
W heaton
M N 991 12
S cab-7
S ym ptom
rating - 3 rd d ay
S ym ptom
rating - 6 th day
1 7.5 a
9.4 c
1 3.2 b
1 2.6 b
6.7 c
0.5 d
0 .85 d
2 5.6 a
23 .0b a
1 6.5 c
1 7.9 bc
1 2.2 c
1.4 d
3.5 d
S ym ptom
rating - 10 th
day
5 9.7 a
5 1.0 b
5 4.7 ba
53 b
49 b
9.0 d
3 3.5 c
S ym ptom rating
- 14 th day
6 5.0 ba
6 7.8 a
6 1.2 ba
6 0.8 b
5 2.7 c
1 9.3 d
5 2.2 c
BT-14-18, TLP transgenic line; 32A, transgenic line co-expressing 383 chitinase/638
glucanase; 32C, transgene-silenced line co-transformed with 383 chitinase/638 glucanase;
‘Bobwhite’, untransformed control; Wheaton, a susceptible check; MN99112, a resistant
check; Scab-7, a resistant check.
Characterization of the apoplastic fluid and in vitro antifungal assays- Western blot
analysis of apoplastic fluid from leaves of the line #32A (see Table 1, with lesion phenotype)
indicated that in addition to the expected transgene-encoded chitinase and β-1,3glucanase bands, these extracts contained several other PR-proteins, including TLP’s. In
the apoplastic (extracellular) fluid prepared from these leaves about 6-10 major bands could
be detected (Fig. 1). Further analyses indicated that the majority of the PR-proteins (>85%)
are secreted and are localized extracellularly. The apoplastic fluid from the chi/glu
transgenic line, 32A, with about 100 µg of total protein showed a distinct inhibitory effect
against F. graminearum in ivitro antifungal assays (data not shown). Less effective mycelial
growth inhibition was detected with equivalent amounts of the apoplastic fluid of the TLP
transgenic line, D34.
A spore germination inhibition assay using the conidial suspension of F. graminearum was
utilized to confirm the results of the mycelial growth inhibition assay. No germination of the
conidia could be detected in the presence of barley chitinase (4 µg) and apoplastic protein
preparation (25 µg protein) after 4 h of incubation, while 75%-100% germination was detected in the presence of non-transgenic apoplast extracts and 4 µg of M. sexta chitinase
(Fig. 2). It is likely that the inhibition of spore germination by the apoplastic fluid might be
due an additive or synergistic effects between chitinase and other PR-proteins including β1,3-glucanase and TLP, or other proteins present in the apoplastic fluid.
Recombinant expression of other PR-protein genes- The successful inhibition of
Fusarium graminearum in the two in vitro assays has prompted us to use an alternative
approach which involves identification of specific combinations of antifungal proteins effecBiotechnology
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2002 National Fusarium Head Blight Forum Proceedings
Western Blot Analysis
97 kDa
30 kDa
45 kDa
30 kDa
20 kDa
30 kDa
30 kDa
20 kDa
20 kDa
Apoplastic fluid fraction
Chitinase
Glucanase
TLP
Figure 1. Detection of PR-proteins in apoplastic fluid prepared from the lesionmimic transgenic wheat plant #32A. M = marker.
tive against scab using in vitro assays prior to the utilization of their genes in transgenic
studies. The coding region fragments (minus signal peptide) were insertd into the E. coli
expression vectors, pQE60 or pTOPO under the control of the lac promoter and colonies
expressing TLP, LTP and 194 chitinase upon induction with isopropyl thiogalactoside (IPTG)
were identified. The recombinant LTP protein appears to be soluble and could be detected
in the supernatant after sonication of the cultures while the rice D34 TLP and 194 chitinase
proteins were in the pellet fraction.
Barley chitinase
(4 µg)
Apoplastic # 32A
(25 µg)
Apoplatic control
(25 µg)
M. sexta chitinase
(4 µg)
Figure 2. Spore germination inhibition assay with different protein preparations. Arrows
indicate the germinating conidiophores.
ACKNOWLEDGMENT
The authors thank J. S. Essig, Dr. M. L. Main, and R. D. Wamsley for wheat transformation
and regeneration. Dr. W. Bockus, Mr. D. Wilson and Mr. M.A. Davis are sincerely acknowledged for their expertise and assistance with scab testing and evaluation.
REFERENCES
Anand, A., Zhou, T., Bockus, B., Muthukrishnan, S., Trick, H.N and Gill, B.S. 2002. Greenhouse testing and
field evaluation of transgenic wheat constitutively expressing different PR-proteins against scab. J. Experimental Botany (communicated).
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2002 National Fusarium Head Blight Forum Proceedings
Anand, A., Janakiraman, V., Zhou, T., Gill, B. S., and Muthukrishnan, S. (2001) Transgenic wheat
overexpressing PR-proteins shows a delay in Fusarium head blight. 2001 National Fusarium Head Blight Forum
Proceedings, 2-6.
Chen, W. P., Gu, X., Liang, G. H., Muthukrishnan, S., Chen, P. D., Liu, D. J., and Gill, B. S. (1998) Introduction
and constitutive expression of a rice chitinase gene in bread wheat using biolistic bombardment and the bar
gene as a selectable marker. Theor. Appl. Genet. 97:1296-1306.
Li, W.L., Faris, J.D., Muthukrishnan, S., Liu, D.J., Chen, P.D., and Gill, B.S. (2001) Isolation and characterization of novel cDNA clones of acidic chitinases and β-1,3-glucanases from wheat spikes infected by Fusarium
graminearum. Theor. Appl. Genet. 102:353-362.
Pritsch, C., Vance, C.P., Bushnell, R.W., Somers, A.D., Hohn, T.M., and Muehlbauer G.J. (2001). Systemic
expression of defense response genes in wheat spikes as a response to Fusarium graminearum infection.
Physiological and Molecular Plant Pathology 58, 1-12.
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VERIFICATION OF MOLECULAR MARKERS LINKED TO FUSARIUM
HEAD BLIGHT RESISTANCE QTLS IN WHEAT
N. Angerer, D. Lengauer, B. Steiner, and H. Buerstmayr*
IFA-Tulln, Institute for Agrobiotechnology, Department of Biotechnology in Plant Production,
Konrad Lorenz Strasse 20, A-3430 Tulln, Austria (website: http://www.ifa-tulln.ac.at)
*Corresponding Author: PH: 43 2272 66280 205; E-mail: buerst@ifa-tulln.ac.at
ABSTRACT
Molecular mapping led to the identification of QTL on chromosomes 3B and 5A of wheat
(Anderson et al. 2001, Buerstmayr et al. 2002). The aim of this work was to verify molecular
markers linked to these QTL in several spring by winter wheat crosses.
Crosses were initiated between FHB resistant spring wheat CM-82036 and several adapted
European winter wheat genotypes. Populations of recombinant inbred lines in F4 to F8
generation were evaluated for resistance to FHB in replicated artificially inoculated field
experiments.
The lines were genotyped with SSR markers mapping to one of the two putative QTL regions, 3B: GWM398, GWM533, GWM493, and 5A: GWM293, GWM304, GWM156.
Depending on testing year and population, markers on 3BS were more frequently associated with FHB resistance reaction than those on 5A, indicating that the QTL on 3BS has a
larger and more consistent effect than the 5A QTL.
Not all 6 markers linked to a QTL in the model spring wheat population showed significant
association in the verification populations. Further analysis should reveal the reasons for
that.
Despite that, marker assisted selection for FHB resistance appears efficient in material
segregating for resistance originating from CM-82036.
REFERENCES
Anderson, J.A., R.W.Stack, S. Liu, B.L. Waldron, A.D. Fjeld, C. Coyne, B. Moreno-Sevilla, J. Mitchell Fetch, QJ.
Song, P.B. Cregan, and R.C. Frohberg. 2001. DNA markers for a Fusarium head blight resistance QTL in two
wheat populations. Theor Appl Genet 102: 1164-1168.
Buerstmayr, H., M. Lemmens, L. Hartl, L. Doldi, B. Steiner, M. Stierschneider, and P. Ruckenbauer. 2002.
Molecular mapping of QTL for Fusarium head blight resistance in spring wheat I: resistance to fungal spread
(type II resistance). Theor Appl Genet 104: 84-91.
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2002 National Fusarium Head Blight Forum Proceedings
WHEAT TRANSFORMATION FOR ENHANCED FUSARIUM
HEAD BLIGHT TOLERANCE
P. Stephen Baenziger1*, A. Mitra2, M.Dickman2, T. Clemente1, S. Sato1, S. Mitra1,
J. Schimelfenig2, and J. Watkins2
Dept. of Agronomy and Horticulture and 2Dept. of Plant Pathology,
University of Nebraska—Lincoln, Lincoln, NE 68583-09815
*Corresponding Author: PH (402) 472-1538; E-mail: pbaenziger1@unl.edu
1
OBJECTIVES
To transform wheat lines with genes that may lead to improved tolerance of Fusarium head
blight.
SUMMARY OF WORK
Fusarium graminearum is an important pathogen of wheat. Infection can result in significant
yield losses and greatly reduce grain end-use quality due to detectable levels of the mycotoxin, Deoxynivalenol. To date, insufficient genetic resistance towards this pathogen has
been identified within wheat germplasm. Biotechnology provides an avenue to introduce
novel genes into elite wheat germplasm to complement an integrated program to manage F.
graminearum pathogenesis. We have developed transgenic wheat with antifungal and antiapoptotic genes using the Agrobacterium-transformation methods. Our preliminary data
indicate that this approach will be effective in controlling FHB in wheat.
Antifungal genes: We have tested an animal lactoferrin gene for potential antifungal
activity to F. graminearum. Lactoferrin (LF), an iron binding glycoprotein has long been
reported to be active against a wide range of microorganisms including fungi (Zhang et. al.
1998). Lethal concentration of bovine LF, for instance, ranged from 18 to 100 µg/ml against
yeast and 2 to 20 µg/ml against filamentous fungi (Bellamy et. al.; 1994). The lethal action of
LF is believed to be due to the binding of the protein to the membrane and subsequent
disruption of proton-gradient across the membrane. This results in membrane leakage and
ultimately cell death. Lactoferrin contains an active antimicrobial domain lactoferricin
(LFcin). Chong and Langridge (2000) recently demonstrated that LF protein expressed in
potato tuber tissues has strong antimicrobial activities. The expression of non-plant antimicrobial genes such as LF in a transgenic plant has potential for broad-spectrum disease
resistance.
In our previous work, the production of LF in tobacco cells indicated a potential for the
development of disease resistant transgenic plants (Mitra and Zhang, 1994, Zhang et. al.
1998). In vitro analyses of total protein extracts from transgenic tobacco demonstrated
strong anti Fusarium graminearum activity. Accordingly, transgenic wheat plants expressing
the LF gene, were generated using both gene-gun and Agrobacterium mediated transformation methods. Among transgenic lines tested, five gene-gun generated lines and eight
Agrobacterium generated lines consistently showed high levels of type II resistance (less
than 10% infection) against scab in greenhouse experiments. Additional transgenic wheat
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plants were generated expressing a short, synthetic LFcin gene (the 41 amino-acid long
peptide from the amino-terminal end of LF) had a much stronger antimicrobial activity in
tobacco than LF. Both LF and LFcin serve as membrane-disruptive agents when interacting
with fungal membranes. Lactoferricin is substantially smaller and more cationic than LF;
features that might help LFcin penetrate the fungal membrane more efficiently and protect it
from further degradation by plant proteases. Nine western positive plants were obtained
and 8-15 progeny of 3 transgenic lines were tested for type-II resistance in greenhouse. The
plants were either 100% susceptible or had a high level of resistance (disease rating 10).
The resistance correlated with the expression of the LFcin protein. In line 1, out of 15 plants,
6 were resistant (disease rating 10%) and 9 were susceptible (disease rating 100%); in line
2, out of 14 plants, 9 were resistant (disease rating 10%) and 5 were susceptible (disease
rating 100%); and in line 3, out of 8 plants, 5 were resistant (disease rating 10%) and 3 were
susceptible (disease rating 100%). Some PCR positive plants were susceptible, however,
no lactoferrin protein could be detected in these plants. We have identified 9 LF and 6 Lfcin
homozygous lines that showed consistent high level scab resistance in green house conditions.
The lactoferrin lines contained a human lactoferrin gene and the lactoferricin lines contained a synthetic human gene sequence. As a result, we decided to delay field trials of
these lines and develop a parallel system using bovine lactoferrin gene. Significant amount
of lactoferrin is present in milk and traces are also found in beef. Bovine lactoferrin will be
safer and more acceptable to consumers and farmers. Accordingly, we have generated 18
primary wheat transformants containing the bovine lactoferrin (blf) gene and 7 primary
wheat transformants with bovine lactoferricin (blfcin) gene. A preliminary assay showed
anti-Fusarium activity of total protein extract from transgenic wheat with bovine constructs.
Homozygous plants of these lines are being created for testing in green house and in field
trials (pending authorization). As for intellectual property rights, we are authorized to use
the full-length LF and the synthetic LFcin, and A-16 promoter belongs to us.
Antiapoptic genes: Our second major set of genes relates to apoptosis, or programmed
cell death (PCD). Apoptosis is a highly regulated process whereby individual cells of multicellular organisms undergo systematic self-destruction in response to a wide variety of
stimuli. Programmed cell death regulates normal cellular turnover, the immune system,
embryonic development, metamorphosis, hormone dependent atrophy, and chemicalinduced cell death (Pennell and Lamb, 1997; Ryerson and Heath, 1996; Jones and Dangl,
1996). It is believed that cell suicide responses evolved in response to viruses, providing a
mechanism for limiting viral replication and spread. Most viruses have evolved genes encoding proteins that effectively suppress or delay PCD long enough for production of sufficient progeny. In addition, a growing number of viruses can induce PCD late in the infection
process, which may promote spread of progeny to surrounding cells, while evading host
immune inflammatory responses and protecting progeny virus from host enzymes and
antibodies.
We have evidence that members of an animal anti-apoptotic gene family (Bcl-2) function in
plants (Dickman et. al., 2001). Transgenic tobacco lines were generated harboring various
anti-apoptotic proteins (human Bcl-2 and Bcl-XL, nematode CED-9, and baculovirus OpIAP). When several economically important fungal and viral pathogens were inoculated
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onto tobacco harboring these transgenes, the plants were highly tolerant and in most cases,
completely resistant. We have now extended these findings to wheat, including having a
number of elite lines expressing resistance to scab. We believe the transgenes are functioning as expected and as they do in other plants for the following reasons. The observed
resistance to the necrotrophic fungal (scab) pathogen is consistent with our previous observations in tobacco (Dickman et al, 2001). In addition, we have resistance in wheat to heat
and salt stress which is also in accordance with results in tobacco. Thus, we are eager to
evaluate these lines under field conditions. We now have homozygous (T5) lines of wheat
carrying Op-IAP. We are creating homozygous lines for Bcl-xl and a mutant transgene Bcl-xl
(G138A). The advantages of Bcl-xl, are: (i) the structure and mode of action is different then
Op-IAP, although both genes are cytoprotective (anti-apoptotic); (ii) antibodies are commercially available for Bcl-xl, and (ii) we have a null mutant construct for comparative purposes.
In addition, we are generating new wheat lines harboring Sf-IAP (Huang et al, 2000). Sf-IAP
from the insect Spodoptera frugiperda is in the same class of proteins as OP-IAP. However,
other transgenic plants (tomato and Arabidopsis) harboring this gene exhibit a number of
interesting phenotypes (eg. delayed fruit ripening, delayed senescence) as well as fungal
disease resistance.
Our Transformation Protocol: Introduction of a maize RIP into wheat as an example: A
maize ribosomal inactivating protein (Genbank accession No. AF233881) was isolated from
maize leaf (cv A188) via PCR. The PCR product was sequenced for authenticity and subsequently subcloned down stream of the maize ubiquitin promoter element coupled with its
5’ intron. The resultant cassette was dropped into the binary plasmid pPZP212
(Hajdukiewicz et al., 1994). The final binary vector is referred to as pPTN285. The binary
plasmid was mobilized into A. tumefaciens strain C58C1 carrying the Ti plasmid pMP90
(Koncz and Schell, 1986) via tri-parental mating (Ditta et al., 1980). Wheat transformations
(cv Bobwhite) were set-up following a modification of the protocol described by Cheng et al.
(1997). A total of ten lines representing eight independent transformants were recovered.
Ten T1 seed per line were sown in the greenhouse. An npt II ELISA was conducted on the
individuals approximately 20 day after planting using a commercial kit (Agdia Cat# 73000/
0480) following the manufacturer’s instructions. Northern blot analysis was conducted on a
T ab le 1. S eg reg ation & N o rth ern b lot (N . b lot) data in T 1 g eneratio n o f w heat lines tran sfo rm ed
w ith pP T N 28 5
L ine
N pt II P os. N pt II N eg .
N . b lot
13 0 -02 -02 -01
10
0
P os
13 0 -02 -05 -01
7
3
P os
13 0 -02 -06 -01
5
5
P os
13 0 -03 -01 -01
4
6
P os
13 0 -03 -02 -01
8
2
P os
ye03 -04 -02 -01
6
4
N eg
ye10 -06 -01 -01
10
0
P os
ye10 -06 -01 -02
5
5
P os
ye10 -06 -01 -03
6
4
P os
ye10 -03 -01 -01
9
1
P os
N pt II P os an d & N eg . colu m n s refer to th e tota l n um ber o f T i in div iduals positive for np t II
ex pression as de term ined b y E L IS A . N . b lo t co lu m n in dicates w hether th e sub set of np t II
po sitive T 1 in divid uals d erived from th e resp ectiv e line w ere ex pressing th e R IP tran scrip t.
Biotechnology
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2002 National Fusarium Head Blight Forum Proceedings
subset of the npt II positive individuals per line. A summary of the data is given in Table 1
below. T2 seed is being increased to identify homozygous lines for future screening.
Field Testing: In 2002, we undertook our first field trials of transgenic wheat. The trials
were done at Mead, NE with APHIS approval. While the lines were misted to induce FHB
infection, the climate was extremely hostile with severe heat after planting which “pushed”
the plants to mature early and produce poor quality seed. We are currently evaluating the
data, but it is expected that the main outcomes of this year’s experiments were increase our
seed and to learn how to meet the necessary regulations to take transgenic lines to the field.
Transfer of transgenes to elite wheat varieties: We are crossing the most FHB tolerant
transgenic lines to Alsen (elite FHB tolerant line), to Wheaton (elite FHB susceptible line),
and to Wahoo or Millennium, and Wesley, popular recent releases. Provided these efforts
show promise, we will expand our crosses to additional lines and market classes. We are
also making crosses among and between our elite antifungal genes and our elite
antiapoptotic transgenic lines. We are interested in determining if pyramiding genes having
similar mechanisms, and transgenes affecting the two mechanisms of antifungal activity that
we are studying in combination may provide added protection against FHB.
REFERENCES
Bellamy, W., M. Takase, K.Yamauchi, H. Wakabayashi, K. Kawase, S. Shimamura, and M. Tomita. 1994. Role of
Cell binding in the mechanism of Lactoferrin action. Lets. in Appl. Microbiol. 18:230-233.
Cheng, M., J.E. Fry, S. Pang, H. Zhou, C.M. Hironaka, D.R. Duncan, T.W. Conner, and Y. Wan. 1997. Genetic
transformation of wheat mediated by Agrobacterium tumefaciens. Plant Physiol. 115:971-980.
Chong, D. K. X. and W. H. R. Langridge. 2000. Expression of full length bioactive antimicrobial human lactoferrin
in potato plants. Transgenic Research 9: 71-78.
Dickman, M.B., Y. K. Park, T. Oltersdorf, W. Li, T. Clemente, and R. French. 2001. Abrogation of disease
development in plants expressing animal anti-apoptotic genes. Proc. Natl. Acad. Sci. 98: 6957-6962.
Ditta, G., S. Stanfield, D. Corbin and D. Helinski. 1980. Broad host range DNA cloning system for gramnegative bacteria: Construction of a gene bank of Rhizobium meliloti. Proc. Natl. Acad. Sci. USA 77:7347351.
Jones, A.M. and J. L. Dangle. 1996. Logjam at the Styx: Programmed cell death in plants. Trends in Plant
Science 1:114-119.
Koncz, C., and J. Schell. 1986. The promoter of TL-DNA gene 5 controls the tissue specific expression of
chimaeric genes carried out by a novel type Agrobacterium binary vector. Mol. Gen. Genet. 204:383-396.
Mitra, A. and Z. Zhang. 1994. Expression of a human lactoferrin cDNA in tobacco cells produces antibacterial
proteins. Plant Physiol. 106: 977-981.
Pennell, R.I. and C. Lamb. 1997. Programmed cell death in plants. Plant Cell 9:1157-1168.
Ryerson, D.E. and M. C. Heath. 1996. Cleavage of nuclear DNA into oligonucleosomal fragments during cell
death induced by fungal infection or by abiotic treatments. Plant Cell 8:393-402.
Zhang, Z., D.P. Coyne, A.K. Vidaver, and A. Mitra. 1998. Expression of human lactoferrin cDNA confers resistance to Ralstonia solanacearum in transgenic tobacco plants. Phytopathology 88: 730-734.
13
Biotechnology
2002 National Fusarium Head Blight Forum Proceedings
MOLECULAR CHARACTERIZATION OF SCAB
RESISTANCE QTL IN WHEAT
G-H. Bai*, A. Bernardo, P-G. Guo, K. Xiao, M. Das, X-Y. Xu and S. R. Gaddam
Dept. of Plant and Soil Sciences, Oklahoma State University, Stillwater, OK 74078
*Corresponding Author: PH: 405-744-9608; E-mail: bai@mail.pss.okstate.edu
Profiling of scab resistance gene expression in wheat
The goals of this project are to discover new genes for scab resistance, identify the pathway
involved in wheat resistance to scab infection and understand the genetic mechanism of
Type II resistance in wheat. Three pairs (forward and reverse) of suppression subtractive
hybridization libraries have been developed from infected spikes harvested 6, 36 and 72
hours after inoculation (HAI) by subtracting cDNA of bulked infected susceptible recombinant inbred lines (RIL) from bulked infected resistant RILs. The RILs generated from the
cross between the resistant cultivar Ning 7840 and the susceptible cultivar Clark. So far,
2,306 differentially expressed sequence tags (ESTs) have been cloned and printed in glass
slides for microarray analysis. About 80 clones were sequenced. For microarray analysis,
several labeling and hybridization kits or protocols were experimented and only the kit from
Genisphere produced reliable result with low background. Infected wheat spikes collected
from both bulks at various time courses after inoculation will be used as probes for array
hybridization to profile the scab resistance gene expression.
Mapping QTL from Wangshuibai
The objectives of this project are to: (1) discover new QTL for scab resistance from the
Chinese landrace Wangshuibai which does not relate to Sumai 3, (2) investigate QTL
effects on type II resistance in Wangshuibai, and (3) develop selectable markers for markerassisted selection. F8 recombinant inbred lines derived from the cross between
Wangshuibai and the susceptible cultivar Alondra’s were evaluated for Type II resistance in
the greenhouse experiment. Total 15 plants were evaluated for each line. The frequency
distribution of percentage of scabbed spikelets among 104 F8 RILs showed continuous
distribution with one peak skewed toward resistant parent (Wangshuibai). The same distribution was observed for DON yield which was evaluated by Dr. Hart from Michigan State
University. About 200 pairs of SSR primers were screened for the parents and about 30%
showed polymophism. Based on the greenhouse scab evaluation, bulked resistant and
susceptible lines were selected and are being used for SSR primers screening. The RILs
will be further evaluated for scab resistance and DON yield next year and more SSR markers will be screened to identify closely linked markers to the QTL for scab resistance in
cultivar Wangshuibai.
Marker-assisted selection
The goals of this project are to increase throughput of marker screening, reduce marker
analysis cost, and develop breeder-friendly STS markers to facilitate marker-assisted selection (MAS). To improve efficiency of MAS, we optimized a fast DNA isolation protocol. In this
Biotechnology
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2002 National Fusarium Head Blight Forum Proceedings
protocol, NaOH is the DNA extraction buffer and Tris is used as DNA storage buffer. The
FastPrep system from Q.Biogene and a mini centrifuge are used for tissue preparation. DNA
can be isolated with this method in any location where electricity is available. This method is
suitable for MAS in conventional breeding programs since costs of equipment and reagents
are low. With this method, about 200 DNA samples can be isolated daily by one person.
Isolated DNA is good for STS and SSR even after 1 year of storage at –20°C. A Li-Cor
Sequencer is used for SSR analysis. To increase throughput and reduce cost, primers of two
flanking markers are labeled with two different fluorescence dyes (IR800 and IR700) and
combined in one PCR reaction. The PCR is analyzed in one gel and produces images of
both flanking markers. Each gel can be reused for 3-4 times. This method significantly
reduces cost of MAS and has been successfully used for marker-assisted backcross to
transfer 3BS QTL to Clark background.
To develop breeder friendly marker, one STS marker tightly linked to 3BS scab resistance
QTL was converted from an AFLP marker and has been released to several breeding
programs. This STS explained about 50% of phenotypic variation for Type II resistance in
the population from the cross of Ning7840/Clark. It can be amplified with crude DNA isolated with the simplified method. An additional STS was developed from another AFLP
marker, but the PCR condition still needs to be optimized before it can be used for MAS.
15
Biotechnology
2002 National Fusarium Head Blight Forum Proceedings
GENETIC DIVERSITY OF NEW FUSARIUM HEAD BLIGHT
RESISTANT BARLEY SOURCES
K.M. Belina, W.J. Wingbermuehle, and K.P. Smith*
Dept. of Agronomy and Plant Genetics, University of Minnesota, St. Paul, MN
*Corresponding Author: PH: (612) 624-1211; E-mail: smith376@tc.umn.edu
OBJECTIVE
To determine the genetic relatedness of new sources of Fusarium head blight resistance in
barley to those already in use by the Minnesota barley breeding program.
INTRODUCTION
Since the outbreak of FHB in the Upper Midwest, there has been a major effort to identify
FHB resistant sources and breed for FHB resistant malting barley. A number of barley
varieties from around the world have been identified as potential sources of FHB resistance.
All of these varieties exhibit partial resistance to FHB and, based on several mapping
studies, it is known that resistance to FHB in barley is a quantitative trait controlled by a
number of genes (Kolb et al, 2001). Thus, to obtain durable disease resistance, the best
breeding strategy is to pyramid genes for FHB resistance from multiple resistant sources
into one or more varieties. Many of the known FHB resistant barley sources are currently in
use in variety development and/or genetic mapping studies. To date, no single source
appears to provide sufficient resistance and additional new sources of resistance with novel
FHB resistance genes are needed.
A major effort to identify new sources was undertaken by researchers at North Dakota State
University (NDSU) who screened of over 8,200 accessions of six-rowed spring barley from
the USDA Small Grains Germplasm Collection (Steffenson, 2002). These researchers
identified some accessions with FHB resistance equal to or greater than that of Chevron;
currently the standard six-rowed resistant check in most barley FHB research (Table 1). To
more effectively utilize these new sources we wanted to determine how related these new
sources are to those already in use.
MATERIALS AND METHODS
The objective of this research was to use SSR markers to determine the genetic relatedness
of new sources of FHB resistance in barley (Table 1) to sources already in use by the Univ.
of MN barley breeding program (Table 2). We obtained seeds of new accessions from Dr.
Brian Steffenson at the Univ. of MN. Dr. Steffenson originally obtained seed from the
USDA’s Germplasm Resources Information Network (GRIN) and performed single head
selection on each line. Plants were grown in the greenhouse and DNA extracted from a
single plant. A genetic diversity study, previously conducted in our lab, included many of the
parents our barley program has used in breeding for FHB resistance (Wingbermuehle,
2002). To combine the information from the Wingbermuehle study with the accessions
screened here for the first time, we selected, for each SSR primer, a representative genoBiotechnology
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2002 National Fusarium Head Blight Forum Proceedings
type for each allele that had been previously identified. Then, for each primer, we re-ran
these representatives along with the new genotypes. In this way, we could unambiguously
assign SSR allele genotypes to each new accession. We used sixty-nine SSR markers in
this study. For two of these markers (HvLTPPB and Ebmatc0028), two loci were used in
analysis for a total of 71 polymorphic loci analyzed. Primers were chosen that both provided
coverage spanning the barley genome and included eight markers that have been previously linked to FHB resistant quantitative trait loci (QTL) (de la Pena et al., 1999; Mesfin et
al., 2003).
Table 1. Recently identified FHB resistant accessions from the spring six-rowed barley
world collection.
Accession
CIho 2492
PI 467654
CIho 6613
PI 371317
PI 371308
PI 370919
PI 370984
Origin
Sweden
Finland
United States
Switzerland
Switzerland
Switzerland
Switzerland
Accession
PI 328642
CIho 9625
PI 383090
CIho 4530
PI 565567
PI 565583
PI 565854
Origin
Romania
Ethiopia
Ethiopia
China
China
China
China
Table 2. Midwest varieties, FHB resistant breeding lines, and FHB resistant varities.
Line
Description
Line
Description
AC Oxbow
FHB resistant variety, 2-row, covered
hull, origin - Canada
FEG4-98
U of MN breeding line, 6-row; FHB
resistance derived from Atahualpa
Atahualpa
FHB resistant variety, 2-row, hulless,
origin - Ecuador
FHB resistant variety, 6-row, covered
hull, origin - Switzerland
FHB resistant variety, 2-row, covered
hull, origin - Japan
FHB resistant variety, 6-row, hulless,
origin - Ukraine
FHB resistant variety, 2-row, covered
hull, origin - USA
FHB resistant variety, 2-row, covered
hull, origin - China
2-row, covered hull, origin – Japan
FEG6-28
M100
U of MN breeding line, 6-row; FHB
resistance derived from Kitchin
6-row malting variety developed at
NDSU
6-row malting variety developed at the U
of MN
6-row malting variety developed by
Busch Agricultural Resources, Inc.
U of MN advanced breeding line, 6-row
M104
U of MN advanced breeding line, 6-row
M105
U of MN advanced breeding line, 6-row
2-row variety developed by North
Dakota State University (NDSU)
6-row malting variety developed at
NDSU
6-row malting variety developed at the
University of Minnesota (U of MN)
U of MN breeding line, 6-row; FHB
resistance derived from AC Oxbow
U of MN breeding line, 6-row; FHB
resistance derived from Zheddar #1
U of MN breeding line, 6-row; FHB
resistance derived from Atahaulpa
M81
U of MN advanced breeding line, 6-row
M84
U of MN advanced breeding line, 6-row
MAS2-002
U of MN breeding line, 6-row; FHB
resistance derived from Kitchin
U of MN breeding line, 6-row; FHB
resistance derived from Kitchin
6-row variety developed by the U of
MN, moderately resistant to FHB
6-row malting variety developed at the U
of MN
Chevron
Frederickson
Hor211
Kitchin
Zhedar1
CIho 9831
Conlon
Drummond
Excel
FEG14-119
FEG2-26
FEG4-66
Foster
Lacey
Legacy
MAS2-054
MNBrite
Robust
17
Biotechnology
2002 National Fusarium Head Blight Forum Proceedings
All gels were run using the Global IR2 collection system with gel images recorded by E-seq
software (LI-COR, Inc). GeneImagIR software (LI-COR, Inc) was used to size bands on gel
images. Final band scores were determined visually with the aid of GeneImagIR. Data
from the two studies were combined and organized using Microsoft Excel. Cluster and
principal coordinate (PCO) analyses were conducted using NTSYSpc software (Exeter
Software). Cluster analyses were calculated using the unweighted pair-group method,
arithmetic average (UPGMA) algorithm.
RESULTS AND DISCUSSION
Using the full set of 71 SSR loci spanning the barley genome, we determined the genetic
relationships of new FHB resistant barley accessions to resistant parent sources already in
use and to existing varieties and breeding lines. From this analysis, larger clustering of
groups is most evident with the PCO (Figure 1), while relationships between subsets of
samples are easier seen with the cluster diagram (Figure 2). Both PCO and cluster analysis
show genotypes falling into two major groups. The first group contains all of the six-rowed
varieties and breeding lines. The second group contains the new resistant sources, the
resistant sources already in use, and the two two-rowed varieties CIho9831 and Conlon.
Particularly interesting with respect to FHB breeding efforts are the relationships in the
second group – those within and between the new resistance sources and those already
being used. Within this cluster there is clearly more variation than within the variety and
breeding line group (Figure 2). In addition, overall clustering does not appear to group
genotypes by origin of accession. Toward the goal of combining different sources of
resistance into a common variety, choosing new parent sources from those most different to
any already being used is likely the best option.
Of the FHB resistance sources already in use as parents, Frederickson and Zhedar 1 (both
two-rowed), are highly similar at 97%. All other parent sources appear to be relatively
different. Based on a similarity index using the 71 SSR marker loci set, all other parent
sources are less than 40% similar to one another (data not shown). Overall, the new FHB
resistant accessions are also not highly similar to one another. This is with the exception of
PI371308 and PI383090, which are 94% similar. Between the sources already in use and
the new sources, two relationships stand out. First, Hor211 clusters closely with PI370919
and PI467654. Second, Chevron is most like accession CIho6613 at 74% similarity. While
genetic dissimilarity does not insure that accessions will contain different genes for FHB
resistance, maximizing the dissimilarity between new parents and those already in use,
should increase the chance of identifying and combining different FHB resistance.
REFERENCES
Kolb, F.L., G-H. Bai, G.J. Muehlbauer, J.A. Anderson, K.P. Smith, and G. Fedak. 2001. Host plant resistance
genes for Fusarium head blight: mapping and manipulation with molecular markers. Crop Sci. 41:611-619.
A. Mesfin, K.P. Smith, R. Dill-Macky, C.K. Evans, R. Waugh, C.D. Gustus, and G.J. Muehlbauer. 2002. Quantitative Trait Loci for Fusarium Head Blight Resistance in Barley Detected in a Two-Rowed by Six-Rowed Population. Crop Sci. (in Press).
Biotechnology
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2002 National Fusarium Head Blight Forum Proceedings
Steffenson, B.J. 2002. Fusarium head blight of barley: impact, epidemics, management, and strategies for
identifying and utilizing genetic resistance. In: K.J. Leonard, W.R. Bushnell (eds.), Fusarium Head Blight of
Wheat and Barley. Amer. Phytopath Soc. Press. St. Paul, MN.
Windels, C.E. 2000. Economic and social impacts of Fusarium head blight: changing farms and rural communities in the Northern Great Plains. American Phytopathological Society 90 (1):17-21.
Wingbermuehle, W.J. 2002. Separating function and form: Using genetic diversity and selective genotyping
to determine if breeding populations are segregating for unknown Fusarium head blight (FHB) resistance genes
in barley. University of Minnesota. MS thesis.
de la Pena, 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.
19
Biotechnology
C3
A xis 3
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20
P I4 67 65 4
0 .1 3
0 .3 4
0 .5 5
G enetic S im ilarity
0 .7 6
0 .9 7
C o p h en etic co rrelatio n r = 0.98
0 .51
0 .51
acco u n t fo r 38.16% o f to talv ariatio n .
0 .28
0 .28
Fig ure 2 . U P G M A clu ster an aly sis o f b arley lin es an d F u sariu m
h ead b lig h t resistan t so u rces,co n d u cted u sin g 71 S S R lo ci.
0 .05
0 .05
A xis 1
M A S 2 -0 54
M A S 2 -0 02
F E G 2-2 6
M 84
M 81
L acey M 1 05
E xcel R o bu st
L eg acy
M 1 00
M 1 04
F E G 1 4 -11 9
F E G 2 -2 6
M 84
E xcel
M 1 00
L acey
M 81
M N B rite
M 1 04
M 1 05
L egacy
R o bu st
F o ster
S tan d er
F E G 6 -2 8
F E G 4 -6 6
M A S 2 -00 2
D ru m m o nd
F E G 4 -9 8
M A S 2 -05 4
C I4 5 3 0
C I2 4 9 2
P I5 6 58 54
P I5 6 55 67
C I6 6 1 3
C h evron
P I3 7 13 08
P I3 8 30 90
P I3 7 13 17
A C O x b ow
C o n lon
F red erick son
Z h edar1
P I3 2 86 42
P I3 7 09 19
H o r21 1
P I4 6 76 54
A tahualp a
K itchin
C I9 6 2 5
C I9 8 3 1
P I3 7 09 84
P I5 6 55 83
1 o f b arley lin es an d F u sariu m h ead
Fig ure 1 . P rin cip alco o rd in ate an alyC sis
b lig h t resistan t so u rces,co n d u cted u sin g 71 S S R lo ci. F irst th ree axes
-0 .19
-0 .19
F rederickso n
Z h ed ar1
0 .07 0 .07
A xis 20 .31 0 .31
0 .550 .55
P I3 28 64 2
P I3 71 30 8 P I3 83 09 0
A tah u alp a P I5 65 56 7
P I3 71 31 7
C h evro n
F E G 4 -9 8 F E G 4-6 6
P I5 65 85 4
M N B rite
P I3 70 98 4
C I6 61 3
F o ster
C I4 53 0
K itch in
F E G 1 4 -1 19
S tan d er
C I9 83 1
F E G 6 -2 8
C I2 49 2
C I9 62 5 A C O xb o w
D ru m m o n d
C o n lo n
-0 .17
-0 .17
.42.42
-0-0
.41.41
-0 -0
C2
.32.32
-0 -0
-0 .02
-0 .02
P I5 65 58 3
0 .207.27
0 .57
H o r2 11
V arieties and breeding lines
F H B resistant sources
P I3 70 91 9
2002 National Fusarium Head Blight Forum Proceedings
2002 National Fusarium Head Blight Forum Proceedings
MAPPING FUSARIUM HEAD BLIGHT RESISTANCE QTL IN THE
CHINESE WHEAT LINE FUJIAN 5114
D.E. Bowen1, S. Liu1, R. Dill-Macky2, C.K. Evans2, and J.A. Anderson1*
1
Department of Agronomy and Plant Genetics and 2Department of Plant Pathology,
University of Minnesota, St. Paul, MN 55108
*Corresponding Author: PH: (612)625-9763; E-mail: ander319@umn.edu
ABSTACT
Breeding for resistance to Fusarium head blight (FHB) is facilitated by the identification of
different resistance lines and resistance QTL. A population of 78 recombinant inbred lines
(RIL) was developed from the cross Fujian 5114/Norm, and was screened for FHB resistance in three field and two greenhouse experiments. Fujian 5114 is a spring wheat cultivar
from the Fujian Province of China. Fujian 5114 has levels of FHB resistance similar to
‘Sumai 3’, but putatively differs from Sumai 3 in some resistance loci. The RIL population
was evaluated for FHB severity and visually scabby kernels (VSK) in mist-irrigated, inoculated field trials in the summers of 2000 and 2001. The population was also evaluated for
spread within the spikelet from point inoculations in two greenhouse trials in 2001. The
results generally correlate well (r = 0.29-0.82 for correlations with p<0.05), with the best
correlations resulting from the greenhouse experiments. In the field study, the proportion of
variance due to RIL was 29% and 30% for field severity and VSK, respectively, and variance
due to RIL X Environment was 34% and 12%. Heritability on an entry mean basis ranged
from 0.90 in the greenhouse to 0.66 in the field FHB severity evaluations. Sixty
microsatellite markers were mapped on the entire population and this information was
combined with phenotypic data for QTL analysis. Interval analysis confirmed the presence
of the 3BS resistance QTL in Fujian 5114. This QTL explained up to 28% of the phenotypic
variation in FHB. An additional QTL was identified on chromosome 5BL, explaining up to
25% of the variation in FHB severity. The R2 values of the two QTLs are higher for the two
greenhouse experiments than those of the field experiments. The QTL on 5BL appears to
be associated with delayed spread of the disease, as the corresponding R2 values were
reduced from the 15 to the 21 day greenhouse evaluations. These results indicate that
Fujian 5114 contains some FHB resistance loci that differ from Sumai 3. Additional investigation of the 5BL QTL for breeding of increased resistance to FHB is warranted.
21
Biotechnology
2002 National Fusarium Head Blight Forum Proceedings
MOLECULAR MAPPING OF QTLS FOR FUSARIUM HEAD
BLIGHT RESISTANCE IN SPRING WHEAT
H. Buerstmayr1*, B. Steiner1, L. Hartl2, M. Griesser1, N. Angerer1
D. Lengauer1, and M. Lemmens1
1
IFA-Tulln, Institute for Agrobiotechnology, Department of Biotechnology in Plant Production, Konrad Lorenz
Strasse 20, A-3430 Tulln, Austria. URL: http://www.ifa-tulln.ac.at; and 2Bayerische Landesanstalt für
Bodenkultur und Pflanzenbau, Vöttingerstrasse 38, D-85354 Freising, Germany
*Corresponding Author: PH: 43 2272 66280 205; E-mail: buerst@ifa-tulln.ac.at
INTRODUCTON
Aim of this work was to detect QTL for combined type I and type II resistance against FHB
and estimate their effects in comparison to the QTL identified previously for type II resistance (Bai et al. 1999, Waldron et al. 1999, Anderson et al. 2001, Buerstmayr et al. 2002).
MATERIALS AND METHODS
Plant materials
The population of F1 derived doubled haploid (DH) lines which was described in
Buerstmayr et al. (2002) was used for this research. The resistant parent was ‘CM-82036’
(originating from Sumai3 x Thornbird) and the susceptible parent was ‘Remus’. In total 364
DH lines were available.
Field experiments for evaluation of Fusarium head blight resistance
The lines were evaluated during two seasons (1999 and 2001) at the experimental field of
IFA-Tulln. Trial location, seed treatment, plot size, sowing density and crop management
were the same as described in Buerstmayr et al. (2002). Inoculation was done in separate
experiments by spraying heads at anthesis with one F. culmorum or one F. graminearum
isolate as described in Buerstmayr et al. (2000). Resistance reaction was assessed out in
percent diseased spikelets per inoculated plot on days 10, 14, 18, 22 and 26 after inoculation.
Genotyping of the DH population with molecular markers
Genotyping of 239 DH lines was performed using 28 RFLP, 267 AFLP, 112 SSR, 3 storage
proteins and one morphological marker. With the markers Barc75, Gwm389, Gwm1034,
Gwm533, Barc133, Gwm493, Barc141, Barc40, Gwm304, Gwm293, Barc117, Barc186, and
Barc1, which appeared to be close to one of the putative QTL regions, additional 122 DH
lines were genotyped and included in the QTL mapping.
Statistical analysis
The FHB severity data were analyzed by ANOVA. Linkage maps were constructed using
MAPMAKER 3.0b for MS-DOS (Lander et al. 1987). QTL analysis was done by simple
interval mapping and composite interval mapping using PLABQTL (Utz and Melchinger
1996).
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2002 National Fusarium Head Blight Forum Proceedings
RESULTS AND DISCUSSION
The population showed significant quantitative variation for FHB severity readings (Figure
1). The genotype by isolate interaction was non-significant underlining the horizontal nature
of FHB resistance in wheat.
QTL analysis revealed that two genomic regions were significantly associated with FHB
resistance in that population, mapping to chromosomes 3B (Qfhs.ndsu-3BS) and 5A
(Qfhs.ifa-5A) respectively (Table 1). The two QTL explained together 47 % of the phenotypic
variance for visual disease severity. The peaks of the LOD profiles obtained by simple and
by composite interval mapping were in the same regions (Figure 2). The two QTL on 3B
and 5A mapped to the same genomic regions as in our previous study for type II FHB
resistance (Buerstmayr et al. 2002), with the exception that we did not find a QTL after spray
inoculation on chromosome 1B. Our results concerning the Qfhs.ndsu-3BS locus are in full
agreement with Waldron et al. (1999), Anderson et al. (2001) and Zhou et al. (2000). A significant QTL in the Qfhs.ifa-5A region was also detected by D. Somers (AG Canada,
Winnipeg, pers. comm.). In the present study using spray inoculation, the effects of the two
QTL were in a comparable range. On the contrary, after single floret inoculation, the 3B QTL
had a much larger effect than the 5A QTL (Buerstmayr et al. 2002). This is an indication that
Qfhs.ifa-5A may contribute more towards type I resistance and to a lesser extent to type II
resistance whereas Qfhs.ndsu-3BS appears to play a role primarily in type II resistance.
For both QTL the allele conferring resistance originated from the resistant parent ‘CM82036’. The association of the two QTL on 3B and 5A with the phenotype is shown in Table
4. Lines with the ‘resistant’ allele (originating from ‘CM-82036’) at both QTL regions had a
mean FHB severity of only 20 % compared to lines with the alleles from susceptible ‘Remus’
which reached on average of 58 % bleached spikelets after 26 days (Table 2).
Both QTL regions are already well covered by SSR markers. Marker assisted selection for
the two major QTL appears therefore feasible and should help breeders to select for improved lines with combined type I and type II resistance.
ACKNOWLEDGMENTS
We thank M Röder (IPK Gatersleben, Germany) for screening SSR markers on the parents
and contributing unpublished SSRs, and P Cregan and Q Song (USDA ARS, Beltsville,
USA) for SSR primers. We are grateful to BS Gill (Kansas State University, USA) and ME
Sorrells (Cornell University, USA) for contributing RFLP clones. This work was supported by
the Austrian Science Fund (FWF) and Probstdorfer Saatzucht, project # P11884-BIO.
REFERENCES
Anderson JA, Stack RW, Liu S, Waldron BL, Fjeld AD, Coyne C, Moreno-Sevilla B, Mitchell Fetch J, Song QJ,
Cregan PB, Frohberg RC (2001) DNA markers for a Fusarium head blight resistance QTL in two wheat populations. Theor Appl Genet 102: 1164-1168.
Bai GH, Kolb FL, Shaner G, Domier LL (1999) Amplified fragment length polymorphism markers linked to a
major quantitative trait locus controlling scab resistance in wheat. Phytopathology 89: 343-348.
23
Biotechnology
2002 National Fusarium Head Blight Forum Proceedings
Buerstmayr H, Steiner B, Lemmens M, Ruckenbauer P (2000) Resistance to Fusarium head blight in two winter
wheat crosses: heritability and trait associations. Crop Sci 40: 1012-1018.
Buerstmayr H, Lemmens M, Hartl L, Doldi L, Steiner B, Stierschneider M, Ruckenbauer P. (2002) Molecular
mapping of QTL for Fusarium head blight resistance in spring wheat I: resistance to fungal spread (type II
resistance). Theor Appl Genet 104: 84-91.
Lander ES, Green P, Abrahamson J, Barlow A, Daley MJ, Lincoln SE, Newburg L (1987) MAPMAKER: An
interactive computer package for constructing primary genetic linkage maps of experimental and natural
populations. Genomics 1: 174-181.
Roeder MS, Korzun V, Wendehake K, Plaschke J, Tixier MH, Leroy P, Ganal MW (1998) A microsatellite map
of wheat. Genetics 149: 2007-2023.
Utz HF, Melchinger AE (1996) PLABQTL: A program for composite interval mapping of QTL. J Agric Genomics.
URL http://www.ncgr.org/research/jag/papers96/paper196/indexp196.html.
Waldron BL, Moreno-Sevilla B, Anderson JA, Stack RW, Frohberg RC (1999) RFLP mapping of QTL for
Fusarium head blight resistance in wheat. Crop Sci 39: 805-811.
Zhou WC, Kolb FL, Bai GH, Shaner G, Domier LL (2002) SSR mapping and sub-arm physical location of a major
scab resistance QTL in wheat. National Fusarium Head Blight Forum, 10-12 Dec 2000, Cincinnati, pp 69-72.
Table 1. QTL estimate for mean values of percentage of infected spikelets on day 26 after
inoculation (FHB-26) over two years of the experiments in Tulln. QTL are described by
chromosome location, logarithm of odds (LOD) and percentage of explained phenotypic
variance (R2). QTL analysis was carried out by composite interval mapping.
FHB-26
Map interval
QTL
2
LOD
R
Xgwm533 - Xgwm493 Qfhs.ndsu-3BS
29.1
31.6
Xgwm293 – Xgwm156 Qfhs.ifa-5A
20.5
23.2
Simultaneous fit
46.9
Table 2. Effect of alternative alleles at two QTL regions for mean percentage of infected
spikelets 26 days after inoculation (FHB-26) for line means obtained in Tulln over two years.
*)
QTL
Qfhs.ndsu-3BS
CM-82036
CM-82036
Remus
Remus
Qfhs.ifa-5A
CM-82036
Remus
CM-82036
Remus
FHB-26
Number of
lines
Median Mean
87
19.8
21.9
74
34.1 34.8
73
37.5 39.6
110
58.3 57.7
Stderr.
8.5
13.6
13.1
16.1
*) Only lines with non-recombined Xgwm533 – Xgwm493 (Qfhs.ndsu-3BS) and Xgwm293 – Xgwm156 (Qfhs.ifa5A) intervals were included in these calculations.
Biotechnology
24
2002 National Fusarium Head Blight Forum Proceedings
50
N u m b e r o f lin e s
M e a n = 4 0.1
L S D 5 % = 1 2.2
C M -8 20 36
40
R em us
30
20
10
0
0
10
20
30
40
50
60
70
80
90
1 00
P erce ntag e of in fe cte d sp ike lets (F H B -2 6)
Figure 1. Frequency distribution of 364 DH-lines for mean values of FHB severity on day
26 after inoculation (FHB-26). Arrows indicate values of the parental lines. The overall
population mean and the least significant difference for comparison of line means (Į = 0.05)
using the genotype by year interaction mean square as an error term are given also.
A
LO D
40
30
20
M a rke r
10
B
cM
0
LO D
30
X b a rc75
0
X g w m 38 9
5 .1
X g w m 10 3 4 5 .1
X g w m 53 3
8 .5
X b a rc13 3
11 .0
X b a rc14 7
11 .8
X g w m 49 3 1 5 .4
X b a rc10 2
20
M a rker
10
X gd m 10 9
X gw m 20 5 a
X ba rc18 0
X bc d 2 07
X ba rc1
X gw m 12 9
X gw m 29 3
X gw m 30 4 a
X gw m 10 5 7
X ba rc11 7
X ba rc18 6
X ba rc56
X ba rc10 0
X ba rc40
X gw m 15 6
X ba rc14 1
X s2 4 m 1 9 _ 5
2 2 .7
X s1 8 m 1 8 -9 4 3 .7
cM
0
0
0
1 3 .5
1 6 .7
1 6 .9
1 8 .4
1 9 .1
1 9 .1
1 9 .1
1 9 .1
1 9 .1
1 9 .1
2 2 .5
2 2 .5
2 3 .9
2 8 .6
3 9 .0
F ig u re 2. Interval analysis o f Q T L fo r percentage o f infected sp ikelets o n day 26 after
ino culatio n (F H B -26) o n linkage gro ups correspo nd ing to chro m oso m es 3 B (A ) and 5A (B ).
L O D values w ere calculated b y co m po site interval m app ing (so lid line) and sim p le interval
m app ing (do tted line).
25
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2002 National Fusarium Head Blight Forum Proceedings
QTL MAPPING AND SSR GENOTYPING OF FUSARIUM HEAD BLIGHT
RESISTANCE IN VIRGINIA TECH WHEAT BREEDING PROGRAM
Chen, Jianli, C, A, Griffey*, M. A. Saghai Maroof, W. Zhao, J. Wilson, and D. Nabati
CSES Department, Virginia Tech, Blacksburg, VA 24061
Corresponding Author: PH: 540-231-9789; E-mail: cgriffey@vt.edu
ABSTRACT
Mapping of quantitative trait loci (QTLs) associated with Fusarium head blight (FHB) resistance and application of marker assisted selection (MAS) can be used to accelerate development of FHB resistant wheat varieties and to provide for a better understanding of the
mechanisms governing resistance. Two F2 and two corresponding F1-derived doubled
haploid populations of Pion2684 x W14 and Madison x W14 were used in discerning the
inheritance and identity of QTLs associated with FHB resistance in wheat line W14. In the F2
populations, two complementary genes with major effects were postulated to govern FHB
resistance. This was confirmed upon subsequent evaluation of doubled haploid populations
in two independent experiments in 2001 and 2002. Microsatellite markers (SSRs) were
used to identify QTLs associated with FHB resistance. Seventy six percent (152 out of 200)
of SSRs detected polymorphism between parents. Among 36 pairs of primers used to date,
a total of 45 loci on three chromosome regions (2BS, 3BS, and 5AL) have been comparatively mapped in one F2 of W14 x Pion2684 and two doubled haploid populations of W14 x
Pion2684 and W14 x Madison. Fifteen markers were significantly (p < 0.05) associated with
FHB resistance, and explained 21%, 36% and 31% of the total variation of disease severity
in F2, F2:3, and DH populations of W14 x Pion2684, respectively. These markers also explained 43% of total variation of disease severity in DH population of W14 x Madison. Nine
of the 15 SSRs were used to genotype in 27 FHB resistant soft red winter wheat lines to
determine the putative contribution of QTLs on these chromosomes to FHB resistance and
the potential for using these SSRs in MAS. Among the nine SSRs loci genotyped, Xgwm
493 in the 3BS QTL region and Xgwm156 in the 5AL QTL region were the most commonly
detected loci having the same fragment size as the FHB resistant wheat lines Sumai 3 and
W14. In contrast, Xgwm 533 was detected in only five of 27 lines, while the other 22 lines
have alleles at 2BS and/or 5AL QTL loci. The contribution of individual QTL towards FHB
resistance will be evaluated further in doubled haploid and near-isogenic line populations.
Biotechnology
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2002 National Fusarium Head Blight Forum Proceedings
INSIGHT IN THE DIFFERENTIALLY EXPRESSED GENES IN
RESPONSE TO FUSARIUM MYCOTOXINS IN FHB
RESISTANCE WHEAT NOBEOKABOUZU-KOMUG
I. Elouafi and T. Ban*
Japan International Research Center for Agricultural Sciences (JIRCAS),
1-1 Ohwashi, Tsukuba, Ibaraki 305-8686, Japan
*Corresponding Author: PH: +81-298-38-6364; E-mail: tomohiro@affrc.go.jp
ABSTRACT
Fusarium head blight (FHB, scab) is a fungal disease of wheat and other small cereals that
is found in both temperate and semi-tropical regions. FHB causes severe yield and quality
losses, but the most-serious concern is the possible mycotoxin contamination of cereal food
and feed. Breeding for FHB resistance by conventional selection is feasible, but tedious and
laborious. This study was conducted to identify the response genes to Fusarium mycotoxins
believed to be held by the Japanese variety Nobeokabouzu-komugi, which is highly resistant to FHB, and to construct a stressed ESTs library from this bread wheat variety. For this
purpose, suppression subtractive hybridization (SSH) technique was used as it combines
normalization (suppression of abundant transcripts and enrichment of rare transcripts) and
subtraction (isolation of differentially expressed transcripts) and as it is a powerful approach
to identify and isolate genes which are transcribed under a certain stress. It may as well
help in understanding the complex regulating mechanism of resistance to FHB.
Nobeokabouzu-komugi seeds were germinated in water; and then transplanted in a phosphorous solid solution with and without metabolite of Fusarium graminearum including 10
ppm DON. The root meristems were collected and used for total RNA extraction. cDNA was
synthesized using Clontech kit and the SSH method was applied to generate differentially
regulated cDNA probes. These cDNA probes were cloned by using TA cloning method.
Approximately one thousand clones were isolated from the subtracted stressed plants. Out
of these clones, 80 random ones were sequenced. Only one duplication was found, meaning that more that 98% are singletons. Further, sequence homology search using BLAST
program from NCBI showed that 19 clones present high homology with some ESTs from
Fusarium infected spike of another FHB resistance variety, Sumai 3; whereas others show
homology to ESTs induced by different stresses specially (vernalization, ABA, dehydration,
salt, etiolating, cold, and drought). These clones will be used for the differential display
analysis by using micro array.
27
Biotechnology
2002 National Fusarium Head Blight Forum Proceedings
CONTROL OF SCAB WITH PUROINDOLINE-CONTAINING
TRANSGENIC WHEAT
S.A. Gerhardt, C. Balconi and J.E. Sherwood*
Dept. Plant Sciences and Plant Pathology, Montana State University, Bozeman, MT
*Corresponding Author: PH: 406-994-5153; E-mail: sherwood@montana.edu
ABSTRACT
Wheat and barley head blight or scab is a fungal disease caused by two species of
Fusarium (F. graminearum and F. culmorum) causing premature ripening and white heads.
The wheat puroindoline proteins (PINA and PINB), which are endosperm-specific and
contribute to grain softness, also have in vitro and in vivo anti-fungal properties. These
studies have been extended to include wheat Fusarium scab. The growth of both Fusarium
species was negatively affected by PIN in in vitro bioassays. Control and transgenic HiLine
wheat varieties that over-express the pinB gene driven by the constitutive maize-ubiquitin
promoter or by the endosperm-specific glutenin-promoter, were inoculated with F. culmorum
in both field (summer 2001) and green-house (2001-2002) studies. The plants were analyzed for scab by visual inspection of the heads. The majority of Hi-Line control plants had
between 40-70% infected spikelets/head. PinB-transgenic lines showed a large increase in
plants with only 0-20% infected spikelets/head, a decrease in both the moderately and
severely infected heads, and a decrease of the percentage of tombstones, when compared
to the control. Experiments are in progress using F. graminearum as the fungal pathogen
causing the scab on Hi-Line controls and on the pinB-containing transgenic wheat. These
data suggest that PIN proteins may provide protection to wheat and barley against Fusarium
scab.
Biotechnology
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2002 National Fusarium Head Blight Forum Proceedings
GENETIC ANALYSIS OF TYPE II FUSARIUM HEAD BLIGHT (FHB)
RESISTANCE IN THE HEXAPLOID WHEAT
CULTIVAR ‘WANGSHUBAI’
Jose L. Gonzalez-Hernandez*, A. del Blanco, B. Berzonsky and S.F. Kianian
Dept. of Plant Sciences, North Dakota State University, Fargo, ND 58105
*Corresponding Author: PH: 701-231-6322; E-mail: jose.gonzalez@ndsu.nodak.edu
ABSTRACT
The dramatic impact of FHB on the wheat production throughout the US has driven breeding and germplasm enhancement projects to search for new potential resistance sources.
Chinese introductions show the most potential for resistance to the spread of the infection
through the head (Type II). ‘Sumai 3’ is the most widely used of these introductions, being
utilized for genetic studies and breeding purposes. In the Wheat Germplasm Enhancement
project at NDSU we are interested in additional resistance sources that can be used in
place of or in conjunction with Sumai 3. One of these new sources is the hexaploid wheat
cultivar ‘Wangshubai’. The level of resistance shown by Wangshubai in our greenhouse
evaluations is 7-11%, compared to 15% for Sumai 3; this is consistent with reported results
of others. Previous genetic diversity studies had detected no genetic relationship between
Sumai 3 and Wangshubai, suggesting different loci or alleles for resistance. In order to
dissect the genetic components of Wangshubai resistance to FHB spread, we have developed a population of 388 F6-derived recombinant inbred lines developed from a cross
between Wangshubai and ND671 (a susceptible elite line from the HRSW breeding program at NDSU). We have phenotypic data from 4 greenhouse and 2 field evaluations. Infection in the greenhouse experiments was achieved through single floret inoculations at
flowering, while the field experiments relied on natural infection. We are using a subset of 88
lines for preliminary QTL analysis. The molecular markers used for this purpose are SSRs
previously mapped to specific wheat chromosomes. For confirmation purposes, the chromosomes will be anchored using RFLP markers. The remaining lines will be used for validation of QTL. Preliminary QTL analysis results using 75 SSR markers covering 13 wheat
chromosomes shows the presence of major QTL in chromosome 3BS located close to the
SSR locus Xgwm533. This QTL explained about 25% of the phenotypic variation, and its
location is similar to that found in Sumai 3. The amount of phenotypic variation explained is
comparable to that explained by the major QTL in Sumai3. However, the level of resistance
in Wangshubai (7 to 11% of spread) is better than in Sumai 3 (15% of spread). This fact
could be because: 1) both sources have different alleles of the gene/s for the QTL found in
chromosome 3BS, or 2) Wangshubai has additional genes contributing to a higher level of
resistance. Our plans include completing the genetic map for the population in this study to
search for additional QTLs explaining more of the phenotypic variation and to study the
possible relationship of resistance to FHB with other traits.
29
Biotechnology
2002 National Fusarium Head Blight Forum Proceedings
IDENTIFICATION OF SCAB RESISTANCE GENE EXPRESSION IN
WHEAT FOLLOWING INOCULATION WITH FUSARIUM
L. Kong1, J.M. Anderson2, and H.W.Ohm1*
1
Agronomy Department, Purdue University, West Lafayette, IN 47907; and 2United States Department of
Agriculture (USDA), Agricultural Research Service (ARS), West Lafayette, IN 47907
*Corresponding author: PH: (765) 494-8072; E-mail: hohm@purdue.edu
ABSTRACT
Fusarium head blight (scab), caused by fungus Fusarium species, is a worldwide disease of
wheat (Triticum aestivum L.). Chinese cultivar, Ning 7480, is one of few wheat cultivars with
resistance to scab. To identify the differentially expressed genes corresponding to scab
resistance of Ning 7840, the pooled cDNA libraries at different time-points, 2hr., 4hr., 6hr.,
12hr., 24hr., 36hr., 72hr. and 96hr., after inoculation with Fusarium were constructed using
glume mRNAs from Ning 7480. We performed a PCR-selected cDNA subtraction using the
pooled glume mRNAs in the tester (Ning 7480 inoculated with Fusarium) and the driver
(Ning 7480 inoculated with water). The cDNA libraries were differentially screened by the
forward subtracted cDNAs (the tester subtracted against the driver) and the reverse subtracted cDNAs (the driver subtracted against the tester) as probes. 24 cDNA clones were
isolated based on their specific hybridization only with the forward subtracted cDNAs, and
not with the reverse subtracted cDNAs. Real-time quantitative PCR showed that the known
defense response protein, chitinase, was induced at 24 hours and reached maximal induction at 72 hours after inoculation with Fusarium. Also, the hypothetical defense response
protein, XP_104345, was induced at 12 hours and showed high levels of induction at 72
hours. Two putative defense response genes, Sigma-E factor and a retroelement, were
down-regulated early from 2 hours after inoculation in the treated tissue with maximal induction occurring around 72 and 96 hours. The slot-blots containing the above putative defense
response genes were probed respectively with the cDNA pools from the tester and driver.
The slot-blot analysis confirmed the presence of the cDNA induced with Fusarium in all of
the four putative defense response genes. The location for these putative genes is proceeding based on nulli-tetrasomics analysis in our lab.
Biotechnology
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2002 National Fusarium Head Blight Forum Proceedings
MAPPING GENES CONFERRING FUSARIUM HEAD
BLIGHT RESISTANCE IN C93-3230-24
K.E. Lamb1, M.J. Green1, R.D. Horsley1*, and Zhang Bingxing2
Dept. of Plant Sciences, North Dakota State University, Fargo, ND, 58105-5051; and
2
Dept. of Plant Protection, Zhejiang University, Hangzhou, Zhejiang, China
*Corresponding Author: PH: (701) 231-8142; E-mail: richard.horsley@ndsu.nodak.edu
1
ABSTRACT
The six-rowed line C93-3230-24, from the cross B2912/Hietpas 5, was identified by researches at Busch Agricultural Resources, Inc. (BARI) to have (FHB) resistance similar to
Chevron, and better FHB resistance than either of its parent in a greenhouse test. The
genetic background of C93-3230-24 appears to be completely different than that of any of
the FBH resistant accessions identified. Thus, this line may have alleles for FHB resistance
and DON accumulation not currently identified. The objectives of this study are: 1) to construct skeletal maps that includes RFLP and SSR markers for an F1-derived DH mapping
population developed from the cross Foster/C93-3230-24 and 2) determine the position of
QTL controlling FHB resistance, DON accumulation, days to heading and maturity, plant
height, and spike nodding angle. Field experiments were conducted in mist-irrigated FHB
nurseries in 2001 and 2002 in North Dakota and Zhejiang Province China using 118 DH
lines and parents. Single locus analysis using available marker data identified six regions
in five chromosomes associated with FHB resistance. The regions are located in chromosomes 2H, 4H, 5H, 6H, and 7H. The region with the largest effect on FHB resistance appears to be in chromosome 2H. Associations between the markers and maturity and/or
plant height were found in the same regions as FHB resistance. Results in this study are
similar to those obtained in studies using the resistant six-rowed cultivar ‘Chevron’ and the
ICARDA/CIMMYT cultivar ‘Gobernadora’. Thus, preliminary results suggest that C93-3230,
Chevron, and Gobernadora may have similar alleles for FHB resistance.
31
Biotechnology
2002 National Fusarium Head Blight Forum Proceedings
TARGETED SATURATION MAPPING OF QFHS.NDSU-3BS USING
WHEAT ESTS AND SYNTENY WITH THE RICE GENOME
S. Liu and J. A. Anderson*
Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, MN 55108
Corresponding Author: PH: (612) 625-9763; E-mail: ander319@umn.edu
ABSTRACT
A major QTL, Qfhs.ndsu-3BS, for resistance to Fusarium head blight (FHB) has been identified and verified by several research groups. However, the DNA marker density near this
major QTL is less than required for map-based cloning. The objective of this project was to
develop STS (sequence tagged site) markers from wheat ESTs to increase the marker
density near this major QTL. On the basis of synteny between wheat chromosome 3BS and
rice chromosome 1S, we initiated a strategy to identify wheat ESTs likely near this QTL. The
sequences of BAC/PAC clones located on the distal portion of rice chromosome 1S were
compared with wheat ESTs in GenBank using BLASTN search. Primers of STS markers
were designed for non-redundant wheat ESTs with E values equal or less than e-15 and the
length of identity greater than 100bp. Using wheat deletion lines for chromosome 3BS, 25
out of 79 STS markers were located to the chromosome bin 3BS 0.78-0.87, where this QTL
is most likely located. Nine STS markers were mapped in a previously reported Sumai 3/
Stoa mapping population. The STS marker XSTS3B-138 explains 55% of the FHB variation of this mapping population. Therefore, this research strategy is useful for developing a
high resolution map of this major QTL region, and may have broad applications for targeted
mapping of other traits in cereal crops.
Biotechnology
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2002 National Fusarium Head Blight Forum Proceedings
IDENTIFICATION OF QTL ASSOCIATED WITH
SCAB RESISTANCE IN ERNIE
Shuyu Liu*, Theresa Musket, Anne L. McKendry, and Georgia L. Davis
Agronomy Department, University of Missouri, Columbia, MO 65211
*Corresponding Author: PH (573) 882-7708; E-mail: sl959@missouri.edu
ABSTRACT
Fusarium head blight (scab) in wheat is a major problem worldwide. No source of complete
resistance is known. Sumai 3, a cultivar from China, is the major resistant resource across
different breeding programs in US. A major QTL conditioning scab resistance in Sumai 3
has been identified on 3BS. Identification of different sources of resistance is critical to
breeding scab resistant wheat to reduce the potential for genetic vulnerability. Ernie, a scab
resistant cultivar, released from the University of Missouri, appears to have a different set of
resistance genes. Using AFLP and SSR markers we have mapped the scab resistant QTL
by 300 F8 recombinant inbred lines (RILs) developed from a cross between Ernie and MO
94-317, a highly susceptible Missouri variety. The scab index (the ratio of infected spikelets
to total spikelets of the inoculated head) in these lines ranged from 15.7 to 75.7%. Eight
EcoRI and 8 MseI primers forming 64 primer pairs were used to screen the parents. Over
80% of these pairs had polymorphic bands. The average number of polymorphic bands was
7 with a range of 2 to 21. Two hundred AFLP and SSR loci were used to construct a linkage
map. MapMaker version 3.0 for Unix was used to construct the linkage map. QTL analysis
was performed on the scab data using QTL Cartographer version 1.16. Two SSR markers
per chromosome were used to anchor the AFLP markers to chromosomes. The QTL information will be useful in developing resistant materials by gene pyramiding.
33
Biotechnology
2002 National Fusarium Head Blight Forum Proceedings
OVER-EXPRESSION OF ANTI-FUNGAL PROTEIN GENES INCREASES
RESISTANCE OF TRANSGENIC WHEAT TO FUSARIUM HEAD BLIGHT
C.A. Mackintosh1*, S.J. Heinen1, L.A. Smith1, M.N. Wyckoff1, R.J. Zeyen2,
G.D. Baldridge2 and G.J. Muehlbauer1
Department of Agronomy and Plant Genetics,and 2Department of Plant Pathology,
University of Minnesota, St. Paul, MN 55108
*Corresponding Author: PH: 612-625-9701; E-mail: caroline_mackintosh@hotmail.com
1
ABSTRACT
Anti-fungal proteins (AFPs) such as ß-1,3-glucanases, chitinases, thaumatin-like proteins
(tlps), thionins and ribosome-inactivating proteins (RIPs) are known to inhibit fungal growth
via different mechanisms. Glucanases and chitinases degrade fungal cell walls, tlps and
thionins degrade fungal cell membranes and RIPs inhibit fungal protein synthesis.
Transgenic wheat (cv. Bobwhite), over-expressing these AFPs, has been generated using
micro-projectile bombardment. We have developed 25, 25, 31, 24 and 15 transgenic wheat
lines carrying a wheat -puro-thionin, a barley tlp-1, a barley ß-1,3-glucanase, a barley RIP
and a barley chitinase, respectively. In addition, we have developed 10, 11 and 11
transgenic wheat lines expressing a combination of chitinase/RIP, chitinase/tlp-1 and RIP/
tlp-1, respectively. These combinations each employ two of the three different mechanisms
of fungal growth inhibition. We screened these lines for resistance to Fusarium head blight.
Four independent glasshouse disease screens have been conducted on the tlp-1 lines and
two of those lines consistently demonstrated an increase in resistance when compared to
non-transgenic controls. Similarly, three disease screens have been conducted with our
glucanase lines and four of these lines have performed well. In four disease screens, one
α−puro-thionin line performed well in three of the screens, and two more lines performed
well in two of the screens, and therefore, have been evaluated further. Molecular characterization of our lines shows that they are genetically independent and that they accumulate
the appropriate AFP. In addition, preliminary disease screen data on the remainder of our
AFP transgenic lines will be presented.
Biotechnology
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2002 National Fusarium Head Blight Forum Proceedings
EFFECT OF CHEVRON ALLELES AT TWO FUSARIUM HEAD BLIGHT
RESISTANCE QTL DETERMINED USING NEAR-ISOGENIC LINES
L. M. Nduulu, A. Mesfin, G.J. Muehlbauer, and K.P. Smith*
Dept. of Agronomy and Plant Genetics, University of Minnesota, St. Paul, MN 55108
*Corresponding Author: PH: (612) 624-1211; E-mail: smith376@tc.umn.edu
OBJECTIVES
To evaluate near-isogenic lines, developed for chromosome 2H and chromosome 6H target
QTL-regions, for FHB resistance and other confounding traits (heading date and plant
height) associated with the disease severity.
INTRODUCTION
Fusarium head blight (FHB), caused primarily by Fusarium graminearum, has caused
significant yield and grain quality losses in barley (Hordeum vulgare L.) since 1993. Phenotypic selection for FHB resistance has been only modestly effective largely because FHB
resistance is highly influenced by the environment and screening methods are laborious
and expensive. Marker-assisted-selection (MAS) is a promising tool to augment current
methods to breed for FHB resistance.
To utilize MAS for FHB resistance, the barley-breeding and genetics program at the University of Minnesota is engaged in mapping and validating QTL for FHB. We have previously
identified FHB resistance QTL distributed across the genome using the source of resistance
Chevron (de la Pena et al., 1999; Canci et al., 2000). We have also used Chevron-derived
populations to validate 3 of the QTL; 2 on chromosome 2H and 1 on chromosome 6H
(Canci, 2001; Gustus et al., 2001). These QTL regions are now candidate targets for MAS,
but a significant problem is that they are coincident with QTL late heading date (HD) and tall
plant heights (HT).
To further understand the association between FHB resistance and these two other traits
(HD and HT) and also elucidate the genetic basis of FHB resistance, it is important to fine
map the associated regions. For this purpose, we developed near-isogenic lines (NILs) for
chromosome 2H and chromosome 6H target QTL-regions using both molecular markerassisted backcrossing and heterogeneous inbred families (HIF) procedures. These lines
will be useful for studying disease resistance. We have also used the BC-derived NILs as
parents to create fine mapping populations.
MATERIALS AND METHODS
Development of the BC3 NILs: We initiated the development of NILs using donor parents
selected from the 101 F4:7 progenies previously used for linkage mapping (de la Pena et al.,
1999). The recurrent parent was the elite line M69. A marker-assisted backcrossing
scheme was used to advance selected lines to the BC3F2 generation. Six BC3F2 lines
35
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2002 National Fusarium Head Blight Forum Proceedings
carrying the FHB-resistance Chevron alleles at each target QTL region were selected and
advanced by selfing to the BC3S4 generation.
Development of HIFs: Among the 101 F4:7 families de la Pena et al. used for mapping
FHB-resistance QTL in 1999, we selected 12 families segregating at marker loci associated
with each target QTL region as HIFs. Ten segregating progeny lines grown from each
selected HIF were genotyped with all available SSR markers in the target QTL region.
Based on the marker data, two NILs contrasting at a specific marker locus were identified
from each HIF and selected as pairs for field evaluation.
Field Evaluations of NILs: The 30 NILs comprising six chromosome 2H BC3-derived NILs,
six chromosome 6H BC3-derived NILs, and 18 HIF-derived NILs together with parental
lines, Chevron and M69, were evaluated at St. Paul and Crookston, Minnesota in the summer of 2002. At each location, entries were planted in 2.4 m long single-row plots spaced at
30 cm apart. The experimental design was a randomized complete block design with three
replications. Nurseries at St. Paul were inoculated using the macroconidia inoculation
technique (Dill-Macky, 2002). The initial inoculation was performed at heading so as to
avoid confounding effect of differences in heading date and potential escape of the pathogen. A second inoculation was repeated three days after initial spraying. At Crookston, a
grain spawn inoculation technique was used (Dill-Macky, 2002). Nurseries were mistirrigated daily after inoculation until soft dough stage. We measured FHB severity, plant
height and heading date. To measure FHB severity, 10 random spikes from each plot were
examined and the number of infected spikelets from each spike counted and expressed as
a percent of the total spikelets present. Heading date was determined as the number of
days after planting to 50% emergency from the boot.
Statistical Analysis: Since all NIL pairs have different alleles only at the target QTL region,
differences in phenotype can be attributed to the genes in those segments. To determine the
effect of each NIL pair, the data were subjected to ANOVA using Proc GLM procedure (SAS
Institute, 2000). Means were separated using LSD. A combined analysis across locations
was conducted to determine genotype x environment interaction. The combined analysis
showed that all measured traits had significant G x E effect. Therefore, results were presented for individual locations. The magnitude of the effect of alleles segregating at the
target QTL regions were determined by comparing the means of NILs carrying different
alleles at each locus.
RESULTS AND DISCUSION
Results of mean separations for the BC3-derived NILs showed that NILs carrying the Chevron allele at the chromosome 2H QTL region reduced FHB by 44% for St. Paul and 41% for
Crookston (Table 1). This same QTL region increased HD by six days confirming previous
studies indicating that the chromosome 2H QTL region is associated with Eam6; a maturity
gene that affects HD. On the contrary, there was no significant effect of the chromosome 6H
region on either FHB or HD. The results for chromosome 2H QTL region are in agreement
with Gustus et al. (2001), however Gustus found a small but significant reduction in FHB
severity with the Chevron allele at the chromosome 6H QTL.
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2002 National Fusarium Head Blight Forum Proceedings
Based on the HIF-derived NILs, the Chevron allele at the marker Bmag0140 on chromosome 2H had a similar effect as the BC-NIL reducing FHB by ~40% (Table 2). However,
these NILs for this marker did not differ for heading date across the 2 locations (data not
shown). Marker Bmag0807 on chromosome 6H was associated with a 31-35% reduction in
FHB. There was no association between HD and any of the analyzed markers on chromosome 6H. However, marker Bmag0807 was associated with plant height in the two locations (data not shown).
The difference in results between the BC3 and HIF NILs for chromosome 6H suggest that
Bmag0807 is closer to the FHB QTL than Bmac0218, which was used to develop the BC
NILs. The general conclusion from this study is that the BC NILs for chromosome 2H should
be useful for fine mapping FHB and HD. Since the BC NILs developed for chromosome 6H
using Bmac0218 did not appear to carry FHB resistance, we are looking back at BC2 lines
generated in this project to see if they carry the Chevron allele at marker locus Bmag0807.
REFERENCES
Canci, P.C., Smith, K.P., Dill-Macky, R., Muehlbauer, G.J., and Rasmusson, D.C. 2000. Validation of fusarium
head blight and kernel discoloration QTLs in barley. American Society of Agronomy Annual Meeting, Minneapolis, MN.
Canci, P.C. 2001. Genetics of Fusarium head blight, kernel discoloration and grain protein content in barley.
Thesis (Ph. D.)—University of Minnesota, 2001.
de la Pena, R.C., Smith, K.P., Capettini, F., Muelhbauer, G.J., Gallo-Meagher, M., Dill-Macky, R., Somers, D.A.,
and Rasmusson, D.C. 1991. Quantitative trait loci associated with resistance to Fusarium head blight and
kernel discoloration in barley. Theor. App. Genet. 99:561-569.
Dill-Macky, R. 2002. Inoculation methods and evaluation of Fusarium head blight resistance in wheat. In: B.
Bushnell (ed.). (In preparation).
Gustus, C. and Smith, K.P. 2001. Evaluating phenotypic and marker assisted selection in the F2 generation for
Chevron-derived FHB resistance in barley. In: Proceedings of the 2001 National Fusarium Head Blight Forum.
Erlanger, KY 12/8/01 - 12/10/01.
SAS Institute, 1999. The SAS system for windows, version 8, SAS Inst., Cary, NC.
T ab le 1. M eans for F usarium h ead bligh t (F H B ), head in g date (H D ) and plant height (H T ) fo r
backcross-derived N IL s an d p arents.
F H B S everity (% )
H ead in g D ate
P lan t H eigh t (cm )
N o. of
G eno typ e L ines
S t. P aul
C ro okston
S t. P aul
C ro okston
S t. P aul C ro okston
C hev ron
1
4.4c
1.7c
56.7a
54.7a
85.3a
105 .5a
M 69
1
55.6a
23.2a
48.0c
48.0b
75.3b
82.5c
B C 3 C hr.2
6
30.9b
11.2b
54.6b
54.2a
76.9b
89.0b
6
65.7a
22.9a
49.6c
47.7b
75.6b
88.9b
B C 3 C hr.6
M eans w ithin th e sam e colum n follow ed b y the sam e letter are not significantly different (P • 0.05).
37
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2002 National Fusarium Head Blight Forum Proceedings
Table 2. Mean FHB severity of NILs derived from HIFs and contrasting at 5 marker loci
FHB Severity (%)
Chromosome No. of NILs
Marker
Genotype
St. Paul
Crookston
2H
6
Bmag0125
A
31.5b
6.9b
B
41.8a
10.8b
2H
6
Bmag0140
A
34.4b
8.3b
B
61.5a
14.1a
2H
6
Bmac0093
A
36.1a
8.8a
B
37.6a
8.5a
6H
6
Bmag0807
A
28.1b
7.0b
B
43.5a
10.2a
6H
6
Bmag0870
A
26.2b
7.8a
B
40.0a
9.1a
A=Chevron; B=M69.
Means within the same column followed by the same letter are not significantly different
(P 0.05).
Biotechnology
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2002 National Fusarium Head Blight Forum Proceedings
SATURATION GENETIC AND PHYSICAL MAPPING OF
CHROMOSOME 3 FUSARIUM HEAD BLIGHT QTL REGION
Deric Schmierer1, Kara Johnson1, Thomas Drader1, and Andris Kleinhofs1,2*
Dept. Crop & Soil Sciences and 2School of Molecular Biosciences,
Washington State University, Pullman, WA 99164
*Corresponding Author: PH: (509) 335-4061; E-mail: dschmierer@wsu.edu
1
ABSTRACT
Complete resistance to Fusarium head blight (FHB), caused by Fusarium graminearum in
the USA, has been a difficult goal to attain. To date, no single gene-for-gene resistance
mechanisms have been discovered. Quantitative trait loci (QTL) for resistance against FHB
have been mapped in several segregating populations. Eighteen of the 21 chromosomes in
wheat and all 7 chromosomes in barley have been reported to be associated with resistance. In three or more mapping studies conducted using Chinese wheat cv. Sumai 3 as the
resistant parent, QTL on chromosomes 3BS and 6BL were discovered. QTL for FHB resistance have also been mapped to chromosomes 2H, 3H, and 1H in several different barley
populations. Because of the high level of synteny between grass species, it was determined that the QTL on 3BS and 3H reside in a syntenous position, between restriction
fragment length polymorphism (RFLP) markers BCD907-ABG471. Using the resources
provided by the Rice Genome Project sequencing effort, we have targeted phage artificial
chromosomes (PACs) from rice chromosome 1 that are located in a syntenous position to
3HS in barley. By using the blastn function on the NCBI web site and limiting the search to
genus Hordeum, barley expressed sequence tags (ESTs) can be identified with homology
to the individual PACs. Eighty-seven unique barley ESTs were identified that covered 18
PACs. To date, 24 ESTs have been screened against the cv. Morex bacterial artificial chromosome (BAC) library. These 24 ESTs identified 193 BAC clones. We have genetically
mapped 12 ESTs to date and 7 mapped in the target region on 3HS in the Steptoe/Morex
DHL population and the Foster/CI4196 RIL population. The ratio of 7 out of 12 mapping to
the target region is sufficient to expect to saturate the region since additional PAC clones
are available from the target region. Another source of ESTs that we are experimenting with
are the wheat ESTs mapped to group 3 using the wheat deletion lines (http://
wheat.pw.usda.gov/cgi-bin/westsql/map_locus.cgi).
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2002 National Fusarium Head Blight Forum Proceedings
MICROSATELLITE GENETIC MAP IN WHEAT
J.R. Shi1, Q.J. Song2, S-Singh3, R.W. Ward1*, P.B. Cregan2, and B.S. Gill3
1
Department of Crop and Soil Science, Michigan State University, East Lansing, MI48824;
2
Beltsville Agricultural Research Center, USDA-ARS, MD20705; and
3
Department of Plant Pathology, Kansas State University, Manhattan, KS 66506
*Corresponding Author: PH: 517-285-9725; E-mail: wardri@msu.edu
ABSTRACT
Genetic maps saturated with informative markers are of great importance for localizing and
manipulating important genes or QTLs. In recent years, microsatellite loci, also referred to
simple sequence repeats (SSRs) have proved to be a valuable source of highly polymorphic DNA markers. SSR polymorphisms are based on differences in the length of simple
sequence repeats at loci defined by locus-specific PCR primers flanking the microsatellite.
Currently, approximately 350 publicly available wheat microsatellite primer pairs have been
reported in the peer reviewed literature (Röder et al. 1998; Korzun et al. 1997; Devos et al
1995; Pestsova 2000; Salina et al 2000). To date, we have developed more than 400 new
SSR primer pairs, 209 of which generate PCR products which map to 225 loci(a) on the ITMI
population. PCR products from an additional 137 primer pairs enable physical mapping of
142 loci. The poster associated with this abstract displays the latest version of a genetic/
physical map containing over 1400 total loci including 367 Xbarc loci. Detailed information
about primer pairs and the loci they amplify will be posted at:
http://wheat.pw.usda.gov/ggpages/genomics.shtml
(a)
Designated with the prefix “Xbarc”, where “barc” in the acronym for “Beltsville Agricultural
Research Center”.
REFERENCE
Devos K. M.; G. J. Bryan, P. Stephenson, and M. D. Gale, 1995 Application of two microsatellite sequences in
wheat storage proteins as molecular markers. Theor Appl Genet 90(2): 247-252.
Korzun V., A. Boerner, A. J. Worland, C. N. Law, and M. S. Röder, 1997 Application of microsatellite markers to
distinguish inter-varietal chromosome substitute lines of wheat Triticum aestivum L. Euphytica 95(2): 149-155.
Pestsova E., M. W. Ganal, and M. S. Röder, 2000 Isolation and mapping of microsatellite markers specific for
the D genome of bread wheat. Genome 43: 689-697.
Röder M. S., V. Korzun, K. Wendehake, J. Plaschke, M. H. Tixier, P. Leroy, and M. W. Ganal, 1998 A
microsatellite map of wheat. Genetics 149: 2007-2023.
Salina E., A. Börner, I. Leonova, V. Korzun, L. Laikova, O. Maystrenko, And M. S. Röder, 2000 Microsatellite
mapping of the induced sphaerococcoid mutation genes in Triticum aestivum. Theor Appl Genet 100(5): 686-689
Biotechnology
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2002 National Fusarium Head Blight Forum Proceedings
STRATEGIES FOR COMBATING FUSARIUM IN BARLEY
THROUGH GENE EXPRESSION TARGETING, METABOLIC
PROFILING AND SIGNALING ANALYSIS
R.W. Skadsen1*, T. Abebe1,2, M.L. Federico1,2, J. Fu3, C. Henson1,
and H.F. Kaeppler2
USDA/ARS Cereal Crops Research Unit, Madison, WI 53726;
Agronomy Dept., University of Wisconsin, Madison, WI 53706; and
3
USDA/ARS Small Grains Germplasm Research Facility, P.O. Box 301, Aberdeen, ID 83210
*Corresponding Author: PH: (608) 262-3672; E-mail: rskadsen@facstaff.wisc.edu
1
2
ABSTRACT
Several basic studies must be undertaken in order to understand the interactions between
Fusarium graminearum and its barley and wheat hosts: 1) Gene promoters are needed to
target the expression of antifungal protein genes to organs that are initially colonized by F.g.,
2) Metabolic profiling must be developed to determine which metabolites are extracted from
host tissues, and 3) It is important to understand the signaling pathway involved in host
perception of F.g. invasion and attempts to mount an effective response. We have previously produced a gene promoter (Lem1) that is specific for the young lemma/palea. More
recently, Tilahun Abebe has used the suppressive subtractive hybridization method to
identify genes expressed in lemmas/paleas but not in flag leaves. This led to the development of the Lem2, which is specific to the lemma/palea of developing seeds during the
period from endosperm elongation through the dough stage. Maria Laura Federico has
developed a promoter (EpiLTP) that has preferential activity in the pericarp epithelium. A
vector (Ala/gfp) was developed by Jianming Fu to test coding sequences of genes in a
transient system, prior to their use in stable transformation. This has been applied to the
expression of the anti-Fusarium gene Hth1 of barley. Portions of the Hth1 coding sequence
were linked to a polyalanine bridge, followed by gfp. This showed that the failure of this
endosperm protein to be produced in lemmas resides with sequences encoding the mature
peptide. GC-MS by Cynthia Henson showed that early infection of the lemma and pericarp
involves accumulation of metabolites that could be essential to fungal metabolism. In particular, metabolites known to be involved in appressorium turgor pressure (trehalose, mannitol and glycerol) were found. Our studies have shown that no alpha-amylase accompanies
infection, even when infections are very heavy. We are examining whether the most obvious
substrate (starch) is ever mobilized during infection, and we are attempting to develop a
metabolic profile for infected tissue. Finally, it is not clear how barley reacts to the F.g. in the
early stages of infection. Initial studies have shown that H2O2 is produced at the site of F.g.
inoculation on the pericarp. Thus, barley may have the beginnings of a productive response
that could be strengthened through breeding/molecular approaches.
41
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2002 National Fusarium Head Blight Forum Proceedings
TRANSGENE EXPRESSION IN SPRING WHEAT (TRITICUM AESTIVUM
L.) TRANSFORMED WITH CANDIDATE ANTI-FUSARIUM GENES
M. Somleva1, P. Okubara2, and A. Blechl1*
USDA-ARS, Western Regional Research Center, Albany, CA 94710; and 2USDA-ARS,
Dept. of Plant Pathology, Washington State University, Pullman, WA 99164
*Corresponding Author: PH: (510) 559-5716; E-mail: ablechl@pw.usda.gov
1
OBJECTIVE
To create transgenic wheat lines carrying novel co-dominant loci with the potential for conferring effective and durable resistance to Fusarium head blight (FHB).
INTRODUCTION
Host plant resistance is the most efficient and cost-effective way to protect the wheat crop
from FHB. Our aim is to create new germplasm sources of Fusarium resistance by using
genetic engineering to introduce novel anti-Fusarium (AF) genes into wheat. We have
designed and constructed a set of transformation vectors that fuse the maize Ubi1 promoter
to AF genes that target either the Fusarium cell walls or membranes or that mitigate the
cellular toxicity of the mycotoxins synthesized by the fungus during infection (Table 1). To
give our candidate AF genes the best chance of protecting the plant, they must be expressed at high levels in tissues encountered by fungus as it invades and spreads, i.e.,
young florets. Here we report semi-quantitative and tissue-specific data for expression of
these transgenes in wheat. We also use a new technique to visualize transgene expression
in wheat tissues and organs.
MATERIALS AND METHODS
Vector and plasmid constructs: The monocot expression vector pUBK (Okubara et al.,
2002) consists of the bar gene conferring resistance to the herbicide bialaphos regulated by
the promoter, first exon, and first intron of the maize Ubi1 gene (UBI) (Christensen and
Quail, 1996). In the AF constructs, the bar gene was replaced by candidate AF sequences
(Table 1).
Generation, selection and progeny analyses of transformants: Transformants made by
particle bombardment of immature embryos of cv. Bobwhite were identified as described
(Okubara et al., 2002). Transgene inheritance was followed and homozygotes were identified using PCR amplification of genomic DNA with a forward primer from the UBI region and
reverse primers specific for each AF coding region.
Transgene expression analyses: Semi-quantitative RT-PCR was carried out with 5-600
ng of total RNA from endosperm (data in Table 1) or other organs (Fig. 1) as described
(Okubara et al., 2002). Transcript-derived cDNA was amplified using a primer specific for the
first exon of the Ubi1 gene and a reverse primer specific for each AF sequence. Actin amplification from 5 to 40 ng of total RNA served as the internal standard for RNA integrity
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2002 National Fusarium Head Blight Forum Proceedings
(Okubara et al., 2002). For visual localization of AF and actin transcripts, we modified a
method for in situ RT-PCR (Kolti and Bird, 2000), adapting it for whole mounts of cereal
organs and tissues (Somleva, unpublished).
RESULTS AND DISCUSSION
We used semi-quantitative RT-PCR to measure transgene expression in endosperm of
hemizygotes and homozygotes from early generations of our initial set of transgenic lines
(Table 1). Levels of transgene steady state mRNA varied among independent transformation
events. In all, 16 hexaploid wheat lines have shown detectable levels of expression of the
AF genes. Lines AB5-126 and C3-9 and C3-10, C1-3 and C9-25, C17-20, and AB8-7 and
AB8-50 express the highest amounts of transgene constructs for tlp-1, FvExo, FvGlu and
FvEndo, respectively. The highest TRI101 transcript accumulation was observed in lines
156 and 176 (Okubara et al., 2002). Transgenes with coding regions of fungal origin were
expressed at least 10-fold lower, on average, than the wheat tlp1 transgenes from the same
promoter. FvGlu exhibited about 100-fold lower expression than wheat tlp transgenes or
endogenous actin.
Because of the possibility of gene silencing, there is no guarantee that primary
transformants showing strong expression will produce progeny with the same characteristics. Therefore, we used semi-quantitative RT-PCR to measure mRNA levels in T4-T7 endosperm from homozygous progeny of some of our lines (Table 1). Nearly all of those tested
had expression levels as high or higher than in earlier generations. Only one line, C17-21,
had lost expression. Evidently transgene silencing did not occur in the majority of our lines,
either in later generations or in the transition from the hemizygous to the homozygous state.
Even transgenics containing the wheat tlp gene, which is completely homologous to wheat
endogenous genes, maintained their expression levels. The increases in transgene expression in lines AB8-7 and C1-3 are higher than can be accounted for by the two-fold increase
in gene copy number in homozygotes compared to early generation hemizygotes. A similar
additive effect of transgene expression has been observed in homozygous rice plants
(James et al., 2002).
To compare transgene expression levels among different parts of the plant, semi-quantitative RT-PCR was performed on mRNA from leaves, endosperm, anthers and ovaries of two
FvExo lines (Fig. 1). Transcript levels were highest in endosperm and lowest in ovaries. This
result shows that measurements of endosperm mRNA levels are not necessarily predictive
of transgene expression in other organs. We plan to measure transcript levels in the outer
tissues of the floret, since that is the first part of the plants encountered by the fungus. In
addition, we are exploring the potential utility of other promoters to support stronger AF
gene expression in floral tissues.
To more precisely localize expression from the UBI promoter, we have adapted a method of
in situ RT-PCR (Koltai and Bird, 2000) for whole mounts of wheat tissues and organs. UBIdriven expression of various AF genes can be detected in lemma, pollen and stigma, but not
in anthers (Fig. 2). These results agree with experiments using GUS fusions to report UBI
activity in transgenic wheat (Stoger et al., 1999). However, the in situ method has the potential for more precise and construct-specific localization.
43
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2002 National Fusarium Head Blight Forum Proceedings
Table 1. AF gene expression in transgenic wheat.
Anti-Fusarium
Expression results6
coding regions Line
RNA analysis in
RNA analysis in
fused to UBI
name
T 1-T 2 generations
T 4-T 7 generations7
wheat tlp1 1
AB5-126
High8
n. a.
C3-9
Medium 9
High (T 5)
C3-10
High
High (T 5)
AB6-74
Low 10, some unspliced
n. a.
Fs TRI101 2
AB6-176
Low-Medium, some unspliced n. a.
AB6-156
Low-Medium, some unspliced n. a.
B65-49
Low, some unspliced
n. a.
AB8-7
Low
High (T 7)
FvEndo3
AB8-15
Low
n. a.
AB8-50
Medium
High (T 5)
AB8-108
Medium
n. a.
4
C1-3
Medium
High (T5)
FvExo
C9-25
Medium
Medium-High (T 5)
AB9-59
Low
n. a.
FvGlu5
C17-20
Low
Low (T 4)
C17-21
Low
n. d.
1
T. aestivum leaf cDNA encoding a thaumatin-like protein (Rebmann et al., 1991); 2Fusarium
sporotrichioides gene encoding DON acetyltransferase (McCormick et al., 1999); 3, 4, 5F.
venenatum cDNAs encoding an endochitinase, exochitinase, and glucanase (Berka,
unpublished); 6 Relative levels based on RT-PCR of endosperm mRNA. 7Homozygous plants.
8
RT-PCR amplification products of similar intensity to those from actin in 5-40 ng total RNA.
9
Detectable expression in 50-200 ng total RNA. 10Expression was only detected in 600 ng RNA.
n. a. = not analyzed; n. d. = not detected.
L
C 1-3
E A
O
L
C 9-25
E A O
4 00 2 00 4 00 4 00
4 00 2 00 4 00 4 00
40
40
F vE xo
20
40
40
20
40
40
A ctin
Biotechnology
44
F ig u re 1. R T -P C R analyses o f F vE xo
expressio n in fo ur w heat organs. (L
leaves, E endosperm , A anthers, O
ovaries). A ctin am p lificatio n w as
used as a stand ard for R N A integrity.
T otal R N A am o unt [ng] used in each
assay is ind icated abo ve the band s.
2002 National Fusarium Head Blight Forum Proceedings
A
C
Figure 2. Localization of transgene
mRNA by whole- mount in situ RT-PCR.
Gene expression is visible as a purple
(dark) precipitate. ( A) Positive control actin expression in a floret from a nontransformed plant (7x); (B) Wheat tlp-1
expressing cells in the lemma (50x); ( C)
TRI101 mRNA in pollen grains (90x);
(D) TRI101 transcripts in the stigma
(90x); ( E) A negative control – no
expression of FvEndo was detected in
palea after in situ RT-PCR without
reverse transcription (7x).
B
D
E
REFERENCES
Christensen, A.H. and Quail, P.F. 1996. Ubiquitin promoter-based vectors for high-level expression of selectable
and/or screenable marker genes in monocotyledonous plants. Transgenic Res. 5:213-218.
James, V.A., Avart, C., Worland, B., Snape, J.W., and Vain, P. 2002. The relationship between homozygous and
hemizygous transgene expression levels over generations in populations of transgenic rice plants. Theor. Appl.
Genet. 104:553-561.
Koltai, H. and Bird, D.M. 2000. High throughput cellular localization of specific plant mRNAs by liquid-phase in
situ reverse transcription-polymerase chain reaction of tissue sections. Plant Physiol. 123:1203-1212.
McCormick, S.P., Alexander, N.J., Trapp, S.E., and Hohn, T.M. 1999. Disruption of the TRI101 gene, encoding 3O-acetyltransferase, from Fusarium sporotrichioides. Appl. Environ. Microbiol. 65:5252-5256.
Okubara, P.A., Blechl, A.E., McCormick, S.P., Alexander, N.J., Dill-Macky, R., and Hohn T.M. 2002. Engineering
deoxynivalenol metabolism in wheat through the expression of a fungal trichothecene acetyltransferase gene.
Theor. Appl. Genet. (in press).
Rebmann, G., Mauch, F., and Dudler, R. 1991. Sequence of a wheat cDNA encoding a pathogen-induced
thaumatin-like protein. Plant Mol. Biol. 17:283-285.
Stoger, E., Williams, S., Keen, D., and Christou, P. 1999. Constitutive versus seed specific expression in
transgenic wheat: temporal and spatial control. Transgenic Res. 8:73-82.
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2002 National Fusarium Head Blight Forum Proceedings
MOLECULAR MAPPING OF RESISTANCE TO FUSARIUM HEAD
BLIGHT IN THE SPRING WHEAT CULTIVAR FRONTANA
B. Steiner*, M. Griesser, M. Lemmens, and H. Buerstmayr
IFA-Tulln, Institute for Agrobiotechnology, Department of Biotechnology in Plant Production,
Konrad Lorenz Strasse 20, A-3430 Tulln, Austria. www.ifa-tulln.ac.at
*Corresponding Author: PH: 43 2272 66280 206; E-mail: steiner@ifa-tulln.ac.at
ABSTRACT
Fusarium head blight (FHB, scab) may cause severe yield losses, but the most serious
concern is the mycotoxin contamination of cereal food and feed. Breeding for FHB resistance by conventional selection is feasible but tedious and expensive. Despite that resistance originating from Sumai 3 is already well characterized (Anderson et al. 2001,
Buerstmayr et al. 2002) only limited molecular genetic information is available on other
sources of resistance.
A population of 210 doubled haploid (DH) lines originating from the F1 of the cross Frontana
(moderately resistant) by Remus (susceptible) were evaluated for the expression of
Fusarium head blight resistance traits in field trials in the seasons 1999 and 2001. Inoculation and evaluation methods used were similar to Buerstmayr et al. (2000).
The population was genotyped with more than 560 markers (SSR, AFLP, RFLP). QTL
analysis revealed significant association of several genomic regions with FHB severity. The
most prominent and consistent QTL effect was detected on chromosome 3A (LOD=5.3 Rsquare=13.3), associated with the SSR markers GWM1110 and GWM1121, and tentatively
named Qfhs.ifa-3A.
REFERENCES
Anderson, J.A., R.W. Stack, S. Liu, B.L. Waldron, A.D. Fjeld, C. Coyne, B. Moreno-Sevilla, J. Mitchel Fetch ,
Q.J.Song, P.B. Cregan, and R.C. Frohberg. 2001. DNA markers for a Fusarium head blight resistance QTL in
two wheat populations. Theor Appl Genet 102: 1164-1168.
Buerstmayr, H., B. Steiner, M. Lemmens, and P. Ruckenbauer. 2000. Resistance to Fusarium head blight in two
winter wheat crosses: heritability and trait associations. Crop Sci 40: 1012-1018.
Buerstmayr, H., M. Lemmens, L. Hartl, L. Doldi, B. Steiner, M. Stierschneider, and P. Ruckenbauer. 2002.
Molecular mapping of QTL for Fusarium head blight resistance in spring wheat I: resistance to fungal spread
(type II resistance). Theor Appl Genet 104: 84-91.
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EXAMINATION OF MOLECULAR VARIABILITY OF
FUSARIUM CULMORUM ISOLATES
B. Tóth1*, Á. Mesterházy1, J. Téren2 and J. Varga3
Cereal Research Non-profit Company, Szeged, Hungary; 2Animal Health and Food Control Station, Szeged,
Hungary; and 3Department of Microbiology, Faculty of Sciences, University of Szeged, Szeged, Hungary
*Corresponding Author: PH: (36) (62) 435-235; E-mail: beata.toth@gk-szeged.hu
1
ABSTRACT
Fusarium head blight is the most important disease of wheat in Hungary. The main causative
agents of this disease are Fusarium graminearum and F. culmorum. Mycotoxin contamination
is the most serious effect of ear fusariosis, since the mycotoxins produced are harmful both
to humans and animals. We examined the mycotoxin producing abilities and molecular
variability of Fusarium culmorum isolates using different techniques. Altogether 11
Hungarian and 28 other F. culmorum isolates were involved in this study, together with F.
graminearum, F. crookwellense and F. pseudograminearum strains. Mycotoxin producing
abilities of the isolates were tested by thin layer chromatography. The mycotoxins tested
involved deoxynivalenol (DON) and its acetylated derivatives, nivalenol (NIV), zearalenone
and fusarenone X. Most of the isolates produced zearalenone. 28 isolates were found to
belong chemotype I (producing DON and 3-acetyl-DON), while 8 represented chemotype II
(producing NIV and/or fusarenone X) according to Miller et al. (1991). Among the Hungarian
isolates, one produced NIV, while all other isolates belonged to chemotype I. Pathogenicity
tests were carried out as described previously (Mesterházy, 1985). Isolates belonging to
chemotype I were in general found to be more pathogenic in in vitro tests than those
belonging to chemotype II. Phylogenetic analysis of random amplified polymorphic DNA
(RAPD) profiles of the isolates obtained by using 40 different random decamers let us
cluster the isolates into different groups, although the variability observed was relatively low.
Most Hungarian isolates formed a well-defined cluster on the dendrogram. Sequence
analysis of a putative reductase gene fragment of the isolates was also carried out. Strong
correlation was observed between the geographic origin of the isolates, and their position
on the cladogram produced based on sequence data. These observations are in agreement
with the previous finding, that a similar correlation between geographic origin and sequence
data exists in the case of F. graminearum isolates (O’Donnell et al., 2000). Correlation was
not observed between sequence relationships and mycotoxin producing abilities or
pathogenicity of the strains. Double-stranded RNA elements indicative of mycovirus
infection were detected for the first time in 5 F. culmorum isolates. The sizes of the dsRNA
elements varied between 0.6-3.9 kbp. Correlation was not observed between the presence
of mycoviruses and geographic origin, mycotoxin production or pathogenicity of the isolates.
Further work is in progress in our laboratory to reveal the structure of Hungarian F. culmorum
populations, and to further characterize their mycoviruses.
B. Tóth was supported by an OTKA postdoctoral grant (D38486).
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REFERENCES
Mesterházy, Á. 1985. Effect of seed production area on the seedling resistance of wheat to Fusarium seedling
blight. Agronomie 5: 491-497.
Miller, J.D., Greenhalgh, R., Wang, Y.Z. and Lu, M. 1991. Trichothecene chemotypes of three Fusarium species.
Mycologia 83: 121-130.
O’Donnell, K., Kistler, H.C., Tacke, B.K. and Casper, H.H. 2000. Gene genealogies reveal global
phylogeographic structure and reproductive isolation among lineages of Fusarium graminearum, the fungus
causing wheat scab. PNAS 97: 7905-7910.
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A NON-CODING WHEAT RNA MAY PLAY AN IMPORTANT ROLE IN
WHEAT RESISTANCE TO FUSARIUM HEAD BLIGHT
D.H. Xing1, Y. Yen1*, and Y. Jin2
Department of Biology and Microbiology and 2Department of Plant Science,
South Dakota State University, Brookings, SD 57007
*Corresponding Author: PH: (605) 688-4567; E-mail: Yang_Yen@sdstate.edu
1
ABSTRACT
To understand the molecular events underlying Fusarium head blight (FHB) resistance in
wheat (Triticum aestivum L.), gene expression profiles (GEP) were compared between the
Fusarium graminearum-inoculated and the water-inoculated (mocking inoculation) spring
wheat cultivar Sumai 3 (FHB—resistant). One GEP, designated as G12 which is specifically
expressed in the pathogen-inoculated Sumai 3, was identified, cloned and sequenced.
Southern blot verified that G12 represented a wheat gene. The corresponding full-length
cDNA, designated as G12-S, was cloned with 5’RACE technology and sequenced.
Bioinformatic analyses indicated that the sequence of G12-S is similar to the minus strand
of the wheat chloroplast gene encoding ATP synthase CF-O subunit I. No significant open
reading frame is found in G12-S sequence, indicating that it may function at RNA level by
directly targeting the complementary transcripts of the chloroplast ATP synthase CF-O
subunit I gene and/or its likes to neutralize their ability to translation.
49
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2002 National Fusarium Head Blight Forum Proceedings
A PUTATIVE ACYL-COA-BINDING-PROTEIN OF FUSARIUM
GRAMINEARUM MAY PLAY AN IMPORTANT ROLE IN
THE FHB PATHOGENESIS IN WHEAT
D.H. Xing1, Y. Yen1*, and Y. Jin2
Department of Biology and Microbiology and 2Department of Plant Science,
South Dakota State University, Brookings, SD 57007
*Corresponding Author: PH: (605) 688-4567; E-mail: Yang_Yen@sdstate.edu
1
ABSTRACT
To identify the genes important to the pathogenesis of Fusarium head blight (FHB) in wheat,
we compared the gene expression profiles (GEP) between Fusarium graminearum (isolate
Fg4) infected and healthy spikelets, as well as between FHB-resistant cultivar Sumai 3 and
FHB susceptible cultivar Wheaton. Several GEPs specific to Fusarium-infected Sumai 3
were identified, cloned and sequenced. Southern analysis indicated that GEP 4CL represents a F. graminearum gene. With 5’RACE technology, corresponding full-length cDNAs
were cloned from the FHB-infected spikelets of Sumai 3 and Wheaton and from F.
graminearum culture, respectively. Sequence polymorphisms were observed in the 5’
untranslated region among the three full-length cDNA clones. Bioinformatic analyses indicated that the cognate gene may encode an acyl-CoA-binding-protein (ACBP) protein. The
possible role of the putative ACBP protein in F. graminearum’s pathogenicity and the importance of the differential mRNA editing to FHB pathogenesis in wheat were discussed.
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IDENTIFICATION OF CHROMOSOME REGIONS ASSOCIATED
WITH FUSARIUM HEAD BLIGHT RESISTANCE IN BREAD
WHEAT CULTIVAR SUMAI 3 WITH ITS SUSCEPTIBLE
NILS BY USING DNA MARKERS
D.H. Xu1, M. Nohda1, H.G. Chen2 and T. Ban1*
Japan International Research Center for Agriculture Sciences (JIRCAS),
1-2 Ohwashi, Tsukuba, Ibaraki 305-8686, Japan; and 2Institute of Plant Protection,
Jiangsu Academy of Agriculture Sciences, Nanjing, 210014, China
*Corresponding Author: PH: +81-298-38-6364; E-mail: tomohiro@affrc.go.jp
1
ABSTRACT
Fusarium head blight (FHB) is one of the most destructive diseases of wheat by reducing
the grain yield and quality. Several types of the host resistance to FHB have been described:
resistance to initial infection (Type I), resistance to spreading of infection (Type II), and
degradation the mycotoxin (Type III). Sumai 3, a Chinese wheat cultivar, is one of the most
widely used resistant resources for FHB resistance in wheat breeding around the world. On
the basis of molecular mapping, chromosomes 5A, 3BS, and 6BS seem to be likely locations for FHB resistant gene from Sumai 3. In this study, we reported the identification of
chromosome regions associated with FHB resistance in Sumai 3 by using its susceptible
near-isogenic lines (NILs).The plant materials used in this study were Sumai 3 and its four
NILs (NILs-1, NILs-2, NILs-3, and NILs-4). The NILs were derived from a cross between
Sumai 3 and Chuan980, a susceptible cultivar, followed by seven-times backcross with
Sumai 3 and screened FHB susceptible lines in each generation by artificial inoculation.
SSR and AFLP analyses were applied to screen the polymorphism between Sumai 3 and
its four NILs. The detected polymorphic markers were mapped using a mapping population
of double haploid lines (DHLs) derived from a cross between Sumai 3 and Gamenya. We
examined 84 SSRs and 107 AFLP primer combinations that produce approximately 900
AFLP markers. Of these markers, two SSR (Xgwm533-a and Xgwm389) and five AFLP
(ACT/CGAC118, AGA/CGAC136, AAC/CGAC285, AGT/CTGA225, and AGA/CTAT304)
markers showed polymorphism between Sumai 3 and its four NILs. The NILs-1, NILs-2, and
NILs-3 have different band pattern from Sumai 3 at all of the seven polymorphic markers
while NILs-4 is different from Sumai 3 only at the AGA/CTAT304 marker, indicating that the
genotypes of the different Sumai 3 NILs with susceptible to FHB is different. Six of the
seven-polymorphism markers were mapped on chromosome 3BS where the resistance
QTLs has been consistently detected in the populations including Sumai 3 or their derivatives. The AGA/CTAT304, which differ the NILs-4 from Sumai 3, was located on chromosome 2AL. The present study revealed that one FBH resistance gene locates on chromosome 3BS in Sumai 3 and Sumai 3 may have other genes that affect the FHB resistance.
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TRANSPOSON-MEDIATED GENERATION OF MARKER-FREE
BARLEY PLANTS EXPRESSING PUTATIVE
ANTIFUNGAL PROTEINS
X-H. Yu1*, P. Bregitzer2, M-J. Cho1, M.L. Chung1, and P.G. Lemaux1
1
Department of Plant and Microbial Biology, University of California, Berkeley CA; and
2
USDA-ARS, Aberdeen, ID
*Corresponding Author: PH: (510)-642-1589; E-mail: xhyu@nature.berkeley.edu
ABSTRACT
The use of transposon-mediated repositioning of transgenes has been proposed as an
attractive strategy to generate transgenic plants free of selectable marker genes (Yoder and
Goldsbrough, 1994). In barley, previous research has demonstrated high transposition
frequencies of a Ds element resulting from crosses of two transgenic plants, one containing
Ds-bar and the other expressing Ac transposase. Expression of the relocated transposonborne transgene is less prone to gene silencing than that of the transgene integrated at the
original site as a result of bombardment (Koprek et al., 2001). Characterization of Ds-delivered transgenes in rice confirmed the stability of insertion site and the expression of the Cry
1B protein during generation advance (Cotsaftis et al., 2002). To date transposon-mediated
repositioning of a value-added transgene has not been demonstrated in barley, although
functionality of the maize As/Ds system as a gene-tagging tool has been described (Koprek
et al., 2001).
Fusarium head blight (FHB), caused by Fusarium graminearum Schwabe (teleomorph
Gibberella zeae) is a major disease for barley and wheat throughout the world (Parry et al.,
1995). Introduction of putative, recombinant antifungal proteins into barley offers the potential to limit pathogen infection and growth. Transformation technologies for barley have been
developed and subsequently improved (Wan and Lemaux, 1994; Cho et al., 1998; Bregitzer
et al., 2000), setting the stage for the introduction of putative antifungal genes. To exploit
potentially useful aspects of the maize Ac/Ds system, we are using this system to produce
transgenic barley plants containing independent insertions of genes encoding putative
antifungal proteins. Crossing of these plants to plants expressing Ac transposase will result
in the excision and reintegration of Ds-bordered transgenes into new locations. This movement will result in the stabilization of transgene expression and the unlinking of the plasmid
and selectable marker sequences needed to identify transgene-containing tissue from the
transgene itself.
The putative antifungal genes chosen, tlp1 (thaumatin-like protein) and tlp4 from oat and
two of the trichothecene pathway genes, Tri101 and Tri12, isolated from Fusarium
sporotrichioides, were placed in a Ds-bordered, maize ubiquitin- or rice actin promoterdriven expression cassette. The resultant tlp constructs, together with pAHC20 (ubiquitin
promoter-bar-nos) or pActHpt4 (actin promoter-hpt-nos), were introduced via bombardment
into scutellar cells of immature embryos or green, regenerative tissues of two spring cultivars of barley, Golden Promise, a 2-rowed variety, and Drummond, an elite 6-rowed variety.
Plants derived from 3 putative DsUbiTlp1 lines and 3 DsUbiTlp4 transgenic GP lines were
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2002 National Fusarium Head Blight Forum Proceedings
positive for bar and further analyses for the presence of tlp and its expression is ongoing.
Three, one, and five hygromycin-resistant lines were obtained from DsActTlp1-, DsActTlp4and DsActTri101- transformed Drummond green tissue, respectively; these lines have been
transferred to regeneration medium. During the transformation process, we found that
bialaphos, used to identify bar- expressing tissue, is not suitable for selection of immature
embryo and green tissue transformants in Drummond. Subsequently, hygromycin was used
for selection. In addition, the Ac-transposase gene driven by its own promoter was transformed into Drummond green tissue; five hygromycin-resistant AcTPase lines were obtained
and are under regeneration. AcTPase was also introduced into Drummond by backcrossing
ubiquitin- and Ac promoter-driven AcTPase-containing Golden Promise lines that were
previously isolated (Koprek et al., 2001).
To assist in characterization of the level of transgene expression, antibodies to TLP1, TLP4,
and the Tri101proteins are being developed. Genes for tlp1, tlp4, tri101 and tri12 were
inserted in vector pGEX-4T3 and TLP1, TLP4 and Tri101 proteins were purified for antibody
preparation. These three antibodies were tested for experimental efficiency. Antibody to
Tri101 was efficient in detecting the Tri101 protein in western blots, while antibodies to TLP1
and TLP4 showed nonspecific binding to barley proteins. Nonspecific antibodies will be
removed by passing serum through an affinity column bound with purified TLP1 and TLP4
proteins produced in E. coli. The Tri12 gene was also cloned into pGEX-4T3, but protein
expression was low. Subsequently the gene was inserted into other vectors pMAL-c2X and
pMAL-p2X; however, the resultant MAL-Tri12 fusion protein expression was still weak. Low
expression of the Tri12 gene product may be due to its 14 transmembrane domains.
REFERENCES
Bregitzer P, Campbell, RD, Dahleen, LS, Lemaux, PG, Cho, M-J (2000) Development of transformation systems for elite barley cultivars. Barley Genet Newsl 30:10-13.
Cho, M-J; Jiang, W; Lemaux, PG. (1998) Transformation of recalcitrant barley cultivars through improvement of
regenerability and decreased albinism. Plant Sci 138(2):229-244.
Cotsaftis O, Sallaud C, Breitler JC, Meynard D, Greco R, Pereira A, and Guiderdoni E (2002). Transposonmediated generation of T-DNA- and marker-free rice plants expressing a Bt endotoxin gene. Mol Breed 10: 165180.
Koprek T, Rangel S, McElroy D, Louwerse JD, Williams-Carrier RE, Lemaux PG (2001) Transposon-mediated
single-copy gene delivery leads to increased transgene expression stability in barley. Plant Physiol 125: 13541362.
McMullen M, Jones R, and Gallenburg D (1997) Scab of wheat and barley: A re-emerging disease of devastating impact. Plant Dis 81: 1340-1348.
Wan Y and Lemaux PG (1994) Generation of large numbers of independently transformed, fertile barlely (Hordeum vulgare) plants. Plant Physiol 104: 37-48.
53
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EFFECT OF BACTERIAL GROWTH MEDIUM COMPOSITION ON
ANTIFUNGAL ACTIVITY OF BACILLUS SP. STRAINS USED IN
BIOLOGICAL CONTROL OF FUSARIUM HEAD BLIGHT
Nichole Baye1, Bruce H. Bleakley1,2*, Martin A. Draper2, and Kay R. Ruden2
Department of Biology/Microbiology and 2Department of Plant Science,
South Dakota State University, Brookings, SD 57007
*Corresponding Author: PH: 605-688-5498; E-mail: bruce_bleakley@sdstate.edu
1
ABSTRACT
Several microbial strains belonging to different taxa, isolated from various parts of the world,
have been shown to have the ability to antagonize Fusarium graminearum to different
extents under various conditions. Some of these microbial strains are being developed as
biological control agents (BCAs) for control of FHB. Different BCAs have different mechanisms of antagonizing FHB, such an enzymes, antibiotics, parasitism, and/or competition for
nutrients. We have studied four different Bacillus sp. strains that show promise for use as
BCAs to control FHB. All these strains seem to belong to a phylogenetic group designated
as the Bacillus subtilis group (group II). Among the many antibiotics that B. subtilis and its
relatives are known to make are cyclic lipopeptides such as iturin. If one or more iturin-like
antibiotics are needed for these bacterial strains to control FHB, it is important that a growth
medium be used for culturing the BCAs that encourages production of such antibiotics. In
previous studies, we have usually grown the four BCAs in potato-dextrose broth (PDB),
which may not have been an optimal growth medium for production of iturin-like antibiotics.
Other researchers working with B. subtilis have found that dextrose (glucose) is not an
optimal carbon source for iturin production, and that the nitrogen source in the growth medium also has a large influence on the amount of iturin produced. All four of our BCAs grew
well in a defined growth medium previously described in the literature that may stimulate
antibiotic production of our BCAs more than does PDB. The defined medium contains
mannitol as a carbon source, and glutamic acid as a nitrogen source, along with inorganic
salts. We have conducted studies with both the broth and agar-solidified form of this medium, finding that the bacteria grow well in both. Plate assays were conducted to test the
ability of the BCAs to antagonize F. graminearum on the agar-solidified form of this growth
medium. Antagonism against the fungus was apparent, suggesting that antibiotic was being
produced in the medium. Presence of iturin in the growth medium will be tested for chromatographically, and compared to amounts produced in PDB. In addition, greenhouse
groundbed trials will compare the effect that BCA cells grown in the defined broth medium
have upon wheat challenged with FHB, to the effect that BCA cells grown in PDB have
upon wheat challenged with FHB. In uniform field trials to compare the ability of different
microbial BCAs to control FHB, it should be recognized that different microbial BCAs can
have different mechanisms of antagonism, and that different growth media may promote
these mechanisms to varying degrees. Formulation and optimization of growth media for
commercial production and application of BCAs to control FHB should also bear this in
mind.
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TAXONOMIC AFFILIATION OF BACTERIAL STRAINS USED IN
THE BIOLOGICAL CONTROL OF FUSARIUM HEAD BLIGHT
SUGGESTS POSSIBLE ROLE OF LIPOPEPTIDE
ANTIBIOTIC IN FUNGAL ANTAGONISM
Nichole Baye1 and Bruce H. Bleakley1,2*
Department of Biology/Microbiology and 2Department of Plant Science,
South Dakota State University, Brookings, SD 57007
*Corresponding Author: PH: 605-688-5498; E-mail: bruce_bleakley@sdstate.edu
1
ABSTRACT
For the last several years, our laboratory has been working with four endospore-forming
bacterial strains (designated as 1B-A, 1B-C, 1B-E, and 1D-3) isolated from South Dakota
wheat foliage and residue that can antagonize Fusarium graminearum in laboratory plate
assays and in greenhouse and field plot trials. We have attempted to identify these bacterial
strains by different techniques, with different identification methods resulting in different
genus affiliations for the strains. In previous work, analysis of membrane fatty acid methyl
esters (FAME analysis) indicated that strains 1B-A and 1D-3 were Bacillus lentimorbus, and
that strains 1B-E and 1B-C were Bacillus subtilis. Sequence analysis showed that all four
strains had identical sequences in the first 500 base pairs of their 16S rDNA genes, and all
were most closely related to Bacillus amyloliquefaciens with less but significant relatedness
to Bacillus atrophaeus. The strains differed in the total number and amount of antibiotic
compounds produced, and their growth curves in potato dextrose broth also differed. In the
work presented here, colonial morphology, microscopic appearance, and 20 different phenotypic traits were evaluated and used to arrive at suggested identities for the strains.
Strains 1B-A and 1B-C had similar colonial morphology, with a shiny and wrinkled appearance, whereas colonies of strain 1B-E were shiny but not wrinkled, and colonies of strain
1D-3 were a dull color with bumps instead of wrinkles. All strains had oval endospores
which did not cause swelling of the sporangium. Results of 20 different phenotypic tests
suggested that all four strains were most closely related to Bacillus firmus. These attempts
to identify the four strains strongly suggest that they are tied to a phylogenetically and
phenetically coherent B. subtilis group (group II). However, the four strains may all belong to
a previously uncharacterized taxon with relatedness to B. amyloliquefaciens and B.
atrophaeus, taxa which were split out of the old Bacillus subtilis taxon. There is a good
amount known about the antibiotics produced by members of the B. subtilis group (group II).
Among the many antibiotics that are known to be produced by B. subtilis and its relatives
are cyclic lipopeptides such as iturin. We are hypothesizing that one or more cyclic
lipopeptides such as iturin are responsible for a significant amount of the biological control
these bacterial strains exert against F. graminearum, and we are presently engaged in
experiments to test this hypothesis.
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JAU 6476 FOR THE CONTROL OF FUSARIUM GRAMINEARUM
AND OTHER DISEASES IN CEREALS
J. R. Bloomberg1*, D.E. Rasmussen2 and T. K. Kroll2
Bayer CropScience, Research Triangle Park, NC; and
Formerly Bayer CropScience, Mahtomedi, MN and Hudson, WI
*Corresponding Author: PH: 816-242-2268; E-mail: jim.bloomberg@bayercropscience .com
1
2
ABSTRACT
JAU 6476 (tested under the code AMS 21619) is a novel broad-spectrum fungicide belonging to the new chemical class of triazolinthione discovered and developed worldwide by
Bayer CropScience. The common name for this molecule is prothioconazole. JAU 6476 is a
systemic fungicide showing excellent efficacy against a broad range of diseases in different
crops, including wheat, barley, peanuts, canola, etc. In cereal crops, JAU 6476 provides
excellent activity against most major diseases, including Fusarium head blight (Fusarium
spp.), leaf blotch diseases (Septoria tritici, Leptosphaeria nodorum, Pyrenophora spp.,
Rhychosporium secalis), rust (Puccinia spp.), powdery mildew (Erysiphe graminis) and
eyespot (Pseudocercosporella herptrichoides). Trial results indicate that JAU 6476 is more
effective than currently tested products for the reduction of deoxynivalenol (DON), a mycotoxin caused by Fusarium graminearum. JAU 6476 applications provide outstanding cereal
disease control along with excellent crop safety to ensure high quality yields.
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EFFECT OF FUNGICIDE TREATMENTS ON FUSARIUM HEAD BLIGHT
AND LEAF DISEASE INCIDENCE IN WINTER WHEAT
A.L. Brûlé-Babel* and W.G.D. Fernando
Department of Plant Science, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2
*Corresponding Author: PH: (204) 474-6062; E-mail: ababel@ms.umanitoba.ca
OBJECTIVES
The objectives of this study were to compare control of Fusarium head blight and leaf diseases of winter wheat with the application of different fungicides and combinations of fungicides.
INTRODUCTION
Winter wheat cultivars grown in western Canada are susceptible to most leaf diseases and
Fusarium head blight (FHB). These diseases can cause losses in yield and quality, which
affects producers and end-users of the grain. Winter wheat producers routinely apply a
fungicide treatment for control of leaf diseases, but not for FHB control. Few fungicides are
registered for FHB control. Those that are registered require different application times for
control of FHB compared to leaf diseases. Timing of fungicide application for FHB control is
critical due to the specific period of host susceptibility. Producers are interested in the
efficacy of fungicides for FHB control, and have questioned whether delaying fungicide
applications to control FHB would compromise their ability to control leaf diseases.
MATERIALS AND METHODS
Trials were conducted at one location in 1999 (Carman, Manitoba), and two locations
(Carman and Winnipeg, Manitoba) in 2000, 2001 and 2002. In 1999 ten cultivars (only
cultivar means will be reported) and eight treatments (Table 1) were evaluated in a split-plot,
four replicate trial. Treatment was the main plot effect and cultivar was the sub plot effect. In
2000, 2001 and 2002 trials with the same treatments were conducted on a single winter
wheat cultivar, “CDC Falcon”, at Carman and Winnipeg, MB.
Where appropriate fungicide treatments were based on label recommendations for FHB
control. Tilt was applied at the boot stage for control of leaf diseases since there is no label
recommendation for FHB control. Bravo and Folicur were applied according label instructions for FHB control. In the Bravo x 2 treatment the first application was made at the recommended time for FHB control and the second application was made two weeks later. All
fungicide treatments preceded FHB inoculation by at least three days.
All plots except for the un-inoculated control were inoculated with a macroconidial suspension of Fusarium graminearum at anthesis and four days after the first inoculation. Mist
irrigation was applied for 5 min./h for 12 h after each inoculation. Eighteen to twenty-one
days after inoculation, 50 spikes/plot were collected from all plots for evaluation of FHB
reaction. The number of infected spikes was determined. Of the infected spikes, the perChemical and Biological Control
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centage of infected spikelets was determined. From this the FHB index was calculated as
(% infected spikes x % infected spikelets)/100.
Percent leaf area affected by leaf spot diseases and leaf rust was evaluated visually on the
flag leaf on a per plot basis in 1999. In 2000-2002, leaf area affected by disease was determined by collecting 20 flag leaves and 20 penultimate leaves per plot and evaluating percent disease through digital imaging technology.
Plot yield was measured at maturity.
T a b le 1: T reatm en ts an d tim in g of treatm ent ap plication to field trials con du cted in 199 9, 2 000 ,
200 1 and 200 2. F H B in oculum w as ap plied to all p lo ts treated w ith fun gicides.
T rea tm en ts an d T im e o f A pp licatio n
U n - inoculated con trol
F H B inoculated control - A n th esis + 4 days later
T ilt - B o ot stag e
B ravo - H ead in g
F olicur - H ead in g
B ravo x 2 - H eading + 2 w eek s later
T ilt (B oo t S tage) + B ravo - H ead in g
T ilt (B oot S tage) + F olicur - H ead in g
RESULTS AND DISCUSSION
1999
Disease levels were high in 1999. Cultivars differed in susceptibility to FHB, leaf rust and
leaf spot (data not reported). The FHB index was higher than the un-inoculated check in all
fungicide treated plots (Figure 1a). Treatment with either Bravo or Folicur reduced the FHB
Index relative to the inoculated check. Plots treated only with Tilt did not differ in FHB Index
compared to the inoculated check. Treatments which included Tilt provided the best control
of leaf spot diseases (Figure 1b) and leaf rust (Figure 1c). All fungicides increased yield
relative to the untreated checks (Figure 1d). The highest yields were obtained with plots
treated with Tilt or combinations of Tilt+Bravo and Tilt+Folicur. Yield was highly negatively
correlated with %leaf spot (-0.98) and %leaf rust (-0.90), but was not significantly correlated
with FHB Index (0.20).
2000-01
Disease levels in trials conducted in 2000 and 2001 were low at both locations. Significant
differences in yield and FHB Index were observed at Winnipeg in 2001, while significant
differences in leaf spot were observed at both locations in 2000. Folicur and Bravo provided
similar levels of FHB control. All fungicide treatments reduced leaf spot relative to the
untreated control. Fungicide treatments did not provide a significant yield advantage compared to the untreated checks.
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2002
FHB levels were high at both locations in 2002. Leaf disease data has yet to be analysed.
At Winnipeg all treatments had higher levels of FHB than the un-inoculated control (FHB
Index =7). The FHB Index of the inoculated control was 41. Folicur (FHB Index = 29) and
Bravo (FHB Index = 26.5) significantly reduced the FHB Index relative to the inoculated
control and were not statistically different from each other. Other treatments were not significantly different from the inoculated control. In Carman, the FHB Index of the fungicide
treatments did not differ significantly from the inoculated check (FHB Index = 26). There
were no significant differences for yield.
The results from these trials show that even when plots are inoculated and mist irrigation is
applied to increase humidity it is difficult to get consistently high levels of FHB on winter
wheat in Manitoba. Lower June temperatures (data not shown) in 2000 and 2001 relative to
1999 appeared to be the main reason for lower disease incidence. Disease forecasts would
be beneficial in this situation.
Under high disease pressure fungicide treatments with either Bravo or Folicur reduced FHB
index. However, the FHB Index of these treatments was still high relative to the un-inoculated control. When either leaf rust or leaf spotting diseases were present, treatments with
Tilt and to a lesser extent, Folicur reduced these diseases. Under low disease pressure,
fungicide treatments provided little advantage. Overall, there was no association between
yield and FHB Index. Leaf diseases appeared to be the main cause of yield differences
observed.
CONCLUSIONS
Under high disease pressure fungicide treatments reduced both FHB and leaf diseases.
Yield differences were primarily associated with differences in leaf disease control. Tilt
provided the best control of leaf diseases. Folicur applied at heading provided some level
of leaf disease control. Folicur and Bravo appear to provide similar levels of FHB control.
Weather conditions during flowering of winter wheat are often not conducive the FHB development. Disease forecasts would be useful to determine whether fungicide application is
necessary in winter wheat.
Chemical and Biological Control
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Leaf Rust (%)
Chemical and Biological Control
60
Check
Tilt
Bravo
Bravo
Treatm ent
x2
Folicur
Treatm ent
Tilt +
Folicur
c
Tilt + Bravo
a
0
1
2
3
4
5
6
7
8
0
5
10
15
20
25
30
35
40
45
Check
Check
Tilt
Tilt
d
Tilt + Tilt +
Bravo Folicur
b
Bravo Folicur Bravo Tilt + Tilt +
Treatm ent x2
Bravo Folicur
Bravo Folicur Bravo
x2
Treatm ent
Figure 1. Effect of fungicide treatment on a) FHB Index, b) % leaf spot, c) % leaf rust and d) yield of winter
wheat grown in trials conducted at Carman, MB in 1999.
0
5
10
15
20
25
Uninoculated
0
5
10
15
20
25
Leaf Spot (%)
Yield (t/ha)
30
2002 National Fusarium Head Blight Forum Proceedings
FHB Index
2002 National Fusarium Head Blight Forum Proceedings
POPULATION DYNAMICS IN THE FIELD OF A BIOCONTROL AGENT
FOR FUSARIUM HEAD BLIGHT OF WHEAT
A.B. Core1, D.A. Schisler2, T.E. Hicks1, P.E. Lipps3*, and M.J. Boehm1
The Ohio State University, Department of Plant Pathology, Columbus, OH 43210;
National Center for Agricultural Utilization Research (NCAUR), USDA-ARS, Peoria, IL 61604; and
3
The Ohio State University/OARDC, Department of Plant Pathology, Wooster, OH 44691
*Corresponding Author: PH: (330) 263-3843; E-mail: lipps.1@osu.edu
1
2
ABSTRACT
Gibberella zeae (anamorph Fusarium graminearum) is the major causal organism of
Fusarium head blight (FHB) on wheat and barley. Wheat is generally most susceptible to
infection at anthesis due to exposed anthers being an important site of infection. Application of a Crytococcus strain, OH 182.9, originally isolated from wheat anthers in Wooster,
Ohio has reduced disease severity by 56% and increased the 100-kernel weight by as
much as 100% in field trials. The goal of this research was to determine the ability of OH
182.9 to survive and possibly reproduce on the anthers in the field. Heads of the soft red
winter wheat cultivar Freedom were marked to distinguish those that had extruded anthers
and those that had no visible anthers. Cells of the yeast antagonist were produced and
harvested after a 48-hour growth in a semi-defined liquid medium at 25ºC in 250 rpm and
applied (1x107colony forming units (CFU)/ml) to thoroughly wet the wheat heads. Nonantagonist/buffer treated plants served as controls. Pathogen inoculum consisted of F.
graminearum colonized corn kernels scattered throughout the plots 3 weeks prior to flowering. Plots were under mist irrigation twice daily throughout anthesis and early grain development growth stages. Anthers were collected for up to 10 days after applying yeast antagonists and CFU per 100 anthers in 0.5 ml buffer were determined. Initial OH 182.9 populations on anthers, at day 0, were 2.6x104 CFU/ml. OH 182.9 population increased to 2.1x106
CFU/ml (80 times) by 6 days after applying the cell suspension. The yeast population was
2.2 x106 by 10 days after application. The population levels were significantly (P<0.05)
greater than those on the control plants on the heads with exposed anthers and heads with
no visible anthers at 6,8,10 days and 8 days, respectively after inoculation. There was no
significant difference in disease severity between OH182.9 treated and untreated plants.
This one season test will be repeated in 2003 to further determine the population dynamics
of OH182.9 on wheat floral structures.
Chemical and Biological Control
61
2002 National Fusarium Head Blight Forum Proceedings
VARIATIONS IN FUNGICIDE APPLICATION TECHNIQUES TO
CONTROL FUSARIUM HEAD BLIGHT
Martha Diaz de Ackermann1*, Mohan Kohli2, and Vilfredo Ibañez1
1
National Agriculture Research Institute, La Estanzuela, CC 39173, Colonia, Uruguay; and
2
CIMMYT, Regional Wheat Program, CC 1217, Montevideo, Uruguay
*Corresponding Author: PH: 001 598 574 8000; E-mail: martha@le.inia.org.uy
ABSTRACT
The frequency and severity of Fusarium head blight (FHB) on wheat have been on increase during
the past decade in Uruguay. Given low level of resistance in the commercial cultivars, chemical
control of the disease is widely adopted. The application of Folicur 430 SC (tebuconazol, 432 g/l) or
Caramba (metconazol, 90 g/l) at the rate of 450 cc/ha and 1000 cc/ha, respectively, is recommended at the beginning of flowering. In order to increase the efficiency of fungicide application,
variations in the spray nozzles and angles were tried.
The treatments, two fungicides (Folicur and Caramba), two types of spray nozzles (hollow cone
spray tips, ConeJet, and twin even flat spray tips, TwinJet, mounted at 0o and 30o angle on the bar),
and two application times (beginning of flowering, Zadoks 61 and mid flowering, Zadoks 65), were
combined in a factorial design with complete blocks replicated four times. All treatments were applied with a CO2 backpack type sprayer. Grain yield, test weight, thousand kernel weight, visual
disease note on a 1-5/1-5 scale, incidence (percentage of diseased spikes) and percentage of
scabby grains were evaluated (Table 1).
The results show that overall Caramba gave better control of the FHB than Folicur. In general, the
early control of the disease at Z61 was superior to FHB control in Z65. The utilization of TwinJet
improved the spike coverage significantly thereby, resulting in better visual score of infection. However, spike infection in the field and grain infection evaluated after harvest demonstrated these
differences more clearly in the case of Folicur than Caramba. Although some advantage in using the
TwinJet at an angle of 30o on the bar was observed, these results need further testing and confirmation. In spite of the fact that grain yield, test weight and thousand kernel weights were affected by
moderate infection of foliar diseases, the utilization of Caramba early on and especially using
TwinJet spray nozzles gave significantly higher grain yield compared to other treatments.
Table 1. Effect of chemical control treatments on FHB infection and grain yield.
Treatments
Application
Time
Z-61
Z-61
Z-61
Z-65
Z-65
Z-65
Z-61
Z-61
Z-61
Z-65
Z-65
Z-65
Spray Degree/
Fungicide nozzle vertical
Caramba
Twin
0º
Caramba
Cone
0º
Caramba
Twin
30º
Caramba
Twin
0º
Caramba
Cone
0º
Caramba
Twin
30º
Folicur
Twin
0º
Folicur
Cone
0º
Folicur
Twin
30º
Folicur
Twin
0º
Folicur
Cone
0º
Folicur
Twin
30º
Check without fungicide
Fusarium infection (%)
Visual
score
22 e
25 de
15 f
28 cd
28 cd
15 f
25 de
35 b
35 b
35 b
32 bc
35 b
52 a
spike
37e
39cde
38de
37e
41cde
36e
55bc
53bcd
55bc
49bcde
64ab
52bcd
78a
Chemical and Biological Control
62
grain
7
7.8
7.3
6
6.9
5.6
8.8
9.6
10.4
9.4
6.8
7.2
10
Grain yield
TKW
Test weight
kg/ha
2415a
2474a
2083ab
1599bc
1863bc
2012ab
1820bc
1759bc
1767bc
1644bc
1691bc
2045ab
1415c
g
27.1ab
28.1a
26.8ab
24.9b
25.7ab
27.1ab
26.2ab
24.4bc
24.8b
24.3bc
24.4bc
26.4ab
21.5c
kg/hl
80.2a
79.6ab
79.6ab
79.5ab
78.5bc
79.5ab
78.2bc
77.6cd
78.6abc
77.0cd
77.3cd
78.6abc
76.4d
2002 National Fusarium Head Blight Forum Proceedings
AERIAL SPRAY COVERAGE TRIALS IN SOUTH DAKOTA – 2002
M.A. Draper1, J.A. Wilson1, B.E. Ruden1, D.S. Humburg2,
K.R. Ruden1, and S.M. Schilling1
1
Plant Science Department and 2Agricultural and Biosystems Engineering Department,
South Dakota State University, Brookings, SD 57007
*Corresponding Author: PH (605) 688-5157, E-mail: draper.marty@ces.sdstate.edu
INTRODUCTION AND OBJECTIVES
In a state such as South Dakota, wheat fields are typically very large and with 2.5 M acres of
wheat and another 400,000 acres of barley, the only practical means of applying fungicide is
from the air. As such it is critical to identify methods whereby applicators and producers can
optimize the application for efficacy and cost effectiveness. These trials were intended to
take initial steps in accomplishing those goals.
MATERIALS AND METHODS
The trial was conducted on August 28, 2002 in collaboration with MJ Aviation, Inc. at
Letcher, SD. Treatments, listed in Table 1 were a comparison of three different brands of
nozzles with varying combinations of modifications. Each treatment was repeated three
times. Airplanes were loaded with water and fluorescent rhodamine dye blended with a pink
foam marker dye.
Table 1: Treatment list of nozzle and spray configurations for aerial coverage trial.
Trt #
GPA
Nozzle
Nozzle
Spacing
Nozzle
Modifications
1
5
Lund
14”
None
2
5
CP
7”
Straight
Stream
3
5
CP
7”
15º
Deflection
4
5
CP
7”
30º
Deflection
5
5
Accu-Flow
0.028
7”
3/32
Restrictor
6
5
Accu-Flow
0.028
14”
3/32
Restrictor
7
5
Accu-Flow
0.028
7”
1/8 Black
Restrictor
8
5
Accu-Flow
0.028
7”
3/32
Restrictor
9
5
Accu-Flow
0.028
7”
3/32
Restrictor
Boo
m
Ht.
16’,
19’,
22’
18’,
15’,
14’
16’,
18’,
19’
18’,
18’,
19’
16’,
13’,
18’
16’,
15’,
18’
18’,
19’,
16’
12’,
14’,
14’
22’,
24’,
23’
Drop
Used
Check
Valve
Expected
Swath
Spray
pressure
(PSI)
Airplane
Speed
No
Brass
TeeJet
60’
39#
130, 130,
130
No
TeeJet
60’
22#
128, 128,
124
No
TeeJet
60’
22#
129, 129,
129
No
TeeJet
60’
22#
125, 128,
128
No
TeeJet
35’
40#
127, 127,
122
No
TeeJet
35’
30#
128, 123,
126
No
Internal
35’
30#
128, 131,
131
6”
TeeJet
35’
30#
126, 125,
125
6”
TeeJet
35’
30#
125, 125,
125
Chemical and Biological Control
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2002 National Fusarium Head Blight Forum Proceedings
Measurements were taken of spray pattern deposition on a string line and measurement of
drift on a string line suspended from an 18 m high drift tower positioned at 46 m from the
center of the spray swath, perpendicular to the prevailing wind. Measurements were also
taken for droplet patterns on water sensitive and chrome coat papers. The rhodamine dye
was used for measurements on the string line tests and the pink foam marker dye was used
for droplet deposition on the chrome coat paper.
Treatment one was applied with an Air Tractor AT-402B Turbo with nozzles spaces every 14
in. and treatments two through nine were applied with an Air Tractor AT-401B radial engine
with nozzles spaces every 7 or 14 in. across the boom.
String line patter and drift tower data were read and analyzed by String Analysis/Graphics
(WRK) and water sensitive and chromecoat paper data was analyzed by Dropletscan (WRK
and DSI). Additional analysis was complied in Excel (Microsoft).
RESULTS AND DISCUSSION
This trial was initially planned for May, but excessive winds through the month prevented
completion of the trial at that time. During the period of the August trial, the wind speed
ranged from two to nine mph and no deposition was measured on the drift tower string line.
One of the most serious problems encountered with aerial application has been incomplete
coverage of the head. One side of the head may receive reasonable coverage while the
opposite side may receive no product. In an earlier trial (Draper, unpublished) CP nozzles
were compared with hollow cones at five or ten gallons of water delivered. In that trial, CP
nozzles gave poor performance for droplet uniformity and head coverage, but increasing the
gallons delivered helped offset the coverage deficiency. Nonetheless, CP nozzles are
preferred by aerial applicators in South Dakota because they work well for herbicide applications. If we are to improve fungicide application by air, we must identify a preferred configuration for optimized coverage. CP nozzles were retained in this study because of their
common usage, Lund nozzles were in place on one of the cooperators airplanes, and the
Accu-Flow nozzles (Bishop Equipment, Inc.) were tested because of their use in orchards
and that they are noted for good patterns with little drift.
No nozzles tested in this trial overcame the problem of poor deposition on the back of the
head.
All Accu-Flow nozzle configurations deposited a slightly narrower swath with less off target
movement than the CP or Lund nozzles.
All Accu-Flow nozzle configurations deposited a more uniform droplet pattern than the Lund
or CP nozzle configurations.
Additional treatments will be competed in the coming year, looking at different orifice size
and other nozzles designed to produce small droplet size with minimal drift.
Chemical and Biological Control
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2002 National Fusarium Head Blight Forum Proceedings
UNIFORM TRIALS FOR BIOLOGICAL CONTROL AGENT
PERFORMANCE IN THE SUPPRESSION OF FUSARIUM
HEAD BLIGHT IN SOUTH DAKOTA – 2002
M.A. Draper1*, B.H. Bleakley1, K.R. Ruden1, N.L. Baye1,
A.L. LeBouc2, and S.M. Schilling1
1
Plant Science Department, South Dakota State University, Brookings, SD 57007; and 2École Nationale
Supérieure Agronomique de Toulouse, Auzeville-Tolosane, France, - F 31326
*Corresponding Author: PH (605) 688-5157, E-mail: draper.marty@ces.sdstate.edu
INTRODUCTION AND OBJECTIVES
Biological control agents (BCAs) have several advantages in the suppression of Fusarium
head blight (FHB or scab). When organic crops are grown, fungicide options are not available and crops such as barley are susceptible over a long period of time following head
emergence and before maturity. As such, biological control has a good fit for FHB management under those conditions.
The objectives to this study were to evaluate the efficacy of various BCAs relative to the
standard fungicide comparisons for the suppression of Fusarium head blight on wheat and
barley.
MATERIALS AND METHODS
‘Robust’ barley was planted in a randomized complete block design with six replications
and ‘Oxen’ and ‘Ingot’ hard red spring wheat were planted in a factorial randomized complete block design with six replications, both at Brookings, SD. Barley was protected with
isolates of Bacillus subtilus-type isolates SDSU-1BA and SDSU-1BC, Cryptococcus
nodaensis OH 182.9, Bacillus-type isolate TrigoCor 1448, Lysobacter sp. strain ‘C3’, Bacillus-type isolate TrigoCor 2, and Bacillus-type isolates BHWJ 4-1 and BHWJ 4-2B. The
BCAs were compared to a standard chemical treatment of Folicur (4 fl/oz/A) with Induce
non-ionic surfactant (0.125%). Spring wheat was protected with Folicur + NIS, Cryptococcus nodaensis OH 182.9, Bacillus-type isolate TrigoCor 1448, isolates of Bacillus subtilustype isolates SDSU-1BA and SDSU-1BC, Lysobacter sp. strain ‘C3’, Bacillus-type isolate
TrigoCor 2, and Bacillus-type isolates BHWJ 4-1 and BHWJ 4-2B
At the time that the heads were completely emerged from the boot, a misting cycle was
started for 5 minutes out of every 20minutes, 24 hours a day. The mist system was turned off
and the BCAs were applied to the heads and allowed to dry before the misting was turned
on again. Two days following inoculation with the BCAs, the crop was challenge inoculated
with 104 macroconidia/ml of Fusarium graminearum ‘Fg4’. The barley plots were misted for
seven days total and the spring wheat plots were misted for three days following anthesis.
Chemical and Biological Control
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2002 National Fusarium Head Blight Forum Proceedings
RESULTS AND DISCUSSION
During the inoculation period, the environment was very hot and dry. And it was difficult to
retain free moisture between misting periods. Very little FHB developed in the either the
wheat or barley plots and no significant differences were detected among the barley treatments for FHB incidence, FHB severity, FHB index (incidence x severity), yield, test weight,
protein, or deoxynivalenol (DON) levels in the harvested grain, even among the challenge
inoculated plots. Only Folicur + NIS resulted in a reduction of any disease component on
spring wheat, although there was no significant FHB that developed on the spring wheat
either. While Folicur reduced overall leaf disease and leaf rust significantly, no biological
controls had a significantly measurable effect on any leaf disease.
Chemical and Biological Control
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2002 National Fusarium Head Blight Forum Proceedings
UNIFORM FUNGICIDE PERFORMANCE TRIALS IN
SOUTH DAKOTA – 2002
M.A. Draper1*, K.D. Glover1, K.R. Ruden1, A.L. LeBouc2,
S.M. Schilling1, and G. Lammers1
Plant Science Department, South Dakota State University, Brookings, SD 57007; and
École Nationale Supérieure Agronomique de Toulouse, Auzeville-Tolosane, France, - F 31326
*Corresponding Author: PH (605) 688-5157; E-mail: draper.marty@ces.sdstate.edu
1
2
INTRODUCTION AND OBJECTIVES
Fusarium head blight (FHB – scab) has been a serious concern for wheat producers in
South Dakota for the past several years. FHB and low market prices are the two reasons
most often cited by producers as they decrease the number of acres they plan to plant to
wheat. Fungicide alternatives for disease control are available to local producers on special
year-to-year labels.
The objectives to this study were to evaluate the efficacy of various fungicides, fungicide
combinations, or biological controls for the suppression of Fusarium head blight and other
wheat diseases.
MATERIALS AND METHODS
Three South Dakota locations were planted to hard red spring wheat and two locations
were planted to hard red winter wheat for inclusion in the Uniform Fungicide Trial for the
suppression of FHB.
Two hard red spring wheat cultivars, Oxen and Ingot, were planted at three South Dakota
locations (Brookings, Groton, and South Shore/Watertown). Two hard red winter wheat
cultivars, Wesley and Arapahoe, were planted at Selby and South Shore/Watertown. Trials
were planted in a factorial randomized complete block design. There were six replications of
spring wheat and four replications of winter wheat. At anthesis, the trial treatments were
applied. The following day, the crop was challenge inoculated with 104 macroconidia/ml of
Fusarium graminearum ‘Fg4’. The plots were misted for three days total.
Sixteen days following treatment, plots were evaluated for leaf diseases, FHB incidence,
FHB head severity, and FHB field severity, Fusarium damaged kernels (FDK),
deoxynivelanol (DON), grain yield, test weight, and protein.
RESULTS AND DISCUSSION
The weather in 2002 was very hot and dry in South Dakota. Grain yields were about half of
normal in much of the state and yields were progressively lower the farther west the fields
were located. In the spring wheat trials at Groton and South Shore/Watertown, very little
disease developed and there were no significant differences among treatments. Similar
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2002 National Fusarium Head Blight Forum Proceedings
results occurred in the winter wheat trial locations. With the supplemental mist irrigation,
while no significant FHB developed, leaf diseases were enhanced and some treatments did
significantly improve results over the untreated control.
No significant differences were detected for FHB incidence, FHB head severity, FHB field
severity, Fusarium damaged kernels (FDK), deoxynivelanol (DON), grain yield, test weigh,
and protein. No significant disease response resulted from challenge inoculation with
Fusarium conidia. However, in greenhouse trials the strain used has been shown to be
highly virulent. Presumably, extremely high temperatures and dry conditions minimized the
conditions for infection. FHB rating was done at five days earlier than normal due to the dry
conditions leading to a rapidly maturing crop.
The presence of a fungicide in the treatment generally resulted in a significant reduction in
leaf disease from the untreated (Table 1). Folicur, BAS 505, and AMS 21619 all resulted in
a reduction in leaf disease. However, BAS 505 and AMS 21619 did not reduce leaf rust in
the trial. The biological control treatments in the trial did not result in reduced disease
unless they were co-applied with Folicur or AMS 21619.
Table 1. Disease categories with a significant response to treatments at Brookings1.
Leaf Disease
Leaf Rust
Whole Plot Leaf
Treatment
Disease Rating2
(% leaf area)
(% leaf area)
Untreated
6.25
65.33
9.18
Folicur + NIS
5.08
36.30
1.12
AMS 21619 + NIS
5.25
34.08
6.63
BAS 505 + NIS
5.50
38.92
9.37
OH 182.9
6.33
62.00
11.77
TrigoCor 1448
6.42
55.08
7.97
TrigoCor + Folicur + NIS
5.08
27.03
0.87
AMS 21619 + Folicur + NIS
5.08
28.02
1.25
LSD (P=0.05)
0.57
14.01
3.86
1
Other measurements of disease and yield were not significant.
2
Green leaf evaluation based on a scale of 0-9 where 0 is disease free and 9 is completely
necrotic.
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2002 National Fusarium Head Blight Forum Proceedings
FUSARIUM HEAD BLIGHT: EPIDEMICS AND CONTROL
S.M. El-Allaf, P.E. Lipps*, and L.V. Madden
Dept. of Plant Pathology, The Ohio State University/OARDC, Wooster, OH 44691
*Corresponding Author: PH: (330) 263 3843; E-mail: lipps.1@osu.edu
OBJECTIVES
i) To document the effect of two biocontrol agents (TrigoCor 1448, and OH182.9) and three fungicides (Folicur, AMS21619, and BAS 505) on disease development, ii) To evaluate the effect of these
materials for managing Fusarium head blight, and iii) To determine the relationships between the
disease, DON and yield.
INTRODUCTION
Fusarium head blight (FHB) or scab, caused by Fusarium graminearum Schwabe (teleomorph
Gibberella zeae) is a major disease in many wheat and barley production regions of North America,
including Ohio, and throughout the world (Bai and Shaner 1994; Parry et al.1995; McMullen et al.
1997). This disease has been difficult to control. Although recent advances in host resistance are
beginning to improve disease management in some wheat production regions, many wheat and
barley producers have few management options. Commonly used methods of disease management
including tillage and crop rotations, have not been effective in eliminating wide spread disease
epidemics (McMullen et al. 1997). Controlling Fusarium head blight will require multiple disease
management strategies, coupled with greater understanding of the epidemiology of the disease (Bai
and Shaner, 1994; Parry, et al., 1995; Shaner and Buechley, 2000).
Effective fungicides could provide growers with management options when susceptible cultivars are
grown, and may help protect yield and grain quality of cultivars with partial resistance under conditions favorable for disease. Although a few fungicides have shown some efficacy against scab, their
results have been inconsistent over locations and years (Parry, et al., 1995; McMullen et al. 1997;
Shaner and Buechley, 1999; Gilbert and Tekauz, 2000). Treatment with some fungicides reduced
DON contamination of grain, but others caused an increase in DON levels (Shaner and Buechley,
1997, 1999 and 2000; Gilbert and Tekauz, 2000).
MATERIALS AND METHODS
Seeds of wheat cultivar Elkhart treated with Raxi-Thiram, were planted using 24 seeds/ft of row on
11 Oct., and 27 Sep., 2000 and 2001, respectively, in Ravenna silt loam soil at the Ohio Agricultural
Research and Development Center, Wooster. For each treatment, there were three replicate plots.
Each plot was 15-ft long, and consisted of 7-rows with 7 in. between rows. Plots were inoculated by
broadcasting colonized corn kernels (0.12 oz/sq ft) over the plot surface on 14 May in 2001, and 30
Apr. in 2002. Plots were misted each day from one week prior to flowering to two week after flowering. Biological agents and fungicides were applied as sprays in 26.2 gal. water/A with a CO- pressurized back pack sprayer at flowering growth stage (GS) 10.5.1. Disease assessments were
made twice a week (June 11 - June 26) in 2001 and three times a week (June 07 - June 21) in 2002
for both incidence and severity in one ft. of row at 15 locations in each plot. Plots were harvested on
17 of July in 2001 and on 11 July in 2002. Yield (bu/A) was determined from harvested grain adjusted to 13.5% moisture, and grain was analyzed for DON content.
Chemical and Biological Control
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2002 National Fusarium Head Blight Forum Proceedings
RESULTS AND CONCLUSIONS
Disease development varied greatly among the different fungicides and biological control treatments
tested in the two years. Based on the coefficient of determination (R2), evaluation of the residual
plots, standard error of estimates (SE) and mean square errors (MSE), the Gompertz model was
appropriate for describing the disease incidence and severity data sets (R2 ranged from 82 to 96%).
The various treatments had a significant effect on disease development. Rates of disease increase
for the various treatments and the control ranged from 0.138 to 0.229 and from 0.054 to 0.129 per
day based on disease incidence, and from 0.093 to 0.172 and from 0.066 to 0.125 per day for
disease severity in 2001 and 2002, respectively (Table 1). Area under the disease progress curve
based on disease incidence (AUDPCI) ranged from 418.0 to 804.2 in 2001, and from 605.2 to 911.8
in 2002; when based on disease severity (AUDPCS) ranged from 125.1 to 315.7 in 2001, and from
176.6 to 383.3 in 2002 (Table 1). Maximum disease incidence (Ymax) for the various treatments
ranged from 55.0 to 89.6%; from 55.1 to 82.5% and Maximum disease severity ranged from 23.9 to
57.9%; from 27.9 to 54.0% in 2001 and 2002, respectively (Table 2).
Plots treated with AMS21619 or BAS 505 had significantly lower rates of disease increase, low
maximum disease, AUDPCI , and AUDPCS values than the untreated control in both 2001 and 2002
(Tables 1and 2). Additionally, plots treated with Folicur had significantly lower rates of disease
progress, low maximum disease, AUDPCI, and AUDPCS values than the untreated control plots in
2002.
Plots treated with AMS21619, and BAS 505 had significantly higher yield in both years, higher test
weight, and lower DON levels than grain from the untreated control plots in 2001 only. However,
plots treated with Folicur had significantly higher yield in 2002. Although the biocontrol agent
OH182.9 did not have a significant effect on reducing disease development, grain harvested from
plots treated with this biocontrol agent had significantly lower DON than grain from the untreated
control plots in 2001. No differences were found among treatments in DON levels, damage kernels,
or test weight in 2002.
There were positive correlations between DON and final disease severity, AUDPCI, AUDPCS. On
the other hand, there were negative correlations between yield and maximum disease severity,
AUDPCI, and AUDPCS.
In conclusion, the treatments exhibited different effects on Fusarium head blight development and
control. Treatments AMS21619 and BAS 505 had low maximum disease, low epidemic rates, and
small AUDPCI and AUDPCS values that were significantly different from the control. On the other
hand, treatments TrigoCor 1448 and OH182.9 had high maximum disease, fast epidemic rates, and
large AUDPCI and AUDPCS values that were not significantly different from untreated control.
These results indicate the AMS21619 and BAS 505 fungicides have greater potential for management of Fusarium head blight than the other treatments tested.
REFERENCES
Bai, G. and Shaner, G. 1994. Scab of wheat: Prospects for control. Plant Dis. 78:760-766.
Gilbert, J., Tekauz, A. 2000. Recent developments in research on Fusarium head blight of wheat in Canada.
Can. J. Plant Pathol. 22:1-8.
Chemical and Biological Control
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2002 National Fusarium Head Blight Forum Proceedings
McMullen, M., Jones, R., and Gallenburg, D. 1997. Scab of wheat and barley: A re-emerging disease of
devastating impact. Plant Dis. 81:1340-1348.
Parry, D. W., Jenkinson, P., and McLeod, L. 1995. Fusarium ear blight (scab) in small grain cereals-a review.
Plant Pathol. 44:207-238.
Shaner, G., and Buechley, G. 1997. Effect of foliar fungicides on control of wheat diseases, 1996. Fungicide
and Nematicide Tests 52:233.
Shaner, G., and Buechley, G. 1999. Control of wheat diseases with foliar fungicides,1998. Fungicide and
Nematicide Tests 54:337-338.
Shaner, G., and Buechley, G. 2000. Control of Fusarium head blight of wheat with foliar fungicides. National
Fusarium Head Blight Forum. Erlanger, KY, December 10-12, 2000. Pages 110-113.
Ta bl e 1 . Fit of mo dels , ep idemic rates , an d area under d isease p ro gress curv e of Fu sa riu m head b light
in cid en ce (A UDPCI) an d s everity (AUDPCS) fo r fun g icides and biocon tro l ag en ts t este d in
Oh io, in 200 1 and 200 2.
___ ___ __ __ ____ ___ ___ ___ _ _____ _ __ _ ____ __ ____ ___ _ _____ _ _____ _ ___ ____ __ ____ _
Year
T rea tme nt
Incid ence
Severit y
&
___ __ __ __ __ __ ___ __ __ __ __ __ __
__ __ __ __ ___ __ __ __ ____ ___ __ __
rate/A
Mo del Fits
Rate AUDPCI
Mo del Fit s
Rate AUDPC S
__ __ __ __ __ __ ___ ___ __ ___ __ ___ __ __ __ __ __ ___ __ __ __ __ __ _____ __ __ __ __ ___ __ __ __ __ ____ __ __ _ __ __
2 00 1
Co n tro l
Go mp ertz
0. 21 2
75 9.3
Go mp ert z
0 .15 9 291 .9
Fo licu r 3 .6 E C 4.0 fl o z
In du ce (0.12 5%, v /v )
Go mp ert z
0.1 94
6 34 .1
Go mpert z
0.14 1
235 .9
AMS2 16 19 48 0SC 5.7 fl o z
Ind uce (0.12 5% ,v/ v)
Go mp ertz
0.1 38*
41 8.0*
Go mp ertz
0 .09 3* 12 5. 1*
BAS 50 5 5 0G 6.2 o z
Go mp ertz
0.143*
46 9.2*
Go mp ertz
0 .1 17* 159 .4*
Trig o Co r 14 48
Go mpert z
0 .23 1
79 8.4
Go mp ertz
0.1 69
31 5.7
OH1 82 .9
Gomp ert z
0.22 9
80 4.2
Go m p ertz
0.17 2 307 .5
__ __ __ __ __ __ ___ ___ __ ___ __ ___ __ __ __ __ __ ___ __ __ __ __ __ _____ __ __ __ __ ___ __ __ __ __ __ ___ __ __ __ ___ _
20 02
Co ntrol
Go mpert z
0 .11 4
91 1.8
Fo licu r 3 .6 E C 4.0 fl o z
In du ce (0.12 5%, v /v )
Go mpe rtz
0. 06 8*
6 55 .4*
Go mpert z
0.0 92*
AMS2 16 19 48 0SC 5.7 fl o z
Ind uce (0.12 5% ,v/ v)
Go mp ert z
0.054 *
6 05.2*
Go mp ert z
0 .06 6* 176 .6*
6 68 .6 *
Gomp ert z
0.0 87*
23 5.0*
Gompertz
0.12 4
33 0.0
BAS 50 5 5 0G 6.2 o z
Go mp ertz
0 .068 *
Trig o Co r 14 48
Go mpert z
0.1 29
81 9.2
Go mp ertz
0 .1 25
38 3.3
21 4.7*
OH1 82 .9
Gomp ert z
0 .10 2
91 2.7
Go mp ertz
0 .11 9 37 4.2
__ __ __ __ __ __ ___ ___ __ ___ __ ___ __ __ __ __ __ ___ __ __ __ __ __ _____ __ __ __ __ ___ __ __ __ __ __ ___ __ __ __ ___ _
* In d icat es means s ign ificant ly d ifferent (P 0.0 5) fro m untreated co ntro l based on Fis her’s LSD.
Chemical and Biological Control
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2002 National Fusarium Head Blight Forum Proceedings
Maximum disease (Ymax) of Fusarium head blight, yield, and DON content of grain for fungicides
and biocontrol agents tested in Ohio in 2001and 2002.
__________________________________________________________________________________________
Year
Treatment
Y max
Damage
Yield
DON Test
&
_________________________ Kernels
(bu/A) (ppm) Weight
Rate/A
Incidence
Severity
___________________________________(%)____________(%)________(%)__________________________
2001
Control
82.5
50.9
61.7
62.3
16.6
56.1
Folicur 3.6 EC 4.0 fl oz
Induce (0.125%, v/v)
75.8
41.5
33.3
66.6
AMS21619 480SC 5.7 fl oz
Induce (0.125%,v/v)
55.0*
BAS 505 50G 6.2 oz
TrigoCor 1448
23.9*
4.3
74.0*
7.2*
59.5
60.1*
28.6*
6.7
77.1*
8.4*
60.0
89.6
57.9
56.0
24.0*
54.7
51.7
12.0
57.7
OH182.9
87.5
51.8
56.7
62.0
13.4
56.7
__________________________________________________________________________________________
2002
Control
81.9
54.0
28.8
43.7
23.0
53.2
Folicur 3.6 EC 4.0 fl oz
Induce (0.125%, v/v)
60.7*
33.5*
23.3
50.3*
13.5
56.0
AMS21619 480SC 5.7 fl oz
Induce (0.125%,v/v)
55.1*
27.9*
16.8
52.9*
13.5
55.8
BAS 505 50G 3.1 oz
62.0*
30.6*
28.3
51.2*
15.5
53.1
TrigoCor 1448
82.5
49.7
48.9
26.0
49.5
51.3
OH182.9
81.3
52.3
33.8
43.7
24.0
53.0
__________________________________________________________________________________________
* Indicates means significantly different (P0.05) from untreated control based on Fisher’s LSD.
Chemical and Biological Control
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EFFECT OF THREE BACILLUS SP. FROM WHEAT
ON FHB REDUCTION
W.G.D. Fernando*, Y. Chen and P. Parks
Department of Plant Science, University of Manitoba, Winnipeg, MB R3T 2N2 Canada
*Corresponding author: PH: (204) 474-6072, E-Mail: d_fernando@umanitoba.ca
INTRODUCTION
Fusarium head blight (FHB) of wheat caused by Fusarium graminearum Schwabe
[Teleomorph = Gibberella zeae (Schwein.) Petch] is becoming one of the most devastating
crop diseases in Canada (Gilbert and Tekauz, 2000). Many reasons contribute to this and
the most important one is the rotations (Dill-Macky and Jones, 2000). In addition, no-till and
minimum till practices also contribute to persistence of the pathogen, and disease spread
(Fernando, 1999). In 2000, FHB damaged 8.5 percent of the total wheat crop in Manitoba
and caused yield losses of about $40 million in total (Tekauz, 2001). At present, available
and affordable traditional disease control options, such as resistant varieties, cultural practices (crop rotations, tillage to destroy residues) and foliar fungicides, are only partially
effective (McMullen et al., 1997). Biological control is an environment-friendly alternative
strategy in FHB management and shows considerable promise for reducing FHB (Khan et
al. 2001). In a bio-ecological view, the understanding of the interactions between FHB and
wheat phyllosphere microbes can be a requisite to finding effective antagonist(s) to the
pathogen.
The objectives of this study are (a) to screen microbes from various plant parts of wheat and
test their ability to inhibit the growth of the pathogen in vitro; (b) to investigate the interaction
between bacterial isolates and the FHB pathogen in plant assays in the greenhouse.
MATERIALS AND METHODS
Microbes originated from the rhizosphere, leaves, leaf sheaths and heads of field wheat.
The bacteria were isolated by serial dilution and single colonies were purified. Bacteria
were identified using the MicroLog system (BiologTM Inc., Hayward CA94545, USA). The
ability of isolates to inhibit radial mycelial growth of Fusarium graminearum was assayed on
PDA and NA plates and percent mycelial inhibition was calculated. Based on in vitro test
results, three Bacillus strains were selected for greenhouse work. In greenhouse (25°C, 14
hrs photoperiod/day), potential FHB antagonistic bacterial strains were individually applied
onto the seeds and heads of highly susceptible cultivar AC-Teal in order to investigate the
microbial interaction between antagonists and the pathogen in vivo. For seed-coating treatment, germinated seeds were immersed into bacterial suspension (4.5 × 108 cfu/ml) for 30
minutes before seeding. When wheat was at 50% flowering, 5 µl of each bacterial suspension was applied onto heads by injecting directly onto the floret. The pathogen macroconidia
(5 × 105/ml) was inoculated into the same spot either before or after bacterial inoculation.
Head inoculation was undertaken as follows: one floret in the middle spike of head was
injected with 2 µl of Fusarium macroconidia suspension (5 × 105 macroconidia/ml and
0.04% Tween 80). After inoculation, wheat plants were incubated in a mist chamber for 72
Chemical and Biological Control
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2002 National Fusarium Head Blight Forum Proceedings
hours at 22°C and transferred to a greenhouse bench. There were six treatments (10 pots/
replicate and 5 plants in each pot): (1) seed coating with bacteria and bacterial application
on head 4 hrs prior to Fusarium inoculation (BST-BBI); (2) seed coating with bacteria and
bacterial application on head 4 hrs post Fusarium inoculation (BST-BAI); (3) seed coating
with bacteria and no bacterial application on head (BST) prior to Fusarium application; (4)
bacterial application on head 4 hrs prior to fusarium inoculation on head and no seed coating of bacteria (BBI); (5) bacterial application on head 4 hrs post Fusarium inoculation and
no seed coating of bacteria (BAI) and (6) no seed coating of bacteria and no bacterial application on head prior to Fusarium application (CK). The FHB incidence (the number of heads
infected) and severity (the number of diseased spikes on each head) were estimated at 16
days after inoculation.
RESULTS AND DISCUSSION
Sixty-one bacterial and five fungal strains were isolated from various parts of the wheat
plant. Forty-nine percent were from rhizosphere, thirty-seven percent from leaves, nine
percent from leaf sheaths and five percent from heads. Only 7% of bacterial isolates inhibited the growth of F. graminearum. Only one phyllosphere fungus, strain L-07-12, inhibited
the growth of the pathogen up to 74%. The inhibitory ability (in vitro) of three bacterial isolates, Bacillus subtilis strain H-08-02 from the head, B. cereus strain L-07-01 from the leaf
and B. mycoides strain S-07-01 from the rhizosphere was 60%, 52% and 55%, respectively.
Microbial interactions in vivo (Table 1) showed that seed coating plus application of bacteria
on head prior to fusarium inoculation (treatment #1) gave the best disease reduction results
for all three bacteria, of which strain H-08-02 performed the best (49.1%). The treatments
with B. subtilis strain H-08-02 significantly reduced disease severity (treatments 1-5). This
means that it will be beneficial if we select the antagonists from wheat heads because the
pathogen and beneficial microorganisms may have co-evolved on heads or the bacterium is
capable of using the head as a niche. This is consistent with other studies on bacterial
population dynamics. In addition; data suggests bacterial application should be done prior to
fungal spore landing and subsequent infection for effective control of the FHB fungus on
heads.
Why do we think biocontrol will work? The wheat plant is most susceptible at anthesis. As
the window of infection that will lead to economic loss is quite narrow, an application of a
biocontrol agent onto heads at or just prior to anthesis should work well. Our results suggests, the antagonist should be applied on heads (infection court) to abort, curtail or delay
germination of spores (mainly ascospores), to achieve control. Though the window of
infection in the barley plant is supposedly a little longer, if optimum conditions and timing of
application are perfected, biocontrol should work. Therefore, our target is to develop a foliar
bio-fungicide that will be effective as a chemical fungicide application in reducing the FHB
incidence and severity on heads, and in turn reduce DON levels. A biological pesticide
capable of reducing initial infection and disease progress should reduce the present economic impact caused by FHB.
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2002 National Fusarium Head Blight Forum Proceedings
ACKNOWLEDGEMENTS
We acknowledge and thank National Science and Engineering Research Council of
Canada for funding this project.
REFERENCES
Dill-Macky, R. and Jones, R. K. 2000. The effect of previous crop residues and tillage on Fusarium head blight
of wheat. Plant Dis. 84: 71-76.
Fernando, W.G.D. 1999. Overview of the Fusarium situation in Canada; in Proceedings of the Canadian Workshop of Fusarium head blight, Nov. 28-30, 1999, Winnipeg, MB. pp. 12-15.
Gilbert, J. and Tekauz, A. 2000. Review: recent developments in research on Fusarium head blight of wheat in
Canada. Can. J. Plant Pathol. 22: 1-8.
Khan, N.J., Schisler, D.A., Boehm, M.J., Slininger, P.J. and Bothast, R.J. 2001. Selection and evaluation of
microorganisms for biocontrol of Fusarium head blight of wheat incited by Gibberella zeae. Plant Dis. 85: 12531258.
McMullen, M., Jones, R. and Gallenberg, D. 1997. Scab of wheat and barley: A re-emerging disease of devastating impact. Plant Dis. 81: 1340-1348.
Tekauz, A. 2001. Pioneering study aims for biological control of Fusarium head blight. Western Grains Research Foundation (WGRF) News Releases, Saskatoon, Sask. March 8, 2001.
Table 1. Effect of three Bacillus strains on FHB infection in vivo
B. mycoides
B. cereus
B. subtilis
(S-07-01)
(L-07-01)
(H-08-02)
severity(%) RB (%)*
c
severity(%)
48.1
a
33.3
b
RB (%)*
45.4
c
RB (%)*
49.1 a
1
49.3
2
90.1 ab
5.9
bc
40.6 ab
36.7 ab
58.9 bc
34.0 abc
3
92.8 a
3.0
c
50.2 ab
21.8 ab
72.0 b
19.3 c
4
63.0 bc
34.2 ab
35.5 ab
44.7 ab
55.6 c
37.7 ab
5
83.6 ab
12.6 bc
59.9 ab
6.7
ab
64.3 bc
27.9 bc
6
95.7 a
0.0
64.2 a
0.0
b
89.2 a
0.0
c
48.1
a
severity(%)
d
Note: * RB = relative control
1 — BST-BBI; 2 — BST-BAI; 3 — BST; 4 — BBI; 5 — BAI; 6 — CK.
The data with the same letter within a column are not significantly different
based on Fisher’s LSD test.
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2002 National Fusarium Head Blight Forum Proceedings
AN EXTENSION AGRONOMIST’S EXPERIENCES WITH FUNGICIDE
APPLICATION TECHNIQUES TO IMPROVE CONTROL OF FHB
T.D. Gregoire
NDSU Extension Service, Devils Lake, ND
Corresponding Author: PH: (701) 662-1364; E-mail: tgregoir@ndsuext.nodak.edu
ABSTRACT
Fusarium head blight has over the years caused billions of dollars in damage to small grain
crops in the U.S. and Canada. Small grain acreage in Northeast North Dakota has declined
38% since 1992 including a 70% decline in durum and barley acres. Genetic resistance is a
few years away and cultural control methods of rotation, residue management and choosing
tolerant varieties have not prevented disease occurrence. Fungicide use has been shown to
significantly reduce Fusarium infection when weather conditions are favorable to disease
development. Fungicides have shown effectiveness in lab and greenhouse and field situations but Fusarium control is often inconsistent and disappointing for growers. Much research has been done since 1993 to improve the effectiveness of fungicides. New fungicides have been labeled for heading application and application techniques have been
examined in detail. Application parameters studied have included the following variables for
ground application of fungicides.
A. Application timing including split application
B. Spray application angle
C. Spray pressure: 30 psi to 90 psi in 10 psi increments.
D. Spray nozzles: Various nozzles studied. Generally smaller orifice nozzles have
better coverage than nozzles providing coarse sprays.
E. Gallons of water per acre (gpa); 9 to 54 gpa.
F. Effects of dew
H. Adjuvants
Techniques learned from these studies and the labeling of more effective fungicides has led
to recommendations that have improved fungicide effectiveness for growers. Fungicide use
has increased as growers have experienced profitable results from fungicide application in
hard red spring wheat. Fungicide effectiveness has been marginal in durum and barley.
Achieving an economic reduction in Deoxynivalenol (DON) content with fungicides is a
continuing problem as reductions in DON are generally small.
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BARLEY CULTIVAR RESPONSE TO FUNGICIDE APPLICATION FOR
THE CONTROL OF FUSARIUM HEAD BLIGHT AND LEAF DISEASE
S. Halley
Langdon Research Extension Center-North Dakota State University, Langdon, North Dakota 58249
Corresponding Author: PH: (701) 256-2582; E-mail: shalley@ndsuext.nodak.edu
OBJECTIVES
To determine if efficacy of fungicides Headline and AMS21619 for the control of Fusarium
head blight (FHB) and leaf disease is different among barley cultivars.
INTRODUCTION
Barley producers in traditional barley producing areas have been frustrated by the
inconsistent performance of fungicides applied to barley. As a result barley acreage has
shifted to different regions of the state where diseases have not been as prevalent.
However, diseases are developing in these regions and disease levels increasing when
environmental conditions are appropriate.
Barley is particularly difficult to research because obtaining adequate disease levels for
effective fungicide evaluation has been inconsistent. The extended period of heading
among barley main stem and tillers, lack of yield loss due to FHB, and an inability to
distinguish losses between leaf diseases and FHB, and near zero tolerance for the
presence of the toxin deoxynivalenol (DON) by the malting industry complicate prioritization
of research goals.
Fungicides are often evaluated on a specifically selected cultivar. Often the first priority of
the cultivar selection is susceptibility to diseases. Little data is available to show that
fungicide performance on a particular cultivar will be similar on all cultivars.
MATERIALS AND METHODS
Five cultivars, Conlon, Drummond, Lacey, Legacy, and Robust were selected for evaluation
in a field at the Langdon Research Extension Center in spring 2002. Seven rows spaced 6inches apart were planted with a double-disk Hege drill in plots 16 ft. long in a RCB design
arranged as a factorial with four replicates. Border plots of Robust barley were planted
between treatment plots to minimize drift potential to adjacent plots. Nutrients were added to
attain a yield goal of 120 bu./acre and recommended production practices were followed.
Three weeks prior to heading a Fusarium spawn grown on spring wheat was hand
broadcast at a rate of approximately 200 grams/plot.
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2002 National Fusarium Head Blight Forum Proceedings
Fungicides and fungicide combination treatments included:
1. AMS 21619 5.7 oz/acre (triazole) and Induce 0.125 % v/v (adjuvant).
2. Quadris (azoxystrobin) 12.3 oz/acre + AMS21619 5.7 oz/acre and Induce 0.125 %
v/v.
3. AMS 21619 5.7 oz/acre and Induce 0.125 % v/v + AMS 21619 5.7 oz/acre and
Induce 0.125 % v/v.
4. Untreated check.
5. Caramba (metconazole) 13.5 oz./acre and Induce 0.125% v/v.
6. Caramba 13.5 oz./acre and Induce 0.125% v/v + AMS 21619 5.7 oz/acre and
Induce 0.125 % v/v.
7. Quadris 12.3 oz/acre.
Treatments were applied by CO2 backpack sprayer at 18 gpa with hydraulic nozzles
XR8002 oriented downward from horizontal at Zadoks growth stage 40 and XR8001
nozzles mounted on a double swivel angled 30 degrees downward and oriented forward
and backward to improve coverage of the target at Zadoks 59. Visual estimation of flag leaf
necrosis, three samples per plot, and FHB incidence and field severity, 20 samples per plot,
(spikelet count per individual head multiplied times FHB infected spikes per head) were
determined. Each plot was harvested with a Hege plot combine and the grain sample
cleaned and processed for yield, plump, and test weight measurement. A sample was
ground for DON analysis at NDSU. Data was analyzed with SAS GLM.
RESULTS AND DISCUSSION
Most of the disease present was Septoria speckled leaf blotch, Septoria passerinni Sacc.
and Stagonospora avenae F. sp. tritica T. Johnson, on the six-row cultivars, Drummond,
Lacey, Legacy, and Robust. Spot blotch, Cochliobolus sativus (Ito & Kirivayashi), was the
most common disease on two-row Conlon. Leaf disease levels on all cultivars were small
and probably did not contribute significantly to yield differences. Lacey had greater levels of
leaf disease than Conlon, Drummond, and Robust (Table 2). Caramba applied alone and
two applications of AMS21619 reduced flag leaf necrosis to levels smaller than the check.
Although there were differences among cultivars and fungicides in % plump, all levels were
excellent.
When yield was compared cultivars responded very differently to fungicide combinations
(Table 1 and Figure 1). Drummond had no significant differences among treatments.
However, Quadris was the only treatment significantly different than the untreated. Cultivars
Lacey and Legacy had a variable response to fungicide. The AMS21619 combination
significantly improved yield over the untreated while other fungicide treatments did not. Yield
of Robust was improved above the untreated by Quadris and the Quadris-AMS21619
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2002 National Fusarium Head Blight Forum Proceedings
combination treatments. Conlon had the greatest yields and responded well to fungicide
treatments. Caramba alone and all other fungicide combinations increased yield over the
untreated in Conlon.
Cultivars had significantly different levels of FHB incidence and severity in untreated plots
but there were no differences among fungicide treatments (Table 2). Lacey had both the
greatest levels of FHB incidence and severity among cultivars. Drummond had the smallest
FHB field severity levels.
Conlon had significantly smaller DON levels than other cultivars (Table 2). Legacy had
smaller DON levels than Drummond, Lacey, and Robust. Drummond had the greatest DON
levels at 27.1 ppm. Caramba applied at Zadoks 59 had DON level of 23.8 ppm, significantly
higher than fungicide combinations that included the AMS21619 fungicide applied at
Zadoks 59. Fungicides applied at Zadoks 40 with AMS21619 at Zadoks 59 tended to
reduce DON levels compared to similar fungicides applied alone. Reduction in DON due to
fungicide application was small (less than 20%) and the reductions would not produce
acceptable malting quality.
SUMMARY
In this trial, cultivars without fungicide treatment had significant differences in leaf disease
and FHB resulting in differing yields, test weights, and % plump. DON levels were different
among cultivars without treatment. Fungicide treatments performed similarly among
varieties for all measured factors except yield. More years of research will be needed to
confirm yield and fungicide response trends among varieties.
C u lt iva r Y i el d R esp o n se to F u n gi c id es
110.0
Yie ld ( bu /ac)
105.0
100.0
Conl on
Dru m mo nd
95.0
La c ey
Le g a cy
Ro bu s t
90.0
85.0
80.0
Un treated AM S 59
Q uadris AM S 40 + Caramba C aramba
40+ AM S
59
AM S 59
59
Q uadris
40 + AM S
59
59
Fu n gi c i d e A p p l ica ti o n a n d T i mi n g (Z o d a k s
G row t h S ta g e)
Figure 1. Cultivar yield by fungicide treatment (2002).
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2002 National Fusarium Head Blight Forum Proceedings
T a b le 1. D is e a s e a n d q u a lity p a ra m e te r re s p o n s e s to fu n g ic id e tre a tm e n ts b y c u ltiva r (2 0 0 2 ).
C ultiva r
C onlo n
D ru m m on d
Lace y
L ega cy
R obu st
C ult*T rt
CV %
F ung icid e
1
2
3
4
5
6
7
1
2
3
4
5
6
7
1
2
3
4
5
6
7
1
2
3
4
5
6
7
1
2
3
4
5
6
7
F la g
Le af
N ecro sis
FH B
Incide nce
%
1.8
1.5
2.3
1 0.0
4.3
1.3
4.3
1.5
9.0
1.3
2.3
1.8
1.8
1.5
3.0
1 4.3
3.0
1 1.3
5.8
7.5
7.0
5.0
3.8
2.5
9.1
1.3
3.0
1 2.3
1 0.5
5.5
2.0
3.0
1.5
2.8
2.8
NS
12 0
%
51
55
50
58
48
56
51
54
55
61
63
54
50
41
91
90
75
74
93
83
89
48
71
53
71
69
66
81
59
65
70
60
56
65
70
NS
21
F ie ld
S everity
%
7.5
6.4
7.5
8.2
6.6
5.9
8.1
4.5
4.8
5.6
7.6
4.3
3.2
3.1
1 8.0
2 1.2
1 3.8
2 0.8
2 2.8
1 4.8
2 1.5
4.7
9.1
5.2
1 4.5
8.6
9.8
1 6.6
6.3
6.9
9.2
6.1
1 1.0
1 0.8
1 0.0
NS
47
Y ie ld
T est
W t.
P lum p
DON*
bu/acre
97 .1
97 .7
98 .3
90 .7
101 .7
108 .2
95 .7
86 .8
87 .4
89 .9
83 .2
83 .9
89 .4
89 .9
95 .2
91 .4
99 .0
91 .1
89 .5
96 .4
88 .5
93 .1
94 .7
100 .7
94 .9
91 .7
92 .7
89 .4
90 .5
98 .6
90 .2
87 .4
90 .8
88 .2
94 .9
6 .6 **
6
lb/b u
5 0.5
5 0.4
5 0.4
5 0.0
4 9.5
5 0.1
4 9.9
4 4.0
4 5.0
4 5.0
4 4.2
4 4.3
4 4.4
4 5.0
4 6.4
4 6.2
4 7.0
4 6.6
4 5.8
4 6.7
4 6.9
4 4.7
4 4.7
4 4.3
4 4.6
4 4.4
4 4.8
4 4.5
4 5.9
4 7.0
4 7.0
4 6.5
4 6.8
4 6.7
4 6.0
NS
2
%
9 6.2
9 5.0
9 5.2
9 4.4
9 5.5
9 5.2
9 4.4
8 2.7
8 3.0
8 5.7
7 6.9
8 3.6
8 0.2
8 1.1
8 8.6
8 8.0
9 1.2
8 3.4
8 6.2
8 5.4
8 7.5
8 7.8
8 9.6
9 0.5
8 9.6
8 6.9
8 6.2
8 4.1
8 8.5
8 6.4
8 9.0
8 5.8
8 5.4
8 7.7
8 6.3
NS
4
Ppm
8.7
8.0
9.2
1 0.2
9.6
9.8
8.6
2 7.3
2 9.3
2 2.8
3 0.8
3 2.1
2 3.8
2 3.7
2 3.5
2 0.3
1 6.9
2 5.2
3 5.1
1 9.0
2 2.2
1 7.4
1 2.7
1 3.5
1 9.6
2 1.8
1 1.9
2 4.1
2 0.6
1 5.4
2 5.2
1 8.1
2 0.5
2 1.8
2 5.3
NS
32
T re a tm e n t 1 A M S 2 1 6 1 9 a p p lie d a t Z a d o k s 5 9 g ro w th s ta g e
T re a tm e n t 2 Q u a d ris a p p lie d a t Z a d o k s 4 0 + A M S 2 1 6 1 9 a t Z a d o k s 5 9 g ro w th s ta g e
T re a tm e n t 3 A M S 2 1 6 1 9 a p p lie d a t Z a d o k s 4 0 + Z a d o k s 5 9 g ro w th s ta g e
T re a tm e n t 4 U n tre a te d
T re a tm e n t 5 C a ra m b a a p p lie d a t Z a d o k s 5 9 g ro w th s ta g e
T re a tm e n t 6 C a ra m b a a p p lie d a t Z a d o k s 4 0 + A M S 2 1 6 1 9 a t Z a d o k s 5 9 g ro w th s ta g e
T re a tm e n t 7 Q u a d ris a p p lie d a t Z a d o k s 5 9 g ro w th s ta g e
* T a c k e , B .K . a n d C a s p e r, H .H . D e te rm in a tio n o f D e o x y n iva le n o l in W h e a t, B a rle y ,
a n d M a lt b y C o lu m n C le a n u p a n d G a s C h ro m a to g ra p h y w ith E le c tro n C a p tu re
D e te c tio n : Jo u rn a l o f A O A C In te rn a tio n a l V o l. 7 9 , N o . 2 . 1 9 9 6 (p .4 7 2 -8 3 0 9 )
** S ig n ific a n t a t 0 .0 5 p ro b a b ility le ve l fo r m e a n c o m p a ris o n s .
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2002 National Fusarium Head Blight Forum Proceedings
T ab le 2 . D isease an d q uality p aram ete r resp on ses b y cultiv ar and fu n gicide treatm en t acro ss
cultiv ars (2 00 2 ).
C u ltiva r
F u n g icid e
C o n lo n
D ru m m o n d
L a ce y
L egacy
R o b u st
F la g
Le af
N e c ro sis
FH B
In c id e n c e
%
3 .6
2 .7
7 .4
5 .3
4 .0
%
5 2 .7
5 3 .9
8 4 .8
6 5 .6
6 3 .6
F ield
S e ve rity
%
7 .2
4 .7
1 9 .0
9 .8
8 .6
Y ie ld
T est
W t.
P lu m p
DO N*
b u ./a c re
9 8 .5
8 7 .2
9 3 .0
9 3 .9
9 1 .5
lb./b u .
5 0 .1
4 4 .5
4 6 .5
4 4 .6
4 6 .6
%
8 1 .9
9 5 .1
8 7 .2
8 7 .8
8 7 .0
Ppm
9 .2
2 7 .1
2 3 .2
1 7 .3
2 1 .0
1
2
3
4
5
6
7
4 .4
6 0 .5
8 .2
9 2 .5
4 6 .3
8 8 .8
1 9 .5
6 .8
6 7 .3
9 .7
9 4 .0
4 6 .7
8 8 .4
1 7 .1
2 .2
6 1 .8
8 .3
9 5 .6
4 6 .8
9 0 .3
1 7 .5
7 .1
6 5 .0
1 1 .5
8 9 .5
4 6 .4
8 6 .0
2 0 .8
2 .9
6 3 .8
1 0 .7
9 1 .5
4 6 .2
8 7 .5
2 3 .8
3 .3
6 4 .0
8 .9
9 5 .0
4 6 .5
8 6 .9
1 7 .3
5 .6
6 6 .5
1 1 .8
9 1 .7
4 6 .4
8 6 .6
2 0 .8
C u ltL S D **
3 .2
6 .6
2 .2
2 .8
0 .4
1 .6
3 .8
T rtL S D **
4 .0
NS
NS
4 .0
NS
2 .2
4 .5 ***
CV %
120
21
47
6
2
4
32
T reatm ent 1 A M S2 1 6 19 ap p lied at Z ad o ks 5 9 grow th stag e
T reatm ent 2 Q u adris ap plied at Z ad ok s 40 + A M S 2 1 6 19 at Z ad o ks 5 9 grow th stag e
T rea tm ent 3 A M S 2 1 61 9 ap p lied at Z ad o ks 40 + Z ad o ks 5 9 grow th stag e
T reatm ent 4 U n treated
T reatm en t 5 C aram b a ap p lied at Z ad o ks 5 9 grow th stag e
T reatm ent 6 C aram b a ap plied at Z ad ok s 40 + A M S 2 16 19 at Z ad o ks 59 grow th stag e
T reatm ent 7 Q u ad ris ap p lied at Z ad o ks 5 9 grow th stag e
* T ack e, B .K . an d C asp er, H .H . D eterm ination o f D eo x yn iv aleno l in W h eat, B arley, an d M alt by C o lu m n C leanu p
and G as C h rom ato grap hy w ith E lectron C aptu re D etectio n: Jo urn al of A O A C In tern atio nal V ol. 7 9, N o. 2 . 19 9 6
(p.4 72 -83 0 9 )
** S ig nificant at 0.0 1 p ro b ab ility level for m ean co m p ariso ns.
** * S ign ifican t at 0.0 5 p ro b ab ility level for m ean co m p ariso ns.
Chemical and Biological Control
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2002 National Fusarium Head Blight Forum Proceedings
ANALYSIS OF THE 2002 UNIFORM WHEAT FUNGICIDE AND
BIOCONTROL TRIALS ACROSS LOCATIONS
D.E. Hershman1* and E.A. Milus2
Dept. of Plant Pathology, University of Kentucky, Princeton, KY 42445; and
2
Dept. of Plant Pathology, University of Arkansas, Fayetteville, AR 72701
*Corresponding Author: PH (270) 365-7541 x 215; E-mail: dhershma@uky.edu
1
OBJECTIVES
Evaluate a common set of foliar fungicide and biological control agent (BCA) treatments,
across a wide range of environments, for effectiveness in managing Fusarium head blight
(FHB) and associated yield and seed quality parameters.
INTRODUCTION
Identifying fungicides and BCA’s that significantly reduce the incidence and severity of FHB
in the field, and mycotoxins in the grain, would have widespread benefits to growers and
end-users of all market classes of wheat. The Uniform FHB Fungicide and BCA Test was
established as a means of rapidly identifying fungicide and/or BCA treatments that are
effective, economical and environmentally safe to use in FHB management programs
across the United States.
MATERIALS AND METHODS
Plant pathologists from 12 states (Table 1) conducted 22 trials across a range of wheat
classes, including durum, hard red spring, soft red winter, and soft white winter wheat. Each
trial evaluated eight uniform treatments (Table 2), including two advanced BCA’s, OH 182.9
Yeast [USDA/ARS] and TrigoCor 1448 bacterium [Cornell University]; three foliar fungicides
(AMS 21619A [Bayer], BAS 505H [BASF], and Folicur [Bayer]); and a non-treated control.
All treatments were applied at early flowering stage using a CO2 pressurized sprayer
equipped with twinjet XR8001 nozzles, mounted at a 60-degree angle forward and backward. Details such as plot size, crop husbandry, spray volume and pressure, sprayer type,
and number of treatment replications varied from location to location. Consult individual
state trial reports for specific details.
Data from individual trials were grouped and statistically analyzed with other winter wheat
or spring wheat trials, respectively. This was done in order to detect any treatment differences that may be linked to production of winter vs. spring wheat, respectively. Treatment
means from each location served as treatment replications. Data summary tables include
treatment means, in actual units measured, as well as means of treatment rankings from
within individual tests. Treatment rankings are provided as an alternative approach to treatment comparison.
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RESULTS AND DISCUSSION
Winter wheat trials– Of 13 winter wheat trials conducted, data are presented for 12 trials
(Tables 3-5); circumstances precluded the collection of disease data at one location in VA.
Table 3 summarizes all FHB data, including a ranked treatment means. Table 4 presents all
yield and seed quality parameters. Table 5 summarizes analyzed disease, yield and seed
quality means from trials that had moderate to severe FHB. All three tables indicate that
treatments involving AMS 21619A were generally superior to other treatments when compared with the check. Most fungicide treatments reduced disease levels compared to the
check plots, but only the treatments involving AMS 21619A translated into statistically
significant yield results in the absence of foliar disease (Tables 4 and 5). Treatments involving Folicur or BAS 505H, although not always as effective as treatments involving AMS
21619A, were often superior to the BCA treatments. Neither of the BCA’s tested, when
applied alone, were statistically different than the check for any parameter except for a more
favorable plot severity ranking (Table 3). Test weights were statistically similar among all
treatments (Table 4). Percent VSK was significantly lower than the check only when AMS
21619A was applied (Table 4, 5). Similarly, DON levels tended to be lowest in treatments
involving AMS 21619A, but differences among treatments were not always significantly
(Tables 4, 5).
Spring wheat trials – Of nine spring wheat trials conducted, four had extremely low levels
of FHB and/or no FHB ratings were collected. These four tests were excluded from this
summary. The results of the remaining five trials (all from North Dakota, and with moderate
to severe FHB levels) are summarized in Tables 6 and 7. When actual data are considered
(Table 6), all solo fungicide treatments provided similar levels of FHB control. In contrast, no
treatment resulted in significantly higher yields compared to the check. Similar results were
seen with treatment mean rankings (Table 7) except that crop yields associated with the
above fungicide treatments ranked significantly higher than the check plots. This may be an
artifact of foliar disease management in those tests, rather than any specific activity against
FHB. Test weights tended to be significantly improved when fungicides were applied. There
was insufficient VSK or DON data collected to make any general comments in regard to
treatment effectiveness. Consult individual state trial reports for further details on VSK and
DON data that was collected.
Summary - In winter wheat trials, treatments involving AMS 21619A were generally superior to the other treatments tested. Neither BCA tested provided control of FHB when compared with the check. Treatments involving Folicur or BAS 505H tended to provide an intermediate level of FHB control. In spring wheat trials, all fungicide treatments performed more
or less similarly. Better results in spring wheat trials for Folicur and BAS 505H may be
related to differences in demands placed on treatments between winter and spring wheat.
BCA’s tested in spring wheat trials were ineffective in managing FHB. Overall, seed quality
parameters associated with FHB from both winter and spring trials were less impacted by
foliar fungicides than were FHB symptoms expression. DON levels were reduced by fungicide treatments in winter wheat trials, but levels were often unacceptably high where moderate to severe FHB existed.
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2002 National Fusarium Head Blight Forum Proceedings
T ab le 1. S tates, p rin cip al in v estigato r (P I), in stitu tio n , n u m b er o f u n ifo rm trials co n d u cted ,
an d w h eat class ev alu ated .
S tate
PI
In stitu tio n
N o . trials
AR
G en e M ilu s
U n iv ersity o f A rk an sas
IL
W ayn e P ed erso n
U n iv ersity o f Illin o is
IN
G reg S h an er
P u rd u e U n iv ersity
KY
D o n H ersh m an
U n iv ersity o f K en tu ck y
M D
A rv G ryb au sk as
U n iv ersity o f M arylan d
M I
P at H art
M ich ig an S tate U n iv ersity
M O
L au ra S w eets
U n iv ersity o f M isso u ri
ND
M arcia M cM u llen
N o rth D ak o ta S tate U n iv ersity
NY
G ary B erg stro m
C o rn ell U n iv ersity
OH
P at L ip p s
O h io S tate U n iv ersity
SD
M arty D rap er
S o u th D ak o ta S tate U n iv ersity
VA
E rik S tro m b erg
V P I an d S U
* S R W W = S o ft red w in ter w h eat
S W W W = S o ft w h ite w in ter w h eat
H R S W = H ard red sp rin g w h eat
1
1
2
1
1
1
2
6
1
1
3
2
W h eat C lass
SR W W *
SR W W
SR W W
SR W W
SR W W
SR W W
SR W W
H R SW
SW W W
SR W W
D u ru m , H R S W
SR R W
Table 2. Treatment, rate, and adjuvant used in the uniform trials in 2002.
#
1
2
3
4
5
6
7
8
Treatment
OH 182……………………
Folicur 3.6F……………….
AMS 21619A 480SC……..
AMS 21619A 480 SC ……
+ Folicur 3.6F
BAS 505F 50WG …………
TrigoCor 1448 …………….
TrigoCor 1448…………….
+ Folicur 3.6F
Non-treated check
Rate of Product/A
varied among locations
4 fl oz
5.7 fl oz
3.6 fl oz + 4 fl oz
6.4 fl oz
varied among locations
varied + 4 fl oz
Chemical and Biological Control
84
Adjuvant
0.125% Induce
0.125% Induce
0.125% Induce
0.125% Induce
0.125% Induce
2002 National Fusarium Head Blight Forum Proceedings
T able 3 . Treatment and rank means for FHB incidence, head severity, and plot severity
from winter wheat trialsa.
Incidence
Head severity
Plot severity
Treatment
(%)
Rank
(%)
Rank
(%)
Rank
OH 182.9……………... 26.4abb 5.5ab
25.5ab 4.6ab
13.2ab 4.2b
Folicur………………….. 22.5ab 3.5cd
24.1ab 3.7bc
9.2bc 3.6b
AMS 21619A…………….18.4b
1.8e
19.8b
3.1bc
6.8c
1.9c
AMS 21619A + Folicur… 18.5b
2.5de
18.8b
2.1c
7.6c
2.1c
BAS 505………………… 21.5ab 2.6de
24.1ab 4.0bc
9.6bc 3.1bc
TrigoCor 1448…………... 28.9ab 4.8a-c
25.4ab 4.6ab
13.9ab 4.4b
TrigoCor 1448 +Folicur… 25.5ab 4.2b-c
22.4ab 4.3a-c
10.2bc 4.1b
Non-treated……………… 30.4a
6.2a
29.7a
6.4a
16.4a
6.1a
a
AR, IL, IN (2 trials), KY, MD, MI, MO (2 trials), NY, OH, VA.
b
Means within a column followed by a common letter are not significantly different
P=0.05, Student-Newman-Keuls; arcsine-transformed percentage data were used in
statistical analyses.
Table 4. Treatment and rank means for yield, test weight, visually scabby kernels (VSK), and
DON from winter wheat trials.
Treatment
Yielda
(bu/A) Rank
Test Weightb
(lbs/bu) Rank
VSKc
(%) Rank
DONd
(ppm) Rank
OH 182.9………………. 59.2nse 5.9a
54.8ns 5.8ns
35.0a 5.2a
11.0ns 5.7a
Folicur………………….. 62.2 4.2ab 55.6 3.7
30.3ab 4.2ab
8.3 2.7cd
AMS 21619A…………... 75.4 2.0c
56.2 2.4
24.0b 2.2c
4.5 1.3d
AMS 21619A + Folicur... 63.1 3.4bc 56.2 3.7
24.3b 2.8c
5.0 2.3d
BAS 505………………... 60.9 4.9ab
55.6 3.8
28.6ab 3.6a-c 7.6 2.8cd
TrigoCor 1448………….. 61.9 4.4ab
54.8 4.2
34.1a 4.6ab 11.3 6.0a
TrigoCor 1448 + Folicur.. 62.5 3.8a-c 55.5 3.7
32.3a 5.4a
9.2 4.0bc
Non-treated…………….. 58.2 5.9a
54.8 4.9
32.8a 4.8ab 10.2 4.3b
a
data from AR, IL, IN, KY, MD, MO(2 trials), NY, OH, VA.
b
data from AR, IN (2 trials), KY, MD, MO (2 trials), NY, OH, VA.
c
data from AR, KY, MO(2 trials), NY, OH, VA.
d
data from IN (2 trials), KY, MD, MO (2 trials), NY, OH, VA.
e
Means within a column followed by a common letter are not significantly different P=0.05,
Student-Newman-Keuls; ns = not significant; VSK percentages were arcsine-transformed for
statistical analysis.
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2002 National Fusarium Head Blight Forum Proceedings
Table 5. Treatment means for FHB incidence, head severity, plot severity, yield, visually
scabby kernels (VSK), and DON from winter wheat trials in AR, IL, KY, MI, NY, and
OH that had moderate to severe levels of FHB and little interference from other diseases.
Treatment
Head
Incidence severity
(%)
(%)
Plot
severity
(%)
Yielda
(bu/A)
VSK
(%)
DONb
(ppm)
OH 182.7………………. 50.4ac
43.0ab
28.3ab
54.3c
38.3a 19.8a
Folicur……………….…. 42.4ab
37.0b
27.5ab
61.0a-c 31.8ab 14.9ab
AMS 21619A………..…. 33.6bc
32.7b
14.0c
66.0a
24.5b
7.8b
AMS 21619A + Folicur…28.6c
31.8b
15.8bc
62.7ab
24.3b
8.5b
BAS 505……………...… 40.0ab
39.0b
20.3a-c 60.0a-c 30.5ab 13.7ab
TrigoCor 1448………….. 49.6a
44.2ab
29.3ab
56.0bc
37.8a 20.4a
TrigoCor 1448 + Folicur.. 41.2ab
37.6b
21.0a-c
61.7a-c 34.0ab 16.7ab
Non-Treated……….….... 51.2a
50.8a
33.3a
54.3c
37.6a 19.7a
a
No yield data for MI.
b
No DON data from AR, IL, and MI.
c
Means within a column followed by a common letter are not significantly different
P=0.05, Student-Newman-Keuls; arcsine-transformed percentage data were used in
statistical analyses.
Table 6. Treatment means for FHB incidence, head severity, plot severity, yield and test
weight from five of nine spring wheat trials that had moderate to severe levels of FHB and
foliar diseases.
Treatment
Head
Incidence severity
(%)
(%)
Plot
severity
(%)
Yield
(bu/A)
Test weight
(lbs/bu)
OH 182.7……………..… 71.0aba
34.5ns
26.5a
40.8ns
56.8bc
Folicur……………….… 66.2a-c
22.8
16.8b
47.0
58.0ab
AMS 21619A………...… 57.2bc
22.8
16.3b
38.0
58.3ab
AMS 21619A + Folicur... 57.6bc
23.0
17.0b
50.3
58.3ab
BAS 505……………...… 51.6c
22.5
15.0b
51.3
59.0a
TrigoCor 1448…….……. 76.8a
32.8
25.8a
41.5
57.0bc
Tr igoCor 1448 + Folicur...71.0ab
32.0
23.5ab
47.8
57.8a-c
Non-treated…………….. 80.8a
34.8
29.3a
39.8
56.3c
a
Means within a column followed by a common letter are not significantly different P=0.05,
Student-Newman-Keuls; arcsine-transf ormed percentage data were used in statistical
analyses; arcsine-transformed percentage data were used in statistical analyses; ns = not
significant.
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2002 National Fusarium Head Blight Forum Proceedings
T able 7 . Average rankings for FHB inci dence, head severity, plot severity, yield, and test
weight from five of nine spring wheat trials that had moderate to severe levels of FHB
and foliar diseases.
Head
Plot
Treatment
Incidence severity
severity
Yield
Test weight
OH 182.7…………....….. 4.2ab a
4.7ns
4.6ab
4.5a
2.8ab
Folicur……………....…... 2.8b
2.4
2.2c
3.5ab
2.0a-c
AMS 21619A…………... 2.4b
3.3
2.8bc
1.5b
1.8bc
AMS 21619A + Folicur... 2.6b
2.3
3.4bc
2.0b
1.5bc
BAS 505…………….….. 2.2b
3.4
2.2c
1.8b
1.3c
TrigoCor 1448………...… 4.8ab
3.9
4.4ab
5.0a
2.8ab
TrigoCor 1448 + Folicur.. 3.8ab
3.4
3.6bc
2.5b
2.3a-c
Non-treated……………... 5.8a
5.0
5.8a
5.0a
3.3a
a
Means within a column followed by a common letter are not significantly different
P=0.05, Student-Newman-Keuls; ns = not significant.
Chemical and Biological Control
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2002 National Fusarium Head Blight Forum Proceedings
MANAGEMENT OF FUSARIUM HEAD BLIGHT IN WHEAT USING
SELECTED BIOLOGICAL CONTROL AGENTS AND
FOLIAR FUNGICIDES, 2002
D.E. Hershman1*, P.R. Bachi1, D.M. TeKrony2 and D.A. VanSanford2
Dept. of Plant Pathology, University of Kentucky, Princeton, KY 42445; and
2
Dept. of Agronomy, University of Kentucky, Lexington, KY 40546
*Corresponding Author: PH: (270) 365-7541 x 215; E-mail: dhershma@uky.edu
1
OBJECTIVES
Evaluate selected foliar fungicides and biological control agents (BCA) for potential use in
soft red winter wheat Fusarium head blight (FHB) management programs in Kentucky. Also,
to generate data as a cooperator in the 2002 National Fusarium Head Blight Uniform Fungicide and Biocontrol Trial.
INTRODUCTION
FHB is a significant disease concern in all wheat and barley producing regions of the
United States. FHB epidemics are rare in Kentucky, but each year some fields are severely
damaged by the disease. Currently, the only options available for the management of FHB
are the use of cultural practices that encourage escape from disease. These include the use
of multiple planting dates and varieties representing different flowering dates and periods.
Moderate resistance is also available in several different wheat varieties, but severe FHB
will occur under conditions that favor FHB. Preliminary studies conducted in various states
indicate that foliar fungicides (Milus, Hershman, and McMullen, 2001) and BCA’s may be
capable of providing safe, effective and economical management of FHB. Nonetheless,
specific and consistent data are lacking in regards to which products and rates are most
suitable for use in FHB management programs. The National FHB Uniform Fungicide and
Biocontrol Test was established as a means of addressing this deficiency in data. This test
involves cooperators at various locations across the county, the use of a standard set of
promising treatments, and a reasonably standardized testing protocol. Each state, including
the one in Kentucky during 2002, also evaluates unique treatments of local interest.
MATERIALS AND METHODS
The test site was established at the University of Kentucky Research and Education Center
in Princeton, KY. The core set of treatments evaluated was determined by collective agreement of the scientists involved in the National FHB Uniform Fungicide and Biocontrol Test.
Treatments included a variety of foliar fungicides and two BCA’s. An additional fungicide
treatment of local interest was also included at the Kentucky trial location. The test site was
planted in a conventionally-tilled seed bed on October 22, 2001. Plots were maintained
according to standard crop husbandry practices for soft red winter wheat production in west
Kentucky (Bitzer and Herbek, 1997). The wheat variety planted was ‘Patton’. This variety
expresses FHB “Type 2” resistance, which is resistance to spread of FHB within a spike.
Maize was the previous crop grown in the test site.
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Plots were inoculated on April 1, 2002 with sterilized, cracked corn infested with a mixture of
several highly pathogenic isolates of Fusarium graminearum, the primary causal agent of
FHB. Test plots were mist-irrigated according to a strict regime in order to encourage the
causal fungus to produce infectious spores and infect the test plots. Between inoculation
and the onset of flowering, plots were mist-irrigated for two hours daily, between 7 pm and 9
pm. Following the onset of flowering, plots were mist-irrigated eight times each day for 15
minutes each misting cycle. Fungicides were applied to plots on April 30, 2002 when the
crop was in the early flowering. Treatments were applied using a CO2-propelled hand-held
sprayer delivering at 40 PSI in 18-20 GPA. The spray boom was equipped with twinjet
XR8001 nozzles oriented at a 60-degree angle forward and backward. FHB incidence,
severity, and field severity data were obtained by collecting, and visually rating, 100 heads
from each test plot. Plots were harvested with a small plot combine and grain yield and test
weight where calculated. Deoxynivalenol (DON) levels were determined for 50-gram grain
subsamples collected from each test plot. DON analyses were conducted at the Michigan
State University Don Testing Laboratory. Tests to ascertain percent seed infected by
Fusarium spp., as determined by plating seed, were conducted at Dr. TeKrony’s Seed Technology Laboratory in Lexington, KY. Percent visually scabby kernel (VSK) percentages were
determined by segregating healthy from scabby kernels for two sets of 100-seed samples
for each treatment replication.
RESULTS AND DISCUSSION
Test conditions were favorable for FHB. Plot yields and test weights were significantly
reduced by excess soil moisture. The two treatments involving AMS 21619A and TrigoCor
1448 when applied alone, significantly reduced FHB incidence compared the check. The
same treatments, plus TrigoCor 1448 + Folicur also significantly reduced FHB plot severity.
No treatment significantly reduced FHB head severity compared with the check. Only
TrigoCor 1448 applied alone, resulted in significantly higher yield compared with the check.
No treatment significantly impacted crop test weight, % visually scabby kernels (VSK),
Fusarium spp. colonization of grain or DON levels. There were no foliar diseases of consequence in this trial.
REFERENCES
Bitzer, M. and Herbek, J. 1997. A comprehensive guide to wheat management in Kentucky. University of
Kentucky Extension Service Publication ID-125, University of Kentucky Press.
Milus, E., Hershman, D., and McMullen, M. 2001. Analysis of the 2001 uniform wheat fungicide trials across
locations. Pages 75-79 in Proceedings of the 2001 Fusarium Head Blight Forum, Erlanger, KY, University Press,
Michigan State University.
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Table 1. Effect of foliar fungicides and biological control agents on FHB, yield, seed quality in
Kentucky, 2002.
FHB Ratings@
Plot
Head
Plot
Yield
Test wt VSK* FC** DON+
Treatment and rate
Inc (%) Sev (%) Sev (%) (bu/A) (lbs/bu) (%) (%) (ppm)
OH 182.7 variable……… 29.8 ab# 10.3ns 3.0ab
41.5ab 50.6ns 28.6ns 66.0ns 1.8b
Folicur 4.0 fl oz
Induce 0.125% v/v……… 27.0ab 10.8
3.0ab
45.3ab 50.2
26.3
76.7
1.8b
AMS 21619A 5.7 fl oz +
Induce 0.125% v/v……… 17.5b
8.5
1.5b
51.2ab
49.9
18.8
63.3
1.9b
AMS 21619A 3.6 fl oz +
Folicur 4 fl oz +
Induce 0.125% v/v……… 19.0b
7.8
1.5b
45.9ab
52.2
16.4
60.0
2.1ab
BAS 505H 6.4 fl oz +
Induce 0.125% v/v……… 26.5ab 11.8
2.8ab
44.5ab
50.7
24.0
72.7
2.0ab
TrigoCor 1448 variable…. 19.8b
9.0
1.5b
55.2a
51.1
19.0
84.0
2.2ab
TrigoCor 1448 variable +
Folicur 4 fl oz +
Induce 0.125% v/v……… 22.8ab
CGA 64250 13.7 fl oz +
Induce 0.125% v/v……… 33.3a
8.8
1.5b
47.5ab
51.5
20.8
76.7
2.0ab
11.0
3.5a
40.5b
50.3
16.9
82.7
3.2a
Non-Treated…………… ..32.0a 11.5
3.8a
42.8b
51.1
25.9 76.6 2.1ab
@: Inc = FHB incidence in plots; Sev = Average severity of FHB for diseased spikes; Plot sev =
Average FHB severity across plot.
* = Visually “scabby” kernels.
** = Seed colonized by Fusarium spp.
+ = Vomitoxin
#Means followed by a common letter are not significantly different P=0.05, Student- NewmanKeuls; ns=no significant differences.
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MULTIPLE INFECTION EVENTS AND SPLIT TIMING OF FOLICUR
FUNGICIDE APPLICATIONS FOR CONTROL OF FHB IN HARD RED
SPRING WHEAT, DURUM WHEAT, AND SPRING BARLEY, 2002
J. Jordahl, S. Meyer, and M. McMullen*
Dept. of Plant Pathology, North Dakota State University, Fargo, ND 58105
*Corresponding Author: PH: (701) 231-7627; E-mail: mmcmulle@ndsuext.nodak.edu
ABSTRACT
Studies at North Dakota State University and at other research locations have indicated that
wheat is most vulnerable to infection by the Fusarium head blight fungus (Fusarium
graminearum) during anthesis, while spring barley cultivars are most susceptible to infection
after the grain head fully emerges from the leaf sheath. However, if environmental conditions are very suitable for disease development over a long time span, multiple infections
may occur up to soft or mid-dough stage, and not just at the most susceptible stage of the
crop. Despite the possibility of multiple infection events, fungicide applications to durum
and barley to control this disease have generally been applied once and targeted to the
single-most critical infection periods. Cost of spray applications and time restraints of producers often prohibit multiple applications. Information was needed on the effect of multiple
infection events on the level of FHB and on the effect of multiple applications of split rate
fungicides in controlling multiple infection events.
A study was established in a controlled greenhouse environment in which spring wheat,
durum wheat, and spring barley were exposed to multiple infection events and treated with
either a single full rate (4 fl oz) or multiple, reduced rate applications of Folicur
(tebuconazole) fungicide. Inoculations and/or fungicide applications were applied at single
or multiple growth stages: Feekes growth stage 10.3 (head half emerged); Feekes 10.5
(head fully emerged but not flowering); Feekes 10.51 (early flowering in wheat); Feekes
10.54 (kernel watery ripe). Ten ml of a dilution of Fusarium graminearum spores (10,000
spores/ml) were atomized onto grain heads at the appropriate growth stage. For fungicide
treatments, Folicur was applied approximately four hours before inoculation, using a track
sprayer equipped with XR8001 flat fan nozzles oriented forward and backward at 600 from
the vertical. FHB incidence, head severity and field severity were determined at the soft
dough stage of kernel development.
Multiple infection events resulted in higher FHB field severities than did a single inoculation
event at the most susceptible growth stage. However, split applications of reduced rates of
Folicur across multiple growth stages generally did not significantly improve disease control
over a single treatment of the full label rate at the most susceptible growth stage. For all
three crops, the least satisfactory control of FHB among fungicide treatments tested was
when a single application of the full label rate of Folicur was applied late, at Feekes 10.54
(kernel watery ripe).
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EVALUATION OF FOLIAR FUNGICIDES AND BIOPROTECTANTS
FOR CONTROL OF FUSARIUM HEAD BLIGHT OF
WINTER WHEAT IN NEW YORK IN 2002
S.O. Kawamoto1, C.A. Stockwell1, D.J. Otis2, W.J. Cox2, M.E. Sorrells3,
and G.C. Bergstrom1*
Departments of Plant Pathology, 2Crop and Soil Science, and 3Plant Breeding,
Cornell University, Ithaca, NY 14853-5904
*Corresponding Author: PH: (607) 255-7849; E-mail: gcb3@cornell.edu
1
OBJECTIVES
To quantify the ability of promising fungicides and bioprotectants, applied to flowering wheat
spikes, to control Fusarium head blight (FHB) and to reduce deoxynivalenol (DON) contamination of the harvested grain.
To assess the efficacy of the bioprotectant TrigoCor 1448 to act synergistically with foliar
fungicides in control of FHB and reduction of DON contamination.
INTRODUCTION
Efforts are being made through the USWBSI to provide safe, affordable and efficacious
fungicides and biological protectants for the integrated management of FHB of wheat and
barley. This study provided a New York site for the Uniform Fungicide and Biocontrol Tests
in 2002. In addition to uniform core treatments, we assessed additional biocontrol agents at
two locations. The reduction of DON contamination of the harvested grain to acceptable
levels remains of critical importance in the management of this disease. We were especially interested in assessing the ability of Bacillus subtilis isolate, TrigoCor 1448, to enhance the reduction of DON when applied to flowering wheat spikes in mixture with fungicides, based on initially promising results with the combination of TrigoCor 1448 with Folicur
3.6F (Stockwell et al., 2001).
MATERIALS AND METHODS
Uniform Fungicide/Bioprotectant Field Trial – Musgrave Farm, Aurora, NY
Twelve treatments were included in the uniform fungicide/bioprotectant trial conducted at
Aurora, NY. Treatments were replicated four times and arranged in a randomized block
design. In addition to AMS 21619A, AMS 21619A plus Folicur, Folicur, BAS 500, TrigoCor
1448 and the USDA/Peoria Yeast which were included as core treatments tested at all
locations, this trial included the commercial Bacillus subtilis bioprotectant product, Serenade (AgraQuest; Davis, CA) and an experimental, endophytic Streptomyces EN27 (courtesy Justin Coombs, Cornell University). Commercial products were applied at labeled
rates. In this same trial, TrigoCor 1448 was combined in treatments with AMS 21619, BAS
500, or Folicur to determine if the combination would give enhanced FHB control over either
bioprotectant or fungicide alone. TrigoCor 1448 was grown for 5 days in nutrient broth with
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yeast extract, NBYE, (2-4 X 108 cfu/ml) and applied undiluted as whole broth. Yeast cells
were supplied as a paste by Dr. Shisler and were suspended in distilled water. Corn grain
infested with G. zeae was scattered in the alleys between the plots one month prior to anthesis. Treatments were applied to wheat at anthesis with a backpack type sprayer at 40 psi,
18-20 gpa using a nozzle arrangement that allowed angled spraying of the heads. After the
heads had dried, they were inoculated with G. zeae at a rate of 2.7 x 1010 macroconidia per
acre. The plots were rated visually for the incidence and severity of Fusarium head blight.
Test weight, yield, % Fusarium damaged kernels (fdk), % seed infection (on SNAWS selective medium) and DON were determined from the harvested grain. Seed from each plot were
sent to Michigan State University for DON analysis.
Bioprotectant Trial - McGowan Field, Ithaca, NY
Five treatments were included in a biocontrol trial conducted at Ithaca, NY on Caledonia
soft white winter wheat. Treatments were replicated 6 times and arranged in a randomized
block design. Wheat heads were sprayed with the treatments on 7 June, 2002. After the
heads had dried, they were inoculated with G. zeae at a rate of 2.7 x 1010 macroconidia per
acre. The plot was wetted for 15 min each afternoon with a fine mist from overhead irrigation. The plots were rated visually for the incidence and severity of Fusarium head blight.
Test weight, yield, % Fusarium damaged kernels (fdk), % seed infection (on SNAWS selective medium) and DON were determined from the harvested grain. Seed from each plot were
sent to Michigan State University for DON analysis.
RESULTS AND DISCUSSION
Uniform Fungicide/Bioprotectant Field Trial – Musgrave Farm, Aurora, NY
Of all the fungicides and bioprotectants tested, only AMS 21619A showed great promise for
control of FHB under the epidemic conditions experienced in New York in 2002 (Table 1).
None of the treatments reduced DON contamination to levels acceptable to the grain trade,
though AMS 21619A reduced DON significantly. The performance of any of the three synthetic fungicides was not increased in combination with the bioprotectant TrigoCor 1448.
Bioprotectant Trial - McGowan Field, Ithaca, NY
None of the biocontrol treatments or the fungicide Folicur controlled Fusarium head blight or
reduced DON contamination in the harvested grain (Table 2).
CONCLUSIONS
If results from other test locations (summary report in this volume by D. Hershman) are
similar to those in New York, extensive evaluation of the foliar fungicide AMS 21619A for its
potential in the integrated management of Fusarium head blight of wheat and barley will be
warranted.
REFERENCE
Stockwell, C.A., Bergstrom, G.C., and Luz, W.C. da. 2001. Biological control of Fusarium head blight with
Bacillus subtilis TrigoCor 1448:2001 field results. Pages 91-95 in: Proc. 2001 National Fusarium Head Blight
Forum, Holiday Inn Cincinnati-Airport, Erlanger, KY, December 8-10, 2001.
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Table 1. Effect of foliar treatment with fungicides and bioprotectants at anthesis on scab
incidence, Fusarium-damaged kernels, yield, test weight, and DON contamination in
Caledonia winter wheat in Aurora, NY in 2002.
T reatm en t an d am ou n t
N o n treated
A M S 2 1 6 1 9 A (5 .7 fl o z/A )
+ In d u ce (0 .1 2 5 % v/v )
A M S 2 1 6 1 9 A (5 .7 fl o z/A )
+ F o licu r 3.6 F (4 fl o z/A )
+ In d u ce (0 .1 2 5 % v/v )
A M S 2 1 6 1 9 A (5 .7 fl o z/A )
+ In d u ce (0 .1 2 5 % v/v )
+ T rigo C or 1 4 4 8
(2 .1 x 10 1 0 cfu /A )
B A S 5 0 0 5 0 W G (0 .4 lb/A )
+ In d u ce (0 .1 2 5 % v/v )
B A S 5 0 0 (0 .1 lb a.i./A )
+ In d u ce (0 .1 2 5 % v/v )
+ T rigo C or 1 4 4 8
(2 .1 x 10 1 0 cfu /A )
F o licu r 3 .6 F (4 fl o z/A )
+ In d u ce (0 .1 25 % v /v )
F o licu r 3 .6 F (4 fl o z/A )
+ In d u ce (0.1 2 5 % v /v )
+ T rigo C o r 1 4 4 8
(2 .1 x 1 0 1 0 cfu /A )
O H 18 2 .9 Y east (2 .2 X 1 0 9
cfu /A )
S erenad e (6 lb/A )
E N 2 7 S trep to m yces (3 .8 x 1 0 9
cfu /A )
T rigoC o r 1 4 4 8 (2 .1 x 10 1 0
cfu /A )
L S D (P = 0.05)
C V (% )
S cab (sp ik e) F u sariu m
in cid en ce on d am aged
k ern els
21 Ju n
(% )
(% )
3 8 .2
1 5 .1
1 4 .0
9 .9
T est w eigh t
@ 13.5%
m oistu re
(lb /b u )
5 0 .9
5 8 .0
Y ield
@ 13.5%
m oistu re
(b u /A )
6 2 .5
7 6 .1
DON
ppm
3 1 .0
8 .0
2 1 .1
1 0 .8
5 7 .6
7 3 .5
1 0 .0
2 3 .4
1 0 .8
5 7 .5
7 6.6
1 2 .0
3 7 .6
1 2 .1
5 5 .3
6 9 .9
2 0 .5
3 2 .1
1 4 .1
5 4 .7
7 0 .4
2 1 .0
3 2 .0
1 2 .8
5 2 .1
6 7 .8
2 9 .5
3 2 .8
1 4 .9
5 3 .8
6 8 .8
2 5 .5
3 7 .7
1 7 .6
5 2 .7
6 3 .9
3 3 .5
4 3 .7
3 5 .9
1 8 .9
1 6 .1
5 1 .0
5 0 .7
5 9 .2
6 9 .0
3 5 .0
3 6 .6
4 3 .2
1 3 .6
5 2 .0
6 1 .0
3 3 .0
8 .6
3 8 .2
0 .4
1 0 .4
2 .5
3 .3
NS
1 2 .8
8 .2
2 3 .1
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Table 2. Effect of foliar treatment with bioprotectants at anthesis on scab incidence,
Fusarium-damaged kernels, yield, test weight, and DON contamination in Caledonia winter
wheat in Ithaca, NY in 2002.
Treatment and amount
Nontreated
Folicur 3.6F (4 fl oz/A)
+ Induce (0.125% v/v)
OH 182.9 Yeast (2.2 X 109
cfu/A)
Serenade (6 lb/A)
TrigoCor 1448 (2.1 x 1010
cfu/A)
LSD (P=0.05)
CV (%)
Scab (spike) Fusarium Test weight
incidence on damaged @ 13.5%
kernels
24 Jun
moisture
(%)
(%)
(lb/bu)
29.9
12.1
53.5
29.7
9.9
53.9
Yield
@13.5%
moisture
(bu/A)
72.1
72.1
DON
ppm
28.0
30.7
28.8
12.0
53.2
68.9
31.0
38.2
31.5
12.5
11.8
51.3
53.2
65.4
70.8
35.0
28.3
6.2
16.5
0.1
15.4
1.6
2.5
NS
12.8
NS
17.8
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HISTORY AND ACCOMPLISHMENTS OF THE USWBSI UNIFORM
FUNGICIDE AND BIOLOGICAL CONTROL TRIALS, 1998-2002
M. McMullen1* and E. Milus2
Department of Plant Pathology, North Dakota State University, Fargo, ND 58105; and
2
Department of Plant Pathology, University of Arkansas, Fayetteville, AR 72701
*Corresponding Author: PH: (701) 231-7627; E-mail: mmcmulle@ndsuext.nodak.edu
1
ABSTRACT
The devastating Fusarium head blight (FHB) epidemics in the US in the early 1990s resulted in intensive individual and regional efforts to evaluate fungicides for control of this
disease. These early evaluations did not use the same treatments and procedures, and this
made comparisons among locations difficult or impossible. A cooperative effort was needed
to assure that tests of chemical and biological control agents (BCAs) would provide useful
information on efficacy and yield parameters each year.
A group of researchers met at the first National Fusarium Head Blight Forum in 1997 in St.
Paul and established the Fungicide Technology Network. This group developed a set of five
uniform fungicide treatments to be tested on three classes of wheat and spring barley in
seven states (IN, KY, MO, MN, ND, OH, SD) during the growing 1998 season. At the 1998
National FHB Forum in East Lansing, Michigan, the Fungicide Technology Network became
part of the Chemical and Biocontrol research area of the USWBSI. At that meeting, plant
pathologists from 14 states (AR, IL, IN, KY, MD, MI, MN, MS, NY, NC, ND, OH, SD and VA)
agreed to cooperate in a uniform trial with a total of seven treatments. In succeeding years,
protocols for applying treatments and recording data were improved and standardized.
Each year, selection of the uniform treatments was decided by the Chemical and Biocontrol
Committee, with new treatments being tested for at least two years. In 2001, the first BCAs
were included in the uniform treatments.
During its first five years, the Uniform Fungicide and Biocontrol Trials have evaluated ten
fungicides provided by six crop protection companies and BCAs from EMBRAPA/Cornell
University and the USDA/Ohio State University. Reductions in FHB field severity across
locations have averaged about 50% and have been as high as 78% with the best fungicide
treatment. Most of the tested treatments have been eliminated from further consideration
because of poor efficacy, tendency to increase DON levels, and/or termination by the crop
protection company. Folicur and AMS 21619A from Bayer have had the most consistent
efficacy against FHB, controlled other important diseases, and generally increased yield
and test weight. Data generated in the Uniform Trials were instrumental for justifying Section 18 registrations for Folicur in several states and likely will be important for any future
registrations. Furthermore, a team of experienced collaborators has been established
across the US that uses common protocols for evaluating fungicides and BCAs across
multiple environments and grain classes, and that readily shares data and ideas for improving the evaluations.
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ND UNIFORM WHEAT FUNGICIDE AND BIOLOGICAL
AGENT TRIALS, 2002
M. McMullen1*, J. Lukach2, K. McKay3, and B. Schatz4
1
Dept. of Plant Pathology, North Dakota State University, Fargo, ND; 2Langdon Research Extension Center,
Langdon, ND; 3North Central Research Extension Center, Minot, ND; and
4
Carrington Research Extension Center, Carrington, ND
*Corresponding Author: PH: (701) 231-7627; E-mail: mmcmulle@ndsuext.nodak.edu
OBJECTIVE
To evaluate registered and experimental fungicides and biological agents for control of
Fusarium head blight (FHB) in hard red spring and durum wheat at multiple locations in ND.
INTRODUCTION
Uniform fungicide trials on wheat in ND in recent years (McMullen et al. 2000, 2001) have
shown statistically significant reductions in Fusarium head blight (FHB) field severity with
some registered and experimental fungicides. Similar results have been observed in the
national uniform trials (Milus et al. 2001). Biological agents tested (from Cornell University
and the USDA at Peoria, Illinois) were less successful in reducing FHB severity (Milus et al.
2001). In 2001, treatments containing the experimental fungicides AMS 21619 or BAS 505
resulted in the lowest FHB field severity and lowest DON levels among treatments in both
the ND trials and the national uniform trial summary. Experiments in 2002 were designed
to further test the efficacy of these two experimental fungicides, applied alone or in combination, and to further evaluate the biological agents, applied alone or in combination with a
fungicide. Tests in ND were established across two wheat classes and four locations to
enhance evaluation across multiple environments and crops.
MATERIALS AND METHODS
A uniform set of four fungicide treatments and three biological agent treatments were evaluated on spring wheat and four fungicide treatments and one biological agent were evaluated on durum wheat in ND in 2002 (Tables 1 and 2). Treatments for each wheat class were
tested across three locations and three cultivars: Oxen spring wheat at Fargo, Russ spring
wheat and Munich durum at Carrington, Ingot spring wheat and Plaza durum wheat at
Langdon, and Ben durum at Minot. Artificial inoculum in the form of inoculated grain was
dispersed in plots at Fargo and Langdon, wheat straw was distributed at Carrington, and
infections at Minot were from natural inoculum. Natural rainfall was augmented by mist
irrigation at Fargo and Langdon and by some overhead irrigation at Carrington.
All treatments were applied at early flowering (Feekes 10.51) with a CO2 backpack type
sprayer, equipped with XR8001 nozzles mounted at a 600 angle forward and backward
toward the grain heads. Spray was delivered in 18- 20 gpa at 40 psi. All treatments were
applied between 6:00 and 8:00 am. Treatments included Folicur (tebuconazole) fungicide,
AMS 21619A (experimental fungicide from Bayer Crop Science), BAS 505 (experimental
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2002 National Fusarium Head Blight Forum Proceedings
fungicide from BASF), a yeast biological (OH 182.9 - Cryptococcus nodaensis) from Dr.
Dave Schilser with the USDA, Peoria, a bacterial biological agent (TrigoCor 1448 - Bacillus
subtilis) from Dr. Gary Bergstrom, Cornell University, a combination treatment of TrigoCor
and Folicur, and a combination treatment of AMS 21619 and Folicur (Table 1). TrigoCor was
not tested on durum wheat at Carrington and Langdon. Fusarium head blight incidence and
head severity and leaf disease ratings were taken at soft dough kernel stage. Plots were
harvested with small plot combines. DON (vomitoxin) data was determined by the NDSU
Toxicology Lab using gas chromatography and electron capture techniques. Plots were in a
randomized complete block design and data were statistically analyzed across locations
using ANOVA.
RESULTS AND DISCUSSION
Hard red spring wheat: All fungicide treatments significantly reduced Fusarium head
blight field severity over the untreated check (47-59%), while treatments with the biological
agents did not (Table 1). DON levels were not significantly reduced by the fungicide or
biological treatments. All fungicide treatments significantly reduced leaf rust severity at
Fargo and Langdon. Leaf rust ratings were a part of overall leaf disease ratings at
Carrington, where leaf rust was much more severe than at Fargo or Langdon. All fungicide
treatments significantly reduced leaf diseases on spring wheat, while the biological
treatments did not (Table 1). Yields were significantly increased by fungicide treatments,
from 18 to 28%. Test weights were increased by fungicide treatments, but not significantly.
Durum wheat: At the Minot site, visible Fusarium head blight (FHB) symptoms were too low
to rate, due to drought and heat stress at that site. However, harvested grain at Minot was
tested for DON and treatments ranged from 0.7 (AMS treatment) to 2.3 ppm (untreated). The
AMS 21619A and BAS 505 fungicide treatments resulted in the lowest FHB field severities,
but differences among treatments were not significant (Table 2). DON levels were
significantly reduced by fungicide treatments containing AMS 21619A or BAS 505. Leaf
spots were significantly reduced by all fungicide treatments but not by the OH 182.9
biological treatment. Yields and test weights were significantly improved by all fungicide
treatments (19% to 32% yield increase and 1 to 1.8 lb test weight increase), but not by the
biological (Table 2). Heat stress in July during the time of flowering and grain development
may have made differences among treatments less significant in 2002 than in previous
years.
ACKNOWLEDGMENTS
The funding for this project was provided by the US Wheat and Barley Scab Initiative.
Fungicides were provided by BASF and Bayer CropScience. Biological agents were
provided by Dr. Gary Bergstrom, Cornell University, and Dr. Dave Schisler, USDA, Peoria,
Illinois.
REFERENCES
McMullen, M., Lukach, J., McKay, K., and Schatz, B. 2001. ND Uniform wheat fungicide and biocontrol trials,
2001. Pages 67-69 in: Proceedings of the 2001 National Fusarium Head Blight Forum, Erlanger, KY, Dec. 8-10,
2001. Michigan State University, East Lansing.
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2002 National Fusarium Head Blight Forum Proceedings
McMullen, M., Schatz, B., and Lukach, J. 2000. Uniform fungicide trial for controlling FHB in Spring wheat,
ND, 2000. Page 98 in: Proceedings of the 2000 National Fusarium Head Blight Forum, Erlanger, KY, Dec. 10-12,
2000. Michigan State University, East Lansing.
Milus, E. A., Hershman, D., and McMullen, M. 2001. Analysis of the 2001 uniform wheat fungicide and
biocontrol trials across locations. Pages 75-79 in: Proceedings of the 2001 National Fusarium Head Blight
Forum, Erlanger, KY, Dec. 8-10, 2001. Michigan State University, East Lansing.
Table 1. Spring wheat: Effect of fungicides and biological agents on Fusarium head blight
(FHB), DON, leaf rust, fungal leaf diseases, yield and test wt. across Fargo, Carrington, and
Langdon , ND, 2002.
Treatment and rate/acre1
Untreated check
Folicur 3.6 F
4 fl oz
AMS 21619A 480 SC
5.7 fl oz
BAS 505 50 WG
6.4 oz
OH 182.9 (Cryptococcus nodaensis)
TrigoCor 1448 (Bacillus subtilis)
TrigoCor 1448 + Folicur
4 fl oz
AMS 21619A 3.6 fl oz + Folicur 4 fl oz
LSD P = 0.05
FHB
FS2
%
17
7
9
7
15
14
12
8
6
DON3
ppm
7.5
6.5
5.3
5.5
6.6
7
6.7
6.3
NS
Leaf rust4
% on flag
5.6
0.4
1.5
1.5
3.5
4.5
0.2
0
1.8
Leaf spot5 Yield
% on flag bu/a
55
39
26
46
22
49
21
50
51
41
45
42
21
46
15
48
19
6
Test Wt.
lbs/bu
56.5
57.8
58.0
58.8
56.8
57.0
57.4
57.6
NS
1
All fungicide treatments had 0.125% Induce added; AMS 21619A an experimental fungicide from Bayer; BAS 505
an experimental fungicide from BASF; OH 182.9 an experimental yeast from the USDA, Peoria; and TrigoCor 1448
an experimental bacterium from Cornell University
2
FHB FS = Fusarium head blight field severity; field severity = incidence x head severity
3
DON levels were not available from Langdon at time of report
4
Leaf rust reported only at Fargo and Langdon
5
Leaf spot diseases primarily tan spot and Septoria leaf spot complex at Fargo and Langdon, but leaf spot readings at
Carrington included leaf rust, which was severe at that site
Table 2. Durum wheat: Effect of fungicides and a biological agent on Fusarium head blight
(FHB), DON, fungal leaf diseases, yield and test wt. across Carrington, Langdon, and Minot,
ND, 2002.
Treatment and rate/acre1
1
Untreated check
Folicur 3.6 F
4 fl oz
AMS 21619A 480 SC
5.7 fl oz
BAS 505 50 WG
6.4 oz
OH 182.9 (Cryptococcus nodaensis)
AMS 21619A 3.6 fl oz + Folicur 4 fl oz
LSD P = 0.05
FHB
FS2
%
36
24
19
21
32
23
NS
DON3
ppm
2.3
1.9
0.7
0.9
2.6
0.8
1.3
Leaf spot4
% on flag
43
12
13
12
35
12
14
Yield5
bu/a
37
45
46
49
39
49
5
Test Wt.5
lbs/bu
59.5
60.5
61.0
61.3
60.0
60.7
1.1
All fungicide treatments had 0.125% Induce added; AMS 21619A an experimental fungicide from Bayer;
BAS 505 an experimental fungicide from BASF; OH 182.9 an experimental yeast from the USDA, Peoria;
TrigoCor 1448 was NOT tested on durums at Carrington and Langdon
2
FHB FS = Fusarium head blight field severity; field severity = incidence x head severity; ratings only from
Carrington and Langdon as Minot did not have enough visible FHB in 2002 due to drought and heat
3
DON levels were available from Carrington and Minot at time of this report; significance at P = 0.1 confidence level
4
Leaf spot diseases primarily tan spot and Septoria leaf spot complex
5
Yield and test weight data from Carrington and Minot only
Chemical and Biological Control
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2002 National Fusarium Head Blight Forum Proceedings
NEW AND EFFECTIVE FUNGICIDES AGAINST THE FHB IN WHEAT
Á. Mesterházy*, T. Bartók and G. Kászonyi
Cereal Research non-profit Co., Szeged, Hungary
*Corresponding Author: PH: 36 (62) 435 235; E-mail: akos.mesterhazy@gk-szeged.hu
OBJECTIVES
To describe the antifusarium effect and efficacy of several novel fungicides including the
four years experiences with the AMS 21619.
INTRODUCTION
Among the fungicides used for the control of FHB until now the tebuconazole, metconazole
and bromuconazole were identified with larger effect against the disease (Mesterházy 1996,
1997, 2001). However in our tests the tebuconazole containing fungicides with higher rate
were the most effective, bromuconazole and metconazole were only of medium effect because the rate used in Hungary were significantly lower than that of suggested in Western
Europe. A part of the results was made public last year (Mesterházy and Bartók 2001). In the
last years extensive investigations were made with the new Bayer experimental fungicide,
signed as AMS 21619 in US or JAU 6476 in Europe. Besides its efficacy the question was
also what would be the best formulation and rating of the product. For this reason also leaf
rust was rated when epidemics occurred.
MATERIALS AND METHODS
The methodology the methods were the same as published last year (Mesterházy and
Bartók 2001). FHB % means disease severity, e. g. the ratio of spikelets showing infection. In
all years three cultivars with differing resistance were used and they were inoculated by two
Fusarium graminearum and two F. culmorum isolates at full flowering, one day after the
fungicide treatment. From 2001 we modified the duration of covering the head of groups by
polyethylene bags from 24 hrs to 48 hrs to allow better disease development under dry
conditions. FHB was rated five times, mean infection severity was calculated together with
AUDPC, but here only arithmetical means are given as the two parameters originating from
the same data show a relationship above 0.998. Leaf rust was rated as ACI, average coefficient of infection, where the coverage of the whole leaf system as a % was multiplied by 1 at
S, 0.8 at MS, 0.6 at MR, 0.4 at R and 0.2 at VR reaction type.
Every year FHB severity, FDK, and relative yield loss were rated. Deoxynivalenol was
measured in 1998 for the AMS 21619, however in 1999 and 2000 the experimental fungicides were not measured, as the formulations tested were not the products yet for commercial use. For this reason no DON data are listed for 1999-2001. In the tables only the averages are given across isolates and cultivars, e. g. the mean of 12 epidemic situations. Active
ingredients of the fungicides used for a L product: Folicur Solo: 250 g tebuconazole, Folicur
Top: 125 g tebuconazole, Falcon 465 EC: 167 g tebuconazole, spiroxamine 250 g +
triadimenole 43, Kolfugo Super carbendazime 200, Caramba SL metconazole 60, Juwel:
Chemical and Biological Control
100
2002 National Fusarium Head Blight Forum Proceedings
Kresoxym-methyl 125 + epoxyconazole 125, Granit SC: bromuconazole 200, Tango:
epoxyconazole 125 + tridemorph 375, Flamenco: fluquinconazole 100, Stratego:
trifloxystrobin 125 + propiconazole 125EC, Sphera: trifloxystrobin 188 + cyproconazole 080
EC.
RESULTS AND DISCUSSION
Table 1 shows the 1998 data. As some lower effective fungicides were mentioned last year,
we present here only the more effective ones to see the performance of the new experimental fungicide. The AMS 21619 was the most effective, 30-50 % better than the second best
fungicide.
In 1999 (Table 2) a wider set of fungicides were tested. Last year we presented only those
that had also DON analysis. Here the whole set is printed to see also products that are
maybe less known in US. In this year we added the carbendazime to 0.8 L/ha Falcon rate (1
L/ha) and this mixture was equivalent with the lower rate (0.8 L/ha) for AMS 21619. Lower
rates of this experimental fungicide produced less control, the situation is the same we hade
with the different tebuconazole containing fungicides, too. The epidemic severity measured
by the Fusarium check or Folicur Solo was about the same, and the best fungicides were
significantly better than this. However the leaf rust epidemic showed that this new product
has only medium or lower protection ability. From the spraying on about three weeks controlled leaf rust well, but thereafter the infection by rust increased rapidly. Other fungicides
like kept their activity against rust until the end of the vegetation period allowing lower than
10 % infection. For this reason the task was to find another fungicide that does not decrease
the antifusarium effect, but increases the efficacy against rust.
For this reason tebuconazole was chosen as partner fungicide in the 2000 trials. The
whether was dryer and warmer, so the infection severity was lower than in 1999 or 1998 that
were favorable for disease development. The AMS concentration was lower and
tebuconazole was also half of the concentration of Folicur Solo, being equivalent with
Folicur Top. The results showed that the new mixtures at 0.8 and 1 L/ha rate were about as
effective as Folicur Solo itself, however slightly lower within the LSD 5 %. All kept FDK
lower than 0.5 %.
In the 2001 trials therefore this new combination was tested at two rates (0.8 and 1 L/ha)
and we applied as check the 0.8 L/ha rate of AMS 21619. The results clearly show that the
new combination is as effective as the AMS 21619, but controls leaf rust as good as Folicur
Solo does. All three AMS fungicides were more effective than Folicur Solo even the disease
development was better than in 2000, but the humidity period was longer. These combinations were also better than our carbendazime-tebuconazole version. It is remarkable that
Caramba at 1.2 L/ha performs better than at 1 L/ha. An increase of the rate to 1.5 L/ha may
provide further improvement. The result support the data of El-Allaf et al. (2001), Hart et al.
(2001), Hershman et al. (2001), McMullen et al. (2001), Milus et al. (2001), however this
positive efficiency could be demonstrated also at higher epidemic severity. This means that
it will be effective also under more severe epidemic conditions than at mostly moderately
infected field trials.
Chemical and Biological Control
101
2002 National Fusarium Head Blight Forum Proceedings
Summary. The data show that AMS 21619 or JAU 6476 combined with tebuconazole
provided more powerful control of FHB and was effective also against leaf rust. At susceptible cultivars a higher dosage (1 L/ha, or somewhat higher) can give hope that food safety
could be secured better until more resistant cultivars will grow on the Great Plain, in Hungary or elsewhere. In Hungary the standard antifusarium fungicide is Falcon at 0.8 L/ha. It is
clear that any of the new combinations decreases at least 50 % the infection severity in
comparison to Falcon 0.8 L/ha. Therefore a change of fungicide should come in the near
future. It is important that the chances of the moderately susceptible or moderately resistant
cultivars will provide higher safety when sprayed with these new fungicides even they have
susceptibility to rust. Novel products are developed also elsewhere, their test will also be
necessary to identify other valuable products. This is necessary to change fungicides not
allowing the selection of fungicide resistant strains in the fungal populations.
REFERENCES
El-Allaf, S. M., Lipps, P. E., Madden, L. W., and Johnson, A. 2001. Effect of foliar fungicides and biological
control agents on Fusarium head blight development and control in Ohio. 2001 National Fusarium Head Blight
Forum, Erlanger, KY, 49-53.
Hart., P., VanEe, G., and Ledebuhr, R. 2001. Uniform fungicide trial study 2001 – Michigan State University.
2001 National Fusarium Head Blight Forum, Erlanger, KY, 54-58.
Hershman, D. E., Bachii, P. R., TeKrony, D. M. and VanSanford, D. A. 2001. Management of Fusarium head blight
in wheat using selected biological control and foliar fungicides. 2001 National Fusarium Head Blight Forum,
Erlanger, KY, 59-63.
McMullen, M., Lukach, J., McKay K. and Schatz, B. 2001. ND uniform wheat fungicide and biocontrol trials,
2001. 2001 National Fusarium Head Blight Forum, Erlanger, KY, 67-69.
MESTERHÁZY Á., BARTÓK, T. 2001. Fungicide control of Fusarium head blight in wheat. 2001 Fusarium Head
Blight Forum, Cincinnati, US. 70-74.
MESTERHÁZY, Á, BARTÓK, T. 1997. Effect of chemical control on FHB and toxin contamination of wheat.
Cereal Res. Comm. 25: 781-783.
MESTERHÁZY, Á., BARTÓK, T., 1996. Control of Fusarium head blight of wheat by fungicides and its effect in
the toxin contamination of the grains. Pflanzenschutz Nachrichten Bayer 49:187-205.
Milus, E. A., Hershman, D., and McMullen, M. 2001. 2001 National Fusarium Head Blight Forum, Erlanger, KY,
75-79.
T ab le 1 . S u m m ary o f fu n g icid e tests ag ain st F H B in w h eat, 1 9 9 8
F u n g icid e an d
rate L /h a
A M S 2 1 6 1 9 2 5 0 E C 1 .0
F o licu r T o p 1 .0 + K o lf.S 1 .5
F o l. S o lo 1 .0
F alco n 0 .8
F alco n 1 .0
F u s. k o n tr.
M ean
LSD 5 %
FH B
sev erity %
5 .0 7
7 .8 8
8 .1 3
9 .8 5
1 1 .6 3
4 1 .5 5
8 .4 1
0 .7 1
Chemical and Biological Control
102
T raits
Y ield lo ss
%
1 1 .0 8
1 6 .0 0
2 0 .8 7
2 0 .5 7
1 8 .4 3
5 0 .3 6
1 3 .7 3
2 .8 9
FD K
%
8 .3 3
1 5 .5 3
1 9 .7 3
2 8 .0 8
2 5 .8 6
5 8 .5 6
1 5 .6 1
3 .3 3
DON
ppm
2 .8 2
4 .1 9
3 .7 9
6 .2 4
5 .7 2
1 1 .3 7
3 .4 1
2 .0 7
2002 National Fusarium Head Blight Forum Proceedings
T ab le 2. F u n g icid es ag ain st F u sariu m h ead b lig h t o f w h eat. S u m m ary o f g en eral m ean s fo r 1 9 9 9 .
F u n g icid e
O rig in al d ata
L eaf ru st
rate L /h a
FH B %
Y ield lo ss %
K ern el in f. %
ACI
F alc.0 .8 + K o lf. 1 .5
1 2 .1 9
2 3 .4 3
1 4 .3 6
0 .4
A M S 2 1 6 1 9 2 5 0 E C 0 .8
1 3 .1 9
2 6 .0 0
1 5 .8 6
3 1 .2
F o licu r S o lo 1 .0
1 3 .4 2
2 7 .1 4
2 2 .3 7
1 .6
A M S 2 1 6 1 9 2 5 0 E C 0 .6
1 4 .5 0
2 8 .2 5
1 8 .2 4
3 4 .5
F alco n 0 .8
1 4 .9 9
2 8 .6 1
2 0 .4 7
1 .8
F o licu r T o p
1 7 .3 8
2 9 .0 3
1 9 .9 1
3 .4
A M S 2 1 6 1 9 2 5 0 E C 0 .4
1 9 .0 0
3 7 .0 5
2 6 .1 3
4 2 .1
C aram b a 1 .0
2 0 .1 2
3 6 .1 7
2 5 .4 2
7 .6
F alco n 0 .6
2 0 .6 8
3 9 .6 9
3 7 .8 6
7 .9
Ju w el 1 .0
2 3 .7 5
3 8 .9 7
3 2 .6 9
9 .5
G ran it 1 .0
2 3 .8 1
3 8 .9 0
3 0 .4 6
2 7 .0
A lert 1 .0
2 4 .8 4
3 6 .3 2
2 7 .9 2
3 4 .2
K o lfu g o S u p er 1 .5
2 5 .1 3
3 9 .9 3
3 1 .3 1
5 8 .2
T an g o 0 .8
2 6 .7 5
3 9 .9 6
2 8 .4 4
4 .6
F lam en co 1 .0
3 3 .5 8
4 8 .7 9
4 3 .4 0
1 4 .2
F u sariu m ch eck
4 1 .9 7
5 6 .1 1
5 8 .7 9
6 4 .0
M ean
2 1 .5 8
3 5 .9 0
2 8 .3 5
2 1 .3 9
LSD 5 %
0 .9 1
2 .9 3
3 .2 1
5 .9 7
T ab le 3. F u n g icid es ag ain st F H B in w h eat. S u m m ary, 2 0 0 0 .
F u n g icid e
rate L /h a
F o licu r S o lo 1 .0
F alco n 0 .8 + K o lf. 1
A M S 2 1 6 1 9 2 5 0 E C 0 .8
A M S 2 1 6 1 9 1 1 2 5 E C + H W G 1 2 5 , 1 .0
F alco n 1 .0
C aram b a, 1 .2
F alco n 0 .8
Ju w el, 1 .0
K o lfu g o , 1 .5
F u sariu m ch eck .
F lam en co , 1 .5
M ean
LSD 5 %
H W G = teb u co n azo le
FH B %
1 .0 6
1 .4 5
1 .5 9
1 .7 9
1 .9 3
2 .5 4
2 .9 6
3 .2 0
4 .5 7
8 .6 7
9 .1 0
2 .4 3
0 .5 0
P aram eters
Y ield lo ss %
6 .7 8
8 .7 4
9 .2 8
9 .4 4
9 .7 0
8 .5 3
1 0 .2 6
1 4 .9 6
1 4 .8 2
2 2 .9 6
2 4 .0 6
8 .7 2
1 .0 6
K ern el in f.%
0 .0 8
0 .4 5
0 .4 2
0 .4 2
0 .8 6
0 .6 8
1 .1 9
1 .6 1
3 .4 5
7 .7 0
1 0 .2 4
1 .6 9
1 .1 0
T ab le 4. F u n g icid e testsag ain st F u sariu m h ead b lig h t in w h eat, su m m ary fo r 2 0 01 .
T reatm en t
FH B %
0 .6 0
0 .9 2
0 .9 9
1 .1 4
1 .3 9
2 .2 8
2 .9 1
3 .0 8
3 .4 0
5 .7 1
1 2 .0 2
2 .6 5
0 .4 9
A M S 2 1 6 1 9 1 2 5 E C + H W G 1 2 5 E C 1 .0
A M S 2 1 6 1 9 2 5 0 E C 0 .8
A M S 2 1 61 9 1 25 E C + H W G 1 2 5E C 0 .8
F o licu r S o lo 1 .0
F alco n 0 .8 + K o lf. S .1 .5
C aram b a 1 .2
F alco n 0 .8
S trateg o 1 .0
S fera 1 .0
K o lfu g o S 1 .5
F u sariu m ch eck.
M ean
L SD 5 %
FD K %
5 .6 1
6 .5 9
7 .4 4
9 .0 8
1 3 .8 0
1 2 .9 4
1 4 .9 1
1 8 .8 1
1 6 .4 1
2 2 .5 6
3 8 .5 4
1 4 .6 9
2 .0 5
O verall m ean s
Y ield lo ss %
3 .9
3 .5
1 .1
4 .8
6 .5
7 .7
1 2 .3
8 .8
1 6 .2
1 7 .0
1 7 .2
1 0 0 .0 0
2 .9 7
L eaf ru st
3 .0 0
3 2 .5 2
2 .5 6
1 .7 4
3 .5 9
6 .7 0
5 .3 7
2 1 .7 8
6 .6 3
5 8 .1 5
7 4 .0 7
1 7 .6 6
3 .4 9
H W G = teb u co n azo le
Chemical and Biological Control
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2002 National Fusarium Head Blight Forum Proceedings
UNIFORM BARLEY FUNGICIDE AND BIOLOGICAL AGENT TRIALS,
FARGO, ND, 2002
S. Meyer, J. Jordahl, and M. McMullen*
Dept. of Plant Pathology, North Dakota State University, Fargo, ND 58105
*Corresponding author: PH: (701) 231-7627; E-mail: mmcmulle@ndsuext.nodak.edu
OBJECTIVE
To evaluate registered and experimental fungicides and biological agents for control of
Fusarium head blight (FHB) in spring barley at Fargo, ND.
INTRODUCTION
Uniform fungicide trials on spring barley in ND in recent years have shown inconsistent
results in reduction of Fusarium head blight (FHB) field severity and DON levels (McMullen
et al., 2000 and 2001). In 2000, fungicides tested reduced FHB field severity by 45 to
66.7%, but differences among treatments were not significant. In 2001, all fungicide treatments significantly reduced FHB field severity, with the experimental fungicide, AMS 21619,
giving the greatest reduction (70.5%). DON levels, however, were not statistically reduced
by treatments in either year. Biological agents were not consistently tested on barley across
locations. An experiment in 2002 at Fargo, ND further tested experimental fungicides,
applied alone or in combination, and evaluated biological agents, applied alone or in combination with a fungicide, for efficacy in controlling FHB in spring barley. Treatments were
the same as those in the uniform trials for wheat.
MATERIALS AND METHODS
A uniform set of four fungicide treatments, two biological agent treatments, and a biological
+ fungicide treatment were evaluated on six row ‘Robust’ spring barley at Fargo, ND in 2002
(Table 1). Plots were planted on May 3, 2002 into wheat stubble that had been chiseled
twice prior to planting. Plants emerged on May 16, but were frosted several times in late
May. Two weeks before head emergence in early July, artificial inoculum in the form of
inoculated corn grain was dispersed uniformly in the plots, approximately 100 g per 162
square foot plot. Natural rainfall was augmented by mist irrigation starting on July 3 and
continuing until July 19.
All treatments were applied at early head emergence (Feekes 10.5) with a CO2 backpack
type sprayer, equipped with XR8001 nozzles mounted at a 600 angle forward and backward
toward the grain heads. Spray was delivered in 18- 20 gpa at 40 psi. All treatments were
applied between 6:00 and 8:00 am. Treatments were: Folicur (tebuconazole) fungicide;
AMS 21619A (Bayer CropScience experimental fungicide); BAS 505 (BASF experimental
fungicide); a yeast biological (OH 182.9 - Cryptococcus nodaensis) from Dr. Dave Schisler,
USDA, Peoria; a bacterial biological agent (TrigoCor 1448 - Bacillus subtilis) from Dr. Gary
Bergstrom, Cornell University; a combination treatment of TrigoCor and Folicur; and a combination treatment of AMS 21619 and Folicur (Table 1). FHB ratings and leaf disease
Chemical and Biological Control
104
2002 National Fusarium Head Blight Forum Proceedings
ratings were taken at soft dough kernel stage. Plots were harvested with a small plot combine. DON (vomitoxin) was determined by the NDSU Toxicology Lab using gas chromatography and electron capture. Plots were in a randomized complete block design and data
were statistically analyzed across locations using ANOVA.
RESULTS AND DISCUSSION
All fungicide and biological treatments significantly reduced FHB head severity and field
severity over the untreated check (Table 1). DON levels were significantly reduced by the
AMS 21619A, BAS 505, the TrigoCor + Folicur and the AMS 21619A + Folicur treatments.
Yields were significantly increased by most fungicide treatments, but not by the biological
treatments. Test weights were significantly increased by only two treatments, the Folicur
alone and the AMS 21619A + Folicur treatment. Although FHB levels were fairly high in this
experiment, late season heat stress and low natural precipitation at this location may have
resulted in poor grain fill, low yields and low test weights, and concomitant smaller differences among treatments.
ACKNOWLEDGEMENTS
The funding for this project was provided by the US Wheat and Barley Scab Initiative. Fungicides were provided by BASF and Bayer CropScience. Biological agents were provided
by Dr. Gary Bergstrom, Cornell University, and Dr. Dave Schisler, USDA, Peoria, Illinois.
REFERENCES
McMullen, M. and Lukach, J. 2000. Uniform fungicide trial for controlling FHB in Barley, ND, 2000. Page 99 in:
Proceedings of the 2000 National Fusarium Head Blight Forum, Erlanger, KY, Dec. 10-12, 2000. Michigan State
University, East Lansing.
McMullen, M., Lukach, J., Jordahl, J., and Meyer, S. 2001. Uniform barley fungicide trials in North Dakota,
2001. Page 66 in: Proceedings of the 2001 National Fusarium Head Blight Forum, Erlanger, KY, Dec. 8-10,
2001. Michigan State University, East Lansing.
T ab le 1 . E ffect of fun gicid es an d bio logical agen ts on F usarium h ead blig ht (F H B ), D O N , yield
an d test w eig ht in ‘R obu st’spring barley, F arg o , N D , 200 2.
FH B
FH B
FH B
H S2
F S2
DON
Y ield
T est w t
I2
%
%
%
p pm
b u /a
lb s/b u
U ntreated ch eck
97
2 5 .0
24
4 .3
50
4 3 .9
F o licu r 3.6 F
4 fl oz
89
8 .7
8
3 .6
55
4 4 .5
A M S 21 6 19 A 48 0 S C
5.7 fl oz
94
7 .3
7
2 .9
54
4 3 .9
B A S 5 05 50 W G
6.4 oz
93
6 .7
6
2 .7
55
4 4 .1
O H 18 2 .9 (C ryptococcu s n od a en sis)
92
8 .8
8
3 .3
50
4 3 .9
T rigo C o r 14 48 (B a cillu s sub tilis)
93
7 .9
7
3 .9
49
4 3 .3
T rig o C or 1 4 48 + F o licu r
4 fl oz
94
6 .9
6
2 .9
52
4 3 .8
A M S 2 16 19 A 3.6 fl oz + F o licu r
4 fl oz
90
7 .9
7
3 .1
57
4 4 .5
L S D P = 0 .05
8
5 .6
6
1 .2
5
0 .6
1
A ll fu ng icid e treatm ents h ad 0 .12 5% Ind u ce add ed; A M S 2 16 1 9A an exp erim en tal fu ng icid e from B ay er;
B A S 50 5 an ex p erim en tal fun gicide fro m B A S F ; O H 1 82 .9 an ex perim ental y east from th e U S D A , P eo ria;
and T rig oC o r 1 44 8 an ex perim ental b acteriu m fro m C o rn ell U niversity
2
F H B I = In cid ence (% tillers sh o w ing sym ptom s); F H B H S = % of k ern els sho w in g sy m p to m s; F H B F S =
F usariu m h ead b ligh t field severity ; field sev erity = in ciden ce x h ead severity
T reatm ent an d rate/acre1
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2002 National Fusarium Head Blight Forum Proceedings
EFFICACY OF FUNGICIDES AND BIOCONTROLS AGAINST FHB
ON WHEAT IN ARKANSAS IN 2002
Eugene A. Milus*, Peter Rohman, and Samuel Markell
Department of Plant Pathology, University of Arkansas, West Lafayette, AR 72701
*Corresponding Author: PH: 479 575-2676; E-mail: gmilus@uark.edu
INTRODUCTION
Identifying fungicides and biocontrols that reduce incidence and severity of Fusarium head
blight (FHB) in the field and levels of damage and mycotoxins in the grain could have widespread benefits to growers and users of all market classes of wheat in the event of FHB
epidemics. This test in Arkansas is part of the Uniform Fungicide and Biocontrol Trial that is
coordinated by the Chemical and Biological Control Committee, and the objective is to
hasten the integration of fungicides and biocontrols that are effective against FHB into costeffective and environmentally-safe wheat disease management strategies.
METHODS
The susceptible wheat cultivar ‘Hazen’ was planted at the University Farm at Fayetteville on
8 October 2001. Seed was treated with Dividend fungicide (1 fl oz / cwt) for loose smut and
seedling diseases and Gaucho insecticide (3 fl oz / cwt) for aphids transmitting barley
yellow dwarf. Individual plots were 7 rows by 13 ft. Plots were fertilized with 80 lb nitrogen
from ammonium nitrate that was applied in equal splits on 28 February and 12 March.
Ryegrass and broadleaf weeds were controlled with recommended herbicides. Infested
corn kernel inoculum was applied to the plots on April 1 and 9 at a total rate of 12 kernels /
sq ft. The mist system operated for eight 10-minute periods between midnight and 8:00 am
for eight nights between 30 April and 8 May. TrigoCor 1448 was grown in broth culture and
OH 182.9 was suspended from frozen paste according to directions supplied with the
biologicals. To determine the concentration of viable cells of each biological agent, the
suspension of each biological was assayed by dilution plating on TSA medium immediately
before application to the plots. Fungicides and biocontrols were applied in a randomized
complete block design with six replications in the late afternoon on 2 May when 50% of the
main stems had begun to flower. Applications were at 20 gal / acre except for one AMS
21619A treatment that was applied at 10 gal / acre. On 23 May, 50 heads per plot were
sampled randomly and evaluated for FHB incidence and head severity, and plot severity
was calculated. Plots were harvested with a plot combine on 14 June, and grain was
passed once through a seed cleaner before test weight and percentage of scabby grain
were measured. Grain samples were sent to Pat Hart’s laboratory for DON analysis.
RESULTS AND DISCUSSION
Except for low levels of barley yellow dwarf from spring infection in some plots, FHB was
the only significant disease. Sixteen days of rain totaling 11.3 inches during April and May
provided very favorable conditions for sporulation on the corn inoculum, infection, and FHB
development. Fusarium head blight was severe by the end of the season, as indicated by
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the high levels of scabby grain (Table 1). Compared to the non-treated check, all fungicides
significantly reduced plot severity and increased test weight, but the two biologicals did not.
However, there were no significant differences among the fungicides. Plots treated with
fungicides had numerically greater yields that the non-treated check or plots treated with
biologicals, but differences were not significant at the 5% level of confidence because of
variability among plots of the same treatment. Poor performance of the biologicals did not
appear to be due to low viability of cells in the suspension applied to the plots. AMS
21619A applied at 10 gal / acre appeared to have greater efficacy than at 20 gal / acre, but
the differences were not statistically significant.
Chemical and Biological Control
107
Chemical and Biological Control
108
*A p p lied in 1 0 gal/a cre
T rigo C o r 14 4 8 (5x 1 0 1 3 cfu )
N o n-trea te d che ck
L S D (P =0.05 )
% cv
P ro d u c t an d ra te p er ac re
A M S 21 61 9 A 4 80S C 5 .7 fl. oz. + 0.1 25 % Indu ce *
A M S 216 19A 48 0S C 5 .7 fl.o z. + 0 .1 % K ine tic
A M S 21 61 9A 4 80 S C 5 .7 fl. oz. + 0 .1 25 % Indu ce
B A S 50 5F 5 0W G 6.4 oz. + 0 .1 25 % Indu ce
A M S 21 619 A 4 80S C 3 .6 fl. oz. + F o licu r 3 .6F 4 fl. oz. + 0 .1 25 % Indu ce
T rigo C o r 14 4 8 (5x 1 0 1 3 cfu ) + F o licu r 3 .6F 4 fl. oz. + 0 .1 25 % Indu ce
A M S 2 1 619 A 4 80S C 5 .7 fl. oz. + S u ccee d 1.17 L /h a
F o licu r 3 .6F 4 fl. oz. + 0 .1 25 % Indu ce
O H 18 2.9 (1.1x 1 0 1 4 cfu )
7.1
7.9
8.5
8.6
8.6
9.3
1 0.9
1 1.0
1 5.4
0 .5 3
0 .5 1
0 .1 4
2 7.6
0 .2 9
0 .3 4
0 .4 4
0 .3 5
0 .3 9
0 .3 8
0 .4 2
0 .4 8
0 .5 2
P lo t s e v erity (% )
1 5.7
1 7.3
5.4
4 2.3
In c id en c e o f
in fec te d h ea d s
T ab le 1. E fficac y of fu n g icide s a nd b io contro ls a ga in st F H B on w h e at in A rkans a s.
Infec ted h e ad
s e v erity (% )
3 0.3
3 3.0
NS
3 1.0
2 5.7
2 0.6
1 9.4
2 3.5
2 1.7
2 4.4
2 7.2
2 2.2
2 9.8
S ca bb y g ra in (% )
67
75
1 0.0
1 4.0
51
60
52
58
52
67
62
65
72
Y ield (b u /A )
5 7.7
5 5.5
NS
1 4.0
7 2.4
6 4.9
6 8.9
6 3.1
5 8.0
6 3.7
6 1.2
6 5.1
5 4.8
T es t w t. (lbs/b u )
4 8.3
4 6.7
2.7
4.6
5 1.9
4 9.9
5 0.4
5 0.5
5 0.8
5 0.1
4 9.6
4 9.6
4 7.6
2002 National Fusarium Head Blight Forum Proceedings
2002 National Fusarium Head Blight Forum Proceedings
PRACTICAL ASPECTS OF GROUND APPLICATION
OF FOLIAR FUNGICIDES
Philip Needham
Opti-Crop consulting (division of Miles Farm Supply), 1401B Springbank Dr., Owensboro,
KY 42304 (website: www.opticrop.com)
Corresponding Author: PH: (270) 926-2420; E-mail: phinee@MILESNMORE.COM
ABSTRACT
While remaining at the forefront of intensive wheat management, Opti-Crop is also an industry leader in providing state-of-the-art consulting services to corn and soybean growers.
Presently, our staff of over 25 Opti-Crop consultants – most with CCA certification – manage
over 200,000 acres of corn, soybeans and wheat in Kentucky as well as parts of Indiana,
Illinois, Tennessee, Kansas, Oklahoma, South Dakota and North Dakota. Opti-Crop also
has divisions that manage over 150,000 acres in Australia, plus consulting operations in
Russia, Romania and Bolivia.
Ground application of foliar fungicides is a very important component of our intensive crop
management program. Our company custom applies over 1,000,000 acres of chemicals and
fertilizer annually, so logistics and timing are always a challenge. We strive to educate and
train our personnel on the latest application technology by conducting field days and training sessions.
Selection of the appropriate fungicide, rates and specific adjuvants has a major impact on
product effectiveness. Correct water volumes and product application timings are also
crucial. We have 8 replicated research sites across the Midwest and Northern Plains, so we
have the luxury of being able to apply different products at different rates and timings to
determine the relative differences and economics of the individual treatments.
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2002 National Fusarium Head Blight Forum Proceedings
EFFICACY OF FUNGICIDES IN CONTROLLING BARLEY FUSARIM
HEAD BLIGHT IN LINES WITH PARTIAL RESISTANCE
J.D. Pederson1, R.D. Horsley1*, M. McMullen2,3, and K. McKay3
1
Dept. of Plant Sciences, 2Dept. of Plant Pathology, and 3North Dakota Extension Service,
North Dakota State Univ, Fargo, ND 58105
*Corresponding Author: PH: (701) 231-8142; E-mail: richard.horsley@ndsu.nodak.edu
ABSTRACT
Research to test the efficacy of fungicides in controlling Fusarium head blight (FHB) and
deoxynivalenol (DON) levels in barley was previously conducted using cultivars (i.e. Robust, Foster, and Stander) that are susceptible to FHB. Results indicate that fungicides had
little to no effect in reducing DON concentration to levels acceptable to the malting and
brewing industry. Minimal information is available on the efficacy of fungicides in controlling
FHB and DON levels on genotypes with partial FHB resistance. The objective of this study
is to determine if the integrated use of fungicides and barley cultivars with partial resistance
to FHB will control FHB severity and accumulation of DON. Experiments were conducted in
the field in North Dakota since 2000 and included genotypes resistant, partially resistant,
and susceptible to FHB. Fungicides used were Folicur in 2000, 2001, and 2002; and
AMS21619 in 2001 and 2002. Folicur did not significantly reduce FHB severity or DON
accumulation in resistant, moderately resistant, or susceptible genotypes. However, genotypes sprayed with Folicur generally had greater yield due to control of septoria speckled
leaf blotch (SSLB), incited by Septoria passerinii. Yield gains due to control of SSLB tended
to be sufficient to cover the cost of Folicur and its application on cultivars developed and
released by upper Midwest barley breeding programs. Preliminary data indicates that
efficacy of AMS21619 was slightly better than Folicur in reducing FHB and DON.
Chemical and Biological Control
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2002 National Fusarium Head Blight Forum Proceedings
AUTOMATED CONTROL OF A WATERING SYSTEM FOR
FUSARIUM HEAD BLIGHT RESEARCH
T. Scherer1*, D. Kirkpatrick1 and M. McMullen2
Dept. of Agricultural and Biosystems Engineering, and 2Dept. of Plant Pathology,
North Dakota State University, Fargo, ND, 58105
*
Corresponding Author: PH: (701) 231-7239, E-mail: tscherer@ndsuext.nodak.edu
1
OBJECTIVES
Use an automated water application control system to create a favorable growth environment for Fusarium head blight in the uniform fungicide trial plots. Evaluate the effectiveness
of the watering system by 1) monitoring the microclimate in the plots and 2) measuring the
FHB field severity levels in the watered control plots and surrounding dryland plots.
INTRODUCTION
To properly evaluate the effectiveness of fungicides, a favorable microclimate for the growth
of FHB must be provided either by nature or artificially. Keeping the grain heads “wet” and/or
maintaining a high humidity during the crucial FHB formation period is important for the
evaluation of fungicides. Many researchers participating in the uniform fungicide trials use
some type of watering system but some do not, relying on natural climatic conditions to
provide the environment for the growth of FHB (McMullen, 2001).
A search of the literature reveals no set protocol for the operation of the watering systems
during the FHB infection period. Most researchers haven’t included a description of the
watering system operation in their reports or research papers. Warnes (1995) used a misting
system with a windbreak around the research plots. He ran the misting system for 20 minutes on even hours from 6 am to 6 p.m. and 10 minutes on even hours from 6 p.m. to midnight. The intended precipitation amount was 0.3 inches per day. Nelson (2000) ran his
misting system for a half-hour at 7 p.m., 11 p.m. and 5 am. Neither researcher explained how
they developed the watering protocol.
Research by Francl (2001) provided a guide for when and how often to operate a watering
system. He is developing a model that uses weather data to predict when the weather
conditions are right for FHB infection. He says, “Details of the interactions among environmental factors, infection, spore survival, etc. are not yet fully understood. As a general guide,
infection is indicated for wet periods longer than nine hours, but this may be substituted for
by a high relative humidity and an average temperature above 60°F.” These guidelines could
be used to operate the watering system using a feedback control system.
For statistical verification of the effectiveness of fungicides, the water application pattern
should be uniform on all plots. The means the frequency, duration and amount of applied
water should be equal for all plots. If too much water is applied, the fungicide could be
washed off, loose its effectiveness and the fungus would overwhelm the plots. If too little
water is applied, the fungus will not grow at an equal rate in all plots. A balance must be
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2002 National Fusarium Head Blight Forum Proceedings
struck that mimics natural conditions that favor the growth of FHB. The watering system must
apply the amount of water that maintains the proper microclimate to grow the fungus without
interfering with the effectiveness of the fungicide.
MICROSPRINKLER WATERING SYSTEM
During the 2000 growing season, a watering system which used microsprinklers was designed and installed in the FHB uniform fungicide trial plots (Scherer, et al., 2000). The
same watering system was used during the 2001 and 2002 growing seasons. The research
field covered about 1.8 acres. About 1.3 acres were planted to one variety of wheat and the
remaining area planted to barley.
The watering system has three zones each 70 feet wide by 360 feet long. Within each zone,
laterals are spaced 10 feet apart to match the plot width. The microsprinklers are spaced 15
feet apart along the laterals. Each lateral has 25 microsprinklers and the total for all three
zones is 528 microsprinkler heads. Two zones had seven laterals and the other zone (a
combination of wheat and barley) had eight laterals. Each zone has its own control valve
and filter. The duration and frequency of the watering system was controlled by a programmed datalogger but could be operated manually.
AUTOMATED CONTROL AND REMOTE MONITORING SYSTEM
The automated control system comprised two sensor stations. One was located in a control
plot in zone 1 and the other in a control plot in zone 2. They were installed on June 28 when
the flag leaf was just starting to show. Each sensor station had a very accurate relative
humidity/temperature sensor and a leaf wetness sensor placed at the same elevation as the
flag leaf on the wheat. The sensor stations were connected to the programmable datalogger
that controlled the watering system and recorded the data. The critical infection period
started on July 1. Relative humidity and temperature was read continuously from each
sensor and an average value recorded every 10 minutes. The leaf wetness sensors were
read continuously to record the “wetness duration” during the critical infection period. An
automated recording rain gage was place in a watered plot and another was placed in an
adjacent non-watered plot to record both watering and rainfall events.
In addition to the sprinkler control sensor system, remote microclimate monitoring stations
were located in four watered control plots and two adjacent dryland plots. In the watered
area, one station was located near the beginning of the sprinkler laterals, one near the end
of the laterals and two were located halfway between. In the dryland area, stations were
placed at one-third and two-thirds the lateral length. Each remote monitoring station had
three self-contained dataloggers to measure relative humidity, wet bulb temperature and dry
bulb temperature (HOBO Pro temperature/RH meters). The three dataloggers at each station
were mounted on a single support pole at 15, 45 and 75 cm (6, 18 and 30 inches) above
ground surface.
They were installed in the plots on June 24 when the wheat was approximately 20 cm (8
inches) tall. They were programmed to record data every 10 minutes. The data were downloaded once per week until July 24 when the wheat had passed the infection stage. A North
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2002 National Fusarium Head Blight Forum Proceedings
Dakota Agricultural Weather Network (NDAWN) weather station is located about 3000 feet
from the research site and weather data for the area is recorded on an hourly basis. These
data will be used to obtain stratified data of the climatic variables in and above the small
grain canopy.
Control Algorithm
Based on recommendations from Dr. Francl and Dr. McMullen, the control system was
programmed to begin watering at 5 p.m. each day if the relative humidity was below 92%.
Each zone was watered for a total of 30 minutes. The first watering cycle ended at 6:30 pm.
At 9 p.m., the relative humidity was checked and if it was below 92%, the watering system
was activated and each zone was misted for 15 minutes. This was repeated every hour on
the hour until 8 am in the morning. This assured at least 9 hours of wet conditions each day.
The dry period during the day allowed the FHB spores to dry and move with the wind to
ensure infection. The watering system was manually tested on June 28 when the wheat
heads were just beginning to emerge. On July 1, the watering system was turned on and
automatic control began. The watering system was under automatic control until July 19
when the system was shut off.
RESULTS AND DISCUSSION
Throughout the control period (July 1 to July 19), the watering system successfully maintained the relative humidity in the plots above 92% from 9 pm to 9 am except on July 5 and
6. On these two days, the wind speed stayed between 20 to 30 miles per hour and the air
temperature between 79 to 93Ú F the entire time. Even under these conditions, the relative
humidity was maintained at slightly over 80 percent.
The readings from the leaf wetness sensors show that the grain heads were wet about 73%
of the time and dry about 27% of the time. By comparison, the wheat heads in the dryland
plots were wet and dry 10% and 90% of the time, respectively. We did not have a leaf wetness sensor in the dryland plots, so these data were estimated using rainfall and relative
humidity readings from the NDAWN station.
Remote Monitoring Sensors
The remote sensors measured the stratification of temperature and relative humidity in both
the watered and dryland plots. One way to determine the wetness of the plots is to measure
the amount of time the relative humidity was at a certain level during the critical infection
Sensor Location
6 inches above ground
18 inches above ground
30 inches above ground
Percent time the RH was
greater than 92% in the
watered plots (average of 4
stations)
80%
65%
45%
Percent of time the RH was
greater than 92% in the
dryland plots (average of 2
stations)
47%
42%
30%
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2002 National Fusarium Head Blight Forum Proceedings
period. The effectiveness of the watering protocol can be verified by examining the relative
humidity data from the four remote monitors in the watered plots and compare that with the
relative humidity data from the dryland plots. These results are shown in the following table.
The relative humidity was above 92% almost 80 percent of the time for the bottom sensors
compared to 48% of the time for the dryland sensors. The difference decreased at the sensor
stations higher in the canopy indicating there was a stratification effect induced by the
watering schedule. It is interesting to note that the top sensor, which is at head height, is
above 92% relative humidity about 50% of the time in the watered plots and 35% in the
dryland plots.
FHB Infection Rates
The objective of this project was to make sure the all the plots had an equal chance for
infection and that the microclimate was conducive to the growth of FHB. The level of infection in each plot was measured by taking 30 wheat heads and using a standardized scale to
rate the severity of infection. The untreated checks in the watered plots had FHB field severity that ranged from 12 to 36 percent with an average of 30%. These levels provided a sufficient infection rate to evaluate fungicide treatments without an overwhelming amount of
FHB. The field severity levels in inoculated dryland plots in adjacent research areas south of
the watered plots (planted with the same variety of wheat) was about 2%.
DISCUSSION
Although the watering system and watering protocol successfully created the microclimate
for the growth of FHB, limitations need to be addressed. The dryland plots, (part of the
fungicide trials where two remote monitor stations were located) were not inoculated with
FHB like the misted plots. We were not able to evaluate the growth of FHB in inoculated
watered plots compared to inoculated dryland plots within the confines of this study. We did
not have a leaf wetness sensor in the dryland plots and therefore had to infer the time of
head wetness. We did not measure the amount of the time the sprinkler system was on and
therefore could not pick out the periods when natural conditions were favorable for the
growth of FHB.
REFERENCES
Francl, L. 2001. Wheat disease forecasting system. NDSU Crop Disease website (www.ag.ndsu.nodak.edu/
cropdisease/wheat/scabmgmt.htm).
McMullen, M. 2001 Personal Communication.
Nelson, G. 2000. Seed distribution, study, Fusarium nursery and general wheat support - Morris. Summary on
the Minnesota Association of Wheat Growers small grains website (www.smallgrains.org).
Scherer, T.F., V.L. Hofman, S. Halley and M. McMullen. 2000. Design of a microsprinkler system for Fusarium
head blight (scab) research of wheat and barley. Paper RRV00-2003 presented at North Central Intersectional
Meeting of the ASAE, Fargo, ND. Sept. 29-30. 8 pgs.
Warnes, D.D. 1995, Management options for reducing severity of wheat scab. Summary on the Minnesota
Association of Wheat Growers small grains website (www.smallgrains.org).
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2002 National Fusarium Head Blight Forum Proceedings
USDA-ARS, OHIO STATE UNIVERSITY COOPERATIVE RESEARCH
ON BIOLOGICALLY CONTROLLING FUSARIUM HEAD BLIGHT 1:
DISCOVERY AND SCALE-UP OF A FREEZE-DRYING PROTOCOL
FOR BIOMASS OF ANTAGONISTCRYPTOCOCCUS
NODAENSIS OH 182.9 (NRRL Y-30216)
D.A. Schisler1*, J.E. VanCauwenberge1, and M.J. Boehm2
1
National Center for Agricultural Utilization Research, USDA-ARS, Peoria, IL 61604; and
2
Dept. of Plant Pathology, The Ohio State University, Columbus, OH 43210
*Corresponding Author: PH: (309) 681-6284; E-mail: schislda@ncaur.usda.gov
OBJECTIVES
To 1) identify cryoprotectant compounds and quantities of these that would enhance the
shelf-life of freeze-dried biomass of OH 182.9 and 2) evaluate the propensity of a superior
cryoprotectant compound to enhance shelf-life and maintain efficacy of OH 182.9 inoculum
produced using precommercial, 100-L fermentor environments.
INTRODUCTION
Fusarium head blight (FHB), primarily incited by Gibberella zeae, can be a devastating
disease of wheat and barley in humid and semi-humid regions of the world. In previous
research, we have demonstrated the potential of several biological control agents to significantly reduce the severity of FHB in greenhouse and field environments (Schisler et al.,
2002). A critical step in producing a commercially available biocontrol product is devising
procedures for stabilizing biomass of the biological agent while maintaining product efficacy.
A product comprised of frozen biomass of our yeast antagonist Cryptococcus nodaensis OH
182.9 was developed and tested at over 15 field sites as part of the U.S. Wheat and Barley
Scab Initiative in the 2001 field season (Schisler et al., 2001, Milus et al, 2001). Though this
product significantly reduced FHB, the development of a dried biocontrol product would
have potential advantages of convenience, ease of handling, favorable economics, and
consumer acceptance. However, dehydration of antagonist biomass can adversely affect its
viability and efficacy.
MATERIALS AND METHODS
Eight cryoprotectant compounds (Fig. 1) were added separately at 25mM to semi-defined
complete liquid medium (SDCL, Slininger et al., 1994), and shake-flask cultures of OH
182.9 initiated. Flasks were maintained at 250 rpm and 25°C for 96 h. Two milliliter aliquots
of colonized broth were placed in 5 ml vials, freeze-dried for 48 h in a 6-L tray freeze-dryer,
and stoppered under vacuum at a final temperature of 4°C. Colony forming units per milliliter (CFU/ml) were determined prior to freeze-drying and for rehydrated freeze-dried products
stored at 24°C for 0, 8 and 37 days.
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The effect of 1 mM to 100 mM concentrations of melezitose (a trisaccharide composed of
two molecules of glucose and 1 molecule of fructose) on OH 182.9 survival and stability
after freeze-drying was studied by adding melezitose and/or 10% (w/v) skim milk to washed
biomass from 48 h shake-flask cultures (Fig. 2). The CFU/ml were determined prior to
freeze-drying and after product storage at 24°C for 0, 6, 13, and 21 days.
Yeast antagonist OH 182.9 was then produced in a B Braun D-100 fermentor charged with
80 L of SDCL medium. To initiate a production run, cells from a log-growth stage SDCL
culture served as a 5% seed inoculum for the D-100 fermentor. Reactor medium pH, temperature, dissolved O2, antifoam dose, and agitation rate were monitored and/or maintained
to insure near identical production runs. After completion of biomass production at approximately 48 h, colonized reactor broth was concentrated into a paste using a Sharples 12-V
tubular bowl centrifuge. The paste was resuspended using buffer containing 25 mM melezitose and 1% skim milk. The cell suspension was then freeze-dried in a 24-L tray freeze
dryer for 48 h and vacuum sealed in mylarfoil bags. The CFU/ml were determined prior to
freeze-drying and after product storage at 4°C for 0, 3, 10, 14, 21, 28, 35 and 42 days (Fig.
3). The effect of the freeze-dried product, freshly produced OH 182.9 cells, and
cryoprotectants alone on FHB severity was determined in greenhouse bioassays after 0, 10
and 28 days storage (data not shown).
RESULTS AND DISCUSSION
Melezitose is characterized, for the first time, as an effective cryoprotectant (Fig. 1). Melezitose and turanose were the most effective in enhancing the survival of freeze-dried biomass
of FHB antagonist C. nodaensis OH 182.9 compared to six other cryoprotectants found to be
effective when drying biomass of other microorganisms.
Melezitose was effective in extending the shelf-life OH 182.9 at 100 mM and 50 mM concentrations but was not at concentrations of 10 mM and lower (Fig. 2). Amending biomass of
OH 182.9 with 10% skim milk was effective in combination with melezitose or alone in
extending OH 182.9 shelf-life.
The precommercial process of producing OH 182.9 biomass in a 100-L fermentor, separating cells from broth using a tubular bowl centrifuge, resuspending the biomass in a solution
containing 25 mM melezitose and 1% skim milk, and freeze-drying the product in a 24-L tray
freeze-drier produced a product that lost more than a log unit of CFU’s during processing
and freeze-drying but then maintained nearly constant CFU’s over the next five weeks (Fig.
3).
Though cell survival of the precommercial product was satisfactory after freeze-drying (Fig.
3), the biocontrol efficacy of this product was less than that of similar concentrations of
freshly produced of OH 182.9 cells in greenhouse bioassays with high disease pressure
(data not shown). A portion of the failure of the freeze-dried product to control disease
appears to be due to 25 mM melezitose and 1% skim milk enhancing disease (data not
shown). Alternative drying methodologies such as air, fluidized bed or spray-drying may be
required to produce a dried OH 182.9 biocontrol product that maintains biocontrol efficacy.
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2002 National Fusarium Head Blight Forum Proceedings
ACKNOWLEDGMENTS
This project was made possible, in part, by funding provided by the U.S. Wheat and Barley
Scab Initiative.
REFERENCES
Milus, E.A., Hershman, D., and McMullen M. 2001. Analysis of the 2001 Uniform Wheat Fungicide and
Biocontrol Trials across locations. Pages 75-79 in: 2001 National Fusarium Head Blight Forum Proceedings.
Kinko’s, Okemos, MI. or see http://www.scabusa.org
Schisler, D.A., Khan, N.I, and Boehm, M.J. 2002. Biological control of Fusarium head blight of wheat and
deoxynivalenol levels in grain via use of microbial antagonists. Pages 53-69 in: Mycotoxins and Food Safety.
J.W. DeVries, M.W. Trucksess, and L.S. Jackson, eds., Kluwer Academic/Plenum Publishers, New York.
Schisler, D.A., Khan, N.I, Iten, L.B., and Boehm, M.J. 2001. USDA-ARS, Ohio State University cooperative
research on biologically controlling Fusarium head blight: pilot-plant-scale production and processing of biomass
of yeast antagonists. Pages 87-90 in: 2001 National Fusarium Head Blight Forum Proceedings. Kinko’s,
Okemos, MI. or see http://www.scabusa.org
Slininger, P. J., Schisler, D. A., and Bothast, R. J. 1994. Two-dimensional liquid culture focusing: A method of
selecting commercially promising microbial isolates with demonstrated biological control capability. Pages 2932 in: Improving Plant Productivity with Rhizosphere Bacteria. M. H. Ryder, P. M. Stephens, and G. D. Bowen,
eds. 3rd International Workshop on Plant Growth-Promoting Rhizobacteria, Adelaide, S. Australia. Graphic
Services, Adelaide, Australia. CSIRO Division of Soils: Glen Osmond.
Log 10 CFU/ml
of Rehydrated Product
11
Arginine
Betaine
Control
Maltose
Turanose
Sucrose
Melezitose
Trehalose
Proline
9
7
5
3
*0
8
37
Days After Freeze-Drying
*F re s h C F U /m l c o u n t ju s t p rio r to in itia tin g a tw o -d a y fre e z e -d ry in g p ro c e s s .
F ig u re 1. In flu e n c e o f c ry o p ro te c ta n ts a d d e d to liq u id p ro d u c tio n m e d iu m in s h a k e -fla s k s o n th e
s u rviva l o f fre e z e -d rie d b io m a s s o f F H B a n ta g o n is t C ry p to c o c c u s n o d a e n s is O H 1 8 2 .9 s to re d a t 2 4 °C .
Chemical and Biological Control
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2002 National Fusarium Head Blight Forum Proceedings
Log 10 CFU/ml
of Rehydrated Product
10
8
6
4
Buffer
Buffer + 10S
1 mM Mz
2
*
10 mM mZ
100 mM mZ
10 mM Mz + 10S
0
6
50 mM Mz
50 mM Mz + 10S
1 mM Mz = + 10S
13
20
Days After Freeze-Drying
*F res h C F U /m l c ou n t ju st p rio r to initiatin g a tw o-d ay fre e ze -dry in g p ro ce ss .
F ig u re 2. In flu e nc e o f a d ding va rio u s c on ce n tra tion s o f m e le zito se (M z ) an d 1 0% s kim m ilk
(10 S ) to s ha k e-fla s k-p rod uc e d, w a sh ed b io m a s s o f O H 1 8 2.9 on the viab ility o f fre ez e-d ried
ce lls sto red a t 2 4 °C .
Log 10 CFU/ml
of Rehydrated Product
10
Freeze-dried cells
Freshly produced cells
6
4
2
*0
3
10 14
21
28
35
42
Days After Freeze-Drying
*F re sh C F U /m l c o un t ju st p rio r to initia tin g a tw o -d a y fre e ze -d ry in g pro ce ss .
F ig u re 3. S urvival of free ze -dried bio m a ss o f F H B a ntag o nis t C ryp toc oc c us n o da en s is O H
18 2 .9 a m e nd ed w ith 25 m M m e le zitos e a nd 1% s kim m ilk after p rod uc tion in a 10 0 L
ferm e ntor a n d stora ge a t 4 °C .
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2002 National Fusarium Head Blight Forum Proceedings
USDA-ARS, OHIO STATE UNIVERSITY COOPERATIVE RESEARCH
ON BIOLOGICALLY CONTROLLING FUSARIUM HEAD BLIGHT 2:
2002 FIELD TESTS OF ANTAGONIST AND ANTAGONIST/
FUNGICIDE MIXTURES
D.A. Schisler1*, M.J. Boehm2, T.E. Hicks2, and P.E. Lipps3
1
National Center for Agricultural Utilization Research (NCAUR), USDA-ARS, Peoria, IL 61604;
2
Dept. of Plant Pathology, The Ohio State University, Columbus, OH 43210; and
3
The Ohio State University/OARDC, Dept. of Plant Pathology, Wooster, OH 44691
*Corresponding author: PH: (309) 681-6284, E-mail: schislda@ncaur.usda.gov
OBJECTIVE
Determine the effect of FHB antagonists, antagonist mixtures, and mixtures of antagonists
and fungicides on FHB symptom development in field tests conducted in Illinois and Ohio
on two cultivars of winter wheat.
INTRODUCTION
Fusarium head blight (FHB) is an important disease of wheat and barley in humid and
semihumid regions of the world (McMullen et al., 1997). Research on optimizing methods
for selectively isolating, mass producing and utilizing microbial antagonists effective against
FHB was initiated in 1997 at the NCAUR in Peoria, IL, in conjunction with The Ohio State
University. Several biological control agents remain under consideration for commercial
development (Schisler et al., 2002). In addition to biological control, promising possibilities
for reducing Fusarium head blight include fungicides and resistant cultivars. Combining
these control measures may provide levels of control superior to that obtained when employing these control measures individually. Disease control measures utilized in various
combinations in field tests conducted in Peoria, Illinois and Wooster, Ohio during the 2002
field season included biocontrol agents, a moderately resistant wheat cultivar, and fungicides.
MATERIALS AND METHODS
A naturally occurring fungicide-tolerant (FT) variant of superior yeast antagonist Cryptococcus nodaensis OH 182.9 (wild type (WT)) was selected from cultures grown in one-fifth
strength Tryptic soy broth amended with 50 ppm of the fungicide BAS 505 50DF. Inoculum
of OH 182.9 WT, OH 182.9 FT and Bacillus subtilis OH 131.1 was produced using a
semidefined liquid culture medium (SDCL) with a carbon:nitrogen ratio of 11 and total
carbon loading of 15 g carbon/liter (Schisler et al., 2002). The soft red winter wheat cultivars
Pioneer 2545 (susceptible) and Freedom (moderately resistant) were used in both locations.
Biomass was harvested from Fernbach shake flasks and applied at the beginning of wheat
flowering (Schisler et al., 2002). Bacterial and yeast suspensions contained 50 % fully
colonized broth (~1x108 CFU/ml and ~5 x 107 CFU/ml, respectively) and were applied at a
rate of 20 gal/acre. The fungicides BAS 505 50DF and Folicur 3.6F were applied at recomChemical and Biological Control
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2002 National Fusarium Head Blight Forum Proceedings
mended rates singly and in combination with microbial treatments (Tables 1 and 2). Controls were untreated plants and plants treated with buffer/wetting agent only. Corn kernels
colonized by Gibberella zeae (Schisler et al., 2002) were scattered through plots (~25-40
kernels/m2) two weeks prior to wheat flowering and mist irrigation provided periodically for
approximately one week after treatment application to promote FHB development. Heads
were scored for disease incidence (presence or absence of disease symptoms) and severity
using a 0-100% scale approximately three weeks after inoculation. Heads were then allowed to dry and threshed. Data for the deoxynivalenol content of grain and 100 kernel
weight is being tabulated (ongoing). Randomized complete block designs were used in
both trials (n=4 in Peoria; n=5 in Wooster).
RESULTS AND DISCUSSION
In Peoria, IL, most single and combination treatments reduced FHB symptoms versus at
least one control on both susceptible cultivar Pioneer 2545 and moderately resistant cultivar
Freedom (Table 1). A combination of yeast OH 182.9 FT and BAS 505 reduced disease
severity by 70% compared to the untreated Freedom control. Combined biological control
agent or biocontrol agent and fungicide treatments did not synergistically interact to reduce
disease to a greater extent than the component parts of the combinations.
In Wooster, OH, on Pioneer 2545, most treatments reduced disease severity compared to
the untreated control with the most effective treatments of BAS 505, OH182.9FT+BAS 505,
OH182.9FT + Folicur and OH131.1+Folicur reducing disease severity by as much as 64%
(Table 2). Treatments did not differ when tested on cultivar Freedom.
Across both locations, the lowest levels of FHB symptom development were found when
two and sometimes three of the available control measures of antagonists, fungicides and
the moderately resistant cultivar were combined. While methodologies for drying biomass
require further development before fresh and dried preparations of OH 182.9 achieve
equivalent efficacy, these results indicate that biocontrol products could play a key role in
the integrated control of FHB.
ACKNOWLEDGMENTS
This project was made possible, in part, by funding provided by the U.S. Wheat and Barley
Scab Initiative.
REFERENCES
McMullen, M., Jones, R., and Gallenberg, D. 1997. Scab of wheat and barley: A re-emerging disease of
devastating impact. Plant Dis. 81:1340-1348.
Schisler, D.A., Khan, N.I., Boehm, M.J., and Slininger, P.J., 2002, Greenhouse and field evaluation of biological
control of Fusarium head blight on durum wheat. Plant Dis. 86:(in press).
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T able 1. 2 0 0 2 field trial re su lts at P eo ria, Illin o is: In flu en ce o f C ryp to co ccu s n o d a en sis
O H 1 8 2 .9 , B a cillu s su b tilis O H 1 3 1 .1 , B A S 5 0 5 5 0 D F , F o licu r 3 .6 F an d co m b in atio n s
th ereo f o n F H B d isease sev erity an d in cid en ce o n tw o cu ltiv ars o f w in ter w h ea t1
W h eat C u ltiv ar
F ree d o m
T rea tm e n t
U n treated co n tro l
B u ffer/tw een 2
B A S 505
3
F o licu r3
O H 1 8 2 .9 W T 4 ,5
P io n eer
2545
% D isease
%
S ev erity
In cid en ce
1 .6
7 .5
% D isease
S ev erity
4 .1
%
In cid en ce
1 9 .6
3 .0
1 5 .0
4 .1
1 5 .0
1 .4
7 .1
1 .3
3 .8
1 .6
9 .6
3 .5
1 2 .1
1 .7
9 .2
2 .5
9 .2
4
3 .0
1 4 .6
0 .8
4 .2
O H 1 8 2 .9 W T + B A S 5 0 5
1 .8
1 0 .8
0 .8
2 .5
O H 1 8 2 .9 W T + F o licu r
4 .0
2 0 .4
3 .1
1 1 .2
O H 1 8 2 .9 F T + B A S 5 0 5
1 .2
6 .2
1 .0
3 .8
O H 1 8 2 .9 F T + F o licu r
2 .2
1 1 .2
2 .8
8 .3
2 .6
1 2 .9
2 .3
9 .2
O H 1 3 1 .1 + B A S 5 0 5
1 .8
9 .6
1 .0
3 .3
O H 1 3 1 .1 + F o licu r
3 .3
1 6 .7
2 .5
7 .9
O H 1 8 2 .9 W T + O H 1 3 1 .1
2 .4
1 2 .9
2 .8
1 1 .2
O H 1 8 2 .9 F T + O H 1 3 1 .1
2 .2
1 1 .2
2 .7
1 0 .8
O H 1 8 2 .9 F T + O H 1 3 1 .1 + B A S
3 .2
1 3 .8
1 .0
3 .8
O H 1 8 2 .9 F T + O H 1 3 1 .1 + F o l
3 .7
1 7 .9
3 .8
1 2 .9
L SD
1 .3
6 .0
1 .5
4 .8
O H 1 8 2 .9 F T
O H 1 3 1 .1
(0 .05)
5
1
W ith in a co lu m n , th e L S D v alu e rep resen ts th e critical v alu e fo r se p aratin g trea tm e n t
m ean s at th e P # 0 .0 5 lev el. D isease sev erity v alu es are arc sin e tran sfo rm ed .
2
W eak P O 4 b u ffer (S ch isler et al., 2 0 0 2 ) an d 0 .0 3 6 % T w een 8 0 .
3
A p p lied at reco m m en d ed lab el rates.
4
W T = w ild ty p e o f strain , F T = F u n g icid e to leran t n atu ral v arian t o f stra in
5
O H 1 8 2 .9 W T an d F T C F U /m l ~ 5 x 1 0 7 , O H 1 3 1 .1 C F U /m l ~ 1 x 1 0 8
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2002 National Fusarium Head Blight Forum Proceedings
T able 2. 2 0 0 2 field trial resu lts at W o o ster, O h io : In flu en ce o f C ryp to co ccu s n o d a en sis
O H 1 8 2 .9 , B a cillu s su b tilis O H 1 3 1 .1 , B A S 5 0 5 5 0 D F , F o licu r 3 .6 F an d co m b in atio n s
th ereo f o n F H B d isease sev erity an d in cid en ce o n tw o cu ltiv ars o f w in ter w h ea t1
W h eat C u ltiv ar
F ree d o m
T rea tm e n t
P io n eer
2545
% D isease
%
S ev erity
In cid en ce
2 0 .4
6 3 .8
% D isease
S ev erity
2 .6
%
In cid en ce
2 5 .0
B u ffer/tw een 2
3 .7
3 0 .7
1 6 .8
5 7 .9
B A S 5053
3 .0
2 5 .0
7 .4
3 2 .1
2 .5
2 5 .3
1 2 .9
4 8 .3
2 .5
2 3 .7
2 1 .4
6 5 .4
O H 1 8 2 .9 F T 4
2 .8
2 8 .0
1 2 .1
4 6 .7
O H 1 8 2 .9 W T + B A S 5 0 5
2 .1
2 1 .3
1 2 .4
4 6 .7
O H 1 8 2 .9 W T + F o licu r
2 .7
2 2 .7
1 3 .4
5 0 .8
O H 1 8 2 .9 F T + B A S 5 0 5
2 .2
2 3 .0
8 .6
3 0 .4
O H 1 8 2 .9 F T + F o licu r
2 .0
1 8 .7
9 .7
3 9 .2
2 .3
2 2 .0
1 4 .9
5 2 .5
O H 1 3 1 .1 + B A S 5 0 5
3 .3
2 3 .3
1 2 .7
4 6 .7
O H 1 3 1 .1 + F o licu r
2 .5
2 2 .7
9 .5
4 2 .5
O H 1 8 2 .9 W T + O H 1 3 1 .1
3 .4
2 6 .0
1 3 .2
5 1 .2
O H 1 8 2 .9 F T + O H 1 3 1 .1
2 .3
2 3 .0
1 5 .4
5 5 .0
O H 1 8 2 .9 F T + O H 1 3 1 .1 + B A S
2 .5
2 2 .0
1 1 .3
4 2 .1
O H 1 8 2 .9 F T + O H 1 3 1 .1 + F o l
2 .4
2 2 .3
1 3 .1
5 2 .9
N SD
N SD
2 .8
8 .8
U n treated co n tro l
3
F o licu r
O H 1 8 2 .9 W T
O H 1 3 1 .1
L SD
4 ,5
5
(0 .05)
1
W ith in a co lu m n , th e L S D v alu e rep resen ts th e critical v alu e fo r se p aratin g trea tm e n t
m ean s at th e P # 0 .0 5 lev el. D isease sev erity v alu es are arc sin e tran sfo rm ed .
2
W eak P O 4 b u ffer (S c h isler et al., 2 0 0 2 ) an d 0 .0 3 6 % T w een 8 0 .
3
A p p lied at reco m m en d ed lab el rates.
4
W T = w ild ty p e o f strain , F T = F u n g icid e to leran t n atu ral v arian t o f stra in
5
O H 1 8 2 .9 W T an d F T C F U /m l ~ 5 x 1 0 7 , O H 1 3 1 .1 C F U /m l ~ 1 x 1 0 8
Chemical and Biological Control
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2002 National Fusarium Head Blight Forum Proceedings
EVALUATION OF FUNGICIDES FOR THE CONTROL OF FUSARIUM
HEAD BLIGHT AND LEAF DISEASES ON ‘ELKHART’ AND
‘PIONEER VARIETY 2540’ WINTER WHEAT IN MISSOURI
L. E. Sweets
Dept. of Plant Microbiology and Pathology, University of Missouri, Columbia, MO 65211
Corresponding Author: PH: (573) 884-7307; E-mail: SweetsL@missouri.edu
OBJECTIVES
To identify fungicides and biological control products that are effective in minimizing the
damage from Fusarium head blight in winter wheat.
INTRODUCTION
The severity of Fusarium head blight epidemics in the United States has caused enormous
yield and quality losses in wheat and barley (McMullen, et al., 1997). The development of
this disease is dependent on host genetics, a range of favorable environmental conditions,
the prevalence of the causal fungus and the survival and spread of the cause fungus
(Sutton, 1982). Control of this disease has been difficult because of the complex nature of
the host/pathogen interaction. In addition to the development of varieties with resistance to
Fusarium head blight, research focusing on fungicide and biological treatments for the
management of Fusarium head blight has been pursued.
In 1998, a Uniform Fungicide Trial was conducted across seven states (McMullen, 1998),
which provided data on efficacy of five products or product combinations in reducing
Fusarium head blight when applied at heading. This Uniform Fungicide Trial permitted
evaluation of the performance of products across numerous states or sites, wheat classes
and environments. Across the test sites that had substantial Fusarium head blight in 1998,
an average of about fifty percent reduction in Fusarium head blight occurred, as well as a
reduction in DON for most products, plus a substantial reduction in wheat leaf diseases.
The Uniform Fungicide Trial has been continued since 1998 with additional test sites in
more states and changes in products tested as new fungicides and biological control agents
have become available. The Uniform Fungicide Trial continues to provide valuable information on efficacy and performance consistency of standard fungicides, new experimental
fungicides and biological control agents. Missouri has participated in the Uniform Fungicide
Trial since 1998 (Sweets, 2000). Results from the 2002 trial are presented in this report.
MATERIALS AND METHODS
Seven fungicide or biological control treatments and an untreated control were evaluated on
‘Elkhart’ and ‘Pioneer variety 2540’ soft red winter wheats at the Bradford Research Center,
near Columbia, MO. ‘Elkhart’ and ‘Pioneer variety 2540’ were drilled directly into soybean
stubble on 12 Oct 01. The soil type at the site was a Putnam silt loam. The planting rate
was 100-lbs of seed/A. The experimental design for each variety was a randomized complete block with 6 replications. Individual plots were 4.5 ft (7 rows) by 30 ft in length. The
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2002 National Fusarium Head Blight Forum Proceedings
entire plot area was fertilized with 30-lbs/A nitrogen pre-plant followed by 90-lbs/A nitrogen
topdressed in the spring. Treatments were applied with a CO2 backpack sprayer with
nozzles directed towards the heads. Treatments were applied in 400 ml of water. Applications were made at Feeke’s Growth Stage (FGS) 10.51 on 14 May 02. Plots were rated for
foliage diseases on 28 May 02. Ratings were done as estimates of the percentage of leaf
area covered with Septoria leaf blotch or leaf rust on each of 10 flag leaves randomly collected from each plot. Fusarium head blight incidence and head severity measurements
were taken 30 May 02. For harvest the plots were end trimmed and individual plot lengths
measured. Plots were harvested on 25 June 02 with a Wintersteiger plot combine. Test
weight and moisture were determined with a Dickey-John GAC 2000 Grain Analyzer.
Samples were submitted to the Veterinary Diagnostic Services Department at North Dakota
State University for DON analysis. Data was statistically analyzed using ANOVA.
RESULTS AND DISCUSSION
Plants emerged well and early stands were uniform. The 2002 season was warm and dry
early; cool and wet as the wheat was flowering and heading; and then hot and dry as the
crop matured. Septoria leaf spot and leaf rust did not begin to develop until late in the
season. When foliage disease ratings were made, the level of leaf rust was very low across
the trial so only Septoria leaf blotch ratings were recorded. Fusarium head blight was also
in very low levels throughout the plot at the time Fusarium head blight incidence and severity ratings were made. However, the number of heads showing symptoms of Fusarium head
blight seemed to increase as the crop matured. At harvest most plots had noticeable
amounts of shriveled, lightweight kernels or tombstone kernels. Barley yellow dwarf was
prevalent throughout the trial. Low temperatures in May caused head damage across the
plot area. Hail on May 12 damaged plants and heads across the plot area.
The yield of the untreated control was significantly lower than the yields for the seven fungicide and biological control treatments on Pioneer variety 2540. There were no statistically
significant differences in yield between the seven treatments and the untreated control on
Elkhart. Septoria leaf blotch ratings were significantly higher for the untreated control than
the seven fungicide and biological control treatments on both Pioneer variety 2540 and
Elkhart. Septoria leaf blotch ratings were significantly lower with TrigoCor 1448 + Folicur
3.6F + Induce on Pioneer variety 2540 and with TrigoCor 1448 + Folicur 3.6F + Induce and
Folicur 3.6F + Induce on Elkhart. Although there were no statistically significant differences
between the untreated control and any of the seven treatments for percent of Fusarium head
blight incidence, percent average head severity, percent field severity or percent of scabby
kernels on Pioneer variety 2540, the untreated control was at the high end of the range for
each of these. The AMS 21619A 480SC + Folicur 3.6F + Induce treatment tended to be at
the low end of the range for percent Fusarium head blight incidence, percent average head
severity and percent field severity. The two AMS 21619A treatments had significantly lower
levels of DON than the untreated control and the other five treatments. On Elkhart there
were statistically significant differences between treatments for percent Fusarium head
blight incidence, percent average head severity, percent field severity, percent scabby
kernels and DON levels. The two AMS 21619A treatments had consistently low ratings for
all of these variables with the combination of AMS 21619A 480SC + Folicur 3.6F + Induce
performing slightly better than the AMS 21619A 480SC + Induce. The OH189.2 biological
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2002 National Fusarium Head Blight Forum Proceedings
control agent had the most variation in results. The OH189 treatment had low percent of
Fusarium head blight incidence, moderate percent of average head severity and percent of
field severity but high percent of scabby kernels and DON levels compared to the other
treatments. The untreated control for Elkhart had the highest percent of Fusarium head
blight incidence, percent average head severity and percent of field severity and among the
highest percent of scabby kernels and DON levels.
REFERENCES
McMullen, M., Jones, R., and Gallenberg, D. 1997. Scab of wheat and barley: a re-emerging disease of
devastating impact. Plant Disease 81: 1340-48.
McMullen, M. 1998. Fungicide technology network of the National FHB Initiative- 1998 Report. Pages 47-49 in:
Proceedings of the 1998 National Fusarium Head Blight Forum, Michigan State Univ., East Lansing, MI.
Sutton, J. C.1982. Epidemiology of wheat head blight and maize ear rot caused by Fusarium graminearum.
Can. J. Plant Pathol.4:195-209.
Sweets, L.E. 2000. Evaluation of fungicides for control of Fusarium head blight and leaf diseases on wheat,
1999. Fungicide and Nematicide Tests 55:357.
T a b le 1. E lk ha rt
T rea tm e nt an d R a te/A
U ntre ate d c on trol
F olicu r 3 .6F 4 .0 fl oz + In du ce 0 .1 25 % v/v
A M S 2 16 1 9A 4 80 S C 5.7 flo z + In d uce
0 .1 25 % v/v
B A S 5 05 F 5 0W G 6.4 o z + In duc e 0 .1 2 5%
v/v
O H 1 82 .9 ~5 x 10 e8 cfu/m l
T rigo C o r 14 48 ~ 7 .5 x 10 ^12^cfu/A
T rigo C o r 14 48 ~7 .5 x 10 ^12^cfu /A +
F olicu r 3 .6F 4 .0 fl oz + In du ce 0 .1 25 % v/v
A M S 216 19 A 48 0S C 3 .6 fl oz +
F o licur 3 .6 F 4.0 fl o z + Ind u ce 0 .1 25 % v/v
L S D (P = 0 .05 )7
% A ve . % F ie ld % S cab D O N
Y ie ld 1 T es t W t. S L B
bu/A
(lb /bu ) R a tin g 2 % F H B 3 H ead S e v.4 S e v.5 K ernels 6 pp m
4 2.8
6 1.0
3 .6 2
18 .3 3
12 .0 5
2 .1 2
1 0.7
2 .2 3
4 2.3
6 1.6
0 .1 7
8 .3 3
5 .0 8
0 .6 8
7.9
1 .6 8
4 5.8
6 1.8
0 .2 7
1 .6 7
1 .1 7
0 .1 2
7.0
1 .1 8
4 5.2
4 4.1
4 2.7
6 1.4
6 1.0
6 0.6
0 .5 2
0 .1 8
0 .5 3
3 .3 3
1 .6 7
3 .3 3
1 .1 7
2 .3 3
2 .3 3
0 .2 3
0 .2 3
0 .2 3
9.1
1 1.8
1 1.0
1 .7 7
2 .2 8
1 .9 0
4 4.4
6 0.4
0 .1 7
8 .3 3
6 .8 3
0 .6 8
9.6
1 .5 3
4 5.0
6 2.1
0 .2 3
0 .0 0
0 .0 0
0 .0 0
7.8
1 .1 0
NS
NS
0 .8 6
6 .8 1
4 .3 5
0 .6 2
2.9
0 .3 6
1
Y ie ld ba se d on 6 0-po und b u she l w e ig h t a djusted to 1 3 % m oistu re con tent
2
S LB ratin g o r S epto ria le a f b lo tch rating b ased o n th e average % of flag le a f sh o w in g sy m p tom s for 10 flag le ave s.
3
% F H B o r p erce nt of F usariu m he a d b lig ht in c id e n ce b ase d o n % o f h ea d s sho w ing sym ptom s for 5 0 h e ad s.
4
% a ve. h ea d sev o r pe rce n t of av era ge h ea d se ve rity b a se d on % of hea d sho w ing F H B sym ptom s for 5 0 h e ad s.
% field s ev or p ercen t field severity ca lc u la ted us ing the fo rm ula (% F H B x % a ve. h e a d se v.)/1 00.
6
% sca b ke rnels or p e rce n t sca b by kerne ls ba se d on % s ca bb y k e rn e ls in a 2 00 kerne lsa m p le.
5
7
D a ta w a s a na lyze d by A N O V A w ith m ea ns se pa rated b y LS D a t P =0 .05.
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2002 National Fusarium Head Blight Forum Proceedings
T able 2.P io ne e r v a rie ty 2 5 40
T rea tm e nt a nd R a te /A
U ntre a ted co n trol
F olicu r 3 .6F 4 .0 fl o z + In du ce 0 .1 2 5 % v/v
A M S 2 1 6 1 9A 48 0 S C 5.7 flo z + Ind u ce
0 .1 2 5 % v/v
B A S 5 05 F 5 0 W G 6 .4 o z + In duc e 0 .1 2 5 %
v/v
O H 1 82 .9 ~5 x 1 0 e 8 cfu /m l
T rig o C o r 1 4 48 ~ 7.5 x 1 0 ^1 2 ^cfu /A
% A ve. % F ie ld % S c ab D O N
Y ie ld 1 T e s t W t. S L B
b u /A
(lb /b u ) R a ting 2 % F H B 3 H ea d S e v.4 S e v.5 K ern els 6 p p m
5 1.8
6 0.2
4 .40
1 0 .0 0
4 .88
0 .84
1 2.7
2 .17
5 6.8
6 0.6
1 .12
6 .67
4 .50
0 .45
1 0.8
1 .62
5 9.4
6 1.0
1 .07
5 .00
7 .00
0 .70
1 1.5
1 .08
6 0.9
5 4.1
5 3.5
6 1.1
6 0.5
6 0.4
0 .97
1 .25
1 .33
6 .67
3 .33
5 .00
3 .60
2 .33
1 .55
0 .88
0 .23
0 .46
9.5
1 0.4
1 1.9
1 .72
1 .72
2 .05
T rigo C o r 14 4 8 ~7 .5 x 10 ^12 ^ cfu /A +
F olicu r 3 .6F 4 .0 fl o z + In du ce 0 .1 2 5 % v/v
5 6.1
6 0.6
0 .80
5 .00
2 .77
0 .44
9.8
1 .63
A M S 21 6 19 A 4 8 0S C 3 .6 fl o z +
F olicu r 3 .6F 4 .0 fl o z + In du ce 0 .1 2 5 % v/v
5 7.6
6 0.8
1 .18
1 .67
2 .00
0 .20
1 1.0
1 .17
NS
NS
NS
0 .26
4.6
NS
0 .67
NS
LS D (P = 0 .0 5 )7
1
Y ield b a se d o n 60 -p o un d b u s he lw eigh t a dju sted to 1 3 % m o is tu re c on ten t
2
S LB ratin g o r S e pto ria le af b lo tch ra tin g b as ed o n th e ave rag e % o f fla g lea f s h o w in g sy m ptom s for 1 0 fla g le av e s.
3
% F H B o r p e rce n t o f F us a riu m h e ad b lig ht in cid en ce b a s ed on % o fh e ad s sh o w in g sym p to m s for 50 he a d s.
% a ve . he a d s ev o r p e rce n to f av e rag e h ea d se verity ba sed on % o f he a d s ho w in g F H B sym p to m s for 50 he a d s.
5
% field sev or p e rc en t fie ld s e verity c a lc u la te d u s ing the fo rm ula (% F H B x % a v e. h ea d se v.)/1 00 .
4
6
% sca b ke rn els or p erce nt sc a bb y ke rne ls ba se d on % s ca b b y k e rne ls in a 2 00 k ern e ls am p le .
7
D a ta w a s a na lyzed b y A N O V A w ith m e a n s se para ted b y L S D a t P =0 .05 .
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REPORT ON INDUCED RESISTANCE AND FIELD BIOLOGICAL
CONTROL OF FUSARIUM HEAD BLIGHT BY
LYSOBACTER ENZYMOGENES STRAIN C3
Gary Yuen* and C. C. Jochum
Department of Plant Pathology, University of Nebraska- Lincoln, Lincoln, NE 68583-0722
*Corresponding Author: PH: (402) 472-3125; E-mail: gyuen1@unl.edu
ABSTRACT
The bacterial biocontrol agent Lysobacter enzymogenes strain C3 was previously reported
to be effective in field tests against a number of fungal pathogens in turfgrass and against
rust in common bean. C3, when applied as a chitin broth culture, also inhibited leaf rust
(Puccinia triticina), spot blotch (Bipolaris sorokiniana), and Fusarium head blight (FHB)
(Fusarium graminearum) on wheat in laboratory and greenhouse experiments. Chitinolysis
was one mechanism by which C3 suppressed a number of pathogens. Induced resistance
involving a heat stable elicitor also is a mechanism in the control of Bipolaris sorokiniana by
C3. One objective in this study was to determine if induced resistance could be involved in
the control of FHB by C3. Another objective was to assess the potential for using C3 to
control FHB under field conditions. Induced resistance was investigated in greenhouse
experiments in which chitin broth culture of C3 was compared with a culture heated to 70°C
for 20 minutes, and with a distilled water control. The heat treatment was intended to kill C3
cells and inactivate lytic enzymes excreted into the broth, but leave the elicitor intact. All
treatments were sprayed onto wheat heads 1 day prior to inoculation with pathogen conidia.
Both C3 treatments significantly reduced scab infection as compared to the distilled water
check, suggesting that FHB inhibition could be due to induced resistance. A field test was
conducted at South Dakota State University in collaboration with Yue Jin to evaluate the
interaction of C3 and spring wheat genotypes in the control of FHB. Three treatments (C3
chitin broth culture, Folicur, and water) were applied at anthesis to four cultivars (Alsen,
Ingot, Russ, and Norm) that differ in susceptibility to FHB. Plots were inoculated with suspensions of pathogen conidia and misted at night to favor development. Disease severity in
three of the four cultivars was reduced by Folicur. C3 significantly reduced FHB severity in
‘Russ’ (39% infected spikelets) as compared to the control (48% infected spikelets), but had
no effect on disease development in the other cultivars. The highest levels of FHB occurred
in ‘Russ’, and thus, lack of C3 efficacy in the other cultivars could be explained in part by
low disease development. Differential C3 activity on different cultivars also is a possible
explanation. C3 colonized wheat heads and increased in numbers to the same extent on all
of the cultivars. This suggests that C3-cultivar interactions may be related to induced resistance rather than antagonism.
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INFLUENCE OF CROP ROTATION AND COVER CROP ON
FUSARIUM HEAD BLIGHT OF WHEAT
H.U. Ahmed1, J. Gilbert2, W.G.D. Fernando1*, A. Brûlé-Babel1,
A. Schoofs1 and M. Entz1
Department of Plant Science, University of Manitoba, Winnipeg, MB, R3T 2N2; and
Cereal Research Centre, Agriculture and Agri-Food Canada, Winnipeg, MB, R3T 2M9
*Corresponding Author: PH: (204) 474-6072; E-Mail: D_Fernando@Umanitoba.ca
1
2
INTRODUCTION
Fusarium head blight (FHB) is a devastating fungal disease affecting the wheat industry
worldwide (Bai and Shaner, 1994, Gilbert and Tekauz, 2002 and McMullen et al., 1997).
FHB reduces yield and grade and may also contaminate the grain with fungal toxins
(vomitoxins) which make grain unfit as food and feed (Gilbert and Tekauz, 2002). Host plant
resistance has been considered as the most economical and environmentally-friendly
means of disease management. Wheat varieties with sufficient resistance to FHB are not
available for cultivation. Fungicide application is the common practice for the control of FHB.
Resistance to some fungicides in the populations of FHB pathogen has been reported
(Zhou et al, 1994). Of concern too, is the fact that chemicals have long-term environmental
consequences. Therefore, alternative disease management options are needed to meet the
immediate need of wheat growers against FHB. The FHB pathogen overwinters mainly on
wheat stubble. We often recommend manipulations of cultivation practices, including crop
rotation with non-host crops for disease management, as a good rotation would allow
enough time for infested residue to decompose before the next cereal crop is seeded. There
is little information available on long-term research data and the benefits of crop rotation
with non-host crops on FHB disease management. FHB disease initiation starts with landing of ascospores originating from infected crop residue left on soil. Therefore, a cover-crop
would likely act as a barrier to ascospore dispersal onto wheat heads. With these objectives
in mind, two long-term experiments were initiated to determine the effect of crop rotation and
cover crop on FHB disease management in wheat.
MATERIALS AND METHODS
Effect of crop rotation
The experiment was conducted at Carman Field Research Station, Carman, MB where
FHB is known to be endemic. It was initiated in 2001 with the establishment of four foundation crops: canola, wheat, oats and peas on four plots of 10 X 60 M (main plot). The four
main plots were separated by a 30 M strip of fall rye. In 2002, the main plots were divided
into four sub-plots (10 X 15 M) where canola, wheat, oats and peas were grown. The crops
included were canola Liberty Link variety 2663 (transgenic for regular herbicide), oats Riel,
peas Carnival and spring wheat CDC Teal. The crops in the rotation were seeded under
zero tillage conditions on the stubbles of previous years’ foundation crops. In 2002, in the
center of the 30 X 60 M barrier strip of fall rye planted in 2001, canola, wheat, oats and peas
were seeded as foundation crops in 10 X 60 M plot for 2003, creating 10 M-wide barrier
strips between the main plots to avoid inter-plot interference. There are 16 crop rotations in
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this trial to be conducted over the years, allowing us to obtain two identical replicates for
statistical analysis. The crops in the rotation were treated with appropriate herbicides for
weed management. At maturity, the crops were combined and harvested and the stubbles
were spread back into plots.
To estimate daily release of ascospores and macroconidia of Fusarium graminearum, one
rotorod spore sampler (Aerobiology, Nepean, ON) was set up in the center of each main
plot. Two other traps were placed outside of the crop plots to determine the background
inoculum. The rods were changed every 24 hrs. The rods were stored in the cold room until
further analysis.
Three 1-meter row wheat head samples were collected from each wheat plot in the rotation,
and the samples were frozen until disease assessment. Percent disease incidence and
disease severity were determined. FHB disease index was calculated as: % incidence X %
severity/ 100. Before combining the harvest, three samples of six 2.5-meter rows were also
hand harvested for yield, FDK and DON analysis.
Effect of cover crop on FHB
This experiment was conducted at the Point Field Research Station, Winnipeg, Manitoba
following a randomized complete block design with four replications. The treatments were i)
no Fusarium inoculum and no cover crop, ii) Fusarium inoculum but no cover crop, iii) no
Fusarium inoculum but cover crop and iv) Fusarium inoculum and cover crop.
Trifolium pratense L. (red clover) was established as the cover crop treatment in plots about
three weeks before wheat seeding. After seeding clover, all the plots including non-cover
crop treatment plots were harrowed once. Usual agronomic practices were performed as
and when necessary for crop management. Fusarium inoculum treatment plots were inoculated about two weeks before anthesis of wheat by spreading 100g Fusarium-infested corn
inocula/M2 (corn spawn). The plots were irrigated with a boom sprayer in the evening for
three days after inoculation to provide high humidity for perithecia development. In each
plot, two rotorod spore samplers were set up at two levels, one just above the height of
cover crop and the other at wheat head height. Four other spore traps were also placed at
two said heights outside of the plots to monitor background inoculum levels.
Data on dry matter of wheat, cover crop and weeds were recoded four times during the
period of spore trapping. Wheat yield and %FDK were also recorded.
RESULTS AND DISCUSSION
Crop rotation and Fusarium head blight
Overall disease incidence and severity of FHB on wheat was low in 2002. However, results
indicated that percent disease incidence on wheat was the highest in pea-wheat crop
sequence (19.25%) followed by canola-wheat (11.66%), wheat-wheat (10.33%) and oatswheat (8.18%), while the percent disease severity was the highest in canola-wheat
(43.09%) followed by oats-wheat (22.71%), wheat-wheat (18.35%), and peas-wheat
(12.64%) crop rotations (Table 1). The FHB disease index on wheat was in the order canolawheat (5.02), peas-wheat (2.43), wheat-wheat (1.89), and oats-wheat (1.85) crop seEpidemiology and Disease Management
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quences. Fusarium damaged kernel (FDK) or tombstone analysis yielded similar results.
The FDK on canola-wheat, peas-wheat, wheat-wheat and oats-wheat were 6.84%, 6.76%,
5.07 and 4.85%, respectively (Table 1). One would not expect more FHB disease when
wheat was followed by non-host crops such as canola or peas. Our data corroborate results
of other crop rotation trials in Brandon, Manitoba where FHB was also higher when wheat
was followed by canola (personal communications with Debbie McLaren, AAFC, Brandon).
We also observed higher FHB disease incidence and severity in a wheat plot few blocks
away from this experimental plot where canola was grown in the preceding year (personal
observation). In Manitoba, Fusarium graminearum is the dominant species associated with
FHB. It is likely that canola and peas are better substrates to harbor and induce perithecium
or ascospore production of F. graminearum, and this warrants investigation. Furthermore,
canola and peas have a closed canopy and leaf defoliation that likely provided an advantageous environment for the establishment of the pathogen. These results are contrary to
accepted theory that a three-year crop rotation with non-hosts including canola, pulses and
forage legumes will reduce the risk of spreading and increasing the disease. However, DillMacky and Jones (2000) reported that FHB disease incidence was the greatest in cornwheat rotation followed by wheat-wheat and the least in soybean-wheat rotation. Our oneyear data is not enough to enable us to draw any conclusion on the benefit of the crop
rotation with non-host crops like canola and peas.
As expected, yield was reduced when the same crop (i.e. wheat-wheat, or canola-canola)
was planted in two consecutive years (Table 2). Wheat yield was significantly higher when
wheat followed another crop. Bourgeois and Entz (1996) have studied the effect of crop
rotation on wheat yields in Manitoba. They found 11% and 8% higher yield of wheat when
wheat was grown after peas and canola, respectively. A similar report has been posted on
the web by Manitoba Management Plus Program (source- Manitoba Crop Insurance Corporation) (www. mmpp.com/Crop_rotation_page.htm).
Cover crop and Fusarium head blight
Results of this experiment are being tabulated and analyzed at the present time. Yield, FDK
and data on spore trapping will be presented at the meeting.
ACKNOWLEDGEMENTS
We acknowledge and thank Agricultural Research and Development Initiative of Manitoba
for funding this project.
REFERENCES
Bai, G. and Shaner, G. 1994. Scab of wheat: Prospects for control. Plant Dis. 78:670-766.
Bourgeous, L. and Entz, M. H.1996. Influence of previous crop type on yield of spring wheat: Analysis of
commercial field data. Can. J. Plant Science 76:457-459.
Dill-Macky, R. and Jones, R. K. 2000. The effect of previous crop residues and tillage on Fusarium head blight of
wheat. Plant Dis. 84:71-76.
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Gilbert, J. and Tekauz, A. 2000. Recent developments in research on Fusarium head blight of wheat in Canada.
Can. J. Plant Pathol. 22:1-8.
McMullen, M., Jones, R. and Gallenberg, D. 1997. Scab of wheat and barley: A re-emerging disease of devastating impact. Plant Dis. 81:1340-1348.
Zhou M. G., Ye Z. Y. and Liu J. F. 1994. The advances in research on fungicide resistance. Journal of Nanjing
Agricultural University 17(3):33-41.
Table 1. Fusarium head blight (FHB) disease incidence, severity and index on wheat following
different foundation crops.
Foundation crop
in 2001
Canola
Wheat
Oats
Peas
Crop in
2002
Wheat
Wheat
Wheat
Wheat
%Incidence
11.66
10.33
8.18
19.25
%Severity
43.09
18.35
22.71
12.64
FHB index
5.02
1.89
1.85
2.43
%FDK
6.84
5.07
4.85
6.76
Three samples of one meter row were randomly chosen from each wheat plot. Data are the mean of
three samples. The samples included 67-120 wheat heads. One hundred grams of seed was used to
determine percent FDK.
Table 2. Yield of canola, wheat, oats and peas following different foundation crops.
Yield ton/ha
Crops in 2002
Foundation crops in
2001
Canola
Wheat
Oats
Peas
Canola
1.47
3.19
2.47
1.36
Wheat
2.20
3.06
2.63
1.01
Oats
2.01
1.30
2.67
2.39
Peas
1.77
1.44
2.75
1.59
Figures are the mean of three samples from each plot, and the sample area was 1.22 X 2.5M (six
rows of 2.5 meter long).
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DETERMINATION OF WETNESS DURATION USING
RADAR-DERIVED PRECIPITATION ESTIMATES
J.A. Andresen*, T.M. Aichele, and A.Pollyea
Michigan Climatological Resources Program, Department of Geography,
Michigan State University, East Lansing, MI 48824
*Corresponding Author: PH: 517-355-0231 ext. 107); E-mail: andresen@msu.edu
ABSTRACT
Fusarium head blight (FHB) of small grains tends to be associated with certain environmental conditions, especially rain-induced wetness periods occurring near anthesis. A Geographic Information System-based model simulation which incorporates 4km resolution
weather radar (NEXRAD)-derived precipitation estimates into a crop canopy energy balance-based scheme to estimate wetness duration periods for small grains on the 4 km
spatial scale has been developed and initially tested, with promising results. Errors found in
the NEXRAD precipitation estimates analyzed during the first and second years of this
project with Michigan precipitation data were less pronounced than previous studies, with
96.3% of the precipitation-hours across the state of Michigan during the 1999 and 2000
growing seasons correctly classified and an overall mean bias and mean absolute precipitation differences of -1.6mm and 2.3mm respectively. An initial validation of simulated leaf
wetness duration in 6 wheat field sites in Lower Michigan at head height during June and
July of the 2002 growing season resulted in mean differences of -0.2 hours and mean
absolute differences of 3.4 hours over 116 separate events associated with dew, precipitation, or both. Mean differences and absolute differences for events associated with precipitation only or with precipitation and dew were +1.5 hours and 3.7 hours, respectively, indicating a slight tendency for overprediction. In an effort to better parameterize the wetness
duration simulation including evaporation rates of dew and total intercepted precipitation, a
field study began in April, 2002 with greenhouse flats planted with spring wheat in individual 10cm pots. Following heading, the flats were monitored with a weighing lysimeter
over time, providing estimates of plant evapotranspiration, dewfall, and interception of
precipitation. Preliminary results from these data suggest a total nightly dewfall ranging from
0.0-0.3mm. To study rainfall interception, wheat heads at the flowering stage were cut and
collected from extra plants in the flats and mounted on 30 cm long, 0.1mm diameter steel
wires. The heads and steel ‘stems’ were in turn mounted on a heavy steel wire frame which
held the mounted wheat heads and wire in a fashion similar to that grown in the field. Rainfall interception totals on the order of 0.1mm to 0.3mm were recorded for 11 events. The
canopy interception was observed to be associated with the drop diameter of precipitation,
with less canopy interception occurring with large droplet diameters and vice versa.
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A SECOND GENETIC MAP OF GIBBERELLA ZEAE
R.L. Bowden1*, J.E. Jurgenson2, J.K. Lee3, Y.-W. Lee4, S-H Yun4,
K. Zeller3, and J.F. Leslie3
USDA-ARS Plant Science and Entomology Research Unit, Manhattan, KS 66506;
Department of Biology, University of Northern Iowa, CedarFalls, Iowa , U.S.A. 50614;
3
Department of Plant Pathology, Kansas State University, Manhattan, KS 66506; and
4
School of Agricultural Biotechnology, Seoul National University, Suwon 441-744
*Corresponding Author: PH: (785)-532-2368; E-mail: rbowden@ksu.edu
1
2
ABSTRACT
We recently reported the construction of a genetic map of Gibberella zeae made by crossing
nitrate non-utilizing (nit) mutants of strains R-5470 (lineage 6 from Japan) and Z-3639
(lineage 7 from Kansas). This genetic map is based on 1048 AFLP markers that have been
assigned to nine linkage groups. The map contains numerous loci with distorted segregation ratios and two possible chromosome rearrangements between the parental strains. The
high degree of polymorphism and high marker density in this linkage map make it very
useful for gene mapping studies. It has been used to map several genes related to
trichothecene toxin biosynthesis and can also be used for QTL analysis. However, the
segregation distortion in this wide cross may limit certain uses. Therefore, we constructed a
second genetic map by making a narrow cross between two lineage 7 strains (Z-3639 and
PH-1 from Michigan). The Z-3639 strain had a deletion in the MAT2 gene, which made it
heterothallic. This avoided the segregation distortion associated with nit markers. In addition
to AFLP markers, we also mapped some nuclear genes using RFLP-PCR. Segregation in
the cross is normal, but marker polymorphism is low so more AFLP primer pairs will be
needed to saturate the map. Loci common to the two genetic maps will allow identification of
the linkage groups and elucidation of the segregation distortion and putative chromosome
rearrangements in the original map.
REFERENCE
Jurgenson, J.E., R. L. Bowden, K. A. Zeller, J. F. Leslie, N. A. Alexander, and R. D. Plattner. 2002. A genetic
map of Gibberella zeae (Fusarium graminearum). Genetics 160:1451-1460.
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WHAT PART DOES PROGRAMMED CELL DEATH PLAY
IN FUSARIUM HEAD BLIGHT?
W. R. Bushnell* and T.M. Seeland
ARS-USDA, Cereal Disease Laboratory, Paul, MN 55108
*Corresponding Author: PH (612) 625-7781; E-mail: billb@umn.edu
ABSTRACT
Deoxynivalenol (DON) is a strong inhibitor of protein synthesis and also induces programmed cell
death (PCD) in animal cells (where it is termed “apoptosis”) (Yang et al., 2000). Results of genetic
manipulation of toxin production by the FHB pathogen, Fusarium graminearum, indicate that DON or
other trichothecene toxins contribute to pathogen virulence in diseased wheat spikes (Desjardins et
al., 1996.). DON is known to be toxic to plant cells but the processes leading to cell death have
been little investigated. In studying the effects of DON in detached leaves of barley (Bushnell et al.,
2002), we obtained preliminary results that support the hypothesis that DON induces PCD: 1) DON
induced a gradual dissolution of chloroplasts (with concomitant loss of carotenoid and chlorophyll
pigments) extending over three to five days before cells collapsed. Mitochondria likewise became
degenerate. The tissues also suffered significant electrolyte loss over the 3-5 day period. Thus, cells
underwent an ordered sequence of autolytic events leading to death, typical of PCD. Furthermore,
the bleached tissues resulting from loss of chlorophyll pigments mimicked lesions of FHB in host
spikes; 2) Like DON, cycloheximide and chloramphenicol, well known inhibitors of protein synthesis,
induced gradual loss of chloroplast pigmentation and of electrolytes preceding cell collapse in the
detached leaf tissues. Cycloheximide and several other inhibitors of protein synthesis have been
reported to induce PCD in animal cells (Kochi & Collier 1993); 3) Ca ++ ions, known to be essential
for PCD in plant cells (Groover & Jones, 1999), markedly accelerated DON-induced loss of both
chloroplast pigments and electrolytes from leaf tissues. Together, these results indicate that DON
induces PCD in leaf tissues and, therefore, may do likewise in FHB-infected spike tissues. We are
following up these experiments by applying treatments to DON-treated leaves that are known to
enhance or inhibit PCD. Further, we will extend these treatments to FHB- infected tissues to obtain
cytological and physiological evidence for a possible role of PCD in FHB pathogenesis.
REFERENCES
Bushnell, W.R., Seeland, T.M., Krueger, D.E. 2002. Light dependent bleaching of detached barley leaf tissue by
deoxynivalenol. Phytopathology 92:S11.
Desjardins, A.E., Proctor, R.H., Bai, G., McCormick, S.P., Shaner, G., Buechley, G. and Hohn, T.M. 1996.
Reduced virulence of trichothecene-nonproducing mutants of Gibberella zeae in wheat field tests. Mol. Plant
Microbe Interact. 9:775-781.
Groover, A. and Jones, A.M. 1999. Tracheary element differentiation uses a novel mechanism coordinating
programmed cell death and secondary cell wall synthesis. Plant Physiology 119:375-384.
Kochi, S.K. and Collier, R.J. 1993. DNA fragmentation and cytolysis in U937 cells treated with diphtheria toxin
or other inhibitors of protein synthesis. Exp. Cell Res. 208:296-302.
Yang, G-H., Jarvis, B.B., Chung, Y-J., and Pestka, J. 2000. Apoptosis induction by the satratoxins and other
trichothecene mycotoxins: Relationship to ERK, p38MAPK, and SAPK/JNK activation. Toxicol. Appl.
Pharmacol. 164:149-160.
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INFLUENCE OF IRRIGATION FOLLOWING DISEASE ASSESSMENT
ON DEOXYNIVALENOL ACCUMULATION IN
FUSARIUM-INFECTED WHEAT
M.D. Culler and R. Dill-Macky*
Department of Plant Pathology, University of Minnesota, St. Paul, MN 55108
*Corresponding Author: PH: 612-625-2227; E-mail: ruthdm@umn.edu
ABSTRACT
A field trial was established in 2002 to evaluate the effect of moisture on deoxynivalenol
(DON) accumulation in Fusarium-infected wheat. The trial was a split-split plot design with
four replicates. Main plots were irrigation levels, subplots were wheat cultivar and subsubplots were inoculation concentrations. Plots of the spring wheat cultivars, Wheaton
(susceptible), Pioneer 2375 (moderately resistant) and Alsen (resistant), were inoculated
with Fusarium graminearum at anthesis (Zadoks growth stage [GS] 61). Two inoculum
concentrations (25,000 macroconidia/ml and 100,000 macroconidia/ml) were used to generate different Fusarium head blight (FHB) severities. Mist-irrigation (3.8 mm/day) was applied uniformly to all plots from anthesis until 15 days after inoculation (DAI) (GS 83). Then,
the different irrigation treatments were imposed. Half of the plots continued receiving irrigation at the initial rate until harvest (35 DAI) and half received no irrigation. FHB severity was
determined 16 DAI as a percentage of symptomatic spikelets for 20 spikes per plot. Sixty
heads per subplot in each of two severity categories (FHB < 50%, FHB > 50%) were tagged
16 DAI. Tagged heads (10/severity category) were harvested for each variety at GS 83
(early dough), GS 87 (hard dough), GS 91 (caryopsis hard) and GS 94 (harvest ripe). Kernels were dissected from collected spikes and assayed for DON concentration using gas
chromatography / mass spectrometry. FHB severities among inoculation concentration
treatments were significantly different (P< 0.001). Mean FHB severities were 30% and 70%
for Wheaton; 29% and 50% for Pioneer 2375; and 26% and 51% for Alsen at the low and
high inoculum treatments, respectively. Data from this experiment should provide an insight
into aspects of DON accumulation in wheat. Sequential sampling following disease assessment may help characterize the timing of DON accumulation during an epidemic. The
influence of irrigation treatments could aid in the prediction of the DON concentration in
grain based on post anthesis weather variables.
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SPATIAL PATTERNS OF FUSARIUM HEAD BLIGHT
IN NEW YORK WHEAT FIELDS IN 2002
E.M. Del Ponte, D.A. Shah, and G.C. Bergstrom*
Department of Plant Pathology, Cornell University, Ithaca NY 14853-5904
*Corresponding Author: PH: 607-255-7849; E-mail: gcb3@cornell.edu
ABSTRACT
Fusarium head blight (FHB), caused by the fungus Gibberella zeae, is a disease of worldwide occurrence that severely reduces the yield, quality, and marketability of wheat. The
spatial pattern of FHB incidence was studied in 60 arbitrarily selected winter wheat fields in
central and western New York at kernel soft dough stages in June 2002. The fields varied in
wheat cultivar, preceding crop, presence of corn residue, and intensity of FHB epidemic.
Incidence of FHB was randomly distributed among 60 sampling quadrats in 55 of the 60
fields. Fields with random FHB ranged from trace to 23% in average incidence of FHB and
followed bean, corn, oat, pea, sorghum, and soybean. The five fields with aggregated FHB
ranged from trace to 27 % in average incidence of FHB and followed bean, corn, oat, and
pea. Mean incidence of FHB was not significantly different between fields with and without
corn residue, though incidence of FHB and aggregation was highest in two fields sown into
standing corn residue without tillage. For eight fields that had corn residue from a corn crop
2 or more years before wheat, there was no evidence of aggregation among all quadrats in
a field or among quadrats with corn stubble; also there was no difference in the mean incidence of FHB between quadrats with and without corn residue. Spatial patterns do not
supply direct proof of inoculum source, but they suggest likely origins of inoculum that can
be confirmed by other observations and experimentation. Based on the predominantly
random patterns of FHB in 2002, we suggest that FHB epidemics in rotational wheat fields
of New York may be initiated by deposition of spores from diffuse atmospheric inoculum.
Over-wintered corn residues are the most prevalent and likely regional source of atmospheric inoculum for FHB in New York.
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INFLUENCE OF CORN RESIDUE AND CULTIVAR SUSCEPTIBILITY
ON THE ACCURACY OF FUSARIUM HEAD BLIGHT
RISK ASSESSMENT MODELS
E. De Wolf1*, P. Lipps2, L. Madden2 and L. Francl1
1
Dept. Plant Pathology, Pennsylvania State University, University Park, PA 16802; and
2
Dept. Plant Pathology, Ohio State University/OARDC, Wooster, OH 44691
*Corresponding Author: PH: (814) 865-9620; E-mail: edd10@psu.edu
OBJECTIVES
To evaluate the performance of forecasting models for Fusarium head blight of wheat in the
United States
INTRODUCTION
Disease forecasting models for Fusarium head blight (FHB) of wheat were developed by a
cooperative effort among researchers in OH, PA, ND, IN, SD, MO, and KS (De Wolf et al.
2000). These forecasting models were based on logistic regression analysis of 50 locationyears of disease observations and predict the probability of a FHB epidemic based on
environmental variables. Members of the cooperative effort are currently evaluating two
models for delivering FHB forecasts at a regional level. For convenience, we will refer to
these models as Model 1 and Model 2.
Model 1 uses weather variables observed during a 7-day period prior to flowering. More
specifically, these models use duration of time (h) that temperature is between 15 and 30°C
and the duration (h) of precipitation (De Wolf et al. 2001). This model correctly predicted
70% of 50 cases used to develop the models. Model 1 correctly predicted 78% of nonepidemic years (FHB severity greater than 10%), but correctly classified only 56% of the
epidemic years.
Model 2 uses environmental variables observed during the 7-days period prior to flowering
and a 10-day period beginning at flower initiation (De Wolf et al. 2001). Variables used by
this model are the duration (h) of temperature between 15 and 30°C for the 7-day period
prior to flowering, and the duration (h) in which temperature is between 15 and 30°C and
relative humidity is greater than 90% during the flowering-time period. Model 2 correctly
classified 84% of the 50 cases used to develop the model with near equal accuracy for both
epidemic and non-epidemic cases.
MATERIALS AND METHODS
Researchers in PA, OH, ND, SD, and IN provided crop growth stage and disease observations from replicated research plots, and this information was combined with hourly measurements of temperature, relative humidity, and precipitation. The presence or absence of
corn residue within the plots was noted at each location. The total data set consisted of 23
location years not used in model development.
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Models 1 and 2 were evaluated for prediction accuracy with the new data, and model accuracy was compared with previous estimates. Model errors were evaluated for trends that
should facilitate application of present models and development of the next generation of
forecasting models.
RESULTS AND DISCUSSION
The total number of cases provided for this project from each state included three cases
from IN, four from ND, six from OH and five from both PA and SD. Disease severity at these
sites ranged from 0 to 74%. Nine of the 23 cases were considered to be epidemics when
converted to the binary scale used by the models (FHB severity greater than 10% = 1).
Seven of 23 cases had significant levels of corn residue within the plots.
Model 1 correctly classified 15 of the total 23 validation cases correctly (Table 1). All eight
errors made by Model 1 were false negatives (incorrectly predicting low disease). In comparison, Model 2 correctly predicted 17 of the 23 cases. Five of the six errors made by
Model 2 were false negatives. These prediction accuracies were lower than previous estimates of model accuracy (De Wolf et al. 2001). The high rate of false negative errors was of
particular concern. However, nearly all of these errors were associated with sites that had
high levels of corn residue, or the highly susceptible spring wheat cultivar ‘Norm’.
Corn residue
When the models were evaluated with sites with little or no corn residue, Model 1 correctly
predicted 11 of the 16 cases, and Model 2 correctly classified 13 of the 16 cases (Table 1).
In contrast, both Model 1 and 2 correctly classified only four out of the seven sites that had
high levels of corn residue within the plots. The reduction in model accuracy in association
with corn residue may, in part, be explained by the high levels of inoculum often associated
with this type of residue (Francl et al. 1999).
Cultivar susceptibility
Cultivar susceptibility also appeared to affect model accuracy. In this analysis, the highly
susceptible cultivar Norm was associated with three of the five errors made by Model 1 for
the low residue data set. All three errors were false negative predictions (incorrectly predicting low disease). Similarly, two of the three errors made by Model 2 for the same data set
involved Norm, and both errors were false negative predictions. The number of errors that
correspond to Norm suggest that highly susceptible cultivars may have an increased likelihood of severe disease that is not considered by the prediction models.
Verification of the prediction accuracy of Models 1 and 2 supports continued deployment in
disease forecasting efforts. However, these results indicate that the models may be less
accurate when wheat is produced in fields with high levels of corn-residue, or when highly
susceptible cultivars are grown. Future modeling efforts will attempt to incorporate potential
inoculum source and cultivar susceptibility into the forecast models.
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Table 1. Prediction accuracy of FHB forecasting models for 23 location-years not used in
model development.
Data set
Model Prediction Accuracy (%)
Model 1
Model 2
Full data set (n=23)
65
74
Location years with low
level of corn residue (n=16)
69
81
Location years with high
level of corn residue (n=7)
57
57
REFERENCES
De Wolf, E. D., Lipps, P. E. and Madden, L. V. 2000. Prediction of Fusarium head blight epidemics. P.131-135,
In: Proceedings of the 1999 National Fusarium Head Blight Forum, Dec. 5-7, Sioux Falls, SD.
De Wolf, E.D., Madden, L. V., and Lipps, P. E. 2001. Fusarium head blight epidemic prediction and risk assessment. Phytopathology 91:S22.
Francl, L., Shaner, G., Bergstrom, G., Gilbert, J., Pedersen, W., Dill-Macky, R., Sweets, L., Corwin, B., Jin, Y.,
Gallenberg, D., and Wiersma, J.1999. Daily inoculum levels of Gibberella zeae on wheat spikes. Plant Dis.
83:662-666.
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EFFECT OF CEREAL RESIDUE BURNING ON THE INCIDENCE AND
STRATIFIED DISTRIBUTION OF FUSARIUM GRAMINEARUM AND
COCHLIOBOLUS SATIVUS IN WHEAT AND BARLEY PLANTS
R. Dill-Macky* and B. Salas
Department of Plant Pathology, University of Minnesota, St. Paul, MN 55108
*Corresponding Author: PH: 612-625-2227; E-mail: ruthdm@umn.edu
ABSTRACT
The effect of residue burning on the stratified incidence of Fusarium graminearum,
Cochliobolus sativus, and other pathogens was studied in barley and wheat planted at three
locations in Minnesota in 2001. Cereal residues were burned 1-5 days after planting using
a propane-powered flame thrower. Subcrown internodes, crowns, nodes, and kernels were
excised from 30 plants collected from each plot at maturity. Tissue segments were surfacesterilized, plated onto half strength PDA (pH=5.5), incubated at 20-24oC under flourescent
lights (12:12 light:dark) for 6-7 days. The observed colonization of tissues showed that
regardless of the host, F. graminearum was mostly associated with kernels, whereas C.
sativus was mostly associated with crowns and the first node. In contrast, Pyrenophora
teres in barley was mostly associated with the third node. Burning significantly reduced
cereal residues (P<0.01), and also significantly reduced the survival of F. graminearum and
C. sativus (P<0.01). The overall incidence of F. graminearum was significantly less (P=0.05)
in wheat plants collected from burned plots (3.3%) in comparison with those collected from
the non-burned plots (5.3%). The effect of residue burning on the incidence of C. sativus
and P. teres was not significant. Our data shows that F. graminearum, C. sativus and P. teres
preferentially colonize certain plant parts and that residue burning may provide an option in
the management of cereal diseases such as Fusarium head blight.
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IDENTIFICATION OF ENVIRONMENTAL VARIABLES THAT AFFECT
PERITHECIAL DEVELOPMENT OF GIBBERELLA ZEAE
N. Dufault1, E. De Wolf1*, P. Lipps2 and L. Madden2
Dept. Plant Pathology, Pennsylvania State University, University Park, PA 16802; and
2
Dept. Plant Pathology, Ohio State University/OARDC, Wooster, OH 44691
*Corresponding Author: PH: (814) 865-9620; E-mail: edd10@psu.edu
1
ABSTRACT
Perithecial development of Gibberella zeae and the severity of Fusarium head blight are
dependent upon favorable environmental conditions. Information about the conditions
favorable to G. zeae perithecial development could be useful in predicting wheat head blight
epidemics. The development of G. zeae perithecia on corn stalk residue was monitored in
replicated plots in wheat fields near Wooster, OH (2000) and State College, PA (2001-02).
Environmental variables including temperature, relative humidity and rainfall were recorded
directly within the plots with an automated datalogger. The moisture levels of the stalks were
monitored with electrical resistance sensors, and the duration of stalk wetness (DSW) was
recorded. Observations of perithecial development were made every five to seven days, and
paired with environmental variables to identify those variables associated with perithecial
development. The rate of perithecial production was the greatest in 2000 and the lowest in
2001. An extended period of DSW was associated with an increase in perithecial development at all locations over in the three years of this study. During this increase in perithecial
development, the 2000 and 2002 years had 14 and 18 days respectively with average
temperatures greater than 15°C. In comparison, only eight days with average temperature
greater than 15°C occurred in 2001. Both the 2000 and 2002 locations received a more than
100 mm of rain during the period of rapid perithecial increase. However, only 60 mm of rain
were recorded in 2001. In 2002, a decrease in the rate of perithecial production was associated with a six-day period of average temperature less than 7°C but the rate increased
again when temperatures increased to greater than 15°C. These results suggest that the
number of perithecia produced and their rate of development are influenced by temperature
and moisture in a wheat field environment. In the future, information relating weather conditions with critical periods for perithecial developmental may improve the accuracy of wheat
head blight forecasting systems.
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RELATIONSHIP OF TEMPERATURE AND MOISTURE TO
GIBBERELLA ZEAE PERITHECIAL DEVELOPMENT
IN A CONTROLLED ENVIRONMENT
N. Dufault1, E. De Wolf1*, P. Lipps2 and L. Madden2
1
Dept. Plant Pathology, Pennsylvania State University, University Park, PA 16802; and
2
Dept. Plant Pathology, Ohio State University/OARDC, Wooster, OH 44691
*Corresponding Author: PH: (814) 865-9620; E-mail: edd10@psu.edu
OBJECTIVES
To examine the relationship between temperature and moisture on Gibberella zeae development in a controlled environment on corn stalk residue.
INTRODUCTION
Fusarium graminearum (G. zeae) survives in association with debris of corn, wheat, barley
and many cultivated grasses (Parry, 1995). These residues are major sources of inoculum
for epidemics of Fusarium head scab of wheat in North America (Francl et al. 1999). A
better understanding of factors affecting survival and reproduction of G. zeae on these
residues is important to the development of new management strategies for head scab of
wheat and barley.
Past research has shown that temperature and osmotic water potential are two important
factors in stimulating growth, reproduction, and sporulation of G. zeae (Sung, 1981;
Tschanz, 1976). This research, however, has only examined temperature and moisture
independently, and has not used crop residues as a substrate. Our objectives were to further
examine the effects of temperature and moisture on the development of G. zeae, and examine these two important factors together using crop residues.
MATERIALS AND METHODS
Inoculation of stalks – Corn stalks collected near State College, PA once the plants had
reached physiological maturity. These stalks were cut into ~30 cm sections, disinfested
twice and placed into cold storage (-10°C) until inoculation. At the time of inoculation, the
stalks were removed from the cold storage, placed into stainless steel trays, covered with
aluminum foil, and disinfested for a third time. A 3mm2 section of G. zeae infested carnation
leaf was placed on the stalks, and stalks were incubated for approximately 14 days at 25°C
in continuous darkness.
Calibration of Sensors with Stalks – After the stalks were infested, six stalks were arbitrarily selected and paired with an electrical resistance sensor. The relationship between
electrical resistance and water content was individually calibrated for each stalk. These
calibrations were done by wetting the stalks and taking repeated measurements of electrical
resistance and stalk water content as the stalks dried. Regression analysis was used to
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develop a calibration curve for each stalk-sensor combination. Sensors were used to monitor and adjust moisture levels within a humidity chamber.
Controlled Environment Chambers – A three-compartment humidity chamber was used
to control the three moisture treatments of dry (< 40% RH), moderate (40-80% RH) and wet
(>80% RH). A layer of disinfested sand, two stalk-sensor pairs, six infested stalks, and a
temperature/humidity probe were placed into each compartment of the humidity chamber.
The temperature for each run was held constant at 15, 25, or 30°C. The number of perithecia
at previously selected points on the six infested stalks were counted every 5 days for a 20day period. Developmental stages of the perithecia were also recorded at this time. All
treatments were repeated at least 3 times and analysis of variance used to identify differences among treatments.
RESULTS AND DISCUSSION
After 11 to 16 days of incubation, the number of perithicia produced at 15 or 25°C was
significantly greater (p = 0.01) than the number produced at 30°C, but there was no significant difference detected between the 15 and 25°C treatments. The number of perithecia was
significantly (p = 0.01) increased at the high moisture level compared to the low moisture
level treatments. There were no perithecia produced with treatments that included 30°C or
the low moisture level (Figure 1). The results indicate the perithecial development of G. zeae
maybe limited by extended periods of residue dryness or temperatures above 30°C.
In these experiments, perithecia with ascospores were produced at 15°C and 25°C (Figure
2). These results do not agree with previous reports that suggest that perithecial development was limited or did not occur at 15°C ( Tschanz 1976). Further experiments are underway to further evaluate temperatures that may limit G. zeae perithecial production.
Figure 1. Mean number of Gibberella zeae perithecia produced on infested sections of corn
stalk incubated at different combinations of temperature and moisture level
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Perithecia growth stages: 2 = perithecia just pigmented; 4 = perithecia beginning to form; 6 = asci formed but
spore development incomplete; 8 = ascospore development complete
Figure 2. Developmental stages of Gibberella zeae perithecia produced at 15 or 25°C at
similar moisture levels
REFERENCES
Francl, L., Shaner, G., Bergstrom, G., Gilbert, J., Pedersen, W., Dill-Macky, R., Sweets, L., Corwin, B., Jin, Y.,
Gallenberg, D., and Wiersma, J. 1999. Daily inoculum levels of Gibberella zeae on wheat spikes. Plant Disease
83:1021-1030.
Parry, D.W., Jenkinso, P., and Mcleod, L. 1995. Fusarium ear blight (scab) in small grain cereals – a review.
Plant Pathology 44:207-238.
Sung, J. M., and Cook, R. J. 1981. Effect of water potential on reproduction and spore germination by Fusarium
roseum ‘Graminearum,’ ‘Culmorum,’ and ‘Avenaceum’. Phytopathology 71:499-504.
Tschanz, A.T., Horst, R. K., and Nelson, P. E. 1976. The effect of environment on sexual reproduction of
Gibberella zeae. Mycologia 68:327-340.
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INCIDENCE-SEVERITY RELATIONSHIPS FOR
FUSARIUM HEAD BLIGHT ON WHEAT
S. M. El-Allaf, L. V. Madden, and P. E. Lipps*
Dept. of Plant Pathology, The Ohio State University/OARDC, Wooster, OH 44691
*Corresponding Author: PH: (330) 263 3843; E-mail: lipps.1@osu.edu
ABSTRACT
The relationship between incidence (proportion of plant units diseased) and severity (the
amount of plant tissue affected by disease) is a valuable tool that is useful for making disease surveys, assessments, evaluating host resistance, and for determining action thresholds for management decisions. The assessment of severity is tedious and time consuming
and may be prone to bias and large experimental error. Therefore, the existence of a quantifiable relationship between incidence and severity greatly facilitates evaluation of disease
intensity for estimates of crop damage. These benefits arise because incidence is determined easily, with more accuracy and precision than severity, and with lower cost. Thus, the
intent of this study was to determine the relationship between incidence (I; percent heads
infected) and severity (S; percent infected spikelets within infected head) of Fusarium head
blight, and determine if severity could be predicted reliably from incidence data. Disease
assessment for both incidence and severity were made visually at several sample sites
(ranged from 45 to 100 sites per field) in artificially and naturally inoculated research plots
and production fields over four years. At each sample site, at least 20 heads were evaluated
for incidence and severity. Incidence of infected heads and the average percentage of
spikelets with disease on each date for each field in each year were analyzed using linear
regression analysis. Ten different, but interrelated, models were fitted to the data and models were compared based on R2, mean square error, and residual plots. Mean disease
incidence and severity varied among data sets, ranging from 28.0 to 75.4% for incidence,
and from 9.1 to 28.2% for severity.
The best fitting model was CLL(S) = α + ß CLL(I), in which CLL is the complementary log log transformation. R2 values ranged from 0.69 to 0.91. Although there was considerable
variability of S for a given I in some years, there was a highly significant relationship between S and I in each year, and the functional relationship was very consistent between
years.
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SPATIAL ASPECTS OF FUSARIUM HEAD BLIGHT
EPIDEMICS ON WHEAT IN OHIO
S. M. El-Allaf, L. V. Madden, and P. E. Lipps*
Dept. of Plant Pathology, The Ohio State University/OARDC, Wooster, OH 44691
*Corresponding Author: PH: (330) 263 3843; E-mail: lipps.1@osu.edu
OBJECTIVE
To quantify the spatial pattern of Fusarium head blight incidence in wheat fields.
INTRODUCTION
Fusarium head blight of wheat (Triticum aestivum L.) caused by Fusarium graminearum
Schwabe (teleomorph Gibberella zeae) is a limiting factor in wheat and barley production. It
reduces wheat yield in many production regions of North America (Bai and Shaner 1994;
Parry et al.1995; McMullen et al. 1997). When environmental conditions are favorable, the
disease can cause yield losses up to $1billion (McMullen et al. 1997). The analysis of
spatial patterns of plant diseases is an important component of epidemiology. Disease
pattern is a useful ecological characteristic that helps define a population such as diseased
wheat heads (Campbell, and Madden, 1990; Madden, et al., 1995).
Despite the economic importance of Fusarium head blight, there is little information showing the spatial patterns (dispersion) of infected heads and the changes in patterns over time
as disease incidence increases. This information would be useful for better understanding
the spatio-temporal dynamics of Fusarium head blight. Additionally, data may help us determine efficient sampling procedures that result in precise estimates of mean disease intensity and help determine the proper statistical analysis for comparing treatments.
MATERIALS AND METHODS
Disease Assessments.
Epidemics of Fusarium head blight of wheat were monitored in four fields in 2001 and in six
fields in 2002. In each field, three transects with 15 sample points per transect, spaced at 1m intervals, for a total of N = 45 sample points per field, or 10 transects with 10 sample
points per transect, spaced at 1-m intervals, for a total of N = 100 sample points per field
were established. Each sample point was marked with a flag that remained in the field
throughout the assessment period. At each sample point, the incidence of scab was recorded for a 1-ft sub-transect across the plant rows.
DATA ANALYSES
Heterogeneity Analyses: Distribution and indices.
The beta-binomial and the binomial distributions were fitted to data on the incidence of
diseased heads per transect for each individual field assessment using the computer program BBD, Version 1.2 (Madden and Hughes, 1994). The beta-binomial has two paramEpidemiology and Disease Management
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eters, p, which is the expected probability of disease (a measure of disease incidence), and
, a measure of the variation (heterogenity or aggregation) in disease incidence per sample
unit. Values ofgreater than 0 indicate aggregation. The binomial has a single parameter
representing the probability of disease. A good fit to the binomial distribution is suggestive
of a random spatial pattern of disease incidence, while a good fit to the beta-binomial is
suggestive of an aggregated (overdispersed) spatial pattern of disease incidence. Standard
X2 goodness-of-fit tests were calculated for each distribution to determine the most appropriate distribution.
For each field and assessment date, the index of dispersion, D, was also calculated. D is
the ratio of the observed variance of incidence among the sampling units to the expected
binomial (i.e., random) variance (Madden and Hughes, 1995).
The effect of disease aggregation is to inflate or increase the observed variance above the
expected binomial variance. Therefore, values of D > 1 suggest spatial aggregation. D has
a X2 distribution under the null hypothesis of randomness. A large test statistic and small
significance level (<0.05) indicate that one should reject the null hypothesis of randomness
(=binomial) in favor of aggregation (overdispersion). Moreover, the so-called C(á) test,
which is more specific than the test of D, was used to test for overdispersion. Here, the
alternative hypothesis is not just overdispersion, but overdispersion described by the betabinomial.
RESULTS AND CONCLUSIONS
Mean disease incidence per field, an estimate of the expected probability of a head being
diseased (p), ranged from 0.018 to 0.693, with a median among fields of 0.024 in
2001(Table 1), and from 0.137 to 0.687,with a median of 0.250 in 2002 (Table 2). As anticipated, p increased over time within all fields.
The program BBD successfully calculated maximum likelihood estimates of p and for all
the data sets in both years. Where there was a sufficient number of disease classes for the
test to be preformed, the frequency distribution of diseased heads could be described by the
beta-binomial distribution in over 75% in 2001 and over 60%of the data sets in 2002, and by
the binomial distribution in 58% and 40% of the data sets in 2001 and 2002, respectively.
The values of ranged from 0.00 to 0.073, with a median of 0.011 in 2001, and from 0.00 to
0.039, with a median of 0.019 in 2002. Estimated in over 90% of data sets were greater
than 0 (Tables 1 and 2) indicating overdispersion.
The index of dispersion D, ranged from 0.88 to 4.50, with a median of 2.22, and from 0.89 to
2.80, with a median of 1.83 in 2001 and 2002, respectively. D and were both positively
correlated with the estimated parameter p.
The X2 test for D (Madden and Hughes, 1995), and the C(α) test both had indicated significant heterogeneity in more than 90% of the data sets (Tables 1 and 2).
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In conclusion, it was found that heads of wheat infected with scab were aggregated within
the wheat fields. Moreover, the degree of aggregation was moderate and increased over
time as incidence increased.
REFERENCES
Bai, G. and Shaner, G. 1994. Scab of wheat: Prospects for control. Plant Dis. 78:760-766.
McMullen, M., Jones, R., and Gallenburg, D. 1997. Scab of wheat and barley: A re-emerging disease of
devastating impact. Plant Dis. 81:1340-1348.
Parry, D. W., Jenkinson, P., and McLeod, L. 1995. Fusarium ear blight (scab) in small grain cereals-a review.
Plant Pathol. 44:207-238.
Campbell, C. L., and Madden, L. V. 1990. Introduction to Plant Disease Epidemiology. John Wiley & Sons, New
York.
Madden, L. V., and Hughes, G. 1994. BBD-computer software for fitting the beta-binomial distribution to incidence data. Plant Dis. 78:536-540.
Madden, L. V., and Hughes, G. 1995. Plant disease incidence: Distributions, heterogeneity, and temporal
analysis. Annu. Rev. Phytopathol. 33:529-564.
Madden, L. V., Hughes, G., and Ellis, M. A. 1995. Spatial heterogeneity of the incidence of grape downy mildew.
Phytopathology 85:269-275.
Madden, L. V., Nault, L. R., Murral, D. J., and Apelt, M. R. 1995. Spatial patterns of the incidence of aster
yellows disease in lettuce. Res. Popul. Ecol. 37(2):279-289.
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Table 1. Statistics for describing the spatial pattern of the incidence of Fusarium head blight in four
wheat fields in Ohio in 2001.
Disease
assessment
Field
date
F 1 (Wooster)
06/11
06/14
06/18
06/21
06/25
F 2 (Wooster)
06/11
06/14
06/18
06/21
06/25
Estimated beta-binomial parametersa
C(Į) testb
p
0.071
0.081
0.204
0.304
0.587
se(p)
0.0092
0.0088
0.0110
0.0097
0.0171
ș
0.053
0.039
0.018
0.021
0.047
se(ș)
0.0179
0.0105
0.0077
0.0089
0.0113
z
8.54
8.32
6.21
2.86
14.91
P(z)
<0.001
<0.001
<0.001
0.002
<0.001
0.018
0.030
0.276
0.635
0.693
0.0033
0.0047
0.0147
0.0162
0.0113
0.011
0.011
0.031
0.047
0.013
0.0090
0.0084
0.0108
0.0108
0.0058
3.01
2.12
6.64
12.01
4.75
<0.001
0.017
<0.001
<0.001
<0.001
-c
-1.30
1.000
23.20
<0.001
F 3 (Hoytville)
06/26
0.047
0.0145
0.000
F 4 (Hoytiville)
06/26
0.623
0.0143
0.073
0.0134
a
p , expected probability of a leaf being diseased, estimated as the mean incidence; ș,
aggregation parameter; se(ș), standard error of designated estimated parameter.
b
z , standard normal statistic of the C(Į) test; P(z ): significance level of z .
se not defined when ș = 0.
c
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Table 2. Statistics for describing the spatial pattern of the incidence of Fusarium head blight in six
wheat fields in Ohio in 2002.
Field
F 1 (Wooster)
F 2 (Wooster)
Disease
Assessment
date
06/10
06/12
06/14
06/17
06/19
06/26
Estimated beta-binomial parametersa
p
0.419
0.523
0.590
0.656
0.687
0.320
se(p )
0.0121
0.0143
0.0155
0.0165
0.0152
ș
0.007
0.017
0.025
0.036
0.039
0.0121
0.000
se(ș)
0.0058
0.0079
0.0097
0.0120
0.0106
-c
C(Į) testb
z
1.69
3.99
5.91
8.32
6.83
-0.85
P(z)
0.045
<0.001
<0.001
<0.001
<0.001
1.000
F 3 (Wooster
06/26
0.363
0.0080
0.008
0.0040
2.65
0.004
F 4 (Wooster)
06/15
06/18
06/21
06/24
06/28
0.137
0.176
0.253
0.342
0.426
0.0072
0.0082
0.0072
0.0084
0.0108
0.029
0.031
0.008
0.012
0.029
0.0066
0.0069
0.0040
0.0045
0.0071
8.89
9.85
2.66
4.02
9.97
<0.001
<0.001
0.004
<0.001
<0.001
F 5 (Hoytville)
06/27
0.285
0.0043
3.44
<0.001
0.0078
0.010
F 6 (Hoytiville)
06/27
0.265
0.0085
0.017
0.0054
6.07
<0.001
p , expected probability of a head being diseased, estimated as the mean incidence; ș, aggregation
parameter; se(*), standard error of designated estimated parameter.
b
z , standard normal statistic of the C(Į) test; P(z), significance level of z.
c
se not defined when ș = 0.
a
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EFFECT OF WHEAT FLORAL STRUCTURE EXTRACTS AND
ENDOGENOUS COMPOUNDS ON THE GROWTH
OF FUSARIUM GRAMINEARUM
Jessica S. Engle1, Patrick E. Lipps1*, Terry L. Graham2 and Michael J. Boehm2
Department of Plant Pathology, OARDC, The Ohio State University, Wooster, Ohio 44691; and
2
Department of Plant Pathology, The Ohio State University, Columbus, Ohio 43210
* Corresponding Author: Phone: (330) 263-3843; E-mail: lipps.1@osu.edu
1
INTRODUCTION
Fusarium head blight (FHB) of wheat, caused by Fusarium graminearum, has become a
wide spread problem in the United States with the increased use of reduced tillage practices (2). Infection of the florets causes sterility, poor seed fill, reduced seed quality and
contamination of grain with mycotoxins (2). Since FHB severity levels are higher when wet
weather coincides with wheat anthesis in the field and when anthers, rather than emasculated spikelets, are inoculated in the greenhouse, it is presumed that anthers are the common route of entry into the plant (2,7). These findings led to the theory that there may be
compounds in anthers that stimulate growth of F. graminearum. The compounds thought to
be responsible for stimulation were choline acetate and glycinebetaine (4,7-9). F.
graminearum has been shown to possess separate constitutive high-affinity transport system that is specific for both choline and betaine, indicating that choline and betaine may be
specifically utilized by F. graminearum (5,6). These compounds may play a role in pathogenesis of F. graminearum or reaction of wheat cultivars to the pathogen.
OBJECTIVES
The first objective of this study was to examine the effect of choline and betaine on spore
germination and hyphal elongation of F. graminearum. The second objective was to determine the relationship between fungal hyphal growth and extracts of different floral structures
from nine wheat genotypes with varying reactions to F. graminearum.
MATERIALS AND METHODS
Rate of hyphal radial growth of three F. graminearum isolates was measured on water agar
and 2% dextrose agar amended with 10nM to 1000µM stock solutions of choline chloride or
betaine hydrochloride in two separate experiments. The control was unamended water agar
and dextrose agar.
Ascospore or macroconidia germination was evaluated on glass slides covered with a
10µM, 100µM, and 1000µM layer of choline, betaine, or an equal molar mixture amended
agar. Unamended agar was the control.
Nine genotypes were selected based on differences in mean FHB severity and incidence
from the 1999 Uniform Winter Wheat FHB Screening Nursery (1), (Table 1). Mean incidence
and severity were based on seven field locations across six states in the United States and
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one nursery in Ontario, Canada. Plants were grown in the greenhouse and spikes were
collected when one floret had extruded anthers. Extracts from anthers, paleas or lemmas
were combined with water agar and rate of hyphal growth of two F. graminearum isolates
were measured.
The percentage of increased growth compared to the unamended control was calculated for
the floral part extracts. Analysis of variance (ANOVA) was conducted using the general
linear model in MINITAB software package for rate of hyphal extension, percentage of
germinated spores and percentage of increased growth compared to the unamended contol.
RESULTS
The three F. graminearum isolates had significantly different (P = 0.05) growth rates, but
growth rate of an isolate was constant across repeats of experiments, with an average radial
growth rate of 0.35 - 0.64 mm/hr on water agar and 0.35 - 0.54 mm/hr on dextrose agar.
Choline had a significant (P = 0.05), but relatively small, effect on radial growth on water
agar, but not on dextrose agar at concentrations from 10nM to 1000nM compared to the
unamended control by 72 hours after plating in the first experiment. However, this small
effect was not observed in the second experiment at concentrations ranging from 10µM to
1000µM.
Betaine had a significant (P = 0.05), but relatively small, effect on radial growth on water
agar, but betaine did not affect growth on dextrose agar at concentrations from 10nM to
1000nM compared to the unamended control by 72 hours after plateing in the first experiment. In the second experiment, betaine did not significantly affect radial growth on the
10µM to 100µM amended agar, although there was inhibition of hyphal growth of all isolates
at the 1000µM concentration compared to the unamended control of both agars in the second experiment.
Likewise, the equal molar mixture of choline and betaine significantly (P = 0.05), although
only slightly, affected the radial growth in concentrations ranging from 10nM to 1000nM in
the first experiment. In the second experiment, the equal molar concentrations of choline
and betaine significantly (P = 0.0001) inhibited hyphal growth at the 1000µM concentration
compared to the unamended control of both agars, but had little effect on hyphal growth with
concentrations ranging from 10µM to 100µM.
Ascospores and macroconidia germinated readily on unamended water and dextrose agar
with 99% germination 24 hours after plating. Germination of ascospores and macroconidia
were not significantly (P = 0.05) affected by 10, 100, and 1000µM concentrations of choline,
betaine, or an equal molar mixture when compared to the unamended control plates over a
24 hour period (data not presented).
Hyphal growth was not significantly affected (P = 0.05) by anther, palea, or lemma extracts
from the resistant or susceptible genotypes when compared to the unamended control (data
not presented), or when growth rate was expressed as a percentage of the control.
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CONCLUSIONS
Macroconidial germination has not been shown to be enhanced by choline or betaine (3,7).
This study agrees with these findings and also shows that ascospore germination was
unaffected. Germination of ascospores or macroconidia appears to be unaffected by the
presence of choline or betaine.
Various in vitro studies have found that choline, betaine and equal molar mixtures of concentrations ranging from 0.1µM to 1mM stimulated, inhibited, decreased hyphal branching,
increased hyphal extension and had no affect on specific growth rate of hyphea of F.
graminearum (3,7,9,11). In the current study, radial growth was found to be enhanced by low
concentrations of choline on water agar, which agrees with previous findings (10), but this
enhancement is not believed to be biologically significant. Regardless of the different isolates of F. graminearum or different protocols used, results of this study are in agreement
with the findings of previous studies (3,11) in that levels of choline or betaine in floral parts
probably have little if any stimulatory effect on growth of F. graminearum.
Results of this study did not show a correlation between FHB resistance reaction of nine
wheat genotypes and rate of radial growth of two F. graminearum isolates on agar amended
with anther, palea or lemma extracts. These findings are in partial agreement with previous
findings (3). Therefore, endogenous compounds of wheat floral structures are not thought to
be important in resistance reactions of wheat genotypes.
Our results indicate that endogenous compounds in wheat floral structures do not enhance
their colonization by F. graminearum and that putative compounds in floral structures have
no substantial role in resistance to F. graminearum.
REFERENCES
Campbell, K. G. and Franchino, B. 1999. Ohio State Univ. Hort. and Crop Sci. Series 690. 21 pages.
McMullen, M., Jones, R. and Gallenberg, D. 1997. Plant Dis. 81:1340-1348.
Nkongolo, K. K., Dostaler, D. and Couture, L. 1993. Can. J. Plant Path. 15:81-84.
Pearce, R. B., Strange, R. N. and Smith, H. 1976. Phytochem. 15:953-954.
Robson, G. D., Best, L. C., Wiebe, M. G. and Trinci, A. P. J. 1992. FEMS Micrological Letters 92:247-252.
Robson, G. D., Wiebe, M. G. and Trinci, A. P. J. 1994. Mycol. Res. 98:176-178.
Strange, R. N. and Smith, H. 1971. Physiol. Plant Path. 1:141-150.
Strange, R. N., Smith, H. and Majer, J. R. 1972. Nature 238:103-104.
Strange, R. N., Majer, J. R. and Smith, H. 1974. Physiol. Plant Path. 4:277-290.
Strange, R. N. and Smith, H. 1978. Trans. Br. Mycol. Soc. 70:187-192.
Wiebe, M. G., Robson, G. D. and Trinci, A. P. J. 1989. J. Gen. Micro. 135:2155-2162.
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A PHENOLOGY-BASED PREDICTIVE MODEL FOR
FUSARIUM HEAD BLIGHT OF WHEAT
J.M.C. Fernandes1* and W. Pavan2
1
Embrapa Trigo, C.P. 451, Passo Fundo, RS, 99001-970, Brazil; 2Universidade de Passo Fundo,
ICEG-CCC, Passo Fundo, RS, 99001-970, Brazil
*Corresponding Author: PH: 011 55 54 311 3444; E-mail: mauricio@cnpt.embrapa.br
OBJECTIVES
Our goal was to establish a model to account for anther extrusion period that could be used
to calculate probabilities of Fusarium head blight incidence as the window of opportunity for
infection advances from beginning of anther extrusion to complete anther fall. The model
combines several elements of meteorology, biology of G. zeae and wheat phenology.
INTRODUCTION
Fusarium head blight (FHB), incited by a fungus (Gibberella zeae (Schwein.) Petch), is an
important disease affecting wheat. Fusarium head blight fungus survives in crop debris and
windborne or splashed spores infects the heads during flowering. Humid weather and
moderate temperatures are favorable for infection (Sutton, 1982). Fusarium head blight can
be devastating to yields if a large proportion of plants are infected. The fungus also can
produce harmful mycotoxins which depreciate grain value (McMullen et al., 1997).
Despite the absence of reliable data for comparing FHB intensity amongst different years in
southern Brazil, it is generally accepted that the disease has been more severe in the last
decade. It is believed that a combination of ‘El Niño’ years and of abundant sources of
inoculum are the cause for such high intensity of FHB in the wheat fields in southern part of
Brazil (Fernandes, 1997).
The relative narrow susceptible phase of wheat and the strong dependence on climatic
requirements for infection success makes the pathosystem suitable for modeling. A realistic
approach should account for availability of susceptible tissue besides weather driven pathogen dynamics.
Process-based models of crop growth and development are exciting tools emerging from
the on-going information technology. Models can improve our understanding of the complex
processes underlying wheat production including Fusarium head blight management. Their
analytical power can help deal with difficult tasks such as predicting the incidence of
Fusarium head blight on wheat.
MATERIAL AND METHODS
Brief Model Description
Model Framework. To develop a wheat simulation model into an Object Oriented environment we started with a small generic crop model. This model is available at
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www.icasanet.org/modular. The model contains three main modules: Soil, Plant and
Weather (Jones, et al., 2001). The model originally written in FORTRAN was converted to
JAVA and followed the principles of Object Oriented approach. The modular structure was
used to depict classes and provide them with the right data behavior. One of key features of
a modular approach is that models should relate to the real world components or processes.
Wheat Simulation Model. Wheat simulation, a process oriented model which is based on
daily time-steps considers 1 m2 area of wheat crop. It simulates the dynamics of wheat
biomass through inputs of historical records of weather data, cultivar coefficients, and soil
properties. The wheat simulation model includes growth, phenology and water balance
routines.
The plant growth module computes crop growth and development based on daily values of
maximum and minimum temperatures, radiation and the daily value of two soil water stress
factors, deficit and surplus. This module also simulates leaf area index (LAI), which is used
in the soil water module to compute evapotranspiration.
Crop development is simulated based on thermal time required to reach specific growth
stages. The model also accounts for simulating the dynamics of heading emergence including extrusion of anthers (flowering). State variables and simulated processes allow accounting for incidence of Fusarium head blight.
The water budget in the model includes precipitation, irrigation, runoff, water infiltration in
the soil profile, crop transpiration, and evaporation. Crop evapotranspiration is determined
from leaf area index.
Fusarium head blight Simulation Model. A module was developed to simulate head infection through inputs of local weather data. The first anthers were empirically set to be extruded on day five after heading emergence. Flowering dynamics was handled as a cohort
of heads exhibiting anthers resulting from simulation and assumed to be a potential infection site.
Predictive modeling tries to match the rules (models) for guessing (predicting) the Fusarium
head blight incidence from weather variables. Stepwise multiple regression procedures
were used to determine the prediction rules. The weather variables examined were solar
radiation, maximum temperature, minimum temperature and precipitation.
Model Inputs
Input data such as location, soil, crop and management files are required to run the model.
An advanced user-friendly interface allows users to easily manipulate input files, create
simulations, execute single and batch run simulations and produce text and graphical
reports. The data base was implemented using PostgreSql and Interbase for remote and
local access, respectively.
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RESULTS AND DISCUSSION
The model predicted reasonable well the phenological stages of the wheat cultivar BR23,
especially at the flowering stage, except at very early or very late sowing dates. In general,
the date for heading stage (50% heads emerged) was predicted within an interval of twothree days around the observed date.
Findings from field experiments revealed that daily number of anthers per head varied
significantly. In general, in a single head flowering lasts from five to eight days. As a contrast,
in a group of heads the course of anther extrusion last from 14 to 18 days. The peak of
number of extruded anthers was observed at six to eight days after the beginning of flowering.
Growth chamber experiments showed that anther extrusion was responsive to temperature.
Rate of extrusion increased proportionally to temperature increments (Vargas et al., 2001).
This conceptual model was translated to the predictive model.
The model attempts to predict the probability of Fusarium head blight based on the weather
variables occurring around flowering. The weather variables inserted in the model are rain
greater than 1mm and maximum temperature. An ascospore cloud is formed every day rain
is greater than 1 mm. The ascospore maturation rate is reduced at temperatures lower than
20°C. Daily ascospore cloud values are summed in simple 4-day moving periods.
If anthers are present infection occurs during a rain event greater than 1 mm. The proportion
of infected heads depends on the time course of anther extrusion and the size of the ascospore cloud.
In the field, the level of Fusarium head blight varied among experiments. The disease intensity was dependent on weather conditions during the flowering stage. Sowing date could
alter flowering date of a cultivar in a particular year causing great differences in disease
levels. As a consequence, fields with distinct sowing date can have a different level of
disease (Figure 1). Thus, to predict Fusarium head blight incidence the simulator first needs
to be very accurate in predicting growth stages of wheat. Any slight deviation from the target
(susceptibility window) may cause a considerable error in predicting Fusarium head blight
incidence. Further studies on wheat phenology are being planned. Hopefully, as more data
becomes available it will be possible to improve the model performance.
The Fusarium head blight predictive model predicts the probability of disease occurrence; it
does not predict level of disease severity. The predictive Fusarium head blight model predicted moderate to high levels of incidence for a majority of simulated wheat fields with
sowing dates in the period of 1998 to 2002, at Passo Fundo, RS, Brazil. This moderate to
high incidence was probably due to the high frequency of rainy days during the flowering
stage of wheat.
In the year 2002, for example, a “El Niño” event occurred. In southern Brazil a “El Niño” year
means precipitation above normal at spring time coinciding with heading stage of wheat.
Thus, diseases such as tan spot, glume blotch and Fusarium head blight are usually severe
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in “El Niño” years. As a consequence during such years wheat yields are degraded (Cunha,
et al., 2001). Besides, rainfall around harvest time may contribute to a lower test weight of
wheat which penalizes profits.
Plans for the future
“As is” the predictive model is a convenient tool for researchers, teachers and students to
use in the study of wheat development and incidence of Fusarium head blight. The model
was developed using up to date technology for Web deployment. Therefore, it can be shared
over the Web with a wide variety of potential users.
So far, this predictive Fusarium head blight model has been developed and tested using the
Brazilian wheat cultivar BR23 and historic weather data from Passo Fundo, RS, Brazil.
Thus, model outputs should be interpreted cautiously avoiding extrapolation to other cultivars and regions before further testing and validation. Nevertheless, the model is suitable
for general research and educational purposes. Hopefully, as more data becomes available,
it can be easily modified to accommodate different cultivars and regions.
In the meantime, aiming to reduce the error in estimating the “window of susceptibility“
model is being modified so that the user can enter the date(s) for any growth stage(s) before
flowering. Finally, the modular structure adopted in the model construction should facilitate
adding new components, as they become available, to expand model capability.
REFERENCES
Cunha, G.R. da, Dalmago, G.A., and Estefanel, V. 2001. El Niño – southern oscillation influences on wheat crop
in Brazil. In: Bedö, Z., and Láng, L. (eds.). Wheat in a Global Environment. Kluwer Academic Publishers. p.445450.
Fernandes, J.M.C. 1997. As doenças das plantas e o sistema plantio direto. In: Luz, W.C. da, Fernandes,
J.M.C., Prestes, A.M. and Picinini, E.C. (Eds.). Revisão Anual de Patologia de Plantas 5:317-352.
McMullen, M., Jones, R., and Gallenberg, D. 1997. Fusarium head blight of wheat and barley: A re-emerging
disease of devastating impact. Plant Disease 81:1340-1348.
Jones, J.W., Keating, B.A., and Porter, C.H. 2001. Approaches for Modular Model Development. Agricultural
Systems 70/2:421-443.
Sutton, J.C. 1982. Epidemiology of wheat head blight and maize ear rot caused by Fusarium graminearum.
Canadian Journal of Plant Pathology 4:195-209.
Vargas, P.R, Fernandes, J.M.C., Picinini, E.C., and Hunt, A.L. 2000. Simulação de epidemia de Gibberella zeae.
Fitopatologia Brasileira 25:497-504.
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2002 National Fusarium Head Blight Forum Proceedings
AFLP-ASSISTED GENETIC CHARACTERIZATION OF FUSARIUM
GRAMINEARUM ISOLATES FROM CANADA
W.G.D. Fernando1*, R. Ramarathnam1, J. Gilbert2 and R. Clear3
Department of Plant Science, University of Manitoba, Winnipeg, MB, Canada R3T 2N2;
Cereal Research Centre, Agriculture and Agri-Food Canada, Winnipeg, MB, Canada, R3T 2M9; and
3
Canadian Grain Commission, 600 – 300 Main St, Winnipeg, MB, R3C 3G8
*Corresponding author: PH: (204) 474-6072, E-Mail: D_Fernando@Umanitoba.ca
1
2
INTRODUCTION AND OBJECTIVES
Fusarium graminearum (Teleomorph - Gibberella zeae) is one of the most important plant
pathogens, which causes fusarium head blight (FHB) in economically important cereals
such as wheat, barley and corn. FHB has caused high losses in yield and grain quality, thus
affecting every aspect of the grain industry (Gilbert et al., 2001. Mycopathologia 153: 209 –
215). Hence, to combat the disease, chemical and biological control, and breeding for
resistance have been the methods of control. But, in the process of developing the various
control measures, we should also bear in mind the natural ability of the pathogen to evolve
over these control strategies. For example in China, resistance of F. graminearum to benzimidazole and cabendiazime fungicides has been reported (Zhou et al. 1994. Journal of
Nanjing Agricultural University 17(3): 33 - 41). Most of our current resistance to FHB has
been traced back to only a few sources (Van Ginkel et al., 1996. Plant Dis. 80: 863 – 867)
and hence, it is possible that F. graminearum could adapt to resistant varieties. Also, this
adaptation of the pathogen could extend to chemical and biological control. A better understanding of the genetic structure would shed more light on the biology of the pathogen,
which would be the key in controlling the disease. DNA-based molecular genetic characterization of the pathogen with molecular marker techniques is better suited for such studies.
Among the various marker techniques available, AFLP fingerprinting is more accurate and
produces a larger number of polymorphic bands with high reproducibility, than the other
techniques such as the slow and laborious RFLP and the less reproducible RAPD. The
objectives of this study were to determine: 1. the genetic diversity of the F. graminearum
isolates; 2. the geographical and host specificity of the isolates; 3. the correlation between
genetic structure and toxin production.
MATERIALS AND METHODS
Fifteen isolates of F. graminearum, isolated from different geographical locations (Alberta,
Manitoba, Ontario and Saskatchewan) and hosts (barley, corn, weed and wheat), were used
for the AFLP analysis. The origin, vegetative compatibility group, aggressiveness and levels
of toxin production of the isolates are presented in Table 1. The mycelia grown in potato
dextrose broth were frozen and ground in liquid nitrogen, and the genomic DNA isolated
with the help of the CTAB method. The standard protocol, (Vos et al., 1995. Nucleic Acid
Res. 23: 4407 – 4414), with a few modifications, was used for the AFLP analysis. The genomic DNA was cut with Eco RI and Mse I restriction enzymes, ligated with Eco RI and Mse
I adapters, preamplified and amplified with five set of selective primers during the AFLP
analysis (Table 2). The polymorphic bands were viewed with the help of silver nitrate stainEpidemiology and Disease Management
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ing (Promega, Madison, WI). The scored polymorphic bands were analyzed using
unweighted pair group mean analysis (UPGMA), in SAHN program of NTSYS- pc 2.1
software package (version 2.1; Exeter Software, Setauket, NY), which was used for the
cluster analysis and the construction of the dendrogram.
RESULTS AND DISCUSSION
The AFLP analysis of the 15 isolates of F. graminearum yielded 105 polymorphic bands
from the five primer sets used. Two isolates, FG8 and FG14, both isolated from corn in
Ontario and belonging to the same VCG-E (Table 1), showed great similarity and the least
genetic distance (Fig 1). FG7, an isolate from corn in Ontario, with significant aggressiveness, produced four toxins, namely DON, 3-ADON, 15-ADON and NIV, and was seen as a
distinct sub-branch in the dendrogram (Fig 1). The geographic location and the hosts from
which the isolates were obtained seem to have had a mild influence on the clustering of the
isolates, as they seem to form small clusters based on either their originating geographic
location or host (Fig 1). Among the isolates that were all isolated from wheat, FG2 isolated
from winter wheat was distinct from the other two isolates, FG1 and FG4, isolated from red
spring wheat, which shared higher genetic similarity (Fig 1). It is interesting to note that
isolates FG1 and FG4 came from Alberta and Manitoba (Table 1), respectively. Among the
isolates from Ontario that formed small clusters in the dendrogram, isolates FG5 and FG10,
isolated from winter wheat, were distinct from the other isolates from corn and barley (Fig 1
and Table 1). Isolate FG13, which was isolated from a weed in Saskatchewan, formed a
distinct sub-branch and thus was distinct from the isolates from Ontario in the other branch
of the cluster (Fig 1).
AFLP analysis showed genotypic diversity between the F. graminearum isolates with reference to their vegetative compatibility groups, and this supported tan earlier work (Bowden
and Leslie, 1992. Exp. Mycol. 16: 308 – 315), which showed genotypic diversity among G.
zeae isolates based on their VCG. The branching of the isolates in the tree was more influenced by the VCG and the levels of toxin production of the isolates (Fig 1). This was clearly
seen in the analysis, as isolates FG8 and FG14 belonging to the same VCG- E showed the
least genetic distance, and isolate FG7 with the highest levels of all the four toxins, formed a
distinct branch in the large upper cluster of the tree (Fig 1). The analysis showed a weak
host or geographic specificity among the isolates, as observed by the weak clustering of the
isolates in the tree based on their geographic location or the host from which they were
isolated. This supported an earlier work (Van Eeuwijk et al., 1995. Theor. Appl. Genet. 90:
221 – 228), which showed non-specificity of resistance in wheat with European strains of F.
culmorum, F. graminearum and F. nivale. The low geographic specificity of the data, as
indicated by isolates FG1 and FG4 isolated from wheat in Alberta and Manitoba, respectively clustering together, seems to suggest the movement of the pathogen to new areas.
The distinct branching of FG2 (from winter wheat) from the cluster of FG1 and FG4 (from
spring wheat), seems to suggest that they produce and release spores at different times in a
season to coincide with the availability of the susceptible growth stage of the respective
host (winter or spring wheat) for infection and colonization.
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It would be interesting to find the lineage of these 15 isolates from Canada that come from
different geographical locations and different hosts. The F. graminearum clade includes
seven distinct lineages (O’Donnell 2000. Proc. Natl. Acad. Sci. 97: 7905 – 7910), with the
isolates from the USA falling within lineage 7. Therefore, it would be useful to find the lineage of the Canadian isolates and to check whether they shared any similarity with their US
counterparts. Isolates of F. graminearum that produce the FHB toxins belong either to the
DON chemotype or the NIV chemotype (Marasas et al. 1984. The Pennsylvania State University Press, University Park, PA.). Most of the isolates from the USA belong to the DON
chemotype (Anne Desjardins - Presentation at the CPS Annual Meeting, Waterton, Alberta,
2002). They produce small amounts of 3ADON and 15ADON. The NIV chemotype produce
DON at only less than one percent of NIV (Anne Desjardins- personal communication). But
two of our isolates, FG7 and FG10, produced significantly very high levels of DON when
compared to NIV, especially FG7, which produced 249 ppm of DON and 1 ppm of NIV. It
would be very interesting to genetically characterize these isolates, which would throw more
light on the biochemistry of toxin production and also help us to understand whether the
pathogen is going through a process of evolution to become a more aggressive form!
ACKNOWLEDGEMENTS
We acknowledge and thank Agricultural Research and Development Initiative of Manitoba
for funding this project.
FG1
FG4
FG2
FG8
FG14
FG10
FG9
UM10
FG15
FG17
FG7
FG5
FG6
FG12
FG13
0.34
0.53
0.72
Coefficient
0.91
1.09
Figure 1. Dendrogram from UPGMA of AFLP data of F. graminearum
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Table 1. Origin, VCG, aggressiveness and toxin production of F. graminearum isolates.
DAOM #
Host
Location
$
VCG
†
Aggressiveness
SFI
‡
Spray
T1*
T2*
T3*
T4*
£
213295 (FG1)
Wheat
AB
J
37
64.9
0.2
0
0.5
0
177409 (FG2)
Wheat
ON
C
17.2
56.4
88.3
8.6
0
0
192132 (FG4)
Wheat
MB
H
32.8
69
2.3
0
5.9
0
177406 (FG5)
Wheat
ON
A
33.2
56.1
10
0
13.1
0
178149 (FG6)
Barley
ON
L
33.4
56.2
27.8
0
24.7
0
170785 (FG7)
Corn
ON
K
31.5
43.6
249
11.7
3.1
1
180378 (FG8)
Corn
ON
E
30.5
45.9
20.5
0
16.2
0
180379 (FG9)
Corn
ON
F
36.3
60.2
80.9
2.8
0.4
0
177408 (FG10)
Wheat
ON
B
29
53.4
53.1
0
44.6
0.3
180377 (FG12)
Corn
ON
M
26.8
39.1
35.3
0
26.9
0
213384 (FG13)
Weed
SK
I
24.8
51
12.1
0
15.4
0
180376 (FG14)
Corn
ON
E
36.9
67.6
21.7
0
17.3
0
192130 (FG15)
Wheat
MB
D
32.2
52.2
6
0
11.6
0
$Location: AB- Alberta; MB- Manitoba; ON- Ontario; SK- Saskatchewan
†Vegetative Compatibility Group
‡Single Floret Inoculation with macroconidia (10µl of 50,000spores/ml)
£Spray inoculation with macroconidia (2 – 3 ml of 50,000 spores/ml)
*T1-DON- Deoxynivalenol; T2- 3ADON- 3-acetyl DON; T3- 15ADON- 15-acetylDON;
T4- NIV- Nivalenol (in ppm)
(Modified from Gilbert et al., 2001. Mycopathologia 153: 209)
T able 2. L ist o f selective p rim ers used fo r the A F L P analysis.
P rim er S et
E co R I en d
M S e I en d
1
AC
A
2
AC
T
3
AA
T
4
AA
AT
5
TG
TT
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ASSESSMENT OF THE DIFFERENTIAL ABILITY OF FUSARIUM
STRAINS TO SPREAD ON WHEAT AND RICE
Rubella S. Goswami1 and H. Corby Kistler2*
1
Dept. Plant Pathology, University of Minnesota, St. Paul, MN 55108; and
2
USDA–ARS Cereal Disease Laboratory, St. Paul, MN 55108
*Corresponding Author: PH: (612) 625-9774; E-mail:hckist@umn.edu
ABSTRACT
In order to understand gene function related to pathogenicity we are evaluating the abilities
of different strains of Fusarium to spread on the hosts and conducting genomic analysis of
these interactions. Several strains selected from each of eight known phylogenetically
distinct lineages of Fusarium graminearum were tested for their ability to spread on Norm, a
susceptible cultivar of wheat, after inoculation of a single central floret. Similar studies were
also conducted using strains belonging to other Fusarium species namely, F. cerealis, F.
pseudograminearum, F. culmorum and F. lunulosoporum. All these strains were found to
differ significantly in both their ability to spread within the wheat head as well as the type
and amount of mycotoxins they produce. A few of the F. graminearum strains were also
tested for their ability to infect rice panicles. These strains caused necrosis in rice, but mycotoxin production was not detected in infected rice florets. Symptom expression, the presence of fungus in each spikelet, as determined by culturing, and mycotoxin concentrations
were recorded from inoculated wheat heads and rice panicles 14 days after inoculation.
Based on these pathogenicity tests one highly aggressive and one less aggressive strain
were chosen for studies conducted with the aim of understanding these variations at the
genomic level. cDNA libraries were created by subtractive hybridization to compare mRNA
populations from wheat heads inoculated with the two strains in order to identify genes
specific to each interaction. Marked differences in the transcript profile of these two interactions was revealed during the initial infection phase.
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DEVELOPMENT OF GIBBERELLA ZEAE ON WHEAT TISSUE
John Guenther and Frances Trail*
Departments of Plant Biology and Plant Pathology, Michigan State University, East Lansing, MI
*Corresponding Author: PH: (517)432-2939; E-mail:trail@msu.edu
ABSTRACT
Fusarium head blight (FHB) is a devastating disease of cereal grains worldwide. In the
United States this disease is mostly attributed to infections by the fungus Gibberella zeae
(anamorph Fusarium graminearum). The disease cycle of FHB is a valuable resource when
considering control of G. zeae. The development of perithecia on wheat residues and the
inoculum produced by perithecia have important impact on disease in reduced tillage
systems. Our objective is to characterize the colonization of vegetative tissue and the subsequent development of perithecia. All plant tissue and cell types are susceptible to ramification by hyphae of G. zeae. However, the colonization of tissues adjacent to cells supporting
perithecium formation is especially significant to the development of perithecia. Chlorenchyma tissue of the internodes and parenchyma tissue of the stem nodes are tissues found
to directly underlie cells that support perithecium development. Perithecia form through
stomates above chlorenchyma of the stem internode and from epidermal cells above the
parenchyma of the stem node region. We are also interested in determining whether head
infections proceed down the stem or if stem tissue is colonized from independent stem
infections. Development of strategies for limiting infection of vegetative tissue is contingent
upon understanding the mode of infection. The results of this study will give insights into the
disease cycle as well as an understanding of infection pathways.
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THE DONCAST MODEL: USING WEATHER VARIABLES PRE- AND
POST-HEADING TO PREDICT DEOXYNIVALENOL
CONTENT IN WINTER WHEAT
David C. Hooker*, Arthur W. Schaafsma, and Lily Tamburic-Ilincic
Ridgetown College, University of Guelph, Guelph, Ontario, Canada. N0P 2C0
*
Corresponding Author: PH: (519)644-2036; E-mail: dhooker@skynet.ca
ABSTRACT
Accurate predictions of deoxynivalenol (DON) concentrations in mature wheat grain are needed at
heading for decisions on whether a fungicide application is necessary to control fusarium head
blight, Fusarium graminearum Scwabe. Our model, now named “DONcast”, was developed using
weather and DON data from 399 farm fields across southern Ontario, Canada, from 1996 to 2000
(Hooker et al., 2002). A web site was launched in 2000 for providing DON predictions (in ìg g-1 of
mature wheat grain) to growers across Ontario http://www.ownweb.ca/models/public/fusarium/
default.cfm?location=none. From 2000 to 2002, DONcast was validated on 121 wheat fields on
private farms across Ontario. All parameters of the first DONcast model were reviewed and other
variables were considered with the addition of both weather and DON data from 2000 and 2002.
DONcast was refined further by considering agronomic influences such as wheat variety, previous
crop, and tillage system from all 520 fields between 1996 and 2002. In the refined DONcast model,
weather was still important between 7 days before heading and 10 days after heading. In the first
period 4 to 7 days before heading, DON generally increased with the number of days with >5 mm of
rain, and decreased with the number of days of <10°C. In the second period 3 to 6 days after
heading, DON increased with the number of days of rain >3 mm, and decreased with days >32°C.
In the third period 7 to 10 days after heading, DON increased with the number of days with >3 mm of
rain. Using multiple regression procedures, the refined model accounted for lower concentrations of
DON when cool temperatures (mean daily temperatures < 15°C) occurred between 3 and 10 days
after heading. Wheat variety susceptibility coefficients from inoculated misting trials were also
included in the refined model, along with a variable for the presence of host crop residue on the soil
surface at wheat planting (Schaafsma et al. 2000). While only one equation is used in each case to
forecast DON, the equation is different depending on the situation. In fields where the previous crop
was not wheat or corn, the refined model explains 78% of the variation in DON using the equation if
rain occurred between 3 and 6 days after heading. If no rain occurred between 3 and 6 days after
heading, then another equation is used, which explains 63% of the variation in DON. In fields where
the previous crop was corn or wheat, another prediction equation explains 86% of the variability in
DON. Using the refined DONcast model, DON concentrations of < 1 ìg g-1 were predicted correctly
on 46 of 52 fields in 2001 and on all 34 fields surveyed around weather stations in 2002.
Concentrations of > 1 ìg g-1 were predicted correctly on 9 of 14 fields in 2001, and on 5 of 11 fields in
2002. Accurate predictions of < 1.0 ìg g-1 suggests that control strategies may not be warranted,
while predictions of 1 to 2 ìg g-1 suggests that control strategies may be warranted to improve the
grade and marketability of wheat.
REFERENCES
Hooker, D.C., A.W. Schaafsma, and L. Tamburic-Ilincic. 2002. Using weather variables pre- and post-heading to
predict deoxynivalenol content in winter wheat. Plant Dis. 86:611-619.
Schaafsma, A.W., D.C. Hooker, L. Tamburic-Ilinic, and J.D. Miller. 2001. AgronomicConsiderations for Reducing
Deoxynivalenol Content in Wheat Grain. Can J. Plant Pathol. 23:279-285.
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FUSARIUM HEAD SCAB RISK FORECASTING FOR OHIO, 2002
Patrick Lipps1*, Dennis Mills1, Erick DeWolf2 and Larry Madden1
1
The Ohio State University/OARDC, Department of Plant Pathology, Wooster, OH 44691; and 2The
Pennsylvania State University, Department of Plant Pathology, University Park, PA 16802
*Corresponding Author: PH: (330) 263-3843; E-mail: lipps.1@osu.edu
ABSTRACT
During the 2002 wheat growing season, head scab risk assessment models were used to
predict the risk of Fusarium head scab in Ohio. This was the second year for testing these
models in the state. Head scab risk assessment probabilities were derived from logistic
models previously developed from hourly weather, crop growth and disease observations
from 50 location-years representing three wheat production regions in the US. Hourly
weather data from 14 weather stations located in Ohio, Indiana and Michigan were used to
determine duration of weather events for the pre- and post-anthesis time periods examined
by the models. Disease risk probabilities were calculated using logistic equations determined by two models representing the critical weather conditions during the time period 7
days prior to anthesis (Model I) and the time period inclusive of the 7 day pre-anthesis plus
10 additional days post anthesis (Model II). Weather conditions in early April were relatively
dry and warm providing conditions for rapid and early development of the crop. Anthesis
dates for wheat fields from south to north in the state varied by more than four weeks (10
May to 9 June) due to cool weather that slowed plant development in May. Precipitation
events became more frequent during late April and throughout May across the state with
most locations reporting up to 32 hours of measurable precipitation during the 7 days prior
to anthesis. However, average daily temperatures for most locations in the state were
generally below 15°C when most of the wheat was in anthesis. Scab risk probabilities were
calculated for early, mid and late anthesis dates for each weather station location. Calculated risk probabilities ranged from 0.00 to 0.81 for Model I and from 0.02 to 0.69 for Model II.
Of 42 location-anthesis date scab risk probabilities calculated, Model I predicted 31 location-anthesis dates with low to moderately low risk and Model II predicted 40 locationanthesis dates with low or moderately low risk. Only one location (Ft. Wayne, IN) had a
moderately high risk prediction for Model I and Model II and another site (Oxford, OH) had a
moderately high risk prediction for Model II. Based on these results, the head scab risk
prediction was reported to be low to moderately low for the majority of locations in the state.
Head scab risk predictions were posted on the Ohio State University Ohio Field Crop Disease web page (www.oardc.ohio-state.edu/ohiofieldcropdisease/) during the critical time of
disease development through harvest. Approximately 14 to 18 days after anthesis 159
fields in 30 counties were surveyed for scab incidence by the OSU Extension Agents. From
1 to 10 fields were surveyed per county. Disease surveys indicated the average incidence
of head scab was 4.1% with a range of 0% to 48.6%. Over 75% of the surveyed fields had
scab incidence levels below 5%, and only 4% of the surveyed fields had incidence levels
above 15.1%. Results of the Scab Risk Assessment Models indicated that they generally
predicted the risk of scab correctly for the majority of locations in the state.
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PRACTICAL APPLICATION OF FUSARIUM HEAD
BLIGHT RISK PREDICTIONS
Patrick Lipps1*, Erick De Wolf2, Dennis Mills1 and Larry Madden1
The Ohio State University/OARDC, Department of Plant Pathology, Wooster, OH 44691; and 2The
Pennsylvania State University, Department of Plant Pathology, University Park, PA 16802
*Corresponding Author: PH: (330) 263-3843; E-mail: lipps.1@osu.edu
1
Fusarium head blight (FHB), primarily caused by the residue-borne fungus Fusarium
graminearum, continues to be an important economic problem in the more humid, temperate
regions of the US and Canada (Bai and Shaner 1994, McMullen et al. 1997). Control of this
disease has been difficult, but progress has been made by scientists funded by the U.S.
Wheat and Barley Scab Initiative (USWBSI) toward the management of FHB. An important
objective of the USWBSI has been the development of adequate disease forecasting systems for FHB.
The most significant purpose of a head scab forecasting system would be to function as an
early warning system. If accurate disease predictions could be made prior to floret infection
at anthesis, then growers could use preventative disease control options, such as chemical
or biological agents, to avert disease and yield loss. Secondly, a timely disease warning
would also provide valuable time for farmers, grain handlers and food processors to deal
with the prognosis of disease and the potential for mycotoxin contaminated grain by establishing the necessary infrastructure to appropriately test for, and manage, damaged grain.
Last year in a presentation at the 2001 National Fusrium Head Blight Forum, Dr. Len Francl
reviewed the various types of forecasting systems available for small grain diseases (Francl
2001). He discussed two FHB forecasting systems that are now being tested. In Ontario,
Canada, Hooker et al (2002) have developed a model that utilizes weather data pre anthesis and weather forecasts post anthesis to predict deoxynivalenol levels in harvested grain.
In Ohio, De Wolf et al, (2001) have developed a disease forecasting system based on risk
assessment. Generally, risk assessment models estimate the probability (i.e., risk) of an
undesirable event occurring at a given location and time (Teng and Yang, 1993). FHB
appears well suited for risk assessment modeling because of the severity of epidemics,
compound losses from mycotoxin contamination and yield loss, and the relatively narrow
time periods of pathogen sporulation, inoculum dispersal, and host infection (De Wolf et al.
1999, Francl et al. 1999).
Our main objectives were to develop relatively simple models using readily accessible
weather variables that would be applicable over a large geographic area including spring
and winter wheat areas. Secondly, to meet the immediate need of farmers we needed to
develop a forecasting system in as short a time as possible. To meet this goal we used
historic disease data and weather records for model development. Dr. De Wolf presented a
description of the initial FHB risk models at the National Fusarium Head Blight Forum
meeting in 2000 (De Wolf et al 2000). We have been testing the models in Ohio during
2001 and 2002 (Lipps and Mills, 2002) and other states (ND, SD, MO, MI and PA) have
tested them during 2002.
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The risk predictions models were developed using historic disease and weather data obtained from cooperators in ND, OH, MO and KS (De Wolf et al, 2000, De Wolf et al, 2001).
Logistic regression models were developed from hourly weather, crop growth and disease
level observations from 50 site-years representing three wheat production regions in the US.
Correlation analysis identified combinations of temperature, relative humidity and rainfall
across time periods 7 days prior to and 10 days after anthesis as significant independent
variables. Of several logistic regression models developed the following two models were
adopted for further testing because of their relatively high prediction accuracies.
Model I predicts the probability of head scab based on the weather that occurs prior to
anthesis. This is the time when fungal inoculum develops. Model I utilizes the duration of
precipitation in hours and the number of hours when the air temperature is between 15 and
30°C for 7 days prior to flowering. Cross validation prediction accuracy for this model was
78% for determining when disease will not be severe (severity < 10%). Its accuracy for
predicting when an epidemic will occur (severity > 10%) was 56%.
Model II predicts the probability of scab based on the weather that occurs 7 days before and
10 days after anthesis. This model addresses the time when the fungus is developing
spores, when infection occurs and when disease develops. Model II utilizes the number of
hours when the air temperature is between 15 and 30°C for 7 days prior to flowering and the
number of hours when the relative humidity is 90% or above and the air temperature is
between 15 and 30°C for 10 days after flowering. Cross validation prediction accuracy for
this model was 83% in determining when disease will be severe (severity >10%).
In order to make the FHB risk forecasting models more user-friendly, Dr. De Wolf and Mr.
Mills developed a Microsoft Excel workbook that contains the various logistic equations.
Probabilities are automatically calculated when the appropriate weather data is entered and
the anthesis date is designated. The Excel file not only calculates the scab risk probability
values, but also graphs the weather variable coordinates and plots them in relation to a risk
threshold curve (logistic regression equation where predicted FHB severity is > 10%). The
distance of weather variable coordinates from the threshold curve defines the relative risk
probability for the weather station location.
There is considerable flexibility for using FHB risk models, especially for processing
weather data and presenting the prediction information to the growers. In Ohio during the
2002 season, hourly weather data from 14 weather stations were used to make risk probability calculations for three anthesis dates (early, mid and late anthesis) for each weather
station location. Actual risk probabilities (as a percentage) were not presented directly to
the public, but numerical probabilities were classified into ‘Risk Levels’ (low, moderately
low, moderately high, and high) based on logistic regression thresholds in order to help
growers better interpret the risk of scab in their area. To facilitate the timeliness of reporting
information during the critical period of scab development, a web page (www.oardc.ohiostate.edu/ohiofieldcropdisease/) was used to deliver scab risk assessments.
Michigan and Pennsylvania, took a similar approaches to providing public access to FHB
risk forecasts and managing the problem of obtaining accurate anthesis date information.
Both reported prediction results on a web site (URL for MI was http://www.cips.msu.edu/
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cips/headblight/index.htm and for PA was http://www.wheatscab.psu.edu/) and used
weather data from multiple locations (18 location in MI and 33 locations in PA). Additionally,
both provided daily risk probabilities for each of the weather locations and presented these
as contour maps of the state with risk probabilities as color-coded areas between contour
lines. This map-based presentation style required the farmer to choose the appropriate
anthesis date for his fields to obtain the FHB risk probability map for that date.
Validation of the FHB risk assessment models in the field is a problem because of the large
area, and consequently large number of fields, for which predictions are made. In Ohio,
thirty County Extension Agents assessed the incidence of head scab in 159 fields by counting the number of diseased and non-diseased heads per foot of row in 10 locations per field.
The FHB risk models predicted low to moderately low scab risk for most of the state in 2002.
The FHB survey indicated that over 75% of the fields had incidence levels below 5% (average for all fields = 4.1%). In Michigan, risk assessment Model II predicted low to moderately
low scab risk for 17 of the 18 weather station locations. A survey of fields in southern Michigan indicated that FHB incidence was low to moderately low and ranged from 0 to 25% in
individual fields. Of 50 grain samples submitted from fields throughout the state all but one
had DON levels between 0 and 0.5 ppm. Risk assessment models were not developed to
predict DON levels in grain. The low DON levels detected in MI were probably due to the
low level of disease and the dry conditions during the grain filling period.
Disease forecasting systems are never 100% accurate because they are mathematical
predictions representing a multitude of variables that determine disease progress. Each of
the variables can have a wide range of values. Problems that limit the accuracy of FHB
forecasting models include variables associated with the pathogen (variation in inoculum
levels in the field due to differences in residue management systems, variation in crop
rotation sequences among fields, wind and rain splash dispersal gradients, pathogen species composition); host (anthesis date differences, susceptibility level, duration of anther
retention, head height); and weather (variation in rain and RH duration across an area,
temperature variation due to topography). Although models are designed to be robust, they
will not accurately describe all possible situations. Risk predictions may be improved by
using weather data from many sites, obtaining more accurate anthesis dates for an area,
averaging risk probabilities over a multiple-day anthesis periods for locations and monitoring inoculum levels. Currently, cooperative research is being conducted in ND, SD, IN, OH
and PA to develop a database of weather, crop development and pathogen inoculum level
information to further validate and improve FHB risk assessment models.
REFERENCES
Bai, G. and Shaner, G. 1994. Scab of wheat: Prospects for control. Plant Dis.78:760-766.
De Wolf, E. D., Lipps, P. E. Francl. L. J. and Madden, L. V. 1999. Role of environment and inoculum level in
wheat Fusarium head blight development. P. 87 - 91, In: Proceedings of the 1999 National Fusarium Head
Blight Forum, Dec. 5-7, Sioux Falls, SD.
De Wolf, E. D., Madden, L. V. and Lipps, P. E. 2000. Prediction of Fusrium head blight epidemics. P. 131-135, In:
Proceedings of the 2000 National Fusarium Head Blight Forum, Dec. 10-12, Erlanger, KY.
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2002 National Fusarium Head Blight Forum Proceedings
De Wolf, E. D., Madden, L. V. and Lipps, P. E. 2000. Risk assessment models for wheat Fusarium head blight.
Phytopathology 90:S19 (Abstract).
De Wolf, E. D., Lipps, P. E. and Madden, L. V. 2000. Crop residue moisture and Gibberella zeae perithecia
development. 2000 National Fusarium Head Blight Forum Proceedings, Erlanger, KY, Dec. 10-12, 2001.
De Wolf, E. D., Madden, L. V. and Lipps, P. E. 2001. Fusarium head blight epidemic prediction and risk assessment. Phytopathology 91:S22 (Abstract).
Francl, L., Shaner, G., Bergstrom, G., Gilbert, J., Pedersen, W., Dill-Macky, R., Sweets, L., Corwin, B., Jin, Y.,
Gallenberg, R. and Wiersma, J. 1999. Daily inoculum levels of Gibberella zeae on wheat spikes. Plant Dis.
83:662-666.
Francl, L. 2001. Past, present, and future of forecasting small grain diseases. P. 123 - 125, In: Proceedings of
the 2001 National Fusarium Head Blight Forum, Dec. 8-10, Erlanger, KY.
Hooker, D. C., Schaafsma, A. W., and Tamburic-Ilincic, L. 2002. Using weather variables pre- and post-heading
to predict deoxynivalenol content in winter wheat. Plant Dis. 86:611-619.
Lipps, P. E. and Mills, D. 2002. Forecasting Fusarium head scab of wheat in Ohio, 2002. Ohio State University,
Ohio Agricultural Research and Development Center, Wooster. Plant Pathology Department Series 117. August
2002.
McMullen, M., Jones, R. and Gallenburg, D. 1997. Scab of wheat and barley: a re-emerging disease of devastating impact. Plant Dis. 81:1340-1348.
Teng, P. S. and Yang, X. B. 1993. Biological impact and risk assessment in plant pathology. Annu. Rev.
Phytopathol. 31: 495-521.
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EPIDEMIOLOGICAL STUDIES ON FUSARIUM HEAD BLIGHT OF
WHEAT IN SOUTH DAKOTA FOR 2002
L. Osborne and Y. Jin*
Plant Science Department, South Dakota State University, Brookings, SD 57007
*Corresponding Author: PH: (605) 688-5540; E-mail: Yue_Jin@sdstate.edu
INTRODUCTION AND OBJECTIVES
South Dakota State University is part of a multi-state collaborative project studying epidemiology of Fusarium head blight (FHB) on wheat under different environments throughout the
upper mid-west. The ultimate goal is to develop a disease risk advisory/forecast system.
Primary objectives include: 1) monitoring inoculum dynamics and disease development in
relation to specific environmental parameters; and 2) to evaluate currently proposed forecast
models.
It has been observed that FHB occurs at epidemic levels when warm, humid conditions and
frequent precipitation have occurred at anthesis (Bai and Shaner, 1994; McMullen et al.,
1997; Parry et al., 1995). By investigating the relationship of FHB incidence and severity to
environmental conditions, a better characterization of the disease can be made. Environmental conditions are thought to influence the FHB disease cycle, but it is not certain which
factors are critical, and which are most predictive of epidemics. By collecting disease and
environmental data for multiple plantings of susceptible wheat across several locations, we
might better characterize both epidemic and non-epidemic conditions for FHB development.
MATERIALS AND METHODS
Spring wheat (cv. ‘Norm’) susceptible to FHB was planted into strips 1.4m by 45m using a 7row grain drill. Two adjacent strips were planted on each of three planting dates (26 April, 6
May, and 22 May, 2002), referred to as planting date (PD) 1, 2, and 3, respectively. Multiple
dates were initially intended to ensure that susceptible host stage and pathogen inoculum
would be present concurrently. Each planting was divided into three replicate plots. Each
plot was further divided into two subplots, one sampled and one unsampled. The
unsampled subplot was used to assess final disease levels for each plot.
Weather and microenvironment data were continuously collected using a datalogger
(Campbell Scientific Inc. model CR10X) and various instruments. Leaf wetness sensors
(Campbell Scientific Inc. model 237) were used to estimate the duration of leaf wetness
within the canopy. Additional sensors were constructed and deployed to detect moisture at
the soil surface (Osborne and Jin, 2000).
Daily airborne inoculum levels were monitored during the sampling period using a Burkhard
Cyclone Sampler (Burkhard Manufacturing). A wash of the cyclone unit was performed daily
to ensure uniform sampling. The sample and wash were plated on Komada’s medium for
spore enumeration (Komada, 1975). Counts were reported as colony forming units (CFU)
per day. Inoculum on wheat spikes was estimated by washing spikes using protocols deEpidemiology and Disease Management
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scribed by Francl et al. (1998), with some modification (sampled spikes were not covered
prior to sampling). On each day, five primary spikes per replicate were collected and placed
in a flask with 50ml of sterile deionized water, shaken vigorously for 60 seconds to dislodge
spores, then discarded. A 0.5ml aliquot of the wash was then spread-plated onto each of
three plates of Komada’s medium. Plates were then incubated 5-8 days. Colonies were
described and counted after incubation. Colonies were reported as CFU per spike per day.
Disease incidence and severity data were collected from each replicate within each planting date three to four times between late anthesis (Zadoks 67) and soft dough stage
(Zadoks 85). In each replicate, 150 spikes from primary tillers were visually rated for FHB.
Severity of FHB for each spike was rated on a 0-9 scale roughly based on percent of the
spike visually blighted (0 to 90+%). Incidence rate was calculated by: number of infected
spikes divided by total spikes counted per replicate. Severity was calculated for infected
spikes by: (sum of spike severity ratings) divided by the number of infected spikes per replicate.
Data from the 2002 FHB monitoring plots will be entered into two FHB risk assessment/
disease forecast models made available by Ohio State University (Ohio I and Ohio II; De
Wolf, et al, 2000). Ohio model I is used to predict risk of a FHB epidemic based on temperature and precipitation variables prior to anthesis. Ohio model II is intended to predict disease risk based on temperature and humidity before and after flowering begins. Model I is
intended to predict epidemics before infection, while Model II is intended to estimate disease risk after infection may have occurred.
RESULTS AND DISCUSSION
Major environmental parameters for each planting date are summarized in Table 1. Generally, dry conditions with warm temperatures were experienced throughout the growing
season in 2002. A short period of three to four days of wet weather was experienced just
prior to flowering of PD 1, but was followed by very warm, dry conditions for several days.
Table 1. Environmental conditions over susceptible periods in each planting date.
a.
b.
Avg. ea
(kPa)
Precip. (mm) /
duration (hrs)
15oC < T < 30oC
(hours, max=168)
RH > 90%
(hours)
T*RH
(hours)
25.7
2.00
0/0
107
10.5
7.5
DOY 180-186
26.4
2.15
0/0
108
9.5
9.5
DOY 190-196
20.5
1.75
2.0 / 2
123
46
27.5
PD
Time period
(susceptible)
Avg. air
temp (oC)
1
DOY 177-183
2
3
a
b
vapor pressure of the air
hours temp is between 15oC and 30oC and RH > 90%
Inoculum level estimates for 2002 are presented in Table 2, and disease levels are presented in Table 3. Inoculum was considered to be moderate as estimated by both the
Burkard spore trap and by the spike-wash method. Disease incidence was much higher
than expected, ranging from 10 to 45% of head affected by FHB. Severity however was very
low in all cases, ranging from 1 to 8% blight on infected spikes, on average.
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Table 2. Inoculum level estimates over susceptible periods in each planting date.
a
Spike-wash
Time period
Burkard Spore Trap
PD
(cfu / spike)
(susceptible)
(cfu / day)
a.
1
DOY 177-183
335
75
2
DOY 180-186
321
105
3
DOY 190-196
215
122
average of 3 reps, 10 spikes per rep.
Table 3. Final disease ratings.
Plant Date 1
Plant Date 2
Plant Date 3
Incidence %
Severity %
Incidence %
Severity %
Incidence %
Severity %
Rep 1
44.7
8.2
43.3
6.1
21.3
2.4
Rep 2
46.7
7.2
38.7
5.0
14.7
1.6
Rep 3
26.7
7.3
37.3
5.6
10.7
1.2
PD Mean
39.3
7.6
39.8
5.6
15.6
1.7
Overall:
Disease Incidence = 20%
Disease Severity = 5%
The high incidence levels, coupled with the moderate levels of airborne and spike-borne
spores suggest that inoculum levels were present at a level conducive to disease, however
environmental conditions experienced during anthesis and after were not considered to be
conducive to FHB development beyond the initial infections.
The results of model validation of Ohio models I and II are given in Table 4. FHB disease
index for all plantings is given as an indicator of overall disease level and is the product of
incidence and severity for each planting date. The probability of an epidemic occurring
based on the two models is also given. Based on this set of validation runs, Ohio model I
was consistent across plantings in relative rank and appear to correlate well to FHB incidence, however, it suggests that an epidemic is likely for PD 1, which had only 2.5% disease. Ohio model II suggests that no epidemic levels would be reached, which corresponds
to the final disease estimates. It is believed that the parameter for precipitation (hours of
precipitation duration) incorporated into Ohio I does not account for precipitation patterns of
the Great Plains, which typically receive large quantities of precipitation in relatively short
periods of time.
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Table 4. Validation of Risk Assessment / Disease Prediction Models (Ohio I and II)
Ohio Model II
Planting
FHB Index
FHB Incidence
Ohio Model I
a
(risk probability)b
Date
(Inc*Sev, %)
(% infected spikes)
(risk probability)
1
2.5
39
0.53
0.08
2
2.2
40
0.31
0.14
3
0.3
16
0.19
0.33
a. epidemic threshold = 0.5
b. epidemic threshold = 0.44
REFERENCES
Bai, G. and G. Shaner. 1994. Scab of wheat: Prospects for control. Plant Disease 78:760-766.
Francl, L. 1998. Development of Fusarium head blight in relation to environment and inoculum. p. 1-3. In Proc.
National Fusarium Head Blight Forum, East Lansing MI. 26-27 Oct. 1998. U.S. Wheat and Barley Scab Initiative,
East Lansing, MI.
Francl, L., G. Shaner, G. Bergstrom, J. Gilbert, W. Pedersen, R. Dill-Macky, L. Sweets, B. Corwin, Y. Jin, D.
Gallenberg, and J. Wiersma. 1999. Daily inoculum levels of Gibberella zeae on wheat spikes. Plant Disease
83:662-666.
Komada, H. 1975. Development of a selective medium for quantitiative isolation of Fusarium oxysporum from
natural soil. Rev. Plant Protec. Res. 8:114-125.
De Wolf, E.D., L.V. Madden, and P.E. Lipps. 2000. Prediction of Fusarium head blight Epidemics. p. 131-135. In
2000 National Fusarium Head Blight Forum, Erlanger, KY. Dec 10-12, 2000. U.S. Wheat and Barley Scab
Initiative, East Lansing, MI.
Osborne, L. and Y. Jin. 2000. A Sensor for Monitoring Wetness at the Soil-Air Interface. Pages 169-172 in: Proc.
2000 National Fusarium Head Blight Forum. Dec. 10-12, 2000, Erlanger, KY.
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.
Parry, D.W., P. Jenkinson. and L. McLeod. 1995. Fusarium ear blight (scab) in small grain cereals-a review.
Plant Pathology 44:207-238.
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FHB INOCULUM DISTRIBUTION ON WHEAT
PLANTS WITHIN THE CANOPY
L. Osborne, Y. Jin*, F. Rosolen, and M.J. Hannoun
Plant Science Department, South Dakota State University, Brookings, SD 57007
*Corresponding Author: PH: (605) 688-5540; E-mail: Yue_Jin@sdstate.edu
ABSTRACT
Fusarium head blight (FHB) of wheat is a potentially devastating disease in many of the
wheat growing regions of the U.S. and Canada. The primary inoculum for this disease is
generally considered to be ascospores of the fungus Gibberella zeae (ana: Fusarium
graminearum). Perithecia of the fungus develop on infected crop residues, especially corn
stalk pieces, remaining on field surfaces. The perithecia forcibly eject ascospores, but their
fate is not certain. A large proportion of ascospores may not be able to contact susceptible
host tissues because the infection window is quite narrow. Instead, these spores may land
on non-susceptible tissues (leaf, stem, etc.). These spores may germinate and reproduce
epiphytically. A study was initiated in the 2002 field season to investigate the types (conidia
or ascospores) and distribution of spores of the FHB pathogen. Wheat plants were collected
from 10 sites (3 groups per site) around the state and subsequently dissected and processed to enumerate conidia (of F. graminearum) and ascospores (of G. zeae) on individual
leaves at specific leaf positions on the plants, as well as on the spikes. Ascospores and
conidia were recovered at levels from 0 to 1500 spores per leaf. Relative ratios of ascospores to conidia varied greatly from 7:1 down to 1:4. Generally, ascospores outnumbered conidia at all leaf positions across most locations, with some notable exceptions. The
results of the sampling show a distinct bimodal distribution pattern for ascospore counts with
higher concentrations (50 to 200% greater) at the upper-most leaf position and the lowermost leaf position within the canopy than at the center leaf position. It is also noted that
conidial distribution among leaves varied widely across locations. In some locations, few
conidia were identified, while at other locations, all leaves were found to hold large numbers
(up to 1500 spores) per leaf. This suggests that the fungus may undergo epiphytic growth
and reproduction, resulting in increased inoculum load within the canopy of a wheat crop.
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SOUTH DAKOTA FUSARIUM HEAD BLIGHT
RISK ADVISORY FOR 2002
L. Osborne and Y. Jin*
Plant Science Department, South Dakota State University, Brookings, SD 57007
*Corresponding Author: PH: (605) 688-5540; E-mail: Yue_Jin@sdstate.edu
ABSTRACT
In 2002, the small grains pathology project at South Dakota State University launched a
web-delivered, weather-based risk advisory for Fusarium head blight (FHB) in northeastern
South Dakota. A thirteen-county area comprising the majority of the spring wheat region in
the state was selected for intensive inoculum, disease and environment monitoring. This
area was selected for a FHB risk advisory to be issued on a county by county basis. Advisory information was to be posted to the internet every one to two days detailing potential
risk of disease to wheat crops in each of the 13 counties. Experimental risk assessment
models (Ohio I and Ohio II) were utilized to provide risk probability based on a few selected
environmental parameters. Model output was considered as part of the overall risk assessment upon which advisories were based. An advisory of ‘high-risk” was issued for the entire
thirteen county region for a three-day period near the end of June, but was downgraded as
weather conditions became unfavorable for disease development. Disease levels were low
in nearly all counties, with levels approaching 5% incidence for small sections of two counties in extreme north and northeast SD. Following the 2002 season, much of the environmental and disease data from the past three years were incorporated into a model development phase resulting in several linear models for the prediction of inoculum, infection and
disease.
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INCIDENCE OF FUSARIUM GRAMINEARUM AND COCHLIOBOLUS
SATIVUS IN WHEAT AND BARLEY CULTIVARS AT
THREE LOCATIONS IN MINNESOTA
B. Salas1, R. Dill-Macky1*, and J.J. Wiersma2
Department of Plant Pathology, University of Minnesota, St Paul, MN 55108; and
Northwest Research and Outreach Center, University of Minnesota, Crookston MN, 56716
*Corresponding Author: PH: 612-625-2227; E-mail: ruthdm@umn.edu
1
2
ABSTRACT
Wheat and barley entries in the 2002 Red River Valley on Farm Yield Trials grown at Perley,
East Grand Forks and Humboldt, MN, were assayed for the colonization of kernels by
Fusarium graminearum and Cochliobolus sativus. The trial consisted of 24 wheat and 8
barley lines-grown in commercial fields in a randomized complete block design with two
replications. At maturity, 100 spikes per plot were arbitrarily collected and threshed. Kernels
(200-400 per treatment) were surface sterilized, plated onto half strength PDA (pH=5.5) and
incubated at 20-24ºC, under fluorescent lights (12:12 light:dark) for 5-6 days. The incidence
of kernels colonized by F. graminearum was highest at Humboldt (18.4%, wheat; 22.2%,
barley). The incidence of C. sativus colonized kernels was highest in wheat at Perley
(30.6%), and in barley at East Grand Forks (23.1%). Ranking of wheat cultivars for kernel
colonization by F. graminearum and C. sativus was significantly affected by the interaction of
cultivar by location, however at all locations, the wheat cultivars Alsen and Gunner had low
levels of F. graminearum and Dandy, Norpro, Pioneer 2375, Oxen and AC Vista were more
highly colonized. Oxen and Gunner generally had low levels of kernel colonized by C.
sativus, while AC Vista and MN97803 showed higher kernel colonization across locations.
The six-rowed barley lines MN109, MN110 and Lacey generally had greater kernel colonization by F. graminearum than Robust, Drummond, Foster and Legacy. The incidence of C.
sativus colonized kernels was similar in all barley entries except Conlon. Kernels of
Conlon, a two-rowed barley, had the lowest incidence of F. graminearum but the highest
incidence of C. sativus. The data suggests that the colonization of wheat kernels by F.
graminearum and C. sativus may be influenced by differences in inoculum availability in a
particular location, and site-specific environmental conditions.
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AIRBORNE POPULATIONS OF GIBBERELLA ZEAE: SPATIAL AND
TEMPORAL DYNAMICS OF SPORE DEPOSITION IN A LOCALIZED
FUSARIUM HEAD BLIGHT EPIDEMIC
David G. Schmale III1, Elson J. Shields2, and Gary C. Bergstrom1*
Department of Plant Pathology and 2Department of Entomology, Cornell University, Ithaca, New York, 14853
*Corresponding Author: PH: (607) 255-7849; E-mail: gcb3@cornell.edu
1
ABSTRACT
Viable propagules of Gibberella zeae (anamorph Fusarium graminearum) were collected
from the air over two wheat fields (spaced 0.5 km apart) in Aurora, New York in May/June
2002. Corn kernels inoculated with a clonal isolate of G. zeae were placed in one of the
fields. Petri plates with Fusarium selective medium were suspended 30 cm above the
wheat canopy. Fields were sampled a total of 20 days before, during, and after wheat anthesis. Ninety six plates were exposed continuously during each day (sunrise to sunset) and
another 96 plates were exposed continuously during each night (sunset to sunrise). Significantly more colonies were collected during the night than during the day. Seven major
deposition events were apparent during the sampling period, and three of these were coincident with rainfall. Three major deposition events occurred during flowering; the largest
occurred two days after anther extrusion. The field bearing the clonal source of G. zeae was
exposed to more colonies than the other field. DNA fingerprinting analyses are being conducted to assess the genetic diversity of airborne populations of the pathogen and contributions from local and regional sources of inoculum.
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DEVELOPMENT OF FUSARIUM HEAD BLIGHT IN INDIANA, 2002
G. Shaner* and G Buechley
Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907-2054
*Corresponding Author: PH: (765) 494-4651; E-mail: shanerg@purdue.edu
ABSTRACT
We are participating in a multi-state cooperative study on the epidemiology of Fusarium
head blight (FHB) of wheat. We monitored weather, inoculum production, and disease
development in order to obtain data that can be used to quantify the effects of weather on
inoculum production and disease development. We planted 3 winter wheat cultivars at 3
dates at the Purdue Agronomy Research Center. Corn residue in the plots served as a
source of inoculum. An automated Campbell station recorded weather data. The dates of
flowering initiation among treatments ranged from 22 to 30 May. There were several days of
unusually warm weather during the third week of April, and then several weeks of cooler
than normal weather. During the 2 wk prior to 22 May, rain fell on 8 days, but mean daily
temperature was above 15 °C on only 16 May. Daily mean temperatures began rising after
26 May, but by then there was little rainfall. Daily airborne spore concentrations estimated
from a Burkard sampler ranged from 0 to 164 cfu.10 m-3 d-1. A second sampler was located in
a field 1.6 km away, but also with corn residue on the surface. There was close agreement
between the numbers of spores collected each day at the 2 sites (r=0.95). Daily variation in
number of airborne spores was large. On only one occasion, 26 and 27 May, were there 2
consecutive days with high counts. Each day we also collected heads at both sites for direct
assay of spores of G. zeae. Spores recovered per head ranged from 0 to 750 d-1. The higher
values occurred later in the season, when wheat was in the grain filling stage. Numbers of
spores recovered from heads at the 2 sites were in general agreement (r=0.75) except for 57 June, when substantially more spores were recovered at the main site. Based on estimates of the volume of air intercepted by a wheat head during 24 h, the Burkard samplers
and the head washing assays gave similar estimates of the number of spores that impact a
head each day, although correlations between daily values were low. Detailed assessment
of incidence and severity of FHB were made at 2- to 3-day intervals in the 2nd planting of
cultivar Elkhart. Incidence and severity both increased linearly from 5 June, when symptoms
first appeared, through 21 June. Incidence increased from 3 to 16% (0.8% per day) and
severity increased from 30 to 83% (3.5% per day). We assessed incidence and severity in
all plots on 21 June. Among the cultivar-planting date treatments, mean FHB incidence
ranged from 1.4 to 9.2%. The effects of cultivar, planting date and their interaction were all
highly significant. For the various cultivar-planting date combinations, there was a significant correlation between incidence of FHB and the number of spores detected with the
Burkard sampler on the 4th or 5th day after beginning of anthesis (r=0.81 and r=0.91, respectively). The correlation between incidence and the sum of spore densities on these 2 days
was 0.97. We used data from this study to evaluate 2 weather-based forecast models developed by DeWolf et al. These models predicted a low probability of a “severe” epidemic,
defined as an incidence of greater than 10%, for all cultivar-planting date combinations,
consistent with what we observed.
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COMPARISON OF SPRAY, POINT INOCULATION METHODS, AND FDK
TO FACILITATE EARLY GENERATION SELECTION FOR FUSARIUM
HEAD BLIGHT RESISTANCE IN WINTER WHEAT
Tamburic-Ilincic, L.1, 2*, Fedak, G.3, and Schaafsma, A.W. 1, 2
Ridgetown College, University of Guelph, Ridgetown, Ontario, Canada;
Department of Plant Agriculture, University of Guelph, Guelph, Ontario, Canada; and
3
Eastern Cereal and Oilseed Research Centre, Ottawa, Ontario, Canada
*Corresponding author: PH: 519/674-1611; E-mail: ltamburi@ridgetownc.uoguelph.ca
1
2
INTRODUCTION
Fusarium head blight (FHB) caused by Fusarium graminearum (Schwabe) is an important
disease of wheat (Triticum aestivum L.) in Canada, and worldwide (Sutton, 1982). Different
types of wheat resistance to FHB have been reported, but in the breeding programs Type 1,
or resistance to initial infection, and Type 2, or resistance to spread of symptoms within the
head are used most often (Schroeder and Christensen, 1963, Mesterhazy, 1995). The most
frequently used source of FHB Type 2 resistance worldwide is Sumai 3.
Several authors reported that Type 1 and Type 2 resistance varied independently among
cultivars (Schroeder and Christensen, 1963; McKendry et al., 2001; Desmeules et al., 2001),
and that selection of genotypes with resistance to FHB depends on the inoculation technique used (Engle et al., 2001; Bockus et al., 2001). Shaner and Buechley (2001) proposed
the expression of severity as the number of spikelets blighted, not the proportion of spikelets
blighted, in order to avoid the influence of spike size on the degree of Type 2 resistance. Van
Saford et al., (1999), and Hall et al., (2000) reported that some genotypes have a lower
kernel infection rate then expected from their spikelets infection rate, and also the opposite.
In order to increase the comprehensive resistant level to FHB, different resistance types to
FHB should be pyramided into improved varieties (Xu et al., 2001).
Even though spray and point inoculation methods are used most frequently as the ways to
estimate wheat resistance or susceptibility to FHB, these two methods are not often directly
compared using segregating populations with known type of FHB resistance.
The objectives of this study are:
-to directly compare FHB severity after spray and point inoculation method using a
segregating population with type 2 FHB resistance,
-to compare Type 1 and Type 2 resistance with Type 4, or resistance to kernel
infection,
-to compare expression of FHB severity as the number of spikelets infected with
expression of FHB severity as the proportion of spikelets infected, in order to examine the influence of spike size on the degree of FHB resistance.
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MATERIALS AND METHODS
The influence of different methods of inoculation on number or proportion of FHB infected
spikelets, and percent of Fusarium damage kernels (FDK), were studied using an F3 population carrying Type 2 FHB resistance. The progeny (n=85) was derived from a cross of
resistant (WEKO60DH3 - a Sumai 3 derivative) and susceptible (AC RON) parents. The
segregating generations, and parents, were planted on October 20, 2000, in 2-m long single
rows, spaced 17.8 cm apart, at Ridgetown, Ontario.
In order to obtain uniform growth stage at the time of inoculation, individual heads, rather
than whole plants or plots, were inoculated at 50 % of anthesis (Zadoks growth stage 60-69)
(Zadoks, 1974). The heads were spray inoculated with 2 mL of the suspension sprayed onto
individual heads, and point inoculated with 10 µl of suspension injected into single florets.
The suspension of macroconidia, including three isolates of F. graminearum, was produced
in liquid shake culture using modified Bilay’s medium, and used at a concentration of
50,000 spores/mL.
Between 10 to 20 plants from each progeny and the parents were inoculated using both
methods of inoculation. Clear plastic bags were placed over the inoculated heads, and left
for 48 hr to maintain humidity. The plots were misted daily with an overhead mister that
delivered about 7.5 mm of water each day. The plots were fertilized and maintained using
provincial recommendations.
The number of diseased spikelets and the total number of spikelets were recorded for each
inoculated wheat head. The spikelet infection rate was calculated as the number of diseased spikelets, or percentage of diseased spikelets of the total number of spikelets. The
average infection rate from each row was calculated. The inoculated heads were hand
harvested separately. Heads from each row, with the same inoculation method, were
threshed together using a single head thresher, retaining all light kernels.
The number of healthy and Fusarium damaged kernels were counted, and percentage of
FDK was calculated for each line and parents, after both methods of inoculation. In order to
avoid the influence of inoculation method on % of FDK, % of FDK after both methods of
inoculation was also averaged for each line, and their parents. FDK were identified as
shriveled kernels, with chalky, pink or white color. The F3 progeny was assigned to phenotypic classes on the basis of their position in the distribution of the proportion of FHB infected spikelets, and % of FDK.
For the proportion of FHB infected spikelets the following classes were used: 1=0-2.5,
2=2.51-5, 3=5.01-7.5, 4=7.51-10, 5=10.01-12.5, 6=12.51-15, 7=15.01-17.5, 8=17.51-20,
9=20.01-22.5, 10=22.51-25, 11=25.01-27.5, 12=27.51-30, 13=30.01-32.5, 14=32.51-35,
15=35.01-37.5, 16>37.51.
For % of FDK, there were the following classes: 1=0-2, 2=2.01-4, 3=4.01-6, 4=6.01-8,
5=8.01-10, 6=10.01-12, 7=12.01-14, 8=14.01-16, 9=16.01-18, 10=18.01-20, 11=20.01-22,
12>22.01.
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Data management and all statistical procedures were completed using SAS v. 6.0. (SAS
Institute Inc, 2001).
RESULTS AND DISCUSSION
Transgressive segregants, with higher levels of resistance than the parents, were found
using visual symptoms and % FDK after both methods of inoculation (Fig. 1-2). Minimum,
maximum, and mean values for % of infected spikelets after point inoculation were 3.7, 32.7,
and 11.4, and lower than after spray inoculation where they were 4.8, 42.0, and 15.4, respectively. This result was expected because this population carrying Type 2 resistance from
Sumai-3. Overall correlation between % of diseased spikelets after spray and point inoculation was positive, but low (r=0.38, P<0.001).
Values for minimum, maximum, and mean percent of FDK after point inoculation were 0,
31.0, and 9.0, and these were higher than % FDK after spray inoculation (0, 20.6, and 6.2,
respectively). AC RON, a FHB susceptible cultivar, had lower scores than WEKO609H3 for
% FDK after spray inoculation with F. graminearum (Fig. 2 B). Correlation between % FDK
and % FHB infected spikelets after the spray inoculation method was significant (r=0.28,
P<0.05), while correlation between % FDK and % FHB infected spikelets after the point
inoculation method was not. When % FDK after both methods of inoculation was averaged
for each line, and correlated with % FHB infected spikelets, the results showed again that %
FDK correlated weakly with % FHB infected spikelets after the spray inoculation method
(r=0.24, P<0.05), but not after the point inoculation method in this population (even while
carrying Type 2 resistance). According to our results, % FDK can be estimated more accurately after spray, than after point inoculation method. It was unexpected that there was no
significant correlation between % FDK after spray, and % FDK after point inoculation
method in this population. When lines were ranked, according to % FDK after different
methods of inoculation, just 2 of the 10 top lines were the same.
The number of infected spikelets correlated well with proportion of infected spikelet (r=0.82,
0.87, P<0.001), after point (Fig. 3A), and spray (Fig. 3B), inoculation method, respectively,
even when several outliers were also identified (Fig. 3). There was a good segregation for
total number of spikelets within the spike in this population, and ranged from 15 to 22. When
lines were ranked, according to proportion of FHB infected spikelets, or number of FHB
infected spikelets, 7 of 10 lines with lowest % of FHB infected spikelets were the same,
confirming that there is no influence of spike size on degree of FHB resistance. We concluded that visual estimate of the proportion of FHB infected spikelets can be recommended
for selection in early generations, rather than the more time and labor intensive counting of
FHB infected spikelets.
This study showed that there is an advantage of using both methods of inoculation, because
progeny lines with higher levels of Type 1, Type 2, and Type 4 were identified, and they
would not have been identified if only one of the methods was used. When these Types of
resistance are identified in an early segregating generation, they should be pyramided
sooner in the improved FHB varieties.
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25
N um b er o f lin es
25
20
15
10
5
20
15
10
5
16
15
14
13
12
10
11
9
O
R
AC
O
K
W
AC
E
R
N
8
7
5
60 6
9
H3
4
16
15
14
12
13
11
9
10
8
7
N
O
5
6
3
4
1
W
E
KO
1
60 2
9H
3
3
0
0
2
N um b er o f lin es
30
% o f F H B in fe cte d sp ike le ts after p o in t in o cu lation
% F H B in fecte d sp ike le ts after sp ra y ino cu lation
25
15
10
5
W
12
11
9
10
8
7
9H
60
W
EK
O
A
AC
O
EK
3
6
5
4
3
N
RO
2
1
0
12
11
10
9
8
7
6
N
20
C
60
RO
5
4
2
3
N um b er o f lin es
16
14
12
10
8
6
4
2
0
1
9H
3
N um b er o f lin es
F igure 1. F req uency d istrib utio n o f p ro p o rtio n o f infected sp ik elets in F 3 generatio n
after po int (A ), and sp ray (B ) inoculatio n w ith F . gram inearum .
% F D K a fte r s p ra y in o c u la tio n
% F D K a fte r p o in t in o c u la tio n
40
30
20
R 2 = 0 .8 2
P < 0 .0 00 1
n= 7 1
10
0
0
2
4
6
N um ber of infected
spikelets
N um ber of in fected
spikelet
F igure 2. F req uency d istrib utio n o f % F D K in F 3 generatio n after p o int (A ), and
sp ray (B ) ino culatio n w ith F . gram inearum .
% of infected sp ikelets after poin t
in oculatio n
50
40
30
20
10
0
R 2 = 0 .87
P < 0 .0 01
n= 78
0
5
10
% of in fected spikelets after spray
in oculatio n
F igure 3. R elatio nship b etw een num b er and p ro p ortio n o f infected sp ik elets in F 3
generation after p o int (A ), and sp ray (B ) ino culatio n w ith F . g ra m in ea ru m .
ACKNOWLEDGEMENTS
This work was funded by partnership between the Canadian Adaptation Council
(CanAdapt) the Ontario Wheat Producers Marketing Board, Bayer Crop Science, Syngenta
Crop Protection, BASF, the Ontario Minstry of Agriculture and Food, Pioneer Hybrid and the
University of Guelph. Technical assistance by Diane Paul, and Todd Phibs is gratefully
acknowledged.
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REFERENCES
Desmeules, J., Paulitz, T., Rioux, S., O’Donoughue, L., Mather, D. 2001. Fusarium head blight symptom development in spring wheat genotypes after inoculation with “point” and “spray” methods. Proceedings of Canadian
Workshop on Fusarium Head Blight. Ottawa, November 3-5, 2001. Pg 50.
Mesterhazy, A. 1995. Types and components of resistance to Fusarium head blight of wheat. Plant Breeding.
114 (5): 377-386.
Shaner, G., Buechley, G. 2001. Estimation of Type II resistance-a dilemma in need of a solution. The 2001
National Fusarium Head Blight Forum Proceedings. Erlanger, KY, December 8-10, 2001. 156-160.
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REMI MUTAGENESIS IN THE WHEAT SCAB FUNGUS
FUSARIUM GRAMINEARUM
Miles Tracy1, Zhanming Hou1, H. Corby Kistler2, and Jin-Rong Xu1*
Dept of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907; and
2
USDA-ARS, Dept. of Plant Pathology, University of Minnesota, St. Paul, MN 55108
*Corresponding Author: PH: 765-496-6918; E-mail: jin-rong@purdue.edu
1
ABSTRACT
Fusarium graminearum is an important pathogen of small grains and maize in many areas
of the world. Infected grains are often contaminated with mycotoxins harmful to humans and
animals. In the past decade, wheat head blight (scab), primarily caused by F. graminearum
in North America, has emerged as a major threat in wheat production. To better understand
the molecular mechanism of plant infection and virulence of F. graminearum, we used the
REMI (Restriction-Enzyme Mediated Integration) approach to generate random targeted
mutants. Over 7000 hygromycin-resistant transformants have been generated by transforming pCB1003 or pCX12 into F. graminearum PH-1. A corn-silk infection assay was devised
to screen for mutants with reduced virulence. Many of the REMI pathogenicity mutants
identified in corn-silk assays were dramatically reduced in their ability to infect and colonize
flowering wheat heads. Genetic analysis and plasmid rescue are underway to identify and
characterize genes disrupted in these mutants.
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THE FUSARIUM GRAMINEARUM GENOMICS PROJECT
Frances Trail1*, Jin-Rong Xu2 and H. Corby Kistler3
Dept. of Botany & Plant Pathology, Michigan State University, East Lansing, MI 48824;
Dept. of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907; and
3
USDA-ARS, Cereal Disease Laboratory, University of Minnesota, St. Paul, MN 55108
*Corresponding Author: PH: (517) 432-2939; E-mail: trail@msu.edu
1
2
ABSTRACT
Fusarium graminearum (teleomorph Gibberella zeae) is a broad host range plant pathogen
that infects many crop plants worldwide. We have taken a genomics approach to better
understand pathogenicity in this fungus. We have generated a collection of ESTs derived
from cDNA libraries generated from cultures grown under several culture conditions and
from infected wheat plants. Sequences were initially assembled into contigs and singletons, based on sequence comparisons, and a putative single gene set was identified.
These sequences were compared to a yeast protein sequence reference set and to the
GenBank non-redundant database using BLASTX. These results can be observed on the
web (see link from www.scabusa.org). Based on presumptive gene function identified by
this process, we were able to compare patterns of gene expression among cDNA libraries.
Homologues of some known fungal virulence and pathogenicity factors and developmentally important genes were identified by this analysis. Funding for the complete genome
sequence has been obtained. A discussion of the availability of tools for genomics and their
potential uses will be presented.
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COMPARATIVE VIRULENCE OF ISOLATES OF FUSARIUM
SPECIES CAUSING HEAD BLIGHT IN WHEAT
A.G. Xue*, K.C. Armstrong, H.D. Voldeng, G.Fedak, Y.Chen and F.Sabo
Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada,
Ottawa, Ontario, Canada K1A 0C6
*Corresponding Author: PH: (613) 759-1513; E-mail: axue@agr.gc.ca
ABSTRACT
Fusarium head blight (FHB) is an important disease of wheat in Canada. To supplement the
development of FHB-resistant cultivars, the virulence of Fusarium isolates representing
eight pathogenic species was investigated. Six wheat genotypes were artificially inoculated
with 12 isolates of Fusarium graminearum and six isolates each of F. acuminatum, F.
avenacium, F. crookwellense, F. culmorum, F. equiseti, F. poae, and F. sporotrichiodes. The
pathogens were isolated from naturally infected wheat, barley, and oat heads collected from
cross Canada from 1965 to 2001. A single spore culture was established for each isolate,
from which spore suspension was produced. Inoculation was performed by spraying spores
over spikes at the 50% anthesis stage. Symptoms of FHB were rated as disease severity
using a 0-9 scale at 4, 7, 14, 21, and 28 days after inoculation; and as percent infected
spikelets after 21 days. All isolates caused visible infections to the six wheat genotypes but
only those of F. graminearum, F. crookwellense, and F. culmorum resulted in severe disease
development and were considered highly pathogenic. Significant differences (P < 0.05)
were also observed among isolates and from genotype x isolate interactions for the three
highly pathogenic species. However, the genotype x isolate interactions were low (< 15%)
compared to differences between isolates or genotypes and did not suggest the occurrence
of pathogenic races. The presesence of different virulence among isolates suggests that
screening for resistance to FHB require a mixture of several isolates of these pathogens to
be included. Wheat genotypes differed significantly (P < 0.001) in susceptibility, and responses of the genotypes to isolates of the highly pathogenic species were generally similar. AC Foremost, CIMMYT11, and Quantum were the most susceptible; FHB37 and HY664
were intermediate; and Sumai 3 was resistant. These results indicate that selection for
resistance to one species in wheat may also confer resistance to the others.
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POPULATION GENETIC DIFFERENTIATION AND LINEAGE
COMPOSITION AMONG GIBBERELLA ZEAE (FUSARIUM
GRAMINEARUM) IN NORTH AND SOUTH AMERICA
K.A. Zeller, J.I. Vargas, G. Valdovinos-Ponce, J.F. Leslie* and R.L. Bowden
Department of Plant Pathology, Kansas State University, Manhattan, KS
*Corresponding Author: PH (785) 532-1363; E-mail: jfl@plantpath.ksu.edu
ABSTRACT
Gibberella zeae (Fusarium graminearum) causes Fusarium head blight (FHB) of wheat and
barley, and has been responsible for severe economic losses worldwide. Sequence analyses of G. zeae have been interpreted to mean that populations of G. zeae are composed of
eight potential phylogenetic lineages, with a phylogeographic structure among these lineages. We used AFLP polymorphisms to compare populations of G. zeae from the United
States, Mexico, Brazil, and Uruguay. We have also examined populations of G. zeae isolated from sorghum seed in Uruguay. Populations of G. zeae causing FHB in the United
States include only a single phylogenetic lineage (Lineage 7). Subpopulations from
throughout the United States have high genotypic diversity, do not deviate from expectations
of random mating, and are interconnected by extensive gene-flow. South American populations of G. zeae from both wheat and from sorghum include a minority component of isolates
that cluster with other phylogenetic lineages (Lineages 1, 2, and 6), but are dominated by
genotypically diverse populations of isolates from Lineage 7. Populations of G. zeae causing FHB on wheat from two locations in Mexico are dominated by isolates from Lineage 3.
Population genetic comparisons of Lineage 7 isolates from North and South America indicate that while intercontinental gene flow may occur, the amount of gene flow between the
continents is much less than that which occurs within each continent.
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METABOLISM OF TRICHOTHECENES BY WHEAT
L.-F. Chen1,2*, H.-Y. Yao2, G. Yu2, W.-P. Xie1, and H.C. Kistler1,3
Department of Plant Pathology, University of Minnesota, St. Paul, MN 55108;
Department of Plant Pathology, Nanjing Agricultural University, Nanjing 210095, China; and
3
USDA ARS Cereal Disease Laboratory, St. Paul, MN 55108
*Corresponding Author: PH: (612) 625 7790; E-mail: lfchen_nau@yahoo.com
1
2
ABSTRACT
Feeding experiments were conducted to determine whether wheat could metabolize exogenously added trichothecenes. Middle spikelets of a moderately resistant wheat cultivar
Su8060 were fed in triplicate, on ten sets of plants, with a fixed amount of deoxynivalenol
(DON, 4000 ng). One day after adding DON, the treated spike on one set of plants and
adjacent tissue was removed for toxin analysis using GC-MS. The remaining nine sets of
plants were fed with DON again. This daily process of toxin addition and sampling was
continued until all spikes were harvested for toxin analysis. The same procedure was carried out with 15-acetyldeoxynivalenol (15ADON, 1000ng), 3-acetyldeoxynivalenol (3ADON,
500ng) or a combination of the three toxins (4000ng of DON, 1000ng of 15ADON and
500ng of 3ADON). The results showed that, when fed DON alone, DON was found in both
the fed spikelets and adjacent fragments of rachis. Additionally in DON treatments, both
15ADON and 3ADON were recovered but only from the fed spikelets. When fed with
15ADON, both DON and 15ADON were recovered from both the fed spikelets and adjacent
fragment of rachis. When fed with 3ADON, both DON and 3ADON were detected only in the
fed spikelets. In all cases, change in the amount of toxins followed the same pattern: reaching the highest cumulative level on the fifth or sixth day of the experiment, and then decreasing to a lower level on the seventh or eighth day despite continued toxin addition. Toxin
levels increased from the ninth day until end of this experiment. When fed with the combination of DON, 15ADON and 3ADON in the ratio of 8:2:1, the relative amounts of the three
toxins recovered from the fed spikelets varied significantly, from 24:3:1 to 80:7:1. Nivalenol
was not detected in any treatment. We hypothesize that the resistant wheat plant metabolizes trichothecenes into different forms as well as other uncharacterized metabolic products. (This study is supported by National Natural Science Foundation of China,
30170601and 39870471)
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YEAST STRAINS ALLOWING PHENOTYPIC DETECTION OF
ESTROGENIC ACTIVITY: DEVELOPMENT OF A SENSITIVE AND
INEXPENSIVE YEAST BIOASSAY FOR ZEARALENONE
R. Mitterbauer1, H. Weindorfer1, N. Safaie2, H. Bachmann1 and G. Adam1*
1
Center of Applied Genetics, University of Agricultural Sciences, Vienna, Austria; and
2
Tarbiat Modarres University, Tehran, Islamic Republic of Iran
*Corresponding Author: PH: 43-1-36006-6380; E-mail: adam@edv2.boku.ac.at
ABSTRACT
Zearalenone (ZON) is a non-steroidal estrogenic mycotoxin produced by plant pathogenic
species of Fusarium. As a consequence of infection with F. culmorum and F. graminearum,
ZON can be found in cereals and derived food products. Since ZON is suspected to cause
human disease such as premature puberty syndrome as well as numerous cases of hyperestrogenism in farm animals, several countries have established monitoring programs and
guidelines for ZON levels in grain intended for human consumption and animal feed. Austria, for instance, has set guideline levels of 60 µg/kg for wheat intended for human consumption and 50 µg/kg for whole feed for breeding pigs. In epidemic situations much higher
levels have been found, for instance average levels of more than 500 µg/kg wheat have
been measured in Northern Germany in 1998. In Northern Iran highly contaminated wheat
has been reported for 1996 (35/35 samples positive, average ZON level of 3,4 mg/kg).
We have developed a low-cost method for monitoring of ZON contamination in grain based
on a sensitive yeast growth bioassay. The indicator Saccharomyces cerevisiae strain
YZRM7 is unable to grow, unless an engineered pyrimidine biosynthetic gene is activated
by the expressed human estrogen receptor in the presence of exogenous estrogenic substances. The deletion of the genes encoding ATP-binding cassette (ABC) transporters
Pdr5p and Snq2p increases net ZON uptake synergistically. Less than 1 µg ZON per liter
medium is sufficient to allow growth of the indicator strain. To prevent interference with
pyrimidines potentially present in biological samples, we have also disrupted the genes
FUR1 and URK1, blocking the pyrimidine salvage pathway. The bioassay strain YZRM7
allows qualitative detection and quantification of total estrogenic activity in cereal extracts
without requiring further clean up steps. The high sensitivity makes this assay suitable for
low cost monitoring of contamination of maize and small grain cereals with estrogenic
Fusarium mycotoxins.
We have furthermore constructed yeast strains allowing phenotypic detection of the estrogenic activity of ZON by engineered ADE2 and MEL1 genes. Together with a positive selection marker (URA3 with estrogen responsive elements in the promoter), such easily
screenable markers are valuable tools for cloning ZON degradation genes by expression of
cDNA libraries in yeast.
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DIAGNOSTIC VOMITOXIN (DON) SERVICES IN 2002/2003 SAMPLES
M.S. Mostrom1*, P. Schwarz2, Y. Dong3 and P. Hart4
1
Dept. of Veterinary Diagnostic Services, 2Dept. of Cereal Science, North Dakota State University, Fargo, ND
58105; 3Dept. of Plant Pathology, University of Minnesota, St. Paul, MN 55108; and 4Dept. of Botany and
Plant Pathology, Michigan State University, East Lansing, MI 48824
*Corresponding Author: PH: (701) 231-7529; E-mail: Michelle.Mostrom@ndsu.nodak.edu
OBJECTIVES
To provide analytical services for deoxynivalenol testing for researchers investigating mitigation of Fusarium head blight in wheat and barley.
INTRODUCTION
Deoxynivalenol (DON or vomitoxin) concentrations in cereal grains are an indicator of
Fusarium head blight (FHB). Researchers evaluating techniques to reduce the adverse
effects of in grains have used DON to determine the resistance in cultivars. Mycotoxin
testing, specifically DON testing, is an important part of the cooperative efforts to reduce
FHB. In 2002, the U.S. Wheat and Barley Scab Initiative provided grants to four regional
DON testing laboratories in Michigan, Minnesota, and North Dakota to analyze for DON in
wheat and barley cultivars.
MATERIALS AND METHODS
The four laboratories involved in DON testing are listed below. The analytical methods used
by the laboratories include ELISA, gas chromatography with electron capture detection, and
gas chromatography with mass spectrometry analysis. The sample preparation method
used for the gas chromatography analysis was developed by Tacke and Casper (1996).
The individual laboratories conduct their own intralab quality control on appropriate control
pools throughout the analysis period for DON testing to ensure quality of the analysis. The
intralab quality control data spans the time from the beginning of DON testing in the labs
through the end of October in 2002. Additionally, a collaborative quality assurance program,
using wheat, was conducted among the laboratories (P. Hart, coordinator). Each laboratory
was requested to perform analyses from a divided sample on two successive days within a
30 day period. The collected data were summarized and sent back to the laboratories.
These check samples allowed each laboratory to evaluate the accuracy and precision of
their system. Data from three check sample tests are included (May through October 2002).
Data (May through October 2002) are also included from a larger collaborative quality
assurance program conducted by North Dakota State University, Department of Cereal
Sciences. All four laboratories participate in this program, which uses malt and barley as
check samples. These data are included for additional matrix quality assurance information.
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Laboratories :
Patrick Hart, Ph.D., Department of Botany & Plant Pathology, Michigan State University,
East Lansing, MI 48824; Phone: 517-353-9428, FAX: 517-353-5598, e-mail: hart@msu.edu
Method: water extraction and DON quantitation with the Neogen Veratox test (ELISA)
Sample types: wheat
Yanhong Dong, Ph.D., Department of Plant Pathology, University of Minnesota, St. Paul, MS
55108; Phone: 612-625-2751, FAX: 612-625-9728; e-mail: dongx001@umn.edu
Method: acetonitrile and water extraction, silylation and quantitation by gas chromatography/
mass spectrometry (GC/MS)
Sample types: wheat, barley, (bulk, single head, single spikelet, single kernel, and small
fragment)
Paul Schwarz, Ph.D., Department of Cereal Science, North Dakota State University, Fargo,
ND, 58105; Phone: 701-231-7732, FAX: 701-231-7723, e-mail:
Paul.Schwarz@ndsu.nodak.edu
Method: acetonitrile and water extraction, silylation and quantitation by gas chromatography/
electron capture detection (GC/ECD)
Sample types: barley, malt, single kernel
Beth Tacke, B.A., Department of Veterinary Diagnostic Services, North Dakota State University, Fargo, ND, 58105; Phone: 701-231-8309, FAX: 701-231-7514, e-mail:
Beth.Tacke@ndsu.nodak.edu
Method: acetonitrile and water extraction, silylation and quantitation by GC/ECD
Sample types: wheat, barley
RESULTS AND DISCUSSION
Table 1 summarizes the participating number of collaborators (principal investigators),
number of states, and estimated number of DON samples to be analyzed in the grant year of
May 2002 through April 2003. Approximately 24,500 cereal grain samples submitted by
about 57 principal investigators in 14 different states investigating FHB will be analyzed for
DON by the four laboratories in 2002/2003.
The intralab coefficient of variation for the four laboratories varies from 6 to 16 % on the
grain control pools that were analyzed with the samples during 2002 (Table 2). The interlab
proficiency check samples for FHB testing show that the four laboratories are determining
similar results, in a wheat matrix, using different analytical methods (Table 3).
Additional interlab check samples were analyzed in malt and barley matrices by the same
four laboratories using the same methodology (Table 4). The data in Table 4 represent the
time frame from May through October 2002, and are part of a two-year check sample program involving a number of participants. The z-value is given for each laboratory by month.
[Note that the z-values were calculated as the lab’s individual value minus the sample mean
divided by the sample standard deviation. A smaller z-value represents less spread of
actual results and higher accuracy and precision.] The data in Table 4 show that the
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z-values for the different laboratories are fairly small (close to zero) and no major differences
were observed in analytical values at lower DON concentrations.
The repeatability of results on successive days reflects the precision of the analysis and
was good for the interlab FHB check samples for those cooperating laboratories. Also, the
intralab coefficients of variations on the control pools were fairly low for the participating
diagnostic lab. These results indicated that the variation in analyses over several days is
low and no major differences in analytical values of check samples occurred between the
four DON diagnostic centers.
Table 1. Estimated DON analyses by laboratories in 2002 through 2003
Number of
Collaborators
20
Number of
States
9
Estimated Number of
Samples Tested in 2002
3,000
MN: Y. Dong
9
3
10,000
ND: P. Schwarz
4
3
7,500
ND: B. Tacke
25
7
4,000
DON Laboratory
MI: P. Hart
Table 2. Intralab quality control data for DON testing through October 2002
DON
Laboratory
MI: P. Hart
MN: Y. Dong
ND: P. Schwarz
ND: B. Tacke
Grain
Wheat
Wheat
Barley
Barley
Barley
Wheat
Barley
Corn
Mean
(ppm)
0.9
7.2
13.8
39.7
2.1
1.8
3.1
4.7
Number
122
30
31
18
9
104
104
104
Standard
Deviation
0.1
0.9
2.0
4.9
0.3
0.1
0.2
0.5
Coefficient of
Variation (%)
12
13
15
12
13
7
6
11
Table 3. Interlab proficiency check samples for DON testing for FHB
DON
Laboratory
MI: P. Hart
MN: Y. Dong
ND: P. Schwarz
ND: B. Tacke
Mean ± std.dev.
DON (ppm)
Grain
Wheat
Wheat
Wheat
Wheat
Test 1
1.0
1.0
0.77
0.6
0.4
0.9
1.0
0.8 ± 0.2
Test 2
4.0
4.4
3.8
3.0
3.2
2.8
2.6
3.4 ±0.7
Test 3
<0.5
<0.5
0.3
0.3
0.4
0.4
0.4 ± 0.1
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Table 4. Interlab check samples for DON in barley (bar) and malt from May through October
2002 by four laboratories (part of a larger check sample program, North Dakota State University, Department of Cereal Sciences)
DON (ppm)
Lab
MI:
P. Hart
MN:
Y. Dong
NDSU:
P.Schwarz
NDSU:
B. Tacke
Sample
MEAN
Sample
Std. Dev.
Z-values
by Lab
MI:
P. Hart
MN:
Y. Dong
NDSU:
P.Schwarz
NDSU:
B. Tacke
May
June
July
August
September
October
Bar.
Malt
Bar.
Malt
Bar.
Malt
Bar.
Malt
Bar.
Malt
Bar.
Malt
5.00
0.60
2.40
0.60
1.70
<0.5
3.20
0.60
4.40
<0.5
4.30
0.70
5.35
0.26
2.57
0.31
1.43
0.13
4.02
0.40
4.58
0.12
18.47 0.39
4.60
0.20
2.20
0.30
1.50
0.15
2.80
0.20
4.00
0.10
20.30 0.50
5.50
0.40
2.40
0.40
1.60
<0.2
3.70
0.40
5.00
0.20
17.30 0.40
5.11
0.37
2.39
0.40
1.56
0.14
3.43
0.40
4.5
0.14
15.09 0.50
0.40
0.18
0.15
0.14
0.12
0.01
0.54
0.16
0.41
0.05
7.30
0.14
Bar.
Malt
Bar.
Malt
Bar.
Malt
Bar.
Malt
Bar.
Malt
Bar.
Malt
-0.28
1.32
0.05
1.42
1.21
-0.43
1.22
-0.23
-1.48
1.41
0.59
-0.59
1.17
-0.66
-1.08
-0.71
1.10
0.0
0.20
-0.38
0.46
-0.75
-1.28
-0.93
-1.27
-0.74
-0.49
0.71
-1.17
-1.22
-1.19
-0.76
0.71
0.02
0.97
0.20
0.05
0.0
0.36
0.50
0.0
1.22
1.13
0.30
-0.68
REFERENCES
Tacke, BK and Casper, HH. 1996. Determination of deoxynivalenol in wheat, barley, and malt by column and
gas chromatography with electron capture detection. J A O A C International 79:472-475.
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HUMAN SUSCEPTIBILITY TO TRICHOTHECENE MYCOTOXINS
James J. Pestka1,2 *, Kristen Penner1 and Jennifer Gray2
Dept. of Food Science and Human Nutrition and 2Dept. of Microbiology and Public Health,
Michigan State University, East Lansing, MI 48824
*Corresponding Author, PH: 517-353-1709; E-mail: pestka@msu.edu
1
ABSTRACT
The trichothecene deoxynivalenol (DON), also given the colloquial name vomitoxin, has
occurred with alarming frequency in wheat, corn and barley produced Michigan and Midwest. A major concern is that, because of the paucity of information on human toxicity, action
levels might be set artificially low, thereby reducing the marketability of Michigan wheat
containing trace levels of DON but posing no risk. Based on studies in the mouse model, we
believe that the most critical step for toxicity induction by DON and other trichothecenes are
their action on leukocytes (white blood cells) either by activation of cellular hormones
known as cytokines or by the induction of programmed cell death (apoptosis). If human
leukocyte cytokine dysregulation and/or apoptosis induction are indeed targeted by the
same levels of DON and other 8-ketotrichothecenes in mice as in the mouse, then the risk of
low ppm levels of DON to humans will be extremely small when one considers the diversity
of the human diet and the actual potential level of DON exposure in human tissues. Two
types of models are being used to test this hypothesis- cloned and primary cells.
DON and other 8-ketotrichothecenes induce, in the U937 human macrophage clonal model
the production of three critical proinflammatory mediators, namely, interleukin-6 (IL-6) and
tumor necrosis factor-α (TNF-α), and the chemokine, interleukin-8 (IL-8). Interestingly, the
higher trichothecene concentrations markedly reduced proliferation and were cytotoxic.
The key signals for cytokine upregulation are likely to involve ribosomal binding DON
ribosome binding â protein kinase R/Hck kinase â MAPKinases â cytokine upregulation.
DON also affected a cloned human T lymphocyte model (Jurkat cells). Although DON stimulated IL-2 production, the four other 8-ketotrichothecenes did not stimulate production of this
cytokine. DON and 15-acetyl DON at 60 to 500 ng/ml and 3-acetyl DON at 600 to 5000 ng/
ml could induce IL-8 production, whereas NIV and FX were not stimulatory. Again, the
higher trichothecene concentrations markedly impaired proliferation and were cytotoxic.
conditions optimized for the primary culture of human leukocytes and conducted preliminary
experiments on the effects of DON.
Two primary leukocyte culture approaches have been evaluated. The first involved direct
culturing of human blood obtained from volunteers. Using the first approach, we have observed that DON will directly induce IL-6. Of particular importance was the finding that some
donors were much more sensitive to DON-induced IL-6 than others in terms of minimum
effective DON concentration and magnitude of response. Furthermore, the doses required
for these effects in primary cells appear to be lower than for cloned cell models suggesting
that human primary cells are slightly more sensitive to DON and potentially other
trichothecenes. Analogous results were found for IL-8 but sensitive donors did not correspond to IL-6 responders. Rather, the low IL-6 responders were high IL-8 responders. The
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second type of primary culture involved leukocytes obtained from processed Red Cross
blood. Similar variability and sensitivity was observed in these cells. However, since we did
not have control of these samples from initial blood draw and know nothing about the donors, it will be difficult to reproduce or interpret findings from with Red Cross materials. Thus,
we will focus all future efforts on human blood culture.
It will be important to ascertain whether blood cells from specific individuals are more or
less sensitive to the toxin and whether these effects are consistent over repeated blood
collections. If so, it will suggest that toxin-susceptible and resistant individuals may exists
among the human population, possibly because of genetic polymorphisms related to toxin
metabolism or cellular target interaction. If responses are variable among the same individual, it will be possibly indicative that a hormonal or environmental factor (eg diet, medication) differentially affects an individual response to DON. Either type of information will be
critical for conducting accurate risk assessments for DON and other 8-ketotrichothecenes.
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USING NEAR INFRARED TRANSMITTANCE AS A
SCREENING TOOL FOR DON IN BARLEY
H. Pettersson1, L. Aberg2, J.A. Persson2, H. Andren2, and M. Matteson3*
Department of Animal Nutrition and Management, Swedish University of Agricultural Sciences, Uppsala,
Sweden; 2Foss Tecator AB, Hoganas, Sweden; and 3Foss North America, Eden Prairie, MN, USA
*Corresponding Author: PH: (952) 974-9892; E-mail: mmatteson@fossnorthamerica.com
1
ABSTRACT
Near infrared transmittance (NIT) spectroscopy has been investigated for rapid estimation of
deoxynivalenol (DON) in whole kernel barley (and wheat). Barley samples, spectra, and
reference analysis data provided by study participants from the USA, Austria, and France.
All spectra (570 – 1100 nm) collected using the FOSS Infratec 1241 Grain Analyzer with the
added color module, and reference analysis using HPLC or GC methods. Calibrations
tested included partial least squares (PLS) and artificial neural network (ANN) for DON
(ppb, ppm) and log (DON ppb). Independent validation data showed best performance
using the ANN calibration log (DON ppb) across locations (N 257, Slope 0.79, Correlation
0.88, SED 0.3024, and Bias –0.0013). Based on current findings, it appears NIT can be
used as a screening tool to measure DON in barley. It is recommended to use a limit of 3.5
(3.2 ppm), values above 3.5 indicate the sample might be infected and should be set aside
for further testing (HPLC, GC). DON calibration development work will continue, by expanding calibration databases to include additional growing seasons, locations, and by addressing issues associated with sampling and laboratory errors, to improve accuracy and precision of DON analysis using NIT.
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STORAGE OF SCABBY WHEAT: FUSARIUM GOES AWAY,
DON DOESN’T
Robert W. Stack1*, Howard H. Casper2, and Dennis J. Tobias1
1
Dept. of Plant Plathology and 2deceased, formerly Dept. of Veterinary Science and Microbiology,
North Dakota State University, Fargo, ND 58105
*Corresponding Author: PH: (701) 231-7077; E-mail: rstack@ndsuext.nodak.edu
ABSTRACT
Some 60 years ago, R. G. Shands at the University of Wisconsin reported that scabby barley
stored for five years (1931-1936) retained its emetic activity when fed to pigs. He proposed
that the effect was due to some “toxic principle”, then unknown, and not to the presence of
the live fungus since the barley remained toxic although Fusarium cultures could no longer
be recovered from it (Phytopathology 27:749-762). Today we would recognize that his
“toxic principle” affecting the pigs was likely DON. Shands did actual feeding experiments
with grain extracts to demonstrate the toxicity of his five-year-old barley samples. The
widespread outbreak of FHB in the northern spring grain region in 1993 was perhaps the
worst since 1928, the one that had prompted Shands’ interest. We had evaluated a large
number of scabby grain samples from the 1993 crop in eastern North Dakota and northwestern Minnesota. Many of these grain samples had DON levels in excess of 10 ppm,
some as high as 50 ppm. Some grain samples from this survey had been retained in storage since 1993. For the present study, we chose 50 of the 1993 wheat samples to reanalyze for DON in 2001. As originally analyzed, the grain samples had contained from
<0.5 ppm to 18 ppm. The same procedure for extraction and analysis by GC-MS was used
in 1993 and in 2001. The level of DON found in the 2001 analysis of these samples was
about 73% of that found in 1993. That ratio of the two analyses was remarkably consistent
for most of the samples (R2 = 0.90). The correlation of DON to presence of tombstone
kernels in grain was nearly the same for ine 1993 as the 2001 analyses (R2 = 0.60, 0.62,
respectively). When cultured in 1994, 65% of the kernels in 1993 grain samples had given
colonies of Fusarium graminearum. Despite the substantial amount of DON remaining in
this grain in 2001, not a single culture of F. graminearum could be recovered when the 660
representative scabby kernels from these samples were plated out on media suitable for
recovery of Fusarium. Our results show that the commonly-held assumption that DON is
long-lasting in grain is correct for wheat as well as barley. The wheat samples tested represented multiple locations in the region and several cultivars; neither site not cultivar showed
any particular deviation from the overall relationship of 1993 to 2001 DON. (This poster
was presented at the 2002 Annual Meeting of the Canadian Phytopathological Society,
Waterton Lakes, Alberta, June 2002.)
Food Safety, Toxicologgy and Utilization
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2002 National Fusarium Head Blight Forum Proceedings
VARIATION FOR RESISTANCE TO FUSARIUM HEAD
BLIGHT IN TRITICUM DICOCCOIDES
H. Buerstmayr1*, M. Stierschneider1, B. Steiner1, M. Lemmens1, M. Griesser1,
E. Nevo2, and T. Fahima2
IFA-Tulln, Institute for Agrobiotechnology, Department of Biotechnology in Plant Production,
Konrad Lorenz Strasse 20, A-3430 Tulln, Austria, (webiste: http://www.ifa-tulln.ac.at); and
2
Institute of Evolution, University of Haifa, Mount Carmel, Haifa 31905, Israel
*Corresponding Author: PH: 43 2272 66280 205; E-mail: buerst@ifa-tulln.ac.at
1
ABSTRACT
Head blight of wheat (FHB, scab) caused by Fusarium spp. is a severe fungal disease
problem worldwide. Apart from yield and grain quality losses, the contamination of the
harvest with toxic fungal metabolites, known as mycotoxins, is of serious impact. In spite of
the fact that several sources for resistance against FHB have been found and utilized in
hexaploid wheat, virtually no resistant tetraploid wheat cultivar has been identified so far.
Wild emmer wheat, Triticum dicoccoides, previously identified as a rich source for disease
resistance genes to several pathogens, was tested for resistance to FHB. Single point
inoculations were applied to evaluate a set of 151 T. dicoccoides genotypes, originating
from 16 habitats in Israel and one habitat in Turkey, for resistance to fungal spread (Type II
resistance) in replicated greenhouse experiments. A considerable level of diversity was
found among the tested genotypes, the broad sense heritability for Type II FHB resistance
was 0.71. Among the eight T. dicoccoides lines with the lowest relative infection rates, five
originated from the Mt. Gerizim population, and three from the Mt. Hermon population. These
two habitats are characterized by a relatively cool and semi-wet climate. Hence, it may be
possible that Fusarium occurrence in these habitats was responsible for natural selection in
favor of resistance.
REFERENCES
Buerstmayr, H., M. Stierschneider, B. Steiner, M. Lemmens, M. Griesser, E. Nevo, and T. Fahima. 2002. Variation for resistance to head blight caused by Fusarium graminearum in wild emmer (Triticum dicoccoides)
originating from Israel. Euphytica, in press.
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2002 National Fusarium Head Blight Forum Proceedings
DESIGNATING TYPES OF SCAB RESISTANCE: A DISCUSSION
W. R. Bushnell
ARS-USDA Cereal Disease Laboratory, 1551 Lindig St., St. Paul MN, 55108
Corresponding Author: PH: 612-625-7781; E-mail: billb@umn.edu
At a meeting of the Germplasm Introduction and Enhancement Group of the U.S. Wheat and
Barley Scab Initiative, held Sept. 12-13, 2002 in St. Paul, MN, the status of terminology for
types of scab resistance was reviewed and discussed. The two principal types of resistance
described by Schroeder & Christensen (1963), resistance to initial infection and resistance
to spread of infection in the head (usually designated types 1 and 2, respectively) have
been widely used. However, several additional types have been postulated without agreement among laboratories on either their definitions or in the sequence of numbering (or
lettering) to be used. Several other factors contribute to confusion among designated types
of resistance (Bushnell 2000). These include:(1) differences among laboratories in the way
disease development, toxin accumulation, and kernel yield and quality are measured; (2)
the need to deduce the amount of some postulated types of resistance from two measured
qualities as, for example, disease severity and yield reduction must be measured to determine tolerance, or toxin concentration and yield loss must be measured to deduce insensitivity to toxin; (3) differences in objectives among laboratories; e.g. a focus on mechanisms
of resistance can lead to postulated types of resistance that are not feasible to measure
routinely in breeding for resistance; (4) uncertainty about the role of trichothecene toxins in
pathogenesis; and (5) limited available information on the physiology and (in most cases)
the genetics of resistance.
Lively and candid discussion by the group led to the following results: About half the participants favored continued use of “type 1” to designate resistance to initial infection and “type
2” for resistance to spread in the head. The remaining participants did not favor use of type 1
or type 2 alone to designate the type of resistance. This subgroup recommended that each
worker describe both what was measured and the inferred type of resistance in words
instead of depending only on use of “type 1” and “type 2”. For resistances other than types 1
and 2, the group was nearly unanimous that it is premature to codify them into a standardized list. Too little is known about them, methods for measuring them are not standardized,
and there is lack of agreement among workers on how to designate them. Postulations of
resistance mechanisms are valuable as a basis for experimental investigation, but should
not be designated by number (or letter) until they are well established and until practical,
uniform methods of measuring them are available. The group hopes these conclusions will
lead to further discussion by the larger FHB research community.
REFERENCES
Bushnell, W.R. 2000. The need for uniformity in designating types of scab resistance. (proceedings) 2000
National Fusarium Head Blight Forum p. 245.
Schroeder, H.W. and Christensen, J.J. 1963. Factors affecting resistance of wheat to scab caused by Giberella
zeae. Phytopathology 53:831-838.
Germplasm Introduction and Enhancement
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2002 National Fusarium Head Blight Forum Proceedings
INHERITANCE OF FUSARIUM HEAD BLIGHT RESISTANCE (TYPE II)
IN NEW WHEAT GERMPLASM CJ 9306 AND CJ 9403
Guo-Liang Jiang1,2* and Richard W. Ward1
Department of Crop and Soil Sciences, Michigan State University, East Lansing, MI 48824-1325; and
2
Wheat Research Laboratory, Nanjing Agricultural University, Nanjing 210095, China
*Corresponding Author: PH: 517-353-0657; E-mail: gljiang@msu.edu
1
ABSTRACT
Fusarium head blight (FHB or scab) caused by Fusarium graminearum is a worldwide
serious disease in wheat. Exploitation and genetic studies of elite resistance sources can
speed up the development of resistant cultivars. Two new resistance sources CJ 9306 and
CJ 9403 developed in a recurrent selection program of Nanjing Agricultural University,
China were crossed to two susceptible cultivars. Experiments with P1, P2, F1, F2, BC1 and
BC2 generations for four crosses and with F6:7 RILs for one cross were carried out in greenhouse to evaluate FHB resistance to fungal spread within a spike. Single-floret inoculation
was conducted at heading and flowering stages, and the inoculated plants were subsequently misted for three days. The number and percentage of scabby spikelets (NSS and
PSS) on the 25th day after inoculation were scored. The frequency distribution in F2s and
BC1s showed continuous with two major peaks and one minor peak between them, indicating that scab resistance in wheat should be a qualitative-quantitative trait. A high level of
resistance in CJ 9306 was mainly attributed to co-presence of two genes. The major gene
expressed at a moderate to resistant level and was the prerequisite for the expression of the
second or minor gene that enhanced the resistance to a high level. In CJ 9403 there might
be three major genes and two to three minor genes governing the resistance. The fittest
genetic model varied depending on specific crosses. A four-parameter model with additive ×
dominance interaction provided the most complete and precise elaboration in the two
crosses with CJ 9306. A simple additive-dominance model was best fitted for the data from
Veery/CJ 9403 and NSS in Norm/CJ9403. For PSS in Norm/CJ 9403, a five-parameter
model with additive × additive and dominance × dominance effects seemed to be more
adequate than others. The additive effects always significantly increased the resistance and
played a major role in the inheritance of scab resistance. The estimates of broad-sense and
narrow-sense heritabilities were 60%-86% and 32%-65%, respectively. As new improved
germplasm of scab resistance, CJ 9306 not only has a high level of Type II resistance as
well as a feature of simpler inheritance, but also possesses well-improved agronomic traits.
Therefore, it should be a good choice for breeding scab resistant cultivars. CJ 9403 could
be directly applied in production in adapted areas and breeding programs because of its
excellent agronomic traits and high yielding potential even if its resistance is a little lower.
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2002 National Fusarium Head Blight Forum Proceedings
SCREENING WINTER AND FACULTATIVE WHEATS FOR
FUSARIUM HEAD BLIGHT INFECTION
Mohan Kohli1*, Martin Quincke1 and Martha Diaz de Ackermann2
CIMMYT, Regional Wheat Program, CC 1217, Montevideo, Uruguay; and
National Agriculture Research Institute, La Estanzuela, CC 39173, Colonia, Uruguay
*Corresponding Author: PH: 598 2 902 8522; E-mail: cimmyt@inia.org.uy
1
2
ABSTRACT
Severe epidemics of Fusarium head blight (FHB) occur regularly in the Southern Cone
region of South America, especially in Argentina, Brazil, Paraguay and Uruguay. Historically
the National Wheat Programs have identified sources of resistance to FHB in the spring
wheats, including Frontana, Alvarez 110, Encruzilhada, Klein Atlas etc. In the recent years,
these have been combined with the Chinese germplasm derived from Sumai#3 and Chuan
Mai#18 to achieve higher level of resistance in the wheat programs.
In order to broaden the spectrum of resistance to include winter and facultative wheats,
CIMMYT’s regional program, based in INIA La Estanzuela, Uruguay, screens local and
introduced germplasm under naturally occurring and artificially inoculated conditions. The
level of naturally occurring FHB infection during 2001 was extremely severe, which allowed
excellent evaluation of the introduced germplasm (Table 1).
Table 1. Classification of winter and facultative wheat germplasm for FHB, 2001.
S o urce
T otal e ntrie s
S ca b cla ssificatio n*
M S
S
VS
4th W O N IR
1 12
3
29
77
10 th F A W W O N
62
8
18
30
G e org ia
86
8
45
27
Lo u isian a **
14
1
4
5
1
K a nsa s
1 18
20
81
15
O kla h om a
30
2
17
9
T exa s
20
3
5
10
M e xico (w inte r)
19
8
5
6
TO TAL
4 61
1
23
56
2 05
1 75
%
1 00
0.2
5.0
1 2.1
4 4.5
3 8.0
*R = R e sistan t; M R = M od .R e sistan t; M S = M o d. S usc ep tible ;S = S usce ptib le; V S = V e ry su scep tible
** C ollectio n o f p are n t line s from the cro ssin g b lo ck
R
MR
2
6
6
3
2
2
2
The evaluation of the CIMMYT international nurseries and winter wheat germplasm from
various collaborators in the US, confirms the presence of large variability in the FHB resistance. In spite of the fact that FHB infection was uniform throughout the cycle, later heading
germplasm tended to show lower levels of infection. The lines from this group will need to
be checked through artificial inoculations. Other lines, early or intermediate for their heading, selected for lower level of FHB infection are presented in Table 2.
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2002 National Fusarium Head Blight Forum Proceedings
While these results confirm the resistance of some parent lines (Shou Chou), they also
demonstrate that other lines such as CIMM1FHB#5, Coker 960208 and ND 2928 are, in
fact, moderately susceptible under Uruguayan field conditions. Two lines (X950412-F-7 and
Pioneer 26R61) were rated at par with local check INIA Tijereta, considered to be moderately resistant and will need to be confirmed in future evaluations. Several other lines
(Bezostaja, Irneria/Mukkab hib., Star/Bwd, OK 98637 and X950446-F-1), rated moderately
resistant to moderately susceptible, represent germplasm with very wide genetic backgrounds which can be useful in the breeding programs.
Table 2. Fusarium head blight reaction of selected facultative and winter wheat lines
Entry
Heading
FHB
(1-5/1-5)
Reaction
I. TIJERETA (Local check)
E*
22
MR**
ND2928
E
24
MS
CIMM1FHB#5
E
25
MSS
SHOU CHOU
E
11
R
OK97508
E
24
MS
TX98V6610
E
23
MS
I. TORCAZA (Local check)
I
32
MS
BEZOSTAJA
I
22
MRMS
STAR/BWD
I
22
MRMS
IRNERIA/MUKKAB HIB.
I
22
MRMS
SULTAN
I
23
MS
93435-1-10
I
13
MS
UGA 931463E27
I
23
MS
PIONEER 26R61
I
12
MR
APD99-5627
I
23
MS
COKER960208
I
14
MS
LA422
I
13
MS
9388D22-1-3
I
23
MS
X950337-II-2
I
23
MS
X950446-F-1
I
22
MRMS
X950412-F-7
I
11
RMR
OK98637
I
22
MRMS
OK95571
I
14
MS
I. GORRION (Local check)
L
45
S
* E= Early (150d), I= Intermediate (160d), L= Late (170d)
Origin
Uruguay
Louisiana
Louisiana
Louisiana
Oklahoma
Texas
Uruguay
10FAWWON
10FAWWON
10FAWWON
10FAWWON
Georgia
Georgia
Louisiana
Louisiana
Louisiana
Louisiana
Louisiana
Kansas
Kansas
Kansas
Oklahoma
Oklahoma
Uruguay
Field screening of facultative and winter wheat germplasm under naturally occurring epidemics of FHB at a hot spot site such as La Estanzuela, Uruguay, provides an excellent
opportunity to identify new sources of resistance. These, in addition, can also be screened
for foliar blights and leaf rust diseases. CIMMYT, in collaboration with the National Agriculture Research Institute, INIA, is trying to incorporate these and other sources of FHB resistance into locally adapted wheats. The preliminary results are very encouraging.
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2002 National Fusarium Head Blight Forum Proceedings
TYPES I, II AND FIELD RESISTANCE TO FUSARIUM HEAD BLIGHT
IN WINTER AND SPRING WHEAT GERMPLASM
Anne L. McKendry*, Kara S. Bestgen, and David N. Tague
Agronomy Department, University of Missouri, Columbia, Missouri 65211
*Corresponding Author: PH: (573) 882-7708; E-mail: mckendrya@missouri.edu
OBJECTIVE
The objective of this research was to evaluate and determine the relationship among Type I, II and
field resistance in spring and winter wheat germplasm that we had previously identified as having
potentially useful levels of resistance to scab.
INTRODUCTION
Fusarium graminearum Schwabe (teleomorph Gibberella zeae (Schwein.), also known as scab, is a
devastating disease of wheat and barley in warm and humid regions of the world. Host plant
resistance has long been considered the most practical and effective means of control but breeding
has been hindered by a lack of effective resistance genes and by the complexity of the resistance in
identified sources. No source of complete resistance is known, and current sources provide only
partial resistance, often in unadapted types. The identification of different sources of resistance in
winter wheat through a systematic evaluation of accessions maintained in the National Small Grains
Collection at Aberdeen, ID has been identified as a key objective of the US Wheat and Barley Scab
Initiative’s (USWBSI) germplasm research area. As such, approximately 4600 winter wheat
accessions have been evaluated at Missouri. Additionally, spring and winter wheat germplasm has
been introduced into the United States through a collaborative effort established between the
USWBSI and CIMMYT. Both initiatives have resulted in the identification and introduction of wheat
germplasm with high levels of Type II resistance. Less is known, however, about the Type I
resistance in these lines, how the two types of resistance are correlated, and whether those types
of resistance relate to field resistance.
MATERIALS AND METHODS
Germplasm: Germplasm was selected for evaluation from two sources. Winter wheat germplasm
was acquired from the National Small Grains Collection in Aberdeen, ID and was kindly provided by
Dr. Harold Bockelman. Germplasm was selected that had functional levels of Type II resistance in
each of 3 successive cycles of greenhouse evaluation. Winter wheat germplasm included was from
China, South Korea, Japan and Italy and included land races, cultivated genotypes and cultivars.
Spring wheat germplasm included most genotypes introduced into Missouri in 2000 through the
CIMMYT/USWBSI collaboration. Lines included were from the CIMMYT breeding program and
included advanced breeding lines and wide crosses. Genotypes also included lines introduced from
China and from Romania. Of the Romanian wheat introduced, 6 had a winter wheat growth habit.
Greenhouse Evaluations: Vernalized seedlings were arranged in a split-plot design with genotype as
the main plot and type of resistance as the sub-plot. For each accession, 10 plants per treatment
were planted and the experiment was replicated six times. For evaluation of Type II resistance,
plants were inoculated at first anthesis with 10µL of a macroconidial suspension of Fusarium
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2002 National Fusarium Head Blight Forum Proceedings
graminearum concentrated to 50,000 macroconidia/mL. Inoculum was placed in a single central
floret at first anthesis using an Oxford 8100™ repeat dispensing syringe. For all inoculations, a single
isolate was used which had been previously determined to be aggressive on the resistant cultivar,
Ernie. Plants were incubated in a mist chamber (100% relative humidity) for 72 h post-inoculation to
promote disease development and then returned to the greenhouse bench. Ratings for Type II
resistance (disease spread in the spike) were made at 21 d after inoculation. A Fusarium head blight
index (FHBI) was also determined at 21 d post-inoculation as the ratio of diseased spikelets to total
spikelets in the inoculated head.
For evaluation of Type I resistance, heads were again inoculated with a macroconidial suspension of
Fusarium graminearum concentrated to 50,000 macroconidia/mL. Inoculum was sprayed directly on
the head at full anthesis using a Pulmo-Aide nebulizer as the power source and an atomizer (Model
163, DeVilbiss Sunrise Medical, Somerset, PA 15501-0635, USA). Inoculum was delivered to each
head, spraying one side and then the other. Plants were incubated in a mist chamber as described
above. At 10 d post-inoculation heads were rated for symptoms of Fusarium head blight. Total
spikelets in the head were recorded followed by the number of spikelets in the head showing
disease. Incidence was determined as the total number of spikelets on the inoculated head showing
disease symptoms. The Type I FHBI rating for each head was determined as the number of
spikelets with disease divided by the total number of spikelets on the head. Ratings were taken
again at 21 d post-inoculation to determine the scab index for the head. The 21-d rating (total number
of infected spikelets/total spikelets in the inoculated head) provided an estimate of severity on the
inoculated head.
Field Evaluations: The field scab index was determined from unreplicated spray inoculations or
winter wheat germplasm. Individual rows were inoculated at 75% anthesis with a macroconidial
suspension of Fusarium graminearum concentrated to 50,000 macroconidia /mL using a CO2
backpack spray system. Plants were maintained under overhead mist irrigation throughout the
inoculation period (approximately 2 wk). Twenty heads from each row were evaluated for symptoms
of scab 18-21 d post-inoculation. Infected spikelets were counted on each head. Incidence was
determined as the number of heads with visible symptoms of disease. Severity was determined as
the ratio of diseased spikelets to total spikelets in the inoculated heads. The field scab index was
calculated as incidence*severity.
RESULTS AND DISCUSSION
Data presented in Table 1 are those from winter wheat germplasm screened with high levels of Type I,
II and field resistance. Superior lines included land races and cultivated lines from China, South
Korea and Italy. Of 45 lines evaluated, 12 lines had good levels of Types I and II resistance, coupled
with good field resistance. Data for spring wheats with good levels of resistance are given in Table 2.
Of 57 wheat genotypes introduced through the CIMMYT collaboration in 2000, 23 genotypes had
excellent levels of Type II resistance (< 10%) while 15 had good Type I resistance (<40%). Nine
genotypes combined good levels of both Type I and Type II resistance. Four of these lines were
introduced from Romania, while two were introduced from China. Type I and Type II resistances
were not highly correlated. Complete data for all lines evaluated will be available at the Scab Forum.
Germplasm Introduction and Enhancement
205
Table 1. Type 1 (greenhouse spray), II (greenhouse point) and field scab resistance for winter wheat germplasm with putative genes for scab resistance. Values that are
bolded do not differ significantly within a column.
Heading Mean spikelets Greenhouse Point Inoculation
Field Scab
Greenhouse Spray Technique
Designation
Origin
date
by accession
21 d
21 d
10 d
10 d
21 d
21 d
Index
Name
Julian
No. spikelets
Spread
Index
Incidence
Index
Incidence
Index
%
c4-3-3
319
15.1
3.7
0.24
6.7
0.44
9.1
0.59
9
CHOW
CULTIVATED China
0.62
0.64
c5-1-1
Kuang tu erh hsiao mai
LANDRACE China
327
14.3
2.1
0.15
8.6
8.9
36
c12-2-3
324
12.8
2.0
0.16
6.8
0.55
7.7
0.62
48
COLOGNA VENETO
LANDRACE Italy
1.8
0.13
6.5
0.46
7.1
0.51
CItr 9428
LANDRACE China
318
13.9
76
70-1-2
71-2-3
316
14.1
3.0
0.22
7.7
0.57
8.9
0.66
14
CItr 9429
LANDRACE China
1.0
0.08
7.0
0.57
0.62
77-1-2
CItr 9445
LANDRACE China
322
13.1
7.7
28
92-2-1
317
12.8
2.3
0.18
7.2
0.57
8.1
0.64
20
CItr 9490
LANDRACE China
1.9
0.59
0.66
15
93-2-1
CItr 9506
LANDRACE China
318
13.1
0.16
7.8
8.8
94-2-2
323
16.0
1.8
0.11
9.0
0.58
10.8
0.70
36
CItr 9507
LANDRACE China
102-3-1
319
13.0
2.7
0.21
7.0
0.54
7.7
0.60
45
CItr 9521
LANDRACE China
122-2-2
315
10.3
1.7
0.18
5.9
0.56
5.7
0.54
15
A1
CULTIVATED China
321
11.5
1.0
0.09
5.9
0.52
6.6
0.57
34
124-2-2
A11
CULTIVATED China
126-4-3
310
10.2
2.6
0.26
5.5
0.53
6.0
0.61
12
A23
CULTIVATED China
312
11.3
4.7
0.42
6.1
0.53
7.2
0.62
35
147-3-4
B22
CULTIVATED China
151-2-4
B36
CULTIVATED China
317
13.3
3.6
0.27
6.0
0.45
7.7
0.58
20
202-2-1
320
15.8
4.8
0.31
10.4
0.65
12.4
0.78
32
D3A
CULTIVATED China
0.61
0.64
244-2-4
D127A
CULTIVATED China
318
15.1
4.9
0.32
9.2
9.7
30
355-2-1
320
12.5
1.5
0.12
8.5
0.68
8.9
0.71
29
COLORBEN 4
CULTIVAR
Italy
1.7
7.5
419-2-4
QUADERNA
CULTIVAR
Italy
311
10.1
0.17
7.3
0.72
0.75
39
433-1-2
314
13.1
3.1
0.23
6.8
0.50
7.7
0.58
36
NORIN 50
CULTIVAR
Japan
1.3
4.6
0.47
5.1
0.53
27
451-1-2
SEU SEUN 6
CULTIVAR
Korea, S
313
9.6
0.14
497-2-1
312
11.3
1.9
0.17
7.4
0.65
8.3
0.73
32
NORIN 96
CULTIVAR
Japan
0.67
17
568-1-4
TRENTO
CULTIVAR
Italy
322
17.3
2.3
0.14
11.7
13.1
0.75
687-2-2
324
14.6
1.8
0.13
7.6
0.50
8.5
0.56
26
CAMPOFIORITO
CULTIVAR
Italy
1.2
0.12
6.0
0.54
6.6
0.59
10
LING HAI MAO YANG MO CULTIVATED China
291
11.2
816-3-4
829-1-2
354
19.2
1.2
0.06
11.2
0.58
12.4
0.64
38
XIN DONG NO. 2
CULTIVATED China
1.6
0.09
0.56
0.60
829-4-1
XIN DONG NO. 2
CULTIVATED China
333
18.8
10.6
11.3
38
870-4-2
316
12.4
1.1
0.09
5.4
0.46
5.9
0.50
18
WAN SHUI BAI
CULTIVATED China
1.6
0.11
0.51
0.59
23
877-1-2
YANG LA ZI
LANDRACE China
320
15.8
8.5
9.8
937-2-3
309
9.4
2.0
0.23
4.1
0.44
4.6
0.49
32
84-5418
CULTIVATED China
6.4
0.60
6.9
0.65
14
Ernie
Resistant Check (early)
Cultivar
USA
315
10.8
2.1
0.20
MO 94-317 Susceptible Check
321
13.3
10.3
0.79
9.9
0.74
12.0
0.89
76
Cultiar
USA
0.7
0.04
0.52
0.56
Sumai 3
Resistant Check (late)
China
339
18.0
9.5
10.3
350
20.5
0.9
0.04
10.0
0.48
10.6
0.51
Ning 7840 Resistant Check (late)
China
Average
320
13.4
2.7
0.21
7.7
0.57
8.6
0.64
30
LSD at 0.05
3.1
1.6
1.3
0.09
2.9
0.23
2.9
0.19
18
c.v.%
3.9
11.0
41.4
37.6
33.3
30.7
29.2
26.6
2002 National Fusarium Head Blight Forum Proceedings
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Table 2. Types 1 (incidence) and II (point) resistance to Fusarium head blight in selected germplasm introduced from CIMMYT in 2000.
Greenhouse spray (incidence)
Greenhouse point
10 Day
21 Day
10 day
21 day
21 day
21 day
CIMMYT 2000 - Pedigree/Designation
# diseased
# diseased
FHBI
FHBI
# diseased
FHBI
spikelets
spikelets
spikelets
SHA3/CBRD
7.2
7.6
0.50
0.52
1.1
0.07
NG8675/CBRD
4.0
5.5
0.22
0.29
1.4
0.07
NS73/PCI//B143.241.2/3/NING8647
13.4
17.1
0.61
0.77
1.7
0.08
MIAN YANG81-5//PC B084.985/JIANZIMAI
13.1
14.5
0.58
0.64
1.6
0.07
MIAN YANG81-5//PC B084.985/JIANZIMAI
8.0
8.8
0.37
0.41
1.0
0.05
PC B084.985/JIANZIMAI//8744
12.0
14.9
0.54
0.67
2.1
0.10
SHANGAI
13.4
14.2
0.68
0.72
0.7
0.04
SHANGAI
11.0
11.9
0.64
0.70
1.0
0.06
GOV/AZ//MUS/3/DODO/4/BOW
6.0
7.6
0.31
0.39
3.6
0.18
RECURRENT SELECTION 1
19.6
20.9
0.91
0.97
2.3
0.11
SODAT/SUM3//NING820/3/NING8626
8.7
10.6
0.47
0.58
1.6
0.09
BCN//DOY1/AE.SQUARROSA (447)
7.5
7.7
0.37
0.38
3.9
0.18
MAYOOR/5/CS/TH.CU//GLEN/3/ALD/PVN/4/CS/LE.RA//2*CS/3/CNO79
2.8
5.3
0.21
0.40
3.2
0.23
BUC//RUFF/AE.SQ/3/MAIZ
4.5
7.6
0.36
0.60
6.3
0.51
FUNDULEA 201 R (Winter)
5.8
6.0
0.34
0.35
0.7
0.04
FUNDULEA 183 P5 (Winter)
10.6
11.7
0.55
0.61
0.7
0.04
FUNDULEA 143-T3-103 (Winter)
5.9
7.7
0.30
0.39
1.2
0.06
TURDA 95 (Winter)
5.0
5.7
0.27
0.31
0.7
0.04
TURDA 195 (Winter)
11.8
14.6
0.57
0.70
1.0
0.05
TURDA 2317-90 (Spring)
6.5
7.4
0.36
0.41
0.5
0.03
NING 896013
1.6
5.1
0.10
0.32
2.0
0.14
NING 894037
13.8
16.6
0.73
0.87
0.9
0.05
MUTANT AT 1
13.7
17.8
0.60
0.78
1.2
0.06
MUTANT AT 2
15.6
17.8
0.68
0.77
1.2
0.06
YANGMAI 9
8.9
10.1
0.43
0.48
1.5
0.07
EMAI 6
5.1
7.9
0.38
0.59
1.7
0.12
ZHONGHUA 1
10.9
12.9
0.57
0.68
1.4
0.07
SUMAI 2
11.5
11.8
0.49
0.50
1.2
0.05
85004/MEXICO 354
5.1
6.0
0.26
0.30
1.6
0.08
Ernie (resistant check)
3.4
3.6
0.26
0.28
2.0
0.17
Sumai 3 (resistant check)
9.5
10.3
0.52
0.56
0.7
0.04
MO 94-317 (susceptible check)
8.7
10.7
0.64
0.79
10.3
0.68
2002 National Fusarium Head Blight Forum Proceedings
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RESISTANCE IN HEXAPLOID WHEAT TO FUSARIUM HEAD BLIGHT
Gregory Shaner
Dept. of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907-2054
Corresponding Author: PH: (765) 494-4651; E-mail: shanerg@purdue.edu
When Fusarium head blight (FHB) re-emerged as a major disease of wheat in North
America about 15 years ago, it was evident that most cultivars were susceptible. Small grain
breeders and pathologists began working with Sumai 3 and related resistant lines from
China as sources of resistance. Although there seem to be no races of Fusarium
graminearum that have adapted to a particular source of resistance, numerous examples
from other pathogens suggest that we should be cautious about reliance on one source of
resistance. Moreover, Sumai 3 is not completely resistant. The discovery of other resistance
genes may allow us to create genotypes with a greater level of resistance than any that are
currently known. To further these objectives, the USWBSI created a Germplasm Introduction
and Enhancement (GIE) research area “to identify new sources of FHB resistance and to
facilitate the utilization of resistant germplasm”.
ACCOMPLISHMENTS
Participants in the USWBSI, as well as workers in other areas of the world, have identified
many wheat lines resistant to FHB (e.g., 2,4,6,9). Snijders hypothesized 3 main pools of
resistant germplasm (12). The GRIN database includes 34 lines that have FHB index values
of 2 or 3. McKendry and Bestgen identified 17 lines from the CIMMYT/USWBSI collaborative germplasm effort that had 1 spikelet or less blighted following point inoculation
(McKendry, personal comm.). No line of common wheat is completely resistant, but several
have a high degree of resistance.
SCREENING TECHNIQUES
Most germplasm screening efforts employ point inoculation, in which an aqueous suspension of conidia is placed in a single floret at the middle or near the tip of the spike. Point
inoculation is designed to detect resistance to spread of the fungus throughout the spike,
based on spread of symptoms, referred to as Type II resistance. Resistance to initial infection (Type I resistance) may be as important as Type II resistance. If weather is favorable for
infection throughout anthesis and early grain fill, there may be multiple primary infection
events, leading to severe head blight without the need for spread of the pathogen through
the rachis. Workers evaluate germplasm for Type I resistance by spraying heads at full
flowering with a spore suspension.
Mesterhazy proposed 5 types of active resistance mechanisms in wheat to Fusarium infection, based on his own and previous work (6). This classification of resistance types (now
referred to as Types I to V) has had a major influence on how researchers view resistance
and its genetic control. As more has been learned about the interaction between Fusarium
and wheat, there are questions about how some of these types of resistance are defined
and measured. Participants at a recent workshop of the GIE group agreed that Type I and
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Type II resistance are reasonably straightforward, as first defined (8). The other 3 types of
resistance (III-V) pose problems.
Type III resistance (resistance to kernel infection) is a valid concept, but operationally it
poses problems. What is the appropriate way to measure it? Point inoculation is not suitable.
If a line has a high degree of Type II resistance, then the kernels from a head on which a
single floret was inoculated will show a low frequency of infection and damage because the
fungus never reached them or reached them too late to cause visible damage. For evaluation of Type III resistance, every kernel evaluated should be exposed to infection. Spray
inoculation might be more suitable, but a line with resistance that impedes progress of the
fungus from the stamen to the ovary could mask whatever resistance or susceptibility kernels might have to invasion. There is a more fundamental question about this kind of resistance. Does it refer to the ability of the fungus to penetrate a kernel or to the degree to which
the fungus ramifies the grain? If Type III resistance is meant to measure differences in the
amount of mycelium in grain, then a test that measures fungal biomass in kernels should be
used.
Mesterhazy defined Type V resistance (active resistance mechanism “e”) as “resistance to
toxins in ears by decomposing them” and cited Miller et al. 1985 (7) and Snijders and
Perkowski 1989 (sic) (13) as sources (The citation of the Snijders and Perkowski paper is
incorrect in Mesterhazy’s paper. The correct citation is given in the reference list below).
Miller at al. suggest 2 reasons for low DON levels in grain: the plant has factors that prevent
formation of toxin, or factors that promote degradation of toxin. Accurate measurement of
resistance to toxin accumulation poses difficulties. If lines have Type I or Type II resistance,
or resistance to invasion of kernels from other tissues in the head, then they would have
lower levels of DON in kernels compared to lines that lacked these forms of resistance.
Detection of resistance to toxin accumulation requires that grain from different wheat lines
has not only been equally exposed to infection, but that fungal biomass in the grain can be
measured and related to DON content (7).
Correlation analysis of DON content versus fungal biomass in kernels may reveal that some
lines have less DON than would be expected. Such lines should be investigated for presence of genes that act to influence the accumulation of DON. It would be desirable to combine these genes with genes for other types of resistance. However, selection for resistance
to DON accumulation against a background of resistance that reduces the frequency or
extent of kernel infection would be difficult. Genes for resistance to DON accumulation
would be ideal candidates for marker-assisted selection.
The concept of tolerance (Type IV, or “d” in Mesterhazy’s list of active defense mechanisms)
has traditionally been applied to foliar or root diseases, in which grain is not directly infected, but its mass and quality are reduced by the stress of infection of vegetative organs. A
tolerant cultivar sustains less yield reduction for a given severity of disease than an intolerant cultivar. What does tolerance mean for a pathogen that infects reproductive organs? If
grain is relatively sound despite severe head blight symptoms, the plant may have a resistance mechanism that interferes with invasion of the grain. Mesterhazy used yield relative to
uninoculated controls as a measure of tolerance, i.e. if a group of lines had equivalent head
blight severity scores, but differed substantially in relative yield, he considered the line(s)
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2002 National Fusarium Head Blight Forum Proceedings
with higher yield to be tolerant. Without direct evidence of comparable timing and extent of
kernel invasion (fungal biomass per kernel), conclusions about tolerance in FHB must
remain tentative.
INHERITANCE OF RESISTANCE
Discovery and phenotypic characterization of resistant lines are only the first steps in using
germplasm. Resistance must be incorporated into cultivars adapted for each region where
FHB is a threat. Breeders can use germplasm without knowing the genetic basis of resistance, but the work is more efficient if they have this knowledge.
Original accessions are often heterogeneous for resistance and it is necessary to reselect
from this germplasm to obtain lines that consistently express resistance. Once this has been
done, genetic studies can be undertaken.
Most studies published so far have evaluated Type II resistance in response to point inoculation. Two or more genes condition resistance in Sumai 3 or its derivative, Ning 7840. A
major QTL has been mapped to 3BS (1,14). Results from a number of studies with various
wheat lines indicate QTLs for FHB resistance may reside on most chromosomes of the
wheat genome (see 14). Many of these genes show additive action. This suggests that as
new genes are identified in other sources of resistance, they will act additively, or in more
complicated manners, with the genes already in hand, to give higher levels of resistance.
Even moderately susceptible lines have contributed useful genes for resistance (1,3,11).
FUTURE DIRECTIONS OF THE GIE PROGRAM
If Types I and II resistance are inadequate to protect the crop, resistance to kernel invasion
or to DON accumulation could provide another layer of protection. The germplasm program
of the USWBSI should give more attention to discovery of germplasm with these other forms
of resistance. With respect to Type I and Type II resistance, emphasis should shift to genetic
characterization of the germplasm already in hand. We need to know more about inheritance of resistance in various sources, the uniqueness of their genes, and how genes interact to affect the resistance phenotype.
We may be approaching the limit of Type II resistance. Several lines show only slight necrosis in the inoculated floret. It will be difficult to detect higher levels of Type II resistance when
such sources are combined. It may be more useful to combine other types of resistance with
a high degree of Type II resistance. Evaluation of the same wheat lines by both point and
spray inoculation suggests that different genes control Type I and Type II resistance. If yet
other genes control resistance to kernel infection and toxin accumulation, then it should be
possible to combine all of these into a single cultivar. For reasons outlined above, phenotypic selection would not work. This is clearly a project that requires marker-assisted selection technology. First we need to carefully characterize, phenotypically and genetically,
resistance other than Type II, and then find reliable markers for these genes.
To accomplish this efficiently within the USWBSI, I propose creation of a coordinated program analogous to programs in other research areas. Many accessions with Type II resistance have been identified in hexaploid wheat. The most resistant accessions, particularly
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2002 National Fusarium Head Blight Forum Proceedings
any that lack the major QTL on 3BS (see e.g., 5), need to be thoroughly studied genetically.
We also need to identify sources of Type I resistance and sources of resistance to kernel
invasion and DON accumulation. For this, we need to develop reliable methods of phenotypic screening. These will likely be more complicated and costly than the point inoculation
technique currently used to identify Type II resistance.
ACKNOWLEDGEMENTS
I thank Bill Bushnell and other participants in the GIE workshop who shared ideas about
evaluating germplasm for resistance to FHB.
REFERENCES
Anderson JA et al. 2001. DNA markers for Fusarium head blight resistance QTLs in two wheat populations.
Theor Appl Genet. 102(8):1164-1168.
Bai GH, Shaner G. 1994. Scab of wheat: Prospects for control. Plant Dis. 78(8):760-766.
Ban T. 2000. Analysis of quantitative trait loci associated with resistance to Fusarium head blight caused by
Fusarium graminearum Schwabe and of resistance mechanisms in wheat (Triticum aestivum L.). Breed Sci.
50(2):131-137.
Ban T, Suenaga K. 2000. Genetic analysis of resistance to Fusarium head blight caused by Fusarium
graminearum in Chinese wheat cultivar Sumai 3 and the Japanese cultivar Saikai 165. Euphytica 113:87-99.
Gupta A et al. 2001. Molecular and pedigree analysis of sources of resistance to FHB in wheat, p. 181 In Canty
SM et al. Eds. 2001 National Fusarium Head Blight Forum Proc.
Mesterhazy A. 1995. Types and components of resistance to Fusarium head blight of wheat. Plant Breed
114:377-386.
Miller JD et al. 1985. Deoxynivalenol and Fusarium head blight resistance in spring cereals. Phytopath Z.
113:359-367.
Schroeder HW, Christensen JJ. 1963. Factors affecting resistance of wheat to scab caused by Gibberella zeae.
Phytopathology 53:831-838.
Shaner G, Buechley G. 2001. New sources of resistance to Fusarium head blight of wheat, p. 203-203 In Canty
SM et al. Eds. 2001 National Fusarium Head Blight Forum Proc.
Snijders CHA. 1990. Diallel analysis of resistance to head blight caused by Fusarium culmorum in winter wheat.
Euphytica 50:1-9.
Snijders CHA. 1990. Response to selection in F2 generations of winter wheat for resistance to head blight
caused by Fusarium culmorum. Euphytica 50:163-169.
Snijders CHA. 1990. Genetic variation for resistance to Fusarium head blight in winter wheat. Euphytica 50:171179.
Snijders CHA, Perkowski J. 1990. Effects of head blight caused by Fusarium culmorum on toxin content and
weight of wheat kernels. Phytopathology 80(6):566-570.
Zhou W, Kolb FL, Bai G, Shaner G, Domier L. 2002. Genetic analysis of scab resistance QTL in wheat with
microsatellite and AFLP markers. Genome 45:719-727.
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NOVEL SOURCE OF TYPE II RESISTANCE TO
FUSARIUM HEAD BLIGHT
Xiaorong Shen*, Lingrang Kong and Herbert Ohm
Agronomy Department, Purdue University, West Lafayette, IN 47907-1150
*Corresponding Author: PH: (765)494-9138; E-mail: xshen@purdue.edu
ABSTRACT
Sources of resistance in wheat (Triticum aestivum) to Fusarium head blight (FHB) of wheat
are limited despite extensive screening of germplasm since Arthur first reported this disease
in 1891. Wheatgrass has been demonstrated to be an important resistance source for wheat
leaf rust, stem rust, and powdery mildew diseases. Here we report the excellent resistance
to FHB of Lophopyrum elongatum (2n = 2X =14, genome EE).
A series of Chinese Spring- L. elongatum substitution lines from 1E(1A) to 7E(7D) except
4E(4D) and 5E(5A) (provided by J. Dvorak, Department of Agronomy and Range Science,
University of California, Davis, CA), were evaluated for Type II resistance to Fusarium
graminearum in a greenhouse, February-April 2002. The recipient parent Chinese Spring
was also included in the experiment. In a completely randomized design, 12 – 24 plants per
line were evaluated for disease severity (DS), the percentage of diseased spikelets in
inoculated spikes. The mean DS of Chinese Spring was 41%. The mean DSs of the substitution lines ranged from 5% - 74%. Pairwise comparisons of means showed that three lines
had significantly higher DSs than Chinese Spring. They are 3E(3D), 2E(2D), and 6E(6A)
with respective DSs of 74%, 71%, and 62%. Three lines had DSs that were significantly
lower than Chinese Spring. They are 7E(7A), 7E(7B), and 7E(7D), with respective DSs of
5%, 5%, and 6%. The disease did not spread beyond the inoculated spike in all tested
plants in these three lines. Our data shows that the 7E chromosome of L. elongatum conditions Type II FHB resistance. Chinese Spring itself has resistance to FHB. The resistance of
Chinese Spring may be located on chromosomes 2D, 3D, and 6A, because when these
chromosomes were replaced with their respective homoeologous L. elongatum chromosome, these substitution lines were more susceptible to Fusarium graminearum than Chinese Spring.
The experiment is being repeated in the greenhouse, October-December, 2002. Results to
date are consistent with our results in the test of February-April, 2002.
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EVALUATION OF THE NATIONAL SMALL GRAINS COLLECTION OF
BARLEY FOR RESISTANCE TO FUSARIUM HEAD BLIGHT
AND DEOXYNIVALENOL ACCUMULATION
L.G. Skoglund* and J.L. Menert
Busch Agricultural Resources, Inc. Fort Collins, CO 80524
*Corresponding Author: PH: (970) 472-2332; E-mail: linnea.skoglund@anheuser-busch.com
INTRODUCTION
Barley is a major crop in the Red River Valley of Minnesota, North Dakota and Manitoba.
Production peaked in the 1980s and decreased to its lowest level in 30 years in 1999.
Many factors have contributed to barley’s decline in the region, including Fusarium head
blight (FHB) (McMullen, et al).
Grain affected by FHB, caused primarily by Fusarium graminearum, has reduced quality
and may be contaminated with unacceptable levels of deoxynivalenol (DON) (Salas, et al.).
With the advent of the FHB epidemics of the 1990s, some brewing companies imposed
strict limits on the levels of DON present in grain going to the malt houses. Barley exceeding the DON specifications is relegated to feed grade with a corresponding drop in price.
The risk involved with potential grade reduction is a primary factor in declining barley
hectarage. This has lead to increased awareness of the problem and a need for barley
varieties that are resistant to FHB and to toxin accumulation.
In 1998, Busch Agricultural Resources, Inc. (BARI) scientists began a study to identify
sources of resistance to FHB and resulting DON accumulation. This effort concentrated on
screening the entire 6-rowed spring barley collection held by the National Small Grains
Collection. Public and private barley breeders may be able to utilize accessions identified
as having high levels of resistance to disease and toxin accumulation to improve malting
barley.
MATERIALS AND METHODS
Initially 7,475 accessions were received from the USDA-ARS, National Small Grains Collection, Aberdeen, Idaho. Field evaluation sites included Casselton, ND and Crookston, MN
in 1998, Park River and Osnabrock, ND in 1999 and 2000, and Park River in 2001. Accessions were planted mechanically as single, non-replicated rows. In July 1998 accessions
with few or no visible symptoms were selected and then hand harvested in August. In 1999,
2000 and 2001 all accessions were hand harvested regardless of visible symptoms. In
2000 percent FHB was determined in July by counting number of infected kernels vs. total
kernels on 10 heads. In all years harvested material was transported to Fort Collins, CO
and threshed. Grain samples were submitted to the Barley DON Diagnostic Laboratory
located in the Department of Cereal and Food Sciences at North Dakota State University for
DON analysis (Tacke and Casper). Selections were made for further testing based on both
percent FHB and ppm DON.
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Selected accessions were planted in the greenhouse in Fort Collins, CO during the winters
of 1999/2000 and 2000/2001. Heads were inoculated at anthesis with an isolate of F.
graminearum collected from Midwest-grown barley. Inoculations were carried out late in the
day and plants placed in clear plastic chambers equipped with a humidifier for approximately 36 hours. Heads were rated for percent FHB by counting the total number of kernels
and the number of visibly infected kernels at 14- and 21-days post inoculation. In the 2000/
2001 season, greenhouse samples also were submitted for DON testing.
RESULTS AND DISCUSSION
A number of the 7,475 rows planted in 1998 were winter, hulless, black, hooded, dwarf, 2rowed or other types of barley and were discarded. Because all the barley at the Crookston
nursery appeared to be free of disease, selections were made and harvested from plants at
the Casselton nursery. The same accessions were harvested at Crookston. Based on
visual selection, the top 98 accessions and 2 checks were submitted for DON analysis from
both locations. DON levels ranged from non-detectable to more than 40 ppm across locations.
Eighty-two accessions were advanced to the 1999 field screening. Toxin levels were generally lower than the previous year and ranged from 0.2 to 11.0 ppm (avg. 2.9 ppm) at Park
River and non detectable to 12.5 ppm (avg. 1.9 ppm) at Osnabrock.
Fifty-six accessions were selected for testing in the greenhouse in 1999/2000. An additional 13 accessions that had been discarded in 1998 but were identified as resistant by
North Dakota State University (NDSU) in 1999 were returned to the study. Twelve accessions did not develop any symptoms of FHB. In all, 9 of the 56 accessions were eliminated
from further testing based on high DON, high FHB or very poor agronomic traits. All of the
NDSU selections that were returned to the study were carried over to the next field season.
In 2000 sixty accessions were planted at Park River and Osnabrock. Toxin analysis and
disease ratings were possible on 48 accessions. Toxin levels averaged 0.3 ppm DON at
Park River but averaged 1.6 ppm at Osnabrock. None of the 12 accessions that had 0%
FHB in the greenhouse remained completely free of disease. However, these and other
accessions low in disease continued to perform well.
A total of 47 accessions were tested in the greenhouse in 2000/2001, 15 from previous
years’ tests and 32 selected by NDSU in 2000. Disease averaged 18% and was high (25 to
95%) in many accessions. Toxin levels ranged from non-detectable to 13.6 ppm with a
mean of 2.1 ppm. All accessions were carried over for another year of field screening.
DON level for 2001 averaged 2.4 ppm. Levels ranged from 0.6 ppm to 3.3 ppm for the 15
accessions remaining in the study.
We have selected 15 accessions to recommend for inclusion in breeding for resistance
(Table 1). Several of these accessions selected were already reported to have resistance
and have been used in various breeding programs over the years (including the 2-rowed
types, Svanhals and Svansota). Steffenson and Scholz also selected a number of accesGermplasm Introduction and Enhancement
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2002 National Fusarium Head Blight Forum Proceedings
sions in their studies (Steffenson and Schulz). As a result of these studies, several new
accessions can be added to the list of resistant germplasm. The geographic sources of
these accessions cover four continents. However, at least 2 accessions from the US have
Chevron in their background. Another US selection has a Svanhals parent. The full diversity of the most resistant accessions needs to be assessed through molecular genetics.
This can further focus breeding efforts on numerous sources of resistance.
T ab le 1. A ccessio n s from th e N atio n al S m all G rain s C o llectio n selected fo r resistan ce to F u sariu m h ead b ligh t
and D eox yn iv alen ol.
A C C E S S IO N
C Ih o
M am m o th W in ter
C Ih o 2 2 0
W isco n sin P ed igree
C Ih o 8 3 5
A b yssin nin an In term ediate C Ih o 2 4 1 4
H ietpas 3
C Ih o 6 6 1 1
S eed S to cks 1 1 4 8 -1
C Ih o 6 6 1 3
M ark h in stz
C Ih o 7 2 7 9
Io w a 5 2 8 6
C Ih o 9 5 3 9
E L S 6 4 02 -3 0 2
C Ih o 1 2 9 04
1 9 48 D
U N A 8 3 92
C ross
C Ih o 2 4 9 2
S v an so ta
C Ih o 1 9 0 7
S v an h als
C Ih o 2 2 7 4
P eatland
C Ih o 2 6 1 3
C hev ron
C Ih o 1 1 1 1
PI
P I 1 4 97 8 2
P I 2 9 87 5 1
P I 3 7 13 1 7
P I 4 7 78 5 4
O R IG IN
U k rain e
U SA
E th io p ia
U SA
U SA
R ussia
U SA
E th io p ia
S w itzerlan d
P eru
S w eden
U SA
S w eden
U SA
S w itzerlan d
P E D IG R E E
S electio n fro m P I 25 6 7 4
S electio n fro m O d erbru cker
M an ch uria, C I 4 47 1 /C h ev ro n
N o . 45 6 /S van hals
S electio n fro m P I 54 7 4
S electio n fro m sam e lan drace as C h ev ro n
S electio n fro m lan d race
REFERENCES
McMullen, M., Jones, R., and Gallenberg, D. 1997. Scab of wheat and barley: A re-emerging disease of devastating impact. Plant Dis. 81:1340-1348.
Salas, B., Steffenson, B. J., Casper, H. H., Tacke, B., Prom, L. K., Fetch, T. G., Jr., and Schwarz, P. B. 1999.
Fusarium species pathogenic to barley and their associated mycotoxins. Plant Dis. 83:667-674.
Steffenson, B. J., and Scholz, U. 2001. Evaluation of Hordeum accessions for resistance to Fusarium head
blight. Pages 208-211 in: 2001 National Fusarium Head Blight Forum Proceedings, Dec. 8-10, 2001, Erlanger,
KY.
Tacke, B. K., and Casper, H. H. 1996. Determination of deoxynivalenol in wheat, barley, and malt by column
cleanup and gas chromatography with electron capture detection. J. A.O.A.C. Intl. 79(2): 472-475.
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FUSARIUM HEAD BLIGHT TYPE II RESISTANCE OF A
SPRING WHEAT POPULATION DERIVED FROM A
HUNGARIAN WINTER WHEAT
R.W. Stack1*, R.C. Frohberg2 and M. Mergoum2
1
Dept. of Plant Pathology, 2 Dept of Plant Sciences, North Dakota State University Fargo, ND 58105
*Corresponding Author: PH: (701) 231-7077; E-mail:rstack@ndsuext.nodak.edu
ABSTRACT
Fusarium head blight (FHB) of wheat, caused mainly by Fusarium graminearum, has
plagued farmers in the spring wheat region for nearly a decade, causing substantial loss.
Disease management by crop rotation, tillage, or fungicides has been marginally successful
at best. The best long term solution to the FHB problem in this region is by incorporation of
resistance into adapted cultivars. Several cultivars resistant or moderately resistant to FHB
have been released in the region over the past several years, and many more advanced
lines are being tested. The resistance to FHB in nearly all such adapted spring wheats has
come from Chinese germplasm sources such as Sumai 3 and its derivatives. Other sources
of resistance need to be explored. In the 1980’s, Akos Mesterhazy in Hungary identified
non-Chinese lines which showed resistance to FHB. He produced several advanced
resistant lines by intercrossing these sources. One of us (RWS) obtained several of his
lines in 1996 and crosses were made to adapted spring wheats. A population derived from
crossing Mesterhazy’s line ‘Ringo Sztarr/Nobeoka Bozu’ by the ND cultivar ‘Grandin’ was
advanced by single seed descent to F-6, selecting only for spring habit. We grew 182 lines
from this population in the greenhouse in two randomized replicates. At anthesis, ten
spikes per replicate were inoculated by single spikelet inoculation and then given 3 days of
intermittent mist treatment. At 3.5 weeks post-anthesis, FHB symptom development on each
spike was scored on a 0-100% severity scale. FHB severity scores of the 182 lines ranged
from 8% to 85%. Based on FHB severity, 14 of the 182 lines were resistant as or better than
our standard resistant check line ‘ND2710’ and other best Sumai 3 derived lines. We
conclude that lines derived from other resistance sources may be as resistant to FHB as
those from presently used Chinese germplasm. This can serve to diversify the germplasm
base for FHB resistance.
(This poster was presented at the Annual Meeting of Amer. Soc. Agron., Indianapolis, IN,
Nov 10-14, 2002)
Germplasm Introduction and Enhancement
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2002 National Fusarium Head Blight Forum Proceedings
PROPOSED CHROMOSOMAL LOCATION OF FHB RESISTANCE
GENES IN ADDITIONAL SETS OF DURUM DISOMIC SUBSTITUTION
LINES DERIVED FROM DIFFERENT T. DICOCCOIDES ACCESSIONS
R.W. Stack1*, J.D. Miller2, and L.R. Joppa2
Plant Pathology Dept., North Dakota State Univ., Fargo, ND; and
2
USDA-ARS, Northern Crop Sci. Lab., Fargo, ND 58105
*Corresponding Author: PH: (701) 231-7077; E-mail: stack@ndsuext.nodak.edu
1
ABSTRACT
Fusarium head blight (FHB), caused mainly by Fusarium graminearum, has been a serious
disease problem on spring wheat in North Dakota and surrounding states for nearly a
decade. North Dakota is the principal durum growing state in the USA and durum has been
especially hard hit by FHB. Development of cultivars with FHB resistance has been much
slower for durum than for the spring bread wheats, in part because the best known sources
of FHB resistance are in hexaploid backgrounds and effective transfer to the tetraploid
durum seems to be difficult. In the 1980’s, USDA geneticist L.R. Joppa had produced a set of
durum disomic chromosome substitution lines derived from a wild emmer (Triticum
dicoccoides, TDIC) selection “Israel A”, identified for high grain protein levels. We recently
reported (Crop Sci. 42:637-642) the finding of FHB resistance on chromosome 3A in the
durum disomic substitution line from this series. Other researchers have found molecular
markers for this gene. In searching for potential sources of FHB resistance, we previously
had screened 290 accessions of TDIC from the USDA world collection, and we had identified several lines with useful levels of FHB resistance. Two of these accessions were used
to produce new sets of chromosome substitution lines in ‘Langdon’ durum following the
method used for the original TDIC chromosome substitution series. The purpose of the
present study was to determine which chromosomes held the resistance loci in these FHB
resistant TDIC accessions. Each substitution line was grown in replicated trials in the
greenhouse and inoculated at anthesis with Fusarium graminearum by the single spikelet
method. FHB response was determined visually 3.5 weeks after inoculation. LDN(DIC)
substitution lines representing five different chromosomes (1A, 3A, 5B, 7A, 7B) had significantly less FHB than the Langdon checks. The other LDN(DIC) lines showed intermediate
responses, not significantly different from the Langdon durum parent. The five TDIC chromosomes substituted in the lines with significantly reduced FHB are proposed as sites of FHB
resistance genes in these two accessions.
Germplasm Introduction and Enhancement
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2002 National Fusarium Head Blight Forum Proceedings
WILD EMMER, TRITICUM DICOCCOIDES, AS A SOURCE OF FHB
RESISTANCE FOR TETRAPLOID AND HEXAPLOID WHEATS
Robert W. Stack1* and James D. Miller2
Dept. of Plant Pathology, North Dakota State Univ. Fargo, ND; and
2
USDA-ARS Northern Crops Research Lab., Fargo, ND 58105
*Corresponding Author: PH (701) 231-7077; E-mail: rstack@ndsuext.nodak.edu
1
ABSTRACT
Wild emmer, Triticum turgidum L. var. dicoccoides (a.k.a ‘T. dicoccoides’) (TDIC), is wild
tetraploid wheat found throughout the Middle East. Because it shares the AB genome with
modern durum (T. turgidum L. var. durum), it readily crosses with it and also crosses with
hexaploid wheat - with some care as to choice of parent. TDIC has long been known as a
source of novel disease resistance including genes for resistance to stem rust, stripe rust,
leaf rust and powdery mildew, among others. Our research with TDIC as a source of FHB
resistance began as two separate lines of inquiry which have since come together. One
area of study was the evaluation of a set of disomic chromosome substitution lines developed by Leonard Joppa in the 1980’s to study a gene for high grain protein. In each of these
lines, one chromosome pair from TDIC replaces the corresponding pair in a durum background. We tested this set of substitution lines for FHB. The entire story of this aspect of the
work was recently reported in Crop Science (42:637-642). We found a major FHB resistance gene on 3A and a major gene on 2A that appears epistatic to FHB resistance. Somewhat less strong resistance was present on 1A and 6B. Another research group at NDSU
has identified molecular markers for the 3A QTL. Research on the 2A epistatic locus is
currently underway. Concurrently, we began screening the USDA world collection of TDIC
for FHB. Between 1995 and 1997 we tested 449 TDIC collections. Of these, 33 (7.3%)
showed levels of FHB substantially lower than durum check lines. About half have held up
as moderately to highly resistant upon repeated testing. In direct crosses between these
TDIC selections and durum, the FHB resistance appears in the offsprings but along with
many undesirable traits. From that point the two lines of research joined together. Two TDIC
accessions from among those confirmed as having FHB resistance were selected upon
which USDA cytogeneticist Leonard Joppa would base new sets of durum disomic substitution lines. An abstract elsewhere in this proceeding describes that process and the results.
From the new series of substitution lines those which showed FHB scores significantly
lower than the Langdon durum background parent were those with TDIC chromosomes 1A,
3A, 5B, 7A, and 7B; however none of these by itself is likely to confer adequate resistance to
FHB. In a diallele study on the original disomic lines, we found that the strong resistance
gene on 3A showed positive combining ability with those on 1A and 6B. In a field trial in
2002 we also confirmed that the FHB resistance on 3A will effectively reduce FHB in a
hexaploid wheat background. Several of the chromosomes in these substitution lines are
not among those previously recognized to bear FHB resistance genes in hexaploid wheat.
Germplasm Introduction and Enhancement
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2002 National Fusarium Head Blight Forum Proceedings
EFFICIENCY AND EFFICACY OF MARKER ASSISTED
SELECTION OVER PHENOTYPIC SELECTION FOR
FHB RESISTANCE IN DURUM WHEAT
B. Suresh*, E.M. Elias, J.L. González-Hernández, and S.F. Kianian
Dept. of Plant Sciences, North Dakota State University, Fargo, ND 58105
*Corresponding Author: PH: 701-231-8441; E-mail: suresh.bhamidimarri@ndsu.nodak.edu
ABSTRACT
We are studying the efficiency of Marker Assisted Selection (MAS) for Type II Fusarium
Head Blight (FHB) resistance in two durum wheat populations derived from a Chinese
bread wheat source ‘Sumai 3’. This study is based on the hypothesis that for a trait such as
FHB, the use of molecular markers for MAS would reduce the time involved in selection
along with a reduction in cost. The first population consisted of 1,814 F2:4 lines that were
developed from crossing a cultivar Ben to Sumai3/Sceptre//D88816 line. The second population consisted of 320 F2:5 that were derived from backcrossing cultivar Lebsock to the line
Lebsock//Sumai3/Lebsock. These two populations were screened for FHB resistance in the
greenhouse in spring 2002 by inoculating the heads with Fusarium graminearum and later
scoring the diseased heads. Screening for the resistance QTL located on the chromosome
3BS was done using the microsatellite locus Xgwm533. In the greenhouse evaluation,
1,124 lines in the first population and 180 lines from the second population were found
resistant with scores of less than 21%. Microsatellite marker identified the resistant QTL in
524 lines from population I and131 lines from population II. Apart from the lines that were
found to be resistant in the presence of marker and susceptible in its absence, some lines
had the marker but were susceptible and some did not have the marker and still were resistant to the disease. Lines representing these four groups will be evaluated in summer 2003
in a replicated scab nursery and the efficacy of both the selection methods will be calculated. In the present study the molecular data showed that using MAS the population size
could have been reduced from 1,814 lines in population I to 524 and 131 from 320 lines in
population II, thus saving a significant amount of greenhouse space, resources and time in
screening. We calculated the efficiency of each selection process so far and found that, with
MAS it took us 44 working days to screen the two populations with an approximate cost of
$1.43 per data point and with phenotypic selection in the greenhouse; it took 141 days with
an approximate cost of $0.99 per data point. In terms of time involved, MAS was found to be
3.2 times quicker saving 97 days. With the use of high throughput non-denaturing gel system, the efficiency of MAS in terms of time and labor will be higher at much reduced cost. In
our next step of study, we plan to advance the agronomically desirable lines by repeated
backcrossing to cultivars Ben and Lebsock. These lines will then be further analyzed for
their FHB resistant phenotype and agronomic performance.
Germplasm Introduction and Enhancement
219
2002 National Fusarium Head Blight Forum Proceedings
PUTATIVE SOURCES OF FUSARIUM HEAD BLIGHT RESISTANCE
IN SPRING WHEAT IDENTIFIED FROM THE USDA
SMALL GRAINS COLLECTION
X. Zhang and Y. Jin*
Plant Science Department, South Dakota State University, Brookings, SD 57007
*Corresponding Author: PH: (605) 688-5540; E-mail: Yue_Jin@sdstate.edu
INTRODUCTION
The use of host resistance will likely be one of the major components in managing
Fusarium head blight (FHB) of wheat. Germplasm improvement and varietal development
for FHB resistance will depend upon continued efforts in discovery and characterization of
diverse resistant sources. Since 1998, we have evaluated 4,400 accessions of spring wheat
from the USDA small grains collection. This report summarizes putative sources of resistance that underwent three consecutive years of field evaluations in replicated trials.
MATERIALS AND METHODS
Spring wheat germplasm from the USDA collection (Aberdeen, ID) were first evaluated in a
preliminary screening nursery (PSN). This is a non-replicated nursery with entries planted
into rows (ca. one meter in length). ND 2710 and BacUp were used as resistant checks and
Sonalika and Wheaton as susceptible checks with a check-to-entry ration of 1:28. The
nursery was inoculated with infected corn grain and conidial suspension. Details in nursery
management, inoculation, and data collection were as described previously (Zhang et al.
2000; 2001). Accessions or plants within an accession with a low FHB index
(incidence*severity) and/or low percentage of Fusarium damage kernels (FDK) were selected. Selections were further evaluated in subsequent years in elite germplasm nurseries
(EGN). Entries of EGN were planted in row-plots with three replicates and arranged into
split-plot design, with maturity as the main plot and genotype as the subplot. Maturity groups
were determined based on days between planting and flowering: early (<55), intermediate
(55-65), and late (>66).
RESULTS AND DISCUSSION
In each of the three evaluation years, high disease pressure was generated by artificial
inoculation and mist-irrigation. FHB indices on the susceptible checks (Wheaton and
Sonalika) consistently exceeded 80%.
Table 1 lists selections with low FHB indices (< 40%) and low FDK (< 40%). This group of
materials generally exhibited stable low FHB reaction over years. Selections with low FHB
indices (< 40%) but high FDK (>40%) or high FHB indices but low FDK (<40%) are given in
Table 2. The first group of materials from Table 2, namely Sin Chunaga, Norin 61 and several other lines originated primarily from Japan, consistently showed lower disease indices,
but high visual FDK ratings. Kernels rated as FDK in this group were mostly bleached, but
remained plump. A recent study on Fusarium infection of seed harvested from the 2002 field
Germplasm Introduction and Enhancement
220
2002 National Fusarium Head Blight Forum Proceedings
FHB screening nursery suggested that discoloration (bleaching) of plump kernels might not
be due to fungal infection (Zhang and Jin, unpublished). Although FHB indices of second
group in Table 2 were high, lines in this group generally had low FDK scores and might
contribute useful resistance/tolerance genes in breeding.
REFERENCES
Zhang, X., Y. Jin, R. Rudd, T. Hall, J. Rudd, and H. >\Bockelman. 2000. Fusarium head blight resistant sources
of spring wheat identified from the USDA collection. Pages 228-233 In: 2000 National Fusarium Head Blight
Forum, The U.S. Wheat and Barley Scab Initiative. Dec. 10-12, 2000. Erlanger, KY.
Zhang, X., Y. Jin, J. Rudd, and H. Bockelman. 2001. Evaluation of USDA Spring Wheat Germplasm for Fusarium
Head Blight Resistance. Pages 220-224 In: 2001 National Fusarium Head Blight Forum Proceedings. Dec. 8-10,
2001, Erlanger, KY.
Table 1. Spring wheat germplasm selections with low Fusarium head blight indices and low
percentage of damaged kernels.
Accession
PI 382161
PI 382154
PI 382153
PI 182568
PI 462151
Citr 12002
PI 345731
PI 519790
PI 434987
CItr 5103
PI 81791
PI 596533
PI 192660
PI 185380
PI 285933
PI 382167
PI 351256
CItr 12021
PI 163429
PI 351221
PI 382144
PI 294975
Citr 13136
PI 264927
PI 104131
Citr 17427
PI 83729
PI 469271
ID
Tokai 66
Nyu Bai
Nobeoka Bozu
ND 2710 (CK)
Sumai 3 (CK)
Norin 43
Shu Chou W. 3
Renacimiento
Tezanos P.P.
274-1-118
Estazuela Young
274
Sapporo H.K.J.
BacUp (CK)
Prodigio Italiano
Prodigio Italiano
Chudoskaja
16-52-9
Japon 2
Centenario
PI 163429
Newthatch Sel.
Encruzilhad
Artemowska
Rio Negro
220
Excelsior
16-52-2
Magyagovar 81
Wheaton (CK)
2000
10.5
13.7
17.0
14.2
15.0
21.5
18.5
25.5
20.2
19.8
22.2
14.5
24.4
35.0
39.0
35.7
36.2
10.9
21.2
32.2
27.0
34.0
29.6
24.5
42.8
31.9
36.3
34.5
51.0
87.6
FHB index (%)
2001
2002
mean
5.9
12.8
9.7
9.4
11.9
11.7
9.8
12.2
13.0
10.2
14.7
13.0
17.0
15.8
15.9
16.1
28.0
21.9
29.8
18.9
22.4
21.2
22.2
23.0
19.3
30.0
23.2
28.9
24.8
24.5
23.4
31.2
25.6
27.2
35.1
25.6
40.0
15.9
26.8
16.5
30.2
27.2
20.2
24.1
27.8
25.0
26.2
28.9
30.5
22.5
29.7
28.5
49.8
29.7
36.2
38.3
31.9
31.8
34.0
32.6
31.8
40.2
33.0
35.2
30.0
33.1
37.3
35.6
34.2
67.0
16.8
36.1
35.2
32.5
36.8
48.3
30.5
36.9
34.2
44.5
38.3
43.5
39.9
39.3
49.8
18.2
39.7
88.8
83.5
86.6
2000
16.0
15.0
13.8
22.8
28.3
46.7
18.8
41.7
20.0
40.0
58.0
19.0
21.7
31.9
22.5
27.5
26.7
23.3
41.7
41.7
30.0
20.0
45.0
20.0
50.0
16.7
21.7
33.3
46.0
93.3
FDK (%)
2001
12.0
15.3
15.7
19.5
25.0
30.0
20.0
36.7
23.0
35.3
15.5
23.3
17.7
17.0
18.7
16.0
21.7
18.0
18.7
25.0
28.7
20.0
33.3
20.0
23.7
20.7
14.0
24.0
22.7
83.7
mean
14.0
15.2
14.7
21.2
26.7
38.3
19.4
39.2
21.5
37.7
36.8
21.2
19.7
24.5
20.6
21.8
24.2
20.7
30.2
33.3
29.3
20.0
39.2
20.0
36.8
18.7
17.8
28.7
34.3
88.5
Germplasm Introduction and Enhancement
221
2002 National Fusarium Head Blight Forum Proceedings
Table 2. Spring wheat germplasm selections with low Fusarium head blight indices and
high percentage of damaged kernels or vice versa.
A ccession
P I 5 9 6 5 33
P I 4 6 9 2 71
P I 4 7 8 2 82
P I 3 8 2 1 40
P I 1 8 2 5 61
P I 1 8 2 5 86
P I 1 9 7 1 28
P I 1 8 2 5 83
P I 4 1 1 1 32
P I 3 5 1 8 16
P I 1 8 2 5 91
P I 1 9 2 6 34
P I 3 5 1 7 43
P I 1 8 5 8 43
P I 3 6 2 4 37
P I 2 6 4 9 98
P I 2 6 4 9 40
P I 1 6 8 7 27
C itr 2 49 2
P I 3 4 4 4 67
P I 2 5 6 9 58
P I 1 6 3 4 39
P I 1 3 2 8 56
P I 3 5 1 9 93
P I 1 6 8 7 16
P I 3 4 9 5 34
P I 3 5 1 4 76
P I 1 8 4 5 12
P I 3 4 4 4 65
P I 1 9 2 2 19
C Itr 1 1 21 5
P I 3 4 4 4 54
P I 3 5 1 1 87
P I 1 1 3 9 49
P I 5 1 9 7 98
P I 2 2 5 1 60
P I 5 8 4 9 34
P I 3 6 2 0 43
P I 3 5 2 0 00
P I 1 9 2 2 29
P I 1 1 3 9 48
ID
N D 2710 (C K )
S u m ai 3 (C K )
B acU p (C K )
W h eaton (C K )
S on alik a (C K )
A b ura
S in C h u n aga
N o rin 43
S h in chu n aga
C h u ko
G o gatsu-K o m u gi
F ro m en t D u Jap on
N o rin 61
T rintecin co
C L U J 4 9-9 2 6
S u rp resa
III/1 4 -B
628
111a
B ah iense
M an chu rian
O n cativo In ta
A cad em ia 4 8
P I 1 6 3 4 39
M en tan a
Z .8 8 .5 4
K lein C on d o r
533B
V aulion
H 51
L au rean o A lv. L .
H atvan i
B elgrad e 4
B u ck A u stral
T aillens V elu S el.
S tep njach ka
P F 7 9 7 82
M en tan a
W hestp h alen
A rn au t d e T oam .
Z .8 9 .3 7
G ran C o m . U n g.
K o operatorka
2000
1 4 .2
1 5 .0
3 5 .0
8 7 .6
8 7 .1
1 5 .1
2 2 .7
3 0 .0
1 7 .0
1 9 .1
2 8 .3
3 2 .0
3 7 .0
5 3 .7
4 0 .2
5 7 .7
3 5 .5
4 3 .7
5 5 .3
3 6 .8
5 7 .0
4 8 .0
4 2 .4
5 9 .0
4 8 .3
5 4 .2
5 4 .8
5 4 .3
5 5 .8
7 6 .7
4 8 .3
4 8 .8
3 9 .6
6 4 .5
4 6 .5
6 3 .0
3 5 .9
3 9 .0
6 2 .5
5 9 .6
5 2 .2
5 7 .7
7 7 .7
FH B
2001
1 0 .2
1 7 .0
1 6 .5
8 8 .8
8 4 .3
1 7 .7
2 2 .0
2 0 .7
3 6 .7
3 9 .8
2 4 .2
2 9 .0
2 9 .2
2 3 .1
6 5 .0
3 9 .7
5 2 .3
4 7 .8
4 7 .7
2 8 .2
2 3 .0
4 5 .3
5 9 .0
3 8 .6
4 1 .8
5 7 .2
6 4 .7
6 7 .3
7 5 .3
3 7 .7
6 2 .5
7 9 .3
8 4 .3
8 1 .5
7 9 .5
7 0 .8
6 7 .0
6 8 .0
6 9 .3
6 1 .3
8 2 .2
7 7 .8
8 8 .3
Germplasm Introduction and Enhancement
222
in dex (%
2002
1 4 .7
1 5 .8
3 0 .2
8 3 .5
8 7 .8
1 7 .5
2 2 .0
2 6 .7
2 7 .8
2 3 .2
3 4 .2
3 3 .0
3 1 .0
4 9 .2
2 1 .5
3 0 .2
4 3 .0
4 0 .5
3 0 .0
6 8 .5
5 6 .0
4 8 .5
4 6 .2
5 1 .0
6 0 .3
3 9 .5
3 2 .2
3 4 .0
3 5 .4
5 2 .5
6 1 .2
4 4 .4
5 4 .3
3 4 .6
5 8 .1
5 0 .7
8 2 .5
8 1 .2
5 8 .0
7 9 .5
6 8 .5
7 4 .5
6 1 .7
)
m ean
13.0
15.9
27.2
86.6
86.4
16.8
22.2
25.8
27.2
27.3
28.9
31.3
32.4
42.0
42.2
42.5
43.6
44.0
44.3
44.5
45.3
47.3
49.2
49.5
50.1
50.3
50.6
51.9
55.5
55.6
57.3
57.5
59.4
60.2
61.4
61.5
61.8
62.7
63.3
66.8
67.6
70.0
75.9
2000
2 2 .8
2 8 .3
3 1 .9
9 3 .3
7 6 .4
3 8 .3
8 6 .7
5 0 .0
8 0 .0
7 8 .8
7 7 .5
7 0 .0
6 6 .7
4 1 .7
2 6 .0
4 2 .5
3 3 .3
3 0 .0
4 1 .7
2 5 .0
2 5 .0
3 8 .8
2 0 .0
4 0 .0
3 6 .7
3 0 .0
3 5 .0
2 6 .7
2 5 .0
3 3 .3
3 6 .7
3 6 .7
3 5 .0
2 8 .8
2 6 .7
3 8 .3
2 7 .5
3 0 .0
4 1 .3
2 3 .3
2 6 .7
3 1 .7
2 6 .3
F D K (% )
2 0 0 1 m ean
1 9 .5
21.2
2 5 .0
26.7
1 7 .0
24.5
8 3 .7
88.5
8 4 .0
80.2
4 7 .3
42.8
7 6 .7
81.7
5 6 .7
53.3
7 6 .7
78.3
7 5 .0
76.9
6 6 .7
72.1
3 3 .3
51.7
4 1 .0
53.8
3 4 .7
38.2
3 0 .0
28.0
2 3 .3
32.9
2 2 .3
27.8
2 6 .7
28.3
1 6 .0
28.8
1 9 .7
22.3
2 3 .5
24.3
3 1 .7
35.2
2 6 .7
23.3
2 7 .7
33.8
2 9 .7
33.2
2 5 .3
27.7
3 1 .0
33.0
2 0 .0
23.3
4 0 .0
32.5
1 9 .0
26.2
3 0 .0
33.3
2 5 .3
31.0
2 9 .0
32.0
3 0 .0
29.4
3 4 .0
30.3
2 4 .7
31.5
2 4 .0
25.8
2 7 .5
28.8
3 1 .7
36.5
3 3 .3
28.3
4 3 .3
35.0
4 0 .0
35.8
4 3 .3
34.8
2002 National Fusarium Head Blight Forum Proceedings
THE DEVELOPMENT OF SCAB (FUSARIUM GRAMINEARUM)
RESISTANT VARIETIES OF WHEAT
P.S. Baenziger1*, Schimelfenig, J.2 and J.E. Watkins2
Department of Agronomy and Horticulture and 2Department of Plant Pathology,
University of Nebraska at Lincoln, Lincoln, NE, 68503-0915
*Corresponding Author: PH: (402) 472-1538; E-mail: agro104@unlnotes.unl
1
OBJECTIVE
The primary objective was to identify and develop elite winter wheat varieties that are tolerant to Fusarium head blight (FHB, scab). The second objective was to field screen the elite
hard winter wheat lines including those in the Regional Germplasm Observation Nursery
(RGON).
INTRODUCTION
Nebraska is second only to California for irrigated crop production. Hence FHB, though a
periodic disease, can be an important disease greatly affecting approximately 35% of
Nebraska’s wheat acreage. As humans consume virtually all of this wheat and over one
half is exported, safe, healthy grain is critical for maintaining the reputation of hard winter
wheat in the domestic and export markets. All winter wheat lines to be released by the
University of Nebraska shall be screened for FHB resistance. This information will be
shared with producers.
The primary objective is to identify and develop elite winter wheat varieties that are tolerant
to Fusarium head blight (FHB, scab), using conventional breeding methods. The second
objective was to screen elite hard winter wheat lines in the Regional Germplasm Observation Nursery (RGON).
MATERIALS AND METHODS
Sources of FHB resistant germplasm originating from our biotechnology efforts, spring and
soft wheat germplasm, and exotic germplasm, were collected for crossing into our elite lines.
F2 and F3 seed produced from these crosses was screened for FHB resistance, in the field
in 2002.
All solutions of inoculum used in the greenhouse and field, were created by combining 6
isolates of Giberella zeae and 5 isolates of Fusarium graminearum to create a 70000
conidia/mL solution. We screened the germplasm for FHB tolerance, in the greenhouse to
allow for better parent identification. Nine replicates of each line were screened in the
greenhouse using a randomized complete block design. One spikelet per head was injected with 0.1 mL of a 70000 conidia/mL solution. The plants were then misted for 72 hrs at
98% humidity. Concerns about induced resistance in response to injury, led us to adjust this
method. In later studies, the replication number was increased from nine to twelve and 2 mL
of 70000 conidia/mL, was sprayed onto the entire head and sealed it in a 16 x 9.5 cm2 snack
Variety Development and Uniform Nurseries
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2002 National Fusarium Head Blight Forum Proceedings
size Ziploc bag for 72 hrs. This procedure avoids false negatives, due to potential induced
resistance from awns being cut or injection.
In the field, twenty six transgenics and eight hundred winter wheat breeding lines, which
included the RGON nursery were planted and screened, against appropriate controls, for
tolerance to FHB, using a system similar to that of Campbell and Lipps (1998). Each variety
was planted in a 10 ft2 plot. Inoculation was carried out in two ways; naturally occurring F.
graminearum infected corn stalks were spread in the field in fall; and 70000 conidia/mL of
inoculum, was sprayed 4 times, at a rate of 50 mL per plot, using a CO2 powered back pack
sprayer, in 2002. This was followed by mist irrigation, using a modified misting system
similar to that employed by Zhang et al. (1999) to mist the plots for 2 minutes at 30 min
intervals. This began 1 week before the plots were inoculated and continued until the first
readings were taken. Bordering the scab nursery with forage triticale provided an excellent
buffer and greatly reduced wind in the misting nursery.
FHB was rated by counting the number of infected spikelets on 30 individual heads (Shaner
and Buechley, 2001). Plot severity or FHB index was calculated by the averaging the 30
FHB ratings. Intensity was calculated by taking a count of the infected heads and dividing it
by 30 the total # of heads scored. The grain, from one of our three most advanced nurseries
was analyzed for Deoxynivalenol (DON) by the Veterinary Diagnostic Service at North
Dakota State University.
RESULTS AND DISCUSSION
Of the eight hundred lines that were screened in 2002, sixty have had extensive FHB
screening in at least 3 mutually exclusive trials, including an independent determination
using different isolates, by South Dakota State University. Fig 1, shows nineteen lines that
have consistently shown significant FHB resistance relative to a FHB susceptible variety
“Wahoo”. These lines will be screened again in the field in a replicated trial in 2003. Of the
RGON lines, 42% show promise and will be screened in a replicated trail in 2003. The
grain, from one of our three most advanced nurseries showed no correlation between levels
of DON and the # of FHB infected florets per head (Fig 2).
CONCLUSION
FHB tolerance in winter wheat breeding nurseries was generally high. F2 and F3 seed
produced from the FHB crosses was screened in the field in 2002. Additional seed will be
produced from the new crosses that have been made for future planting in the field. As soon
as we have recovered bulks with the level of agronomic performance required to survive our
winters, are resistant to stem rust (P. graminis Pers. : Pers. f. sp. tritici Eriks & E. Henn), and
yield well, head row selection for elite line identification, will begin.
In the 2002-2003 cycle, the most FHB tolerant transgenics will be crossed to varieties
having some FHB tolerance, and to Wesley, a very widely grown, but FHB susceptible line.
We will screen 420 lines from our elite germplasm (our three most advanced nurseries), 46
lines from the FHB screening nursery, 20 - 50 transgenic spring wheat lines (initially) from
our biotechnology efforts, and 277 lines coming from the RGON in the field.
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2002 National Fusarium Head Blight Forum Proceedings
ACKNOWLEDGEMENTS
Funding for this research was provided by the Scab Initiative. We would like to thank J.
Stack, A. Sparks and S. Ali for their G. zeae and F. graminearum samples. G Shaner, J. Yue,
and X. Zhang for their advice about inoculation procedures And Dr. Boosalis for his assistance making field inoculum.
REFERENCES
Campbell, K. A. G., and P. E. Lipps. 1998. Allocation of resources: sources of variation in Fusarium head
blight screening nurseries. Phytopathology 88:1078-1086.
Shaner, G., and G. Buechley. 2001. Estimation of type II resistance – a dilemma in need of a solution. 2001
National Fusarium Head Blight Forum Proceedings. Pp:156-160.
Zhang, X., Y. Jin, R. Rudd, and H. Bockelman. 1999. Screening of spring wheat scab resistance from the
USDA germplasm collection. 2001 National Fusarium Head Blight Forum Proceedings. Pp:140-142.
Variety Development and Uniform Nurseries
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Variety Development and Uniform Nurseries
226
Fig 1. FHB Severity v/s Wheat Lines, avaraged across at Least 3 Indipendent Trials conducted by the
University of Nebraska at Lincoln and varified by South Dakota State University,
4
1
3
9
9
4
1
7
8
5
8
6
2
4
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A
Y
S
R
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69
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AR
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IO
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N
C
Lines of FHB Resistant Wheat and Wahoo a FHB Susceptible Line.
27
37
47
57
67
77
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2002 National Fusarium Head Blight Forum Proceedings
FHB Head Severity (% LSMeans)
Variety Development and Uniform Nurseries
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# infected florets or DON (ppm)
0
2
4
6
8
10
N97V121
NE00403
NE97465
Wheat Lines
Fig 2. DON and the # FHB Infected Florets per Head for
Wheat Lines in the NIN Field Nursery in 2003
NE98466
WESLEY
NUPLAINS
NE00687
NE00698
NE98692
NE00439
NE00469
NE00611
NI98414
CHEYENNE
NE99554
NE97638
NE99469
NIOBRARA
DON ppm
# infctd flrts/Head
CULVER
SCOUT66
WAHOO
2002 National Fusarium Head Blight Forum Proceedings
2002 National Fusarium Head Blight Forum Proceedings
NUMBER OF LOCATION-YEARS NEEDED TO DETERMINE
THE REACTION OF WINTER WHEAT CULTIVARS
TO FUSARIUM HEAD BLIGHT
William W. Bockus*, Mark A. Davis, and Karen A. Garrett
Department of Plant Pathology, Throckmorton Hall, Kansas State University, Manhattan, KS
*Corresponding Author: PH: (785) 532-1378; E-mail: bockus@plantpath.ksu.edu
ABSTRACT
Fusarium head blight (FHB) is a serious disease of wheat and barley that is best controlled by host
resistance. In KSU Extension publications, the reaction to FHB for winter wheat cultivars is reported to producers using a 1-9 scale where 1-3 = resistant, 4-6 = intermediate, and 7-9 = susceptible. This research sought to determine how many location-years are needed to accurately determine the reaction of a cultivar to FHB. Twenty-nine different winter wheat cultivars were screened 212 times in 12 field nurseries over a 3-yr period (n=133). Experimental design for each location-year
was a randomized complete block with four replications. Corn grains, colonized by Fusarium
graminearum and spread on the soil surface, followed by sprinkler irrigation were used to produce
the epidemic. FHB index (% diseased spikelets) was determined for each cultivar between four and
six times for each experiment and averaged. To compare data across location-years, linear regression was used to fit a 1-to-9-scale model to the data for each location-year. To produce the model, an
index value of zero was assigned a scale value of 1 and the highest index value in a location-year
was assigned a scale value of 9. The model was then used to calculate scale values for all other
cultivars in that location-year. A mean scale value was calculated for each cultivar (n=2-12 locationyears) and an overall standard deviation for all cultivar-location-years (n=133) was calculated using
the departure from the mean for each scale value for each cultivar-location-year. To estimate the
number of location-years needed to determine the reaction of a cultivar to FHB, the formula x-bar +/t(alpha/2) * s / sqroot(n) was used to calculate 95% confidence intervals. In this formula, x-bar is the
observed mean scale value (1-9 scale) of a cultivar, t(alpha/2) is the t-value corresponding to the
desired alpha level (0.05) divided by 2, s is the standard deviation among location years, and n is the
sample size (number of location years). If an observed mean scale value is within +/- 0.5 units of the
correct value, it will be rounded to the correct scale value. Required standard deviations to produce
a mean within +/- 0.5 units were calculated for samples of n=2-20 (not shown). If an observed mean
scale value is within +/- 1.5 units, it will be rounded to a scale value that is +/- 1 unit from the correct
scale value. To achieve a mean scale value within +/- 1.5 units, required standard deviations for
samples of n=2, 3, 4, and 5 are 0.167, 0.604, 0.943 and 1.208, respectively. In our data, the overall
standard deviation for departure from the mean for all cultivar-location-years (n=133) was 1.05.
Based upon the overall standard deviation, 20 location-years would be needed to have a mean scale
value that would have a 95% chance of being within +/- 0.5 units of the correct mean and, therefore,
rounded to the correct scale value. Based upon the overall standard deviation (1.05), 5 locationyears would be needed to have a mean scale value that would have at least a 95% chance of being
within +/- 1.5 units and, therefore, rounded to +/- 1 unit of the correct scale value. For most purposes, +/- 1 unit is sufficient accuracy for producers; therefore, we recommend that reactions of
winter wheat cultivars that are reported to producers be based upon data from at least five location
years.
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IDENTIFICATION OF DNA MARKERS FOR FUSARIUM HEAD
BLIGHT RESISTANCE OF WHEAT LINE HUAPEI 57-2
William Bourdoncle and Herbert W. Ohm*
Department of Agronomy, Purdue University, West Lafayette IN 47906
*Corresponding Author: PH: (765) 494-8072; E-mail: hohm@purdue.edu
ABSTRACT
Fusarium head blight or scab greatly affects grain yield and quality of wheat (Triticum
aestivum L.). Because it is a trait of low heritability and costly to evaluate, marker assisted
selection is particularly attractive for breeding programs. To identify DNA markers for
Fusarium head blight resistance, a population of 163 recombinant inbred lines was developed by single seed descent from the cross between the resistant line ‘Huapei 57-2’ and the
moderately susceptible cultivar ‘Patterson’. All lines including parents were evaluated in one
field experiment and two greenhouse tests for resistance to spread of disease (Type II
resistance). Based on phenotypic data, extreme lines were selected to initiate bulked segregant analysis using microsatellites. Markers suggesting association with a putative quantitative trait locus (QTL) were then tested on the entire population to confirm the linkage. A
major QTL was identified on the chromosome 3BS in a region well known from previous
studies. Additional QTLs were also found on chromosomes 3A, 3BL and 5B.
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COORDINATED FUSARIUM HEAD BLIGHT SCREENING NURSERY
FOR WHEAT BREEDING PROGRAMS IN WESTERN CANADA
A.L. Brûlé-Babel1*, W.G.D. Fernando1, P. Hucl2, G. Hughes2, S. Fox2, R. DePauw3,
M. Fernandez3, J. Clarke3, R. Knox3, J. Gilbert4, G. Humphreys4 and D. Brown4
Department of Plant Science, University of Manitoba, Winnipeg, Manitoba; 2Crop Development Centre,
University of Saskatchewan, Saskatoon, Saskatchewan; 3Semiarid Prairie Agricultural Research Centre,
Agriculture and Agri-Food Canada, Swift Current, Saskatchewan; and
4
Cereal Research Centre, Agriculture and Agri-Food Canada, Winnipeg, Manitoba
*Corresponding Author: PH: (204) 474-6062; E-mail: ababel@ms.umanitoba.ca
1
ABSTRACT
Fusarium head blight (FHB) continues to be a serious disease of wheat in western Canada and in
particular, the eastern prairies. Screening for FHB resistance has been difficult for breeders working
outside of the eastern prairie region. As a result, breeders and pathologists entered into a collaborative agreement to establish a common FHB screening nursery at Carman, Manitoba. This region is
known to provide an environment that is more conducive to FHB development. The nursery was
established in 2001, and lines were evaluated in 2001 and 2002. Advanced lines that are in the final
stages of testing for cultivar registration were evaluated in replicated rows, while earlier generation
breeding lines were evaluated in non-replicated rows. In 2001 approximately 6000 1 m row plots
were grown in the nursery. In 2002 the number of plots was increased to approximately 9900. Five
checks were placed every 50 plots within the nursery. In 2001 the checks were AC Morse, AC
Vista, CDC Teal, FHB 37 and Glenlea. The same checks were used in 2002 with the exception that
CDC Teal was replaced by AC Cora to provide a check with an intermediate FHB reaction. In 2001
FHB infected corn inoculum was applied to plots two to three weeks prior to anthesis. Date of
heading and anthesis were recorded for each plot. A macroconidial suspension of F. graminearum
was applied to plots at 50% anthesis and again three to four days later. After each macroconidial
inoculation, plots were irrigated with a mist irrigation system to maintain high humidity. Eighteen to
21 days after inoculation each plot was visually rated for incidence (% of spikes infected) and
severity (% area of spike infected) of FHB infection and an FHB index was calculated. In 2002 corn
inoculum was not applied to the plots but all other inoculation and evaluation protocols were similar
to 2001. In 2001 conditions in the nursery were highly conducive to FHB development. The mean
FHB index on susceptible checks ranged from 28 to 41. The resistant check had an FHB index of 5.
This provided a good distinction between susceptible and resistant lines. In 2002, weather conditions were drier and cooler than in 2001 and FHB levels were lower in the nursery, overall. The
mean FHB index for the susceptible checks ranged from 11 to 21. The intermediate check produced a mean FHB index of 6, while the mean FHB index of the resistant check was 0.3. Therefore, there were still clear distinctions between resistant and susceptible lines. However, disease
levels were higher in groups of lines that were inoculated earlier in the season, when conditions
were warmer and more humid, than those inoculated later in the season. The variability noted in
the nursery indicated that there may be more escapes in the 2002 nursery and that lines with intermediate reactions may be difficult to separate from resistant lines. This emphasizes the need for
multi-year testing to fully characterize FHB reaction. In general this nursery is providing useful
information to plant breeders and will facilitate development of FHB resistant cultivars.
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TIMING OF INOCULATIONS OF DRYLAND WHEAT PLOTS AND THE
EFFECT ON FUSARIUM HEAD BLIGHT (FHB) SEVERITY
AND MYCOTOXINACCUMULATION DUE TO
FUSARIUM GRAMINEARUM INFECTION
C. K. Evans* and R. Dill-Macky
Department of Plant Pathology, University of Minnesota, St. Paul, MN 55108
*Corresponding Author: PH: (612) 625-4271, E-mail: evans035@umn.edu
ABSTRACT
Eight wheat (Triticum aestivum) genotypes were assessed for reaction to FHB in a dryland
inoculation study. The test was a randomized complete split-split block design. Main-plots
were timings of inoculation (TOI), sprayed at anthesis (SAA), 3 days post-anthesis (DPA),
and 6 DPA. Sub-plots consisted of two spray-inoculation treatments, inoculated once or
twice, and sub-sub plots were the eight wheat genotypes. Significant differences were
found among TOI treatments for FHB severity (7.1 % SAA, 7.7 % sprayed 3 DPA, and 0.6 %
sprayed 6 DPA with l.s.d.0.05 = 2.42) and deoxynivalenol (DON) accumulation (3.0 ppm SAA
and 3 DPA, and 0.3 ppm sprayed 6 DPA with l.s.d.0.05 = 0.60). Average DON ppm accumulations in grain of wheat genotypes were 0.2 for BacUp, 0.2 for ND2710, 0.2 for Ingot, 0.2 for
Forge, 0.8 for Oxen, 1.4 for Parshall, 5.3 for Norm, and 8.6 for Wheaton (l.s.d.0.05 = 1.07). Our
data demonstrate that dryland inoculations of wheat can be useful for screening germplasm
for reaction to FHB.
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VARIETY DEVELOPMENT AND UNIFORM NURSERIES:
FHB RESISTANCE IN BARLEY
J.D. Franckowiak
Department of Plant Sciences, North Dakota State University, Fargo, ND 58105
Corresponding Author: PH: (701) 231-7540; E-mail: j.franckowiak@ndsu.nodak.edu
ABSTRACT
Acknowledgements: This report is an overview of research progress made on FHB resistance by researchers of cooperating barley improvement programs in the upper Midwest.
Development of barley (Hordeum vulgare) cultivars having resistance to Fusarium head
blight (FHB), incited primarily by Fusarium graminearum, is the main goal of barley improvement programs in the upper Midwest. Unlike many other disease problems, strategies for
control of FHB did not exist prior to 1993 when epidemics began to occur annually. Initial
goals included: 1) identification of accessions resistant to FHB, 2) establishment of screening procedures, 3) determination of inheritance patterns for FHB resistance, and 4) development of more focused breeding schemes. Nearly ten years later, what have we accomplished? Or more important, have we developed strategies for control of FHB?
FHB screening nurseries were established in North Dakota (ND) and Minnesota using
prepared inoculum and mist irrigation systems to enhance disease development. Cooperative nurseries were developed in Eastern China where natural inoculum and favorable
weather often cause high levels of FHB. Greenhouse tests using various inoculation procedures were conducted. Laboratory testing of cultivars and breeding lines for deoxynivalenol
(DON) content helped determine the effectiveness of genetic and cultural controls of FHB.
We have learned that the FHB problem on barley in the upper Midwest has both regional
and international aspects. Midwest spring barley cultivars, both two- and six-rowed, have a
unique genetic system for control of maturity and plant height. FHB is a problem in barley
growing areas where these adaptation genes are used: the Canadian Prairies, Central
Mexico, and Uruguay. Thus, the cooperative regional FHB nursery (MinnDak) was expanded in 2002 to include a Canadian cooperator and a test site at Brandon, Manitoba, and
renamed the North American Barley Scab Evaluation Nursery (NABSEN). Agreements to
have the ICARDA/CIMMYT barley program in Mexico as a full participant in the NABSEN
nursery for 2003 season are in place. Contacts have been made regarding a participant in
Uruguay. Data from the MinnDak and NABSEN nurseries suggest that progress in development of FHB resistant cultivars has been slow. Differential heading dates or photoperiod
responses across sites contribute to the variable data obtained on FHB incidence and DON
values. Expanded cooperation on FHB testing should improve our understanding of these
problems.
Barley accessions from southern Germany and eastern China were identified as having the
high levels of FHB resistance. Some resistance is also present in current Midwest barley
cultivars. Cultivars from Brazil (PFC88209) and Mexico (Atahualpa) are being used as
sources of FHB resistance. Evaluations of mapping populations have found QTL for FHB
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resistance on all seven barley chromosomes. The largest ones were consistently located
on chromosome 2H near loci that control spike type (vrs1), spike length (lin1), plant height
(hcm1), and heading date (Eam6) in Midwest barley cultivars. Most FHB resistant accessions differ from Midwest cultivars in the alleles present at these four loci. Since at least
three QTL for FHB resistance on chromosome 2H may be involved, a major breeding problem exists. This linkage group help explain why many FHB resistant selections have tworowed spikes and are tall and late. A further complication is the lack of recurrent parents
having good resistance to leaf spot diseases incited by Cochliobolus sativus, Pyrenophora
teres, and Septoria passerinii.
Utilization of the two-rowed cultivar Conlon as a malting barley has provided barley growers
in ND with some relief from FHB epidemics. Conlon often has slightly lower FHB readings
and significantly lower DON values than other malting barleys recommended in ND. Yet,
much higher levels of FHB resistance are needed to keep malting barley as major crop in
the upper Midwest. Progress is being made in developing breeding lines with more FHB
resistance, accessions as Shenmai 3 with more resistance to FHB are being identified
among early-heading two-rowed lines from eastern China, alternative genes for control of
plant height and maturity are being investigated, alternative breeding strategies are being
evaluated, and results from marker assisted selection experiments are positive. None of
these studies, however, offers a quick, easy solution to the FHB problem in barley.
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A FUSARIUM RESISTANCE GENE AND AN AWN PROMOTOR ARE
ASSOCIATED ON CHROMOSOME 5A OF SPRING WHEAT
Richard C. Frohberg1, Robert W. Stack2* and S.S. Maan1
1
Dept. of Plant Sciences and 2Dept. of Plant Pathology, North Dakota State University, Fargo, ND 58105
*Corresponding Author: PH: (701) 231-7077; E-mail: rstack@ndsuext.nodak.edu
ABSTRACT
Fusarium head blight (FHB) has been a serious concern in the spring wheat region of the
northern Great Plains for nearly a decade. Most spring wheat cultivar are moderately to
highly susceptible to FHB, and no lines are completely immune to infection. Farmers in the
region continue to grow susceptible cultivars, in part because most of the lines released as
having resistance to FHB have other more severe defects. One North Dakota line,
‘ND2710’, derived from the Chinese wheat Sumai 3, has shown high resistance to FHB in
many environments. Our objective was to determine the chromosome(s) where the FHB
resistance gene(s) in ND2710 reside using the set of 21 monosomic lines developed by
S.S. Maan in the 1960’s and based on the hard red spring wheat cultivar ‘Chris’. For the
present investigation, the entire set of 21 Chris monosomic plants as female were crossed
to ND2710. The monosomic F1’s were identified and 20 or more monosomic F1-derived F2’s were grown and advanced to F2:5 by single seed descent. Seed from the F5 lines was
planted in a field FHB testing nursery in 1999. Plots of the parents and a non-monosomic
Chris X ND2710 check population also were present. In this FHB nursery, disease was
produced by inoculation with Gibberella zeae and regular mist irrigation. At 3.5 weeks post
anthesis, spikes were cut and frozen for later scoring. Individual spikes were scored using
a 0 - 100% scale for FHB severity and plot means calculated. On average there were 24
spikes per plot and 45 plots per monosomic cross. In 2000, there were 15 spikes scored per
plot and 50 plots per monosomic cross. The control cross Chris (tip awned spikes) X
ND2710 (awned spikes) produced F1’s with all tip awned spikes, as did 19 of the 21 monosomic F1’s. In subsequent generations these displayed segregation for awns. Crosses of
monosomic 2A and monosomic 5A produced disomic and monosomic F1’s which had
awned spikes. Subsequent generations also had awned spikes. In 1999 and 2000, the
Chris/ND2710 check population had a mean FHB severity score of 40%, just midway between the scores of the parents Chris (62%) and ND2710 (22%). This is typical of FHB
scores in such resistant by susceptible crosses. Several of the monosomic crosses, however, had FHB severity scores significantly lower; in particular, ChrisM5A/ND2710 was
nearly as low as the resistant parent ND2710. When the co-occurrence of awn type and
FHB score was tested, they were found to be associated and not independent. We suggest
that a FHB resistance gene is associated with an awn promotor in chromosome 5A of
ND2710. The type of association between these two remains to be determined but we
propose that Chris mono 5A has a T 2A 4A translocation chromosome and ND 2710 has an
awn promoter on 5A. Chromosome banding is presently being used to verify the translocation. (This poster was presented at the 2001 ASA Annual Meeting, Charlotte NC Nov.
2001)
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A HISTORICAL ANALYSIS OF THE UNIFORM REGIONAL SCAB
NURSERY FOR SPRING WHEAT PARENTS
D.F. Garvin1* and J.A. Anderson2
USDA-ARS, Plant Science Research Unit, St. Paul, MN 55108; and
Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, MN 55108
*Corresponding Author: PH: 612-625-1975; E-mail: garvi007@umn.edu
1
2
OBJECTIVES
We sought to evaluate progress that has been made by spring wheat breeding programs
seeking to enrich their germplasm pool for resistance to Fusarium head blight. Fusarium
head blight resistance data of spring wheat germplasm representing several breeding
programs in the Upper Midwest and Canada is available through annual reports of the
Uniform Regional Scab Nursery for Spring Wheat Parents (URSN). We sought to use the
data in these reports to monitor progress in scab resistance enhancement within spring
wheat germplasm during the last seven years, during which time breeding programs in the
spring wheat region have focused intensively on achieving this goal.
INTRODUCTION
The severe epidemics of Fusarium head blight, or scab, in the 1990’s have caused economic losses that measured in the billions of dollars in the spring wheat region encompassing North Dakota, Minnesota, and South Dakota (McMullen, 1997; Nganje, 2001). As a
result of these epidemics, concerted efforts to develop scab-resistant wheat varieties were
accelerated in several regions of the U.S. and continue today. A component of these efforts
was the implementation of regional nurseries specifically devoted to assessing new wheat
germplasm emerging from breeding programs for scab resistance at multiple locations. For
the northern spring wheat region, Dr. Robert Busch established the Uniform Regional Scab
Nursery for Spring Wheat Parents (URSN) in 1995. It has since been conducted annually,
with support from the U.S. Wheat and Barley Scab Initiative.
The benefits of the URSN and similar nurseries for other market classes of wheat are twofold. First, these nurseries provide a vehicle for obtaining multi-site scab resistance data,
which is important given the large environmental effect on scab development and severity
(Groth et al., 1999). Second, these nurseries provide a means of germplasm exchange
among wheat breeding programs. An additional benefit of these nurseries is that they
provide a record of progress in enhancing scab resistance in wheat germplasm over time.
The goal of this paper is to provide a historical review of progress in developing scabresistant germplasm in the northern spring wheat region, based on several years of URSN
data.
MATERIALS AND METHODS
Data on scab resistance were obtained for the years 1996 through 2001. Although 1995
was the first year that of the URSN was run, consistent protocols for rating scab (disease
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2002 National Fusarium Head Blight Forum Proceedings
index = incidence x severity) and kernel damage (tombstones or vsk) started in 1996. Thus,
the analysis was restricted to data for the years 1996-2001. Entry lists and complete data for
each year of the URSN are available at gopher://greengenes.cit.cornell.edu:70/11/.Performance/.hrswregional/Uniform%20Regional%20Scab%20Nursery/. For purposes of analysis, BacUp and ND 2710 were designated as resistant checks because they were grown in
each of these years. For each of these years, the grand mean for disease index across
entries (excluding checks, durums, and plant introductions) was calculated. A standardized
disease index for each year was obtained by dividing each grand mean by the disease
index grand mean for BacUp and ND 2710 in the same year. This same procedure was
also completed for tombstone frequency.
RESULTS
Since 1995, the URSN has been grown at six different locations in Minnesota, North Dakota, South Dakota, as well as Canada (attempts to grow the nursery further south in Iowa
were attempted for a few years, but were not successful and thus were not continued). The
number of entries in each year has increased relatively steadily during this period, and is
now averaging over 40 per year. These entries include common wheat, durum wheat and
more recently, plant introductions. The entries have come from public breeding programs in
the abovementioned states and Canada, as well as from private breeding enterprises.
Several resistant and susceptible check varieties were included in each nursery. General
information on the URSN from 1996-2001 is summarize in Table 1.
Table 1. Summary statistics for the URSN common wheat entries, 1996-2001.
Disease
Disease
Number of
Number of
Index
Range
Year
Locations
Entries1
1996
6
31
38
12-73
1997
6
31
38
20-65
1998
6
25
40
21-62
1999
7
30
42
24-63
2000
7
26
25
11-39
2001
7
29
32
16-55
1
Excludes checks, durum entries and unimproved introductions.
Mean
Tombstone
24
29
25
28
21
27
Tombstone
Range
7-59
15-45
13-40
16-42
12-32
14-47
URSN entries were categorized by their resistance source(s) by examination of their pedigrees (Table 2). The number of “native” resistance sources (i.e. no identifiable scab resistance source in their pedigree) dropped from 17 of 31 entries in 1996 to only 5 of 25 entries
in 1998. This corresponded with an increase in the number of entries with parentage tracing
to Sumai 3 and its derivatives. In recent years, entries with South American and European
sources of scab resistance were entered in the nursery. Including composite crosses, the
number of entries with multiple resistance sources (excluding native sources) in their pedigree have been limited, averaging three per year. The majority of entries in 1995-1997
containing a defined resistance source were 1/4 to 1/2 by pedigree of that resistance source.
By comparison, the 2002 nursery contained only one entry that is 1/4 Sumai 3 by pedigree,
with all other entries 1/8 or less.
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2002 National Fusarium Head Blight Forum Proceedings
Table 2. Fusarium head blight Scab resistance source of entries in the URSN common
wheat entries, 1995-2002.
Resistance Source1
Year
No. Entries
“Native”
Sumai 3
Nyu Bay
S. Amer.
Europe
1995
28
16
11
1
0
0
1996
31
17
13
1
0
0
1997
31
10
17
0
4
0
1998
25
5
18
2
0
0
1999
30
9
18
3
0
0
2000
26
5
20
1
0
0
2001
29
4
21
2
2
3
2002
27
5
17
2
0
2
1
“Native” sources include those present in the germplasm prior to 1990 and not containing an
identifiable scab resistance source in its pedigree; Sumai 3 includes its derivatives, Ning 8331 and
Ning 7840.
Evaluating historical trends in scab resistance and related traits within the URSN over time
provides a means of assessing progress by spring wheat breeding programs attempting to
enrich for scab resistance in their germplasm. We assessed this progress by calculating
standardized disease indices and tombstone frequencies, relative to the resistant checks
BacUp and ND 2710. The results of this analysis suggest that the relative scab resistance
of the germplasm entries has increased substantially since 1996. In 1996, the disease
index of entries was approximately 216% of the mean of BacUp and ND 2710. However,
between 1997 and 1999, this decreased significantly, with relative disease levels dropping
to 128% of the resistant checks by 1999 (Table 3). This trend reversed somewhat in 2000
and 2001 however, with the relative disease index of entries rising to approximately 153% of
the resistant check means in 2001. The standardized tombstone frequencies of entries also
exhibited consistently lower values between 1997 and 2001, relative to 1996 (Table 3).
Table 3. Standardized scab disease indices and tombstone frequencies for URSN common
wheat entries, 1996-2001.
Standardized
Standardized
1
Year
Disease Index
Tombstone1
1996
216
228
1997
151
123
1998
129
138
1999
128
144
2000
149
138
2001
153
143
1
values are the % of the mean of BacUp and ND 2710.
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2002 National Fusarium Head Blight Forum Proceedings
DISCUSSION
The major benefit that the URSN provides to wheat breeders is that it permits germplasm to
be evaluated in multiple locations under different environmental conditions. Given the large
effect that the environment has on expression of scab resistance, evaluation of a genotype
in multiple environments ensures that useful data are obtained. However, the data obtained
by the URSN is also useful for assessing overall progress by several different breeding
programs each seeking to develop resistance to the same disease. The results of our
analysis suggest that the different breeding programs that, as a whole, contribute contributing entries to the URSN for evaluation, as a whole, have made substantial progress in
enhancing scab resistance in their germplasm over the last 6 years. The challenge that
remains is to determine whether the plateau of resistance that has been obtained to date,
principally by deploying Sumai 3-derived resistance, can be reduced further by incorporating new sources of resistance. Entries in the nursery the past few years also have improved
agronomic qualities and resistance to rust diseases as the proportion of the pedigree, by
resistance source, has dropped to less than 1/8 for the majority of entries. Incorporating
additional novel sources of resistance and combining it with the Sumai 3 resistance is
necessary to provide higher levels of resistance and genetic diversity.
REFERENCES
Groth, J.V., Ozmon, E.A., and Busch, R.H. 1999. Repeatability and relationship of incidence and severity
measures of scab of wheat caused by Fusarium graminearum in inoculated nurseries. Plant Dis. 83: 1033-1038.
McMullen, M.P., Jones, R., and Gallenberg, D. 1997. Scab of wheat and barley: a reemerging disease of
devastating impact. Plant Dis. 81: 1340-1348.
Nganje, W.E., Johnson, D.D., Wilson, W.W., Leistritz, F.L., Bangsund, D.A., and Tiapo, N.M. 2001. Economic
impacts of Fusarium head blight in wheat and barley: 1998-2000. Agribus. Appl. Econ. Rep. No. 464. North
Dakota State University. 49 pp.
Variety Development and Uniform Nurseries
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2002 National Fusarium Head Blight Forum Proceedings
GENES WITH MAJOR EFFECTS ON FHB RESISTANCE
PROMISE EASY MARKER APPLICATION
L. Gilchrist, M. van Ginkel*, R. Trethowan, and E. Hernandez
CIMMYT, APDO. POSTAL 6-641, Mexico, D.F., MEXICO 06600
*Corresponding Author: PH: 52-55-5804-2004; E-mail: m.van-ginkel@cgiar.org
OBJECTIVES
To determine the inheritance of resistance to FHB in three resistant bread wheat lines, and
describe the implication of this finding.
INTRODUCTION
Chromosomes and genes with major and minor effects - Many chromosomes have
been implied to carry genes conferring resistance to Fusarium head blight (FHB). However,
some contribute more to overall resistance than others. Likewise many genes have been
reported, but there is evidence that gene effects may vary, with some genes having a stronger effect on phenotype. Molecular analyses also indicate that many different genes are
available in genetic stocks (Anonymous, 2001).
The inheritance of FHB resistance in some of the key progenitors, such as Frontana, Sumai
3 and Ning7840, used by wheat breeding programs around the world is likely to be controlled by two or three genes with major effects (Ban and Suenaga, 2000; Singh et al.,
1995a; van Ginkel et al., 1996). The latter study also found evidence that the four genes
were distinct, providing promise that pyramiding these genes may result in increased resistance (Singh and van Ginkel, 1997).
In this study we determined the mode of inheritance of FHB resistance in three wheats
considered to be genetically distinct based on their pedigrees.
MATERIALS AND METHODS
Genetic materials - We used the following three lines expressing similar FHB resistance
but of distinct parentage (see Table 1).
Table 1. Parental lines used in the inheritance study.
G O V /A Z //M U S /3 /D O D O /4/B O W
C A T B IR D
B A U /M IL A N
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2002 National Fusarium Head Blight Forum Proceedings
Some of these lines, such as Gov/Az//Mus/3/Dodo/4/Bow and Catbird-derived lines, have
been successfully used by participating breeders the U.S. Wheat and Barley Scab Initiative
in crosses to locally adapted germplasm (Bacon et al., 2000).
Random F2-derived F8 progenies of crosses among the three resistant parents were used
in this inheritance study.
Inoculation methodology - Inoculation was carried out at anthesis, using the so-called
‘cotton method’ to detect Type II resistance (Gilchrist et al., 2002, this Proceedings). Evaluation of infection was carried out by counting the total number of spikelets per spike and the
number of spikelets infected; an infection percentage was then obtained.
RESULTS
The distributions of the 200 F8 lines per cross displayed discrete classes in each case and
X2 analyses confirmed a relatively simple gene inheritance. The data confirmed a preliminary analysis in 2001 (van Ginkel et al., 2001).
1. In the crosses of BAU/MILAN with GOV/AZ//MUS/3/DODO/4/BOW and BAU/MILAN with
CATBIRD, two major genes segregated.
2. In the cross of CATBIRD and GOV/AZ//MUS/3/DODO/4/BOW, four genes of major effect
segregated.
3. A total of four loci were involved.
DISCUSSION
With some luck and perseverance, FHB may become a textbook example of the application
of markers to breeding. Why? Two points in favor need to be considered.
1. It is clear from the literature that while many chromosomes have been implicated and
many genes have been described as contributing to FHB resistance, a few key genes often
explain most of the variation observed. Their gene action is frequently observed to be additive. This study of just three parents found up to four genes controlling resistance, confirming
that genes for scab are not uncommon in wheat. Genes with such major effects should be
targeted for use in the application of markers in wheat breeding.
2. The evaluation of germplasm for FHB response is cumbersome, requiring several rounds
of inoculation, a minimum of 5-10 inoculated spikes per plot, and replication at each site and
across years, to show consistency in resistance response. Frequently, germplasm that is
resistant one year may no longer be so the following year. Only after 3-4 years of testing can
resistance patterns be confirmed, and confident statements made about resistance/susceptibility. The reason lies in the significant interaction between infection processes and climatic
factors. This is not very encouraging for a breeding program hoping to make rapid progress.
Variety Development and Uniform Nurseries
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2002 National Fusarium Head Blight Forum Proceedings
Simple genetic resistance (point 1) is confirmed through a time-consuming and extremely
laborious process (point 2). Hence, developing and then linking phenotypic data with molecular data is a protracted undertaking requiring great precision. However, unlike the traditional breeding process where progeny from every cross must be screened for the disease,
markers promise a quick test for the presence/absence of desired alleles with major effects
on resistance.
Environment-neutral marker systems will provide significant savings in time and costs. The
liberated funds could be spent more effectively on marker development than on perpetual
resistance screening of segregating populations in the field or greenhouse.
Clearly there is no lack of genetic diversity for FHB. It is also clear that some of these genes
have major effects, particularly with regards to Type II resistance. It should not be difficult to
develop markers for such major genes, and they would have ready application in any
wheat-breeding program.
REFERENCES
Anonymous, 2001. National Fusarium Head Blight Forum Proceedings, Holiday Inn Cincinnati-Airport, Erlanger,
Kentucky, USA, December 8-10, 2001.
Bacon, R.K., E.A. Milus, J.T. Kelly, C.T. Weight, and P.C. Rohman. 2000. Development of FHB resistant cultivars of the mid-south. 2000 National Fusarium Head Blight Forum. Holiday Inn Cincinnati-Airport, Erlanger, KY.
December 10-12, 2000.
Ban, T., and K. Suenaga. 2000. Genetic Analysis of resistance to Fusarium head blight caused by Fusarium
graminearum in Chinese wheat cultivar Sumai 3 and the Japanese cultivar Saikai 165. Euphytica 113:87-99.
Singh, R.P. Hong Ma, and S. Rajaram. 1995a. Genetic analyses of resistance to scab in spring wheat cultivar
Frontana. Plant Disease 79:238-240.
Singh, R.P., M. van Ginkel. 1997. Breeding strategies for introgressing diverse scab resistances into adapted
wheats. Pp. 86-92 in: Fusarium Head Scab: Global Status and Future Prospects. H.J. Dubin, L. Gilchrist, J.
Reeves, and A. McNab (eds.). Mexico, D.F.: CIMMYT.
Van Ginkel, M., W. van der Schaar, Yang Zhuping, and S. Rajaram. 1996. Inheritance of resistance to scab in
two wheat cultivars from Brazil and China. 1996. Plant Disease 80:863-867.
Van Ginkel, M., L. Gilchrist, and C. Velazquez. 2001. Newly accumulated resistance in CIMMYT bread wheat
germplasm. Pp 168-170 in: Proceedings of the Warren Kronstad Symposium. J. Reeves, A. McNab, and S.
Rajaram (eds), Mexico, D.F.: CIMMYT.
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2002 National Fusarium Head Blight Forum Proceedings
SOURCES OF COMBINED RESISTANCE TO FUSARIUM HEAD
BLIGHT, STRIPE RUST, AND BYD IN TRITICALE
L. Gilchrist1*, A. Hede1, R. Gonzalez2 and R.M. Lopez1
1
CIMMYT, Mexico D.F., Mexico; and 2INIFAP, Campo Experimental Morelia, Mich., Mexico
*Corresponding Author: PH: 52-55-5804-2004; E-mail: l.gilchrist@cgiar.org
OBJECTIVES
1) To identify triticale lines that possess high levels of FHB resistance under Mexican conditions and thus could be used as resistance sources in breeding programs, and 2) to find out
whether the lines also carry resistance to yellow rust and BYD.
INTRODUCTION
Fusarium head blight (FHB, caused by Fusarium graminearum) infects small-grain cereals
during flowering and grain filling in temperate and humid weather conditions. It causes
considerable yield losses and contaminates grain with mycotoxins, which are harmful to
both human and animal health. In general, triticale has been reported to be resistant to
many pathogens, but under Mexican conditions, it has shown susceptibility to different
Fusarium species (F. nivale, F. graminearum, F. avenaceum, F. culmorum, F. poae, and F.
equiseti) that cause fusarium head blight (FHB), as well as to yellow rust (Puccinia
striiformis) and barley yellow dwarf (BYD), two other serious diseases affecting triticale
crops today. In the past few years, changes in the pattern of yellow rust races that attack
triticale have been observed in high altitude locations in different parts of the world, including Mexico. Though not a problem every year, BYD has the potential to cause substantial
losses when present. For this reason, combined resistance to FHB, stripe rust, and BYD is
considered to be highly useful in environments where all three can be present at the same
time.
The objectives of this study were 1) to identify triticale lines that possess high levels of FHB
resistance under Mexican conditions and thus could be used as resistance sources in
breeding programs, and 2) to find out whether these lines also carry resistance to yellow
rust and BYD.
MATERIALS AND METHODS
Adequate levels of genetic variation for FHB resistance have been found in primary triticales, which can be used as basic breeding materials through a pre-breeding scheme
(Dorman and Oettler, 1993).
The CIMMYT triticale program routinely evaluates advanced hexaploid triticale lines for
FHB resistance under natural infection in two hot-spot locations in Mexico: Toluca (state of
Mexico) and Patzcuaro (state of Michoacan). Breeders observed signs of FHB resistance in
the test triticale lines and pre-selected them for use in this study.
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2002 National Fusarium Head Blight Forum Proceedings
Twenty-six advanced hexaploid triticale lines were planted in small plots under artificial
inoculation in Toluca, located in the central highlands of Mexico. During three cycles (2000,
2001, and 2002), the triticale lines were inoculated, evaluated, and characterized for different types of Fusarium resistance: penetration (Type I), spread (Type II), toxin content (Type
III), and grain filling (Type IV) (Gilchrist et al., 1997; Gilchrist, 2001). The methodology and
procedures used for inoculating and evaluating the lines have been described by Gilchrist
et al. (1997). Two triticale varieties, IAPAR 23 and BR 2, reported as FHB resistant by Capan
et al. (1987), were included as checks.
The test triticale lines and the resistant checks were also planted in Patzcuaro in 2001 and
2002 to confirm the results obtained in Toluca. To that end, two FHB readings under natural
infection were conducted at the milk grain stage.
Toxin analysis of triticale grain was carried out in CIMMYT’s toxin laboratory following the
FluroQuant Romer procedures. Barley yellow dwarf was evaluated using a scale 1 to 9
(Bertschinger, 1994).
RESULTS
Fusarium head blight symptoms are more difficult to observe in triticale than in wheat.
Mainly due to the gray-green color of triticale, it is very easy to confuse FHB symptoms with
other diseases, and only for a few days is it possible to observe the infected spikelets, which
show premature darkening of the straw color. However, if the spikes are carefully evaluated
at the correct time and appropriate check cultivars are used for comparison, valuable information can be collected that will allow the identification of triticale lines with superior resistance to FHB.
Sixteen of the twenty-six test lines consistently showed resistant reactions to FHB during
the three cycles in Toluca. Results of characterizing the lines for different types of FHB
resistance and of evaluating them for resistance to yellow rust and BYD are shown in Table
1. It should be noted that some lines showing FHB resistance were also resistant to stripe
rust in the year 2001. A new stripe rust race was detected in 2002, and the damage increased in some of the lines that had shown a resistant reaction the year before. Glume
damage was also observed in some lines.
Barley yellow dwarf is not common in Toluca in summer, but a dry period at the beginning of
the cycle in 2002 caused a considerable natural increase in the number of aphid vectors of
MAV and PAV strains of the BYD virus. This in turn raised the level of BYD infection and
provided an ideal testing ground for the disease.
DISCUSSION
In studies by Maier and Oettler (1993), triticale appeared to have higher levels of FHB
resistance than the resistant wheat cultivar Frontana. In the evaluations carried out in Toluca
and Patzcuaro, a great majority of the lines showed a superior level of FHB resistance. As
for the check lines IAPAR 23 and BR2, only the latter proved to have intermediate resistance in both Toluca and Patzcuaro; IAPAR 23 was susceptible in Toluca and the second
Variety Development and Uniform Nurseries
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2002 National Fusarium Head Blight Forum Proceedings
and third years in Patzcuaro. This is an indication that FHB resistance expressed in one
location is not necessarily effective in all places where FHB is a problem.
CONCLUSION
The combined resistance to FHB, stripe rust, and BYD that we found in triticale has good
potential as a source of resistance in improvement programs that apply a plant breeding
strategy in which different types of resistance are combined in a single genotype.
REFERENCES
Bertschinger, L. 1994. New procedure for the effective field screening of cereals for symptomatic tolerance to
barley yellow dwarf luteovirosis. In: L. Bertschinger (ed.), Barley Yellow Dwarf Newsletter. Vol. 5, p.14. Mexico,
D.F. CIMMYT.
Dormann, M. and Oettler, G. 1993. Genetic variation of resistance to Fusarium graminearum (head blight) in
primary hexaploid triticale. Third European Fusarium Seminar. IHAR Radzikow. Hod Rosl. Aklim. Nasien.(Special
Edition) 37(3):121-127.
Capan, M.I.S., Nunes, R.M.R. and Baier, A.C. 1987. Evaluation of earliness and scab resistance in triticale,
wheat and rye. EMBRAPA-CNPT. Passo Fundo, Brazil.
Gilchrist, L., Rajaram, S., van Ginkel, M., Kazi, M. and Franco, J. 1997. Characterizing Fusarium graminearum
resistance of CIMMYT bread wheat germplasm. Fifth European Fusarium Seminar. Szeged, Hungary. Vol 25
No.3/2.
Gilchrist, L. 2001. Perspectives on Fusarium head blight Resistance in Barley. In: Vivar, H.E. and McNab, A.
(eds.). Breeding Barley in the New Millenium. Proceedings of an International Symposium. Mexico,
D.F.:CIMMYT.
Maier, F.J. and Oettler, G. 1993. Selection for the Fusarium toxin deoxynivalenol in callus cultures of triticale.
Third European Fusarium Seminar. IHAR Radzikow. Hod Rosl. Aklim. Nasien.(Special Edition) 37(3):43-49.
Variety Development and Uniform Nurseries
244
Table 1. Characterization of advanced triticale hexaploid lines for different types of fusarium head blight (FHB) resistance: I (penetration), II (spread), III (toxin content), IV (grain losses and grain filling)
3.49
5.47
4.13
0.81
1.13
2.67
2.6
3.94
2.14
2.28
3.86
2.19
4.55
3.14
3.05
0.25
7.28
2.69
1.68
1.65
3.78
2.49
3.15
3.81
2.06
1.18
1.34
3.65
2.78
2.53
5.57
3.45
6.64
2.95
ARDI 1/TOPO 1419//ERIZO/ 9/3/SUSI 2
ERIZO 10/BULL 1-1/5/TAPIR/YOGUI
1//2*MUSX/3/ERIZO 7/4/FARAS 1
FAHAD 4/FARAS 1
LIRON_2-1/3/MUSX/LYNX//STIER_12-3
MAH 17486.3/3/HARE 132/CIVET//STIER
28/4/CAAL
MORSA/COPI 1
PACA 2/3/MUSX/LINX//STIER 12-3
MASSA/NIMIR 3/3/YOGUI 1 /TARASCA 87
3//HARE 212/4/ ANOAS 3/STIER 6
IAPAR 23 (S-MS)
BR 2 (R-R)
Checks
KER 6/FARAS 1//BULL 2/ 4/TAPIR/ YOGUI
1//2*MUSX/ 3/BAGAL 2
KISSA_7/POLLMER_4//ERIZO_10/BULL_1-1
HIPO 2/ASAD/4/2*BGL/ CIN//MUSX/3/TESMO
8
KER 6/FARAS 1//BULL 2
4
3.45
4.25
1.79
150./2WALRUS 1//ERIZO 6/NIMIR 4
7.83
5.05
2.73
5.81
5.77
5.93
4.82
5.96
5.71
5.66
5.95
4.18
6.27
5.05
4.95
6.4
5.03
1.08
5.14
1.84 17.08
0.76
0.5
0.48
1.01
0.41
0.5
0.21
0.22
0.5
0.23
0.39
0.5
1.13
2.56
0.43
0.7
0.85
2000 2001 2002 2000
%
Toxin
Type III
losses
Type IV
TOLUCA
4.42
5.41
8.45
5.25
4.7
7.08
4.37
1.97
5.07
5
6.81
10.1
7.24
6.38
7.8
9.52
7.71
5.73
7.03
6.45
5.42
5.26
4.82
4.33
10.11 4.25
7.02
6.78
5.51
5.95
5.68
6.47
8.7
7.49
11.09 N.A.
6.79
6.46
0.24
0.69
0.65
x
0.22
0.73
0.74
0.61
0.89
0.59
0.53
0.17
0.88
0.96
0.42
0.49
0.68
0
0
0.49
0.28
0.22
0.29
0.3
0.3
0.29
0.4
0.23
0.24
0.43
0.47
0.27
0.71
0.23
3.27
8.64
5.24
1.01
6.76
8.41
3.97
6.13
4.51
1.99
13.88 3.11
4.09
2.94
4.26
4.63
2.58
10.8
6.93
7.17
9.48
1.2
3.96 11.07 2.71
8.01 24.73 9.65
4.28 14.72 3.97
4.88
4.13
3.33
2.29 11.65 3.97
4.6
1.84
5.49 13.15 5.91
3.46 11.91 4.69
7.19
5.2
7.98
4
6.22 10.74 6.47
9.63
2.76
6.69 13.01 1.48
3
5
2
1
1
1
2
2
1
2
2
1
2
1
2
2
1
2
1
%
(ppm)
%
2001 2002 2000 2001 2000 2001 2002 2000
damage
damage
150./2WALRUS 1//ERIZO 6/NIMIR4
/4/GNU/ASAD//ARDI/3/MANATI 1
804/BAT/4/ERIZO 11/3/BGL/JLO// YOGUI
1/5/JIL96/6/GAUR 3/ANOAS 2/ / BANT-1
ANOAS-1/2*BULL 1-1//ERIZO 11/YOGUI
3/3/ANOAS 5/STIER 13
ANOAS 5/STIER 13//ANOAS 5/ FARAS 1
Crosses
Type II
Type I
Toluca was resistance to yellow rust (in 2001, 2002) and BYD (in 2002).
3
5
1
1
2
1
1
1
1
2
2
1
2
1
1
3
1
2
1
2001
Grain
(1-5)
Type V
4
3
2
1
2
2
3
3
2
3
2
1
1*
1
2
1
3
2
2002
40MR
10MR
40MS
TR
0
0
0
0
0
0
10MS
0
10MS
0
0
TR
TS
40MR
20MR
20MR
20MR
40MR
20MR
40MR
40MR
TR
40MR
TR
20MR
40MR
20MR
TR
10MR
10MR
10MS-MR 20 MR
10MS-MR 60 MS
damage
2001
2002
Leaf
Stripe rust
4
1
1
0
2
0
1
0
0
1
2
1
0
0
0
2
2
0
1
2002
damage
glumes
%
infection
FHB
NA
NA
1
1
2
2
1
5
4
7
6
5
1
5
6
5
2
1
1
20
15
3
30
8
20
10
50
3
60
10
5
3
40
5
3
5
5
3-
20
80
3
25
5
25
30
3
30
25
5
3
3
3
5
5
3
3
2002 2001 2002
BYD
PATZCUAR
under artificial inoculation during three cycles (2000, 2001, 2002) in Toluca and % FHB infection during two cycle (2001,2002) under natural conditions in Patzcuaro (Michoacan). Also evaluated in
2002 National Fusarium Head Blight Forum Proceedings
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2002 National Fusarium Head Blight Forum Proceedings
PROGRESS IN BREEDING FUSARIUM HEAD BLIGHT
RESISTANCE IN SOFT RED WINTER WHEAT
C.A. Griffey*, J. Wilson, D. Nabati, J. Chen, and T. Pridgen
CSES Department, Virginia Tech, Blacksburg, VA 24061
*Corresponding Author: PH: 540-231-9789; E-mail: cgriffey@vt.edu
ABSTRACT
A primary goal of our breeding program is to accelerate the development of adapted and
commercially viable Fusarium head blight (FHB) resistant SRW wheat varieties by identifying and incorporating diverse types of resistance into elite genotypes. Breeding methods
being used to accomplish this goal include topcrossing, backcrossing, doubled haploid
techniques and molecular marker genotyping. In 2002, 229 segregating populations were
evaluated in a mist-irrigated FHB nursery, inoculated using colonized maize seed, at Mt.
Holly, VA. Seventy-seven of these populations (34%) were advanced on the basis of FHB
incidence and severity, agronomic traits, and resistance to other prevalent diseases such as
powdery mildew. In field tests, approximately 4500 headrows (F3-F8 and various backcross
generations) were evaluated for agronomic traits and resistance to diseases other than FHB
at Warsaw, VA. In addition, approximately 2800 F5-F7 headrows were evaluated for FHB
resistance and agronomic traits in an inoculated, mist-irrigated nursery at Blacksburg, VA.
From these headrows, 32 backcross-derived lines and 26 topcross-derived lines were
selected for further testing in our scab nursery at Blacksburg and in Observation yield tests
at two locations in 2003. Twelve lines from the 2001-02 Observation yield test were selected for further testing in Preliminary wheat trials. Four elite lines were selected for testing
in our Advance yield trial, and two elite lines will be tested in Virginia’s official variety trial.
Twelve lines will be tested in the 2002-03 Uniform Winter Wheat FHB Nurseries. Two newly
released varieties from the Virginia Tech Small Grains Program, ‘McCormick’ and ‘Tribute’,
possess a significant level of scab resistance. Progress in transferring type II resistance into
SRW wheat genotypes has been accelerated via use of the wheat by maize doubled haploid (DH) system. One DH line, VA01W-476, developed from the cross ‘Roane’/W14, was
found to have good scab resistance in greenhouse and field tests and also has major genes
for scab resistance as determined by DNA analysis this spring. A total of 135 doubled
haploid lines derived from nineteen 3-way crosses consisting of diverse scab-resistant
parents were selected on the basis of field and greenhouse tests this year and will be evaluated for scab resistance in our inoculated, mist-irrigated nursery at Blacksburg and for
agronomic traits at Warsaw. Type II resistance from five different sources (Futai8944,
Futai8945, Shaan85, VR95B717 and W14) has been backcrossed into seven adapted SRW
wheat backgrounds, and two of the recurrent parents (Roane and Ernie) possess FHB
resistance other than Type II. A total of 180 BC4F2 and BC5F2 individuals were selected on
the basis of scab severity in greenhouse tests and will be evaluated for scab resistance in
our inoculated, mist-irrigated nursery at Blacksburg and for agronomic traits and similarity to
the recurrent parent at Warsaw. Near-isogenic SRW wheat lines with Type II resistance are
being developed and will facilitate pyramiding of different types of FHB resistance.
Variety Development and Uniform Nurseries
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2002 National Fusarium Head Blight Forum Proceedings
COMPARISON OF FHB DEVELOPMENT ON HARD WINTER
WHEAT USING DIFFERENT PLANTING SCHEMES
D.M. Gustafson*, A.M.H. Ibrahim, and L. Peterson
Plant Science Department, South Dakota State University, Brookings, SD 57007
*Corresponding Author: PH: 605-688-4764; E-mail: dawn_gustafson@sdstate.edu
ABSTRACT
Fusarium head blight (FHB) is a destructive disease of wheat causing yield loss and poor
grain quality. Winter wheat producers in South Dakota have adopted a reduced tillage
cropping system and have increased production of winter wheat in traditional corn-soybean
rotations. These practices could very well lead to an increase in FHB severity. The winter
wheat breeding program at South Dakota State University has established a proactive effort
to develop FHB-resistant hard winter wheat varieties. Transplanted hill nurseries have been
screened since 1999 utilizing an established mist-irrigated field screening nursery designed
to test cultivars, elite lines, and preliminary lines for resistance to FHB. However, transplanting winter wheat is a time consuming process because it involves the vernalization of seedlings in cold chambers, proceeded by hand planting. The root system is far from established
in transplanted wheat, often leading to poor plant development. The laborious transplanting
process also does not follow the conventional direct seeding method followed by wheat
producers. This has led to the investigation of planting schemes to determine if direct
seeded row materials are affected differently than transplanted hill plots when they are
inoculated with FHB. In October 2000, several multi-location winter wheat trials, including
the South Dakota Crop Performance Trials (CPT), were directly seeded into the FHB nursery. The CPT trials were also vernalized and transplanted in May 2001. Significant correlations between the two types of planting techniques were observed for FHB severity and
disease indices. However, FHB incidence for the direct seeded rows was low and was not
significantly correlated with the incidence levels in the transplanted hills. This was perhaps
due to the early flowering of the direct seeded materials. The cooler temperatures at anthesis may have inhibited FHB development. In 2002, we investigated transplanted seedling
performance in comparison to delayed seeded CPT lines. The CPT and several other trials
were directly seeded on November 26, 2001. This planting scheme helped delay flowering
by approximately two to three weeks compared to conventional timely seeding. In May
2002, the CPT trial was transplanted into the mist-irrigated field nursery. Significant correlations (P < 0.05) between the two types of planting techniques in 2002 were observed for
FHB severity, incidence, and disease indices. Correlations between the different planting
types across years were also highly significant. These results suggest that delayed direct
seeding could replace transplanting. However, transplanted hills should be used if improper
weather conditions prevent a successful direct seeded nursery.
Variety Development and Uniform Nurseries
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2002 National Fusarium Head Blight Forum Proceedings
STABILITY OF TYPE II RESISTANCE AND DON LEVELS ACROSS
ISOLATE AND SOFT RED WINTER WHEAT GENOTYPE
Anne L. McKendry*, Kara S. Bestgen, David N. Tague, and Zewdie Abate
Agronomy Department, University of Missouri, Columbia, Missouri 65211
*Corresponding Author: PH: (573) 882-7708; E-mail: mckendrya@missouri.edu
ABSTRACT
Fusarium graminearum Schwabe (teleomorph Gibberella zeae (Schwein.), also known as
scab, is an important disease of wheat world-wide. Although host plant resistance has long
been considered the most practical and effective means of control breeding has been hindered by a lack of effective resistance genes and by the complexity of the resistance in
identified sources. No source of complete resistance is known, and current sources provide
only partial resistance. The identification of new sources of resistance and their incorporation into adapted wheat varieties provides the most economical solution to this problem.
Within both germplasm screening programs and breeding programs, the goal is to identify
resistance that is stable over genotypes and across geographical areas. Choice of isolate
may be an important factor in accomplishing this goal. We evaluated the effect of 5 diverse
Fusarium graminearum isolates on type II resistance and DON levels in adapted winter
wheat germplasm entered into the Northern Uniform Scab Nursery in 1999. Genotypes were
planted in the greenhouse in a split-plot arrangement with genotypes as the main plot and
isolates as the sub-plot. The experiment was replicated six times. Five plants per isolate
per replication were inoculated at first anthesis with 10µL of a macroconidial suspension of
Fusarium graminearum concentrated to 50,000 macroconidia/mL. Plants were then incubated in a mist chamber for 72 h and rated for type II resistance at weekly intervals postinoculation. At maturity, inoculated heads were harvested, hand-threshed and seed were
bulked for deoxynivalenol (DON) analyses. Results indicated significant differences in the
aggressivity of the isolates used with the Missouri isolate being the most aggressive across
all genotypes. Mean DON levels varied significantly ranging from 160 ppm to 3 ppm. Significant genotype by isolate effects were evident for DON production.
Variety Development and Uniform Nurseries
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2002 National Fusarium Head Blight Forum Proceedings
DEVELOPING FHB-RESISTANT CULTIVARS AND
GERMPLASM FOR THE MID SOUTH
E.A. Milus1*, R.K. Bacon2, S.A. Harrison3, P. Rohman1, S. Markell1, and J. Kelly2
1
Dept. of Plant Pathology and 2Dept. of Crop, Soil and Environmental Science,
University of Arkansas, Fayetteville; and
3
Dept. of Agronomy, Louisiana State University, Baton Rouge
*
Corresponding Author: Phone 479 575-2676; E-mail gmilus@uark.edu
INTRODUCTION
Growing resistant cultivars likely will be the primary component of any management strategy
for Fusarium head blight of wheat in the Mid South. None of the currently grown cultivars
have a high level of FHB resistance, but a collaborative program has identified promising
resistant lines that are agronomically adapted and have resistance to other important diseases.
MATERIALS AND METHODS
A crossing program was initiated in 1991 between adapted genotypes and various sources
of resistance. These populations were advanced as bulks for 5 years then lines were developed using pedigree selection. These lines have been evaluated for FHB resistance in an
inoculated screening nursery along with a FHB resistant check (Ernie) to allow for FHB
evaluation in a natural epidemic. These F7 derived lines have been selected for FHB resistance, yield and other agronomic traits. Additionally, the most advanced lines from the
University’s wheat breeding program are being screened to determine the level of resistance (or susceptibility). For future development, many different sources of resistance have
been used to develop over 450 populations (F1- F4) from which lines will be selected.
Ninety-three F7, topcross F6, or topcross F6 germplasm lines were evaluated for FHB resistance in the greenhouse and in inoculated and misted screening nurseries at Fayetteville
and Kibler. The lines also were evaluated for resitance to other diseases that are important
in the Mid South and for spring freeze damage, vernalization, lodging, and agronomic
phenotype.
RESULTS AND DISCUSSION
Yield plots harvested in June at Stuttgart and Marianna, AR indicated several high-yielding
lines in the scab resistant nursery (Table 1). Of the 34 lines tested at two locations, 17 were
not significantly different than Ernie for FHB. Five of these lines actually had lower numerical ratings than Ernie at both locations. Of those five lines, two were not significantly different in yield than the high-yielding check ‘Pat.’ Four lines were tested in the 2001-02 Southern Winter Wheat Scab Nursery. Field results across eight reporting locations indicated that
all four Arkansas lines had ratings for FHB Index that were not significantly different than the
resistant check Ernie. Percentage scabby kernels for all the Arkansas lines was also not
different than Ernie. The 18 best FHB breeding lines from the 2002 FHB yield test and
Variety Development and Uniform Nurseries
249
2002 National Fusarium Head Blight Forum Proceedings
8 lines from Milus’ germplasm enhancement will be tested in replicated inoculated yield
trials at two Arkansas locations in 2003. Four new lines were entered in the 2002-03
Southern Winter Wheat Scab Nursery. Fifty F1 populations will be grown in the greenhouse
for seed increase. The more advanced populations, which include 100 F2, 254 F3, and 49 F4
populations, were planted at Stuttgart and will be grown in inoculated blocks to help natural
selection shift the populations towards resistance.
Thirteen germplasm lines (Table 2) were selected based on level of FHB resistance, parentage, resistance to other diseases, and agronomic characteristics, and these would be useful
parents in breeding programs. Five of the selected lines were entered in the 2003 Southern
Uniform Winter Wheat FHB Nursery. For evaluation and crossing, seed of all selected lines
were provided to Dr. Lucy Gilchrist of CIMMYT and Barton Fogleman of Agripro, and seed of
selected lines were provided to Dr. Mohan Kohli of CIMMYT in South America. Seed of
lines with Karnal bunt resistance in their parentage were sent via Dr. Art Klatt to CIMMYT for
Karnal bunt screening. To determine which lines carry different genes for FHB resistance,
lines are being screened by Dr. Guihua Bai for a QTL associated with FHB resistance, and
six lines are included in a diallel genetic study.
Variety Development and Uniform Nurseries
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2002 National Fusarium Head Blight Forum Proceedings
T ab le 1. P e rfo rm an ce o f lin es in ino cu lated sca b trials at M ariann a and S tuttg art, A rk an sas w ith
F H B ratin gs fro m F ayettev ille and K ib le r, A rk ansa s in 20 0 1-0 2 .
_ __ _ __ __ _ __ _ __ __ _ __ __ ___ _ __ _ __ __ __ __ _ _ __ ___ __ __ _ _ __ __ __ _ __ __ _ ___ __ __ _ __ __ _ __ _ __ __ ___ __ _ _ __ __ __ __ _ __ _ ___ __ __ _ _ ___ _ __ _ __
E ntry
Y ield
b u/A
T est H eading M atu rity
w eig h t
d a te
d ate
lb /b u
P at (check)
A R 9309 5-4-1
A R 9303 5-4-1
A R 9303 5-4-3
A R 9303 5-4-4
E rnie (check)
A R 9303 5-4-2
A R 93188-12-1-1
A R 9303 5-7-1
A R 9310 8-8-1
A R 9310 8-1-3
A R 9318 9-3-1
A R 9318 8-1-1
A R 9309 1-4-2
A R 9318 9-4-1
A R 9310 8-9-1
A R 9318 9-7-1
A R 93187-6-1
A R 9310 8-3-2
A R 9306 9-5-1
A R 9301 9-2-1
A R 9304 8-8-2
A R 9318 8-7-1
A R 9303 2-6-1
A R 9310 8-1-2
A R 9300 1-3-2
A R 878 -2-1
A R 9308 1-2-1
A R 9310 8-8-2
A R 857 -1-2
A R 9318 7-4-2
A R 9310 8-4-1
A R 857 -1-1
A R 880 -5-1
A R 9303 5-1-1
A R 922 -5-1
79.9
75.8
75.7
74.7
71.5
70.5
69.1
68.2
67.8
66.7
65.8
65.6
65.1
65.0
63.7
63.6
62.9
62.6
62.5
62.1
62.1
61.7
60.9
60.4
60.3
59.6
59.5
57.5
57.2
57.0
56.7
56.3
54.1
53.2
50.1
46.9
56.9
56.1
56.6
57.5
55.9
55.3
55.6
55.2
55.2
52.8
54.1
55.5
53.9
56.8
54.5
52.9
55.0
54.0
56.3
58.3
57.5
51.9
53.9
56.9
53.1
56.9
56.1
53.1
51.6
54.2
54.8
51.4
55.0
52.9
56.0
57.3
M ean
C V (% )
L S D 05
63.2
11.9
8.5
55.1
5 .4
3 .4
4/2 0
4/1 8
4/1 8
4/1 7
4/1 7
4/1 5
4/1 7
4/1 8
4/1 7
4/1 6
4/1 8
4/1 9
4/1 9
4/1 9
4/2 0
4/1 5
4/1 8
4/2 0
4/1 4
4/1 8
4/2 1
4/1 6
4/2 0
4/1 6
4/1 7
4/1 7
4/1 5
4/1 8
4/1 7
4/1 6
4/1 9
4/1 6
4/1 6
4/1 8
4/1 8
4/1 7
5/22
5/21
5/22
5/22
5/21
5/20
5/22
5/20
5/22
5/18
5/22
5/20
5/20
5/22
5/21
5/20
5/21
5/21
5/19
5/21
5/21
5/19
5/22
5/20
5/19
5/21
5/20
5/20
5/19
5/20
5/21
5/20
5/21
5/21
5/20
5/20
P la nt
ht
in .
39
38
35
36
35
34
34
34
35
36
34
33
35
39
35
35
34
34
36
37
40
35
34
37
36
36
42
40
37
37
34
34
35
37
37
36
FH B
FH B
F ayett. K ib le r
%
%
2.0
2.5
1.5
1.3
1.3
4.0
2.0
21.3
2.3
30.0
7.5
17.5
11.3
7.5
18.8
12.5
16.3
26.3
8.8
10.0
1.6
16.3
15.0
13.8
23.8
1.4
2.5
15.0
22.5
0.1
10.0
13.8
0.0
2.5
3.5
3.5
1.8
2.8
5.5
5.5
4.3
4.8
6.3
33.8
7.5
17.5
6.3
21.3
10.0
2.5
15.0
12.5
37.5
26.3
8.0
11.7
1.5
18.8
17.5
21.3
8.8
5.0
5.5
7.5
2.0
12.5
17.5
0.5
7.5
5.5
9.3
5.1
7.0
_ _ __ _ __ _ _ _ __ __ __ _ _ __ __ __ _ __ _ __ _ __ _ _ __ _ __ __ _ _ __ __ __ __ _ _ __ ___ _ _ __ _ __ _ __ _ ___ _
Variety Development and Uniform Nurseries
251
0.4
0.6
0.2
0.5
Fayetteville
0.4
0.1
0
0
Mason/Catbird (G93)
Mason/Catbird (G95)
Freedom/Catbird (G82)
Variety Development and Uniform Nurseries
252
2.0
1.5
1.5
0.7
Mason/3/Freedom//Clark*4/N7840
P2684/3/N7840//Parula/Veery#6
2
2
1
0
1
2
1
Winnsboro, LA1
45
50
2
2.5
4.3
22.8 26.4 62.2
Mason
P2684
89
75
1
4.5
1
% Scabby Seed
Fayetteville
47.5
55.0
42.5
25.0
15.0
17.5
35.0
55.0
35.0
26.3
20.0
30.0
20.0
6.5
15.0
21.3
42.5
60
Fayetteville
50
40
50
60
22.5
Kibler
0
0
0
0
0
0
3.0
1.4
0.8
1.0
1.3
1.3
Greenhouse3
22.5
0
3.1
55
55
26.3
45
49.3 3.8
30
28.5
40
15
22.5
1
2
0
4.5
Baton Rouge
0
0
25
0
0
0
0
0
0
0
% Stripe rust
3.0
3.0
0.0
4.0
0
0
0
0
4.0
5.0
4.0
4.0
3.5 4.0
1
25
0
Spindle Streak4
0
15
2
4.1
2.2
5.8
0.3 18.5
3.2
2.6
0
0
0
4.0
0.0
1.5
0.7 81.5 7.5 1.5
77.5 22.5 22.5 3.0
60
63.8 22.5 18.5 2.6
70
50
50
65
65
51.3 3.3
56
Kibler - LR DSN
43.8 22.5
Kibler - FHB Test
49.3 4.4
73.8
70
55
60
70
50
60
68.8
60
55
60
65
3.0
1.5
5.0
4.0
3.0
5.5
1.5
5.5
5.0
4.0
1.5
5.0
5.0
Soilborne +
spindle streak4
2
0-9 Scale, 0=No symptoms.
Septoria tritici blotch was the principal leaf disease, but also some stripe rust, leaf rust, and spring infection of barley yellow dwarf.
3
Inoculated with race TNRL. Infection type on flag leaves rated on 0-9 scale, 0=no symptoms.
4
0-9 Scale, 0=No symptoms.
5
0-9 Scale, 0=No Lodging.
6
0-2 Scale, 0=Not Vernalized.
7
0-9 Scale, 0=Excellent.
8
0-2 Scale, 0=No Damage.
9
0-9 Scale, 0=No Damage.
10.9 64.4
15.2 55.5
2
10.9 59.5
17.8 37.5
26.6 52.5
20.8 67.5 1.5
18.4
10.2
25.0 47.5 2.5
22.5 56.7
2
1
20
12.7 47.5
9.3
25.8 11.3
11.0 42.5
12.3 52.5
Patton
3.5
Kibler - Late
26.5 47.5
Greenhouse
Ernie
3.6
2.0
Checks
1.8
0.8
0.7
2.5
0.5
Mason*2//Sha3/Super Kauz
P2684/Er-Mai 9
2.3
Mason/Yu-Mai 7
1.8
1.1
1.4
Mason//Freedom/Super Zlatna
Mason//Freedom/Super Zlatna
0.7
2.8
0.1
0.9
Freedom/Catbird (G82)
Mason//Sha 3/Catbird
Parentage
Kibler - Early
Mason/Catbird (G49)
1.8
Lodging - Kibler5
2.4
1.4
5.6
4.9
6
3.3
3.8
1.3
0
2
4.5
1.3
4.3
4.8
0
3.3
Vernalization
Baton Rouge6
2
1
1.5
2
0.5
2
1
0
1
0.5
1
1
1.5
2.5
Phenotype
Winnsboro, LA7
1.5
3
4
2.5
5
4
3.5
2.5
4.5
3
2
2.5
0.1
Fayetteville8
0
2
0
0
0.0
0.0
0.5
0.5
0.0
0.5
0.0
0.3
0.3
0.0
0.1
0.1
1.5
5
1
3
1
1
1
1
1
1
1
1
1
1
Baton Rouge9
Table 2. Disease and agronomic ratings for F7, topcross F6, and backcross F6 germplasm lines selected during the 2002 season, compared to FHB resistant and
susceptible checks.
Spring Freeze
2
% Green leaves
Damage
FHB-% Florets Infected
% Leaf Rust
2002 National Fusarium Head Blight Forum Proceedings
2002 National Fusarium Head Blight Forum Proceedings
UNIFORM SOUTHERN SOFT RED WINTER WHEAT FUSARIUM
HEAD BLIGHT SCREENING NURSERY
J.P. Murphy1*, R.A. Navarro1 and D.A. Van Sanford2
Dept. of Crop Science, North Carolina State University, Raleigh, NC; and
2
Dept. of Agronomy, University of Kentucky, Lexington, KY.
*Corresponding Author: PH: (919) 513-0000; E-mail: njpm@unity.ncsu.edu
1
ABSTRACT
The Third Uniform Southern Soft Red Winter Wheat Fusarium head blight (FHB) Screening
Nursery comprised 28 advanced generation breeding lines and two check cultivars. Five
public (Univ. of Arkansas, Univ. of Georgia, Univ. of Maryland, N.C. State Univ., and Virginia
Tech) and two private (Syngenta Seeds and AgriPro) cooperators submitted entries. Ten
cooperators submitted field, greenhouse, and SSR data for the annual report. Significant
genotype and genotype by location variation was observed for FHB incidence, head severity, index, and percent scabby seed in combined analyses of field data and for head severity
in greenhouse evaluations. No significant correlations were observed between plant height
or head emergence and any of the FHB variables. Matrices containing the means of the 30
genotypes at each location for each FHB variable were subjected to GGE biplot analyses to
provide insight into the underlying causes of the genotype by location interaction and to
identify consistently superior genotypes across test locations. A single megaenvironment
encompassing eight locations was observed for FHB incidence. Two megaenvironments
(LA and Bay-AR versus OH, IL, KY, VA, NC, MD, Fayetteville-AR, and Kibler-AR) were
observed for head severity. Nevertheless, there was a high degree over overlap among the
most resistant genotypes in both megaenvironments. Two megaenvironments were observed for percent scabby seed (NC versus Fayetteville-AR, Bay-AR, IL, KY, and VA) and
two megaenvironments were observed for greenhouse estimates of head severity (NC
versus Bay-AR, MO, IL, and KY). Again, there was a high degree of overlap amongst the
most resistant genotypes in both sets of megaenvironments. VA01W476, a doubled haploid
line from the cross between the moderately resistant ‘Roane’ and the resistant Chinese line
W14, was the most resistant genotype overall. It rated most resistant for FHB incidence,
severity, index in field tests, and head severity in greenhouse evaluations overall.
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2002 National Fusarium Head Blight Forum Proceedings
DEVELOPED EVALUATION METHOD OF FUSARIUM HEAD BLIGHT
(FHB) RESISTANCE IN WHEAT BY CONTINUOUS SIMULATED
RAINFALL AND DIVERSITY OF FHB RESISTANCE
IN DOMESTIC WHEAT
Zenta Nishio1*, Kanenori Takata1, Tadashi Tabiki1, Tomohiro Ban2
National Agricultural Research Center for Hokkaido Region, Shinsei, Memuro, Hokkaido 082-0071, Japan;
and 2Japan International Research Center for Agricultural Sciences (JIRCAS),
1-2 Owashi, Tsukuba, Ibaraki 305-8686, Japan
*Corresponding Author: PH: 81-155-62-9210, E-mail:zenta@affrc.go.jp
1
ABSTRACT
In the Fusarium head blight (FHB) resistance evaluation test of wheat, variances and errors
of FHB severities in every test are still big issues for resistance breeding. For this problem,
we developed a method by continuous simulated rainfall system with injection inoculation to
reduce environmental error factors of each inoculation. Sprinkler system has equipped to
cover all the test plots and simulated rainfall were operated every 5 minutes for 60 seconds
to keep spikes wet at all time of disease developing. Suspension of Fusarium graminearum
spore in distilled water (1x105/ml) was injected into a single sipikelet of middle spike on the
each material’s flowering day and FHB disease severities were investigated after 21 days of
inoculation. The correlation coefficient between field test and greenhouse test were
r=0.84(n=70, P<0.01, 2001), r=0.71(n=185, P<0.01, 2002) respectively, they showed high
values for 2 years. The correlation coefficient between every year’s average FHB severities
in the field test and the greenhouse test was r=0.71(n=30, P<0.01, 2001-2002), it also
showed high value. While FHB inoculation, leaf disease by F.graminearum was observed,
so the relationship between FHB and leaf disease was investigated. The correlation coefficient between FHB severities and leaf disease severities was significant, r=0.38(n=70,
P<0.01), but it seemed to be difficult to presume FHB resistance from leaf disease severities. The highest resistance cultivar of FHB was Sumai 3 Austria line, a derivative of Sumai
3. Sumai 3 (Kyusyu) and Sumai 3 (CIMMYT) showed a little different plant height, but there
observed not so much differences in FHB resistance. The relationship of FHB resistance of
domestic cultivars has been investigated. 4 major cultivars of Hokkaido (Takunekomugi,
Horoshirikomugi, Chihokukomugi, Hokushin) showed stable FHB severities for 2 years, so
they were employed as standard cultivars. Takunekomugi showed highest resistance
among of them, as the same resistance as Saikai 165 in Kyusyu. Saikai 165 was bred from
a cross of Sumai 3/Asakazekomugi for the purpose of improved FHB resistant line, but the
resistance was a little inferior to Sumai 3. Hokushin has a good quality for white salt noodle
(Udon), and it is a leading cultivar of Hokkaido at the present time, but it was most susceptible among Hokkaido’s materials. We found Kachikei 28, showed more FHB resistance
than Takunekomugi, but both of their parents were susceptible to FHB. We developed a high
reliability evaluation method for FHB resistance, and elucidated the relationship of FHB
resistance in domestic wheat and Sumai 3.
Variety Development and Uniform Nurseries
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2002 National Fusarium Head Blight Forum Proceedings
PHENOTYPIC EFFECTS OF QFHS.NDSU-3BS ON FUSARIUM HEAD
BLIGHT RESISTANCE IN NEAR-ISOGENIC WHEAT LINES
M.O. Pumphrey and J.A. Anderson*
Dept. of Agronomy and Plant Genetics, University of Minnesota, St. Paul, MN 55108
*Corresponding Author: PH: (612) 625-9763; E-mail: ander319@umn.edu
OBJECTIVE
To use QTL near-isogenic lines to evaluate phenotypic effects of Qfhs.ndsu-3BS in multiple
genetic backgrounds
INTRODUCTION
Despite high levels of FHB resistance identified in some wheat cultivars, progress in breeding for FHB resistance has been relatively slow due to complex inheritance, large environmental influences on disease development, and the resources required to conduct successful breeding nurseries. Recent QTL studies have identified chromosomal regions carrying
putative genes for FHB resistance by exploiting statistical associations between molecular
markers and resistance phenotypes. A major QTL, Qfhs.ndsu-3BS, associated with reduced pathogen spread (Schroeder and Christensen, 1963) has been identified from Chinese wheat cultivar ‘Sumai 3’, or its derivatives, in several studies (Waldron et al. 1999;
Anderson et al. 2001; Buerstmayr et al. 2002; Zhou et al. 2002).
The consistent ability to detect Qfhs.ndsu-3BS and the magnitude of effect in each mapping
population imply that it should be useful for marker-assisted selection (MAS). However, to
justify MAS for the QTL region, increased levels of resistance due to this QTL should be
observed in multiple genetic backgrounds. To test the robustness of Qfhs.ndsu-3BS, nearisogenic lines (NILs) contrasting for the QTL region were tested in greenhouse point-inoculation experiments and field FHB screening nurseries. NILs are particularly effective genetic stocks to study FHB resistance that is attributable to a QTL because NILs standardize
the genetic background, morphology, and agronomic characters that may influence disease
assessment.
MATERIALS AND METHODS
Plant materials. Co-dominant microsatellite markers gwm493, barc133, and gwm533
(Roder et al., 1998; Cregan and Song, 2002) were selected to develop NILs with alternate
marker alleles for Qfhs.ndsu-3BS. Homozygous near-isolines were identified by genotyping
the progeny of self-pollinated, heterozygous, F3:4 lines from 17 unique cross combinations
that were grown in summer breeding nurseries in 2000 and 2001. Each of the 17 selected
populations had a FHB resistant parent with Sumai 3 in its pedigree and correct marker
alleles that are unique to this region (Liu and Anderson, in press). In total, 33 QTL-NIL pairs
were produced.
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2002 National Fusarium Head Blight Forum Proceedings
Field Screening. In the summer of 2002, all entries were tested at St. Paul and Crookston
misted and inoculated nurseries. Check cultivars and NIL parents, including: ‘Sumai 3’
(resistant), ‘Roblin’ (susceptible), ‘Wheaton’ (susceptible), ‘Alsen’ (moderately resistant), ‘ND
2710’ (resistant), ‘ND 2603’ (resistant), and ‘Bacup’ (resistant) were included to represent a
range of maturity, height, and disease resistance phenotypes. NIL pairs were randomized
with check cultivars in a complete block design with 4 replications.
At St. Paul, macroconidia [1x105/ml] of Fusarium graminearum were applied at anthesis at a
rate 30 ml m-1 row, followed by a second application 3 days later. At Crookston, infested
corn-kernel inoculum was spread evenly throughout the field at a rate of 112 kg ha-1 at the 5
leaf stage. For both nurseries, the number of infected spikelets were counted on 20 spikes
per row approximately 20 days post-inoculation. Rows were harvested by hand using a
sickle and then 30 spikes per row were threshed from two replications in a manner that
retained diseased kernels. The weight of seed from 30 threshed spikes was measured.
Percent visually scabby kernels (VSK) was estimated based on the scale of Jones and
Mirocha (1999). Data analyses were performed using SAS PROC GLM. QTL alleles were
considered as fixed effects. Replications, NIL pair, and NIL pair by QTL interaction were
considered random effects.
Greenhouse Screening. Approximately 15 plants per genotype were tested in two experiments by inoculation of 10µl macroconidia [1x105/ml] into a central spikelet at anthesis.
Plants were incubated in a dew chamber (100% RH, 20°C) for 72 hours after inoculation.
The number of symptomatic spikelets and total spikelet number were counted at 21 days
post-inoculation. The difference in number of symptomatic spikelets between near-isolines
was analyzed using t-tests.
RESULTS AND DISCUSSION
Field. Statistical analysis of individual field experiments revealed that error variances were
not homogeneous; therefore, locations were not combined for ANOVA. Significantly lower
disease pressure at the Crookston nursery and different inoculation methods between the
two locations are the most probable causes (Table 1). The effect of Qfhs.ndsu-3BS was
highly significant (P<0.01) for 5 of 6 trait by location combinations (Tables 2 & 3). The marginal significance (P=0.046) of Qfhs.ndsu-3BS in increasing 30-spike seed weight at
Crookston is likely due to low disease pressure. As expected when sampling lines from
diverse populations, the effect of NIL pairs was highly significant (P<0.001) for all traits in
each location. The interaction between Qfhs.ndsu-3BS and genetic background (NIL pair)
was only highly significant for disease severity at St. Paul and was marginally significant for
30-spike seed weight at Crookston. The average reduction in disease severity with
Qfhs.ndsu-3BS present was 22% at St. Paul and 14% at Crookston. The average reduction
in VSK was 19% at St. Paul and 24% at Crookston (data not shown).
Greenhouse. Sixteen of twenty-nine pairs had significantly reduced spread within the
spike in isolines with Qfhs.ndsu-3BS across two point-inoculation experiments (Figure 1).
Pairs with no statistically significant difference generally had lower disease levels, but the
trend was towards more resistant genotypes with Qfhs.ndsu-3BS. The average reduction in
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2002 National Fusarium Head Blight Forum Proceedings
symptomatic spikelets with Qfhs.ndsu-3BS present was 27% in the first experiment and
36% in the second.
These results confirm that selecting for Qfhs.ndsu-3BS with molecular markers should
enhance FHB resistance in breeding populations. The absence of consistent QTL by NIL
pair interaction across 17 different cross combinations indicates that Qfhs.ndsu-3BS should
increase FHB resistance independent of genetic background. We are producing fine mapping populations developed from the most promising NIL pairs to further define this QTL
region.
T able 1. T rait m eans fo r check cu ltivars an d Q fh s.nd su -3B S N IL p airs at tw o n ursery locatio ns.
S t.P aul
C ro ok ston
D isease
V SK
30 -S p ike S eed
D isease
V SK
30 -S p ike S eed
E n try
S everity (% )
(% )
W eig ht (g )
S everity (% )
(% )
W eig ht (g )
A lsen
33
20
9 .5
16
7
1 7 .9
B acU p
24
20
1 4 .2
12
9
2 0 .6
H J98
55
19
1 2 .5
19
19
1 9 .9
Iv an
34
38
9 .0
9
25
1 8 .5
N D 2 6 03
18
12
1 7 .2
8
6
2 2 .3
N D 2 7 10
10
14
2 2 .4
4
3
2 8 .1
R eed er
58
40
7 .6
18
15
1 8 .9
P arsh all
39
30
9 .9
17
13
2 0 .9
R o b lin
74
40
9 .4
54
12
1 8 .8
S u m ai3
5
2
2 1 .5
3
2
1 9 .2
V erd e
42
24
1 1 .1
19
18
1 7 .9
W heaton
75
45
1 3 .7
53
40
1 8 .9
N IL p airs
28
20
1 4 .5
15
11
2 3 .1
T able 2. A N O V A of Q fhs.nd su -3 B S N IL s fo r th ree F H B traits at S t.P aul field n ursery .
S o urce
R ep lication
N IL -P air
Q fhs.nd su -3 B S
N IL -P air*Q T L
E rro r
* , ** , ** *
D isease S ev erity
df
M S
3
0 .0 7 ** *
39
0 .1 2 ** *
1
0 .41** *
39
0 .0 1 ** *
S K 's
df M S
1 172*
39 23 8 ** *
1 67 7 ** *
39 14
2 41
0.0 05
81 28
E ffect sign ifican t at P < 0.05 , 0 .0 1, an d 0.00 1 , resp ectiv ely
S p ike S eed W t
df M S
1 0 .1
39 5 4 .6 ** *
1 32 .6 * *
39 4 .1
81 4 .4
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2002 National Fusarium Head Blight Forum Proceedings
T able 3. A N O V A of Q fhs.nd su-3 B S N IL s fo r th ree F H B traits at C ro ok sto n field n ursery .
S o urce
D isease S ev erity
V S K 's
30 -S p ike S eed W t
df
M S
df
M S
df
M S
R ep lication
3
0.01 * *
1
2
1
1 2 .7
N IL -P air
39
0 .0 3 ** *
39
13 2 ** *
39
6 0 .4 ** *
Q fhs.nd su -3 B S
1
0 .0 3 ** *
1
35 9 ** *
1
1 9 .1 *
N IL -P air*Q T L
39
0.0 03
39
19
39
7 .2 *
2 39
0.0 03
81 18
E ffect sign ifican t at P < 0 .0 5 , 0 .0 1 , an d 0 .0 0 1, respectiv ely
81
4 .7
E rro r
* , ** , ** *
*
1 00
*
% In fec ted S p ike lets
80
*
*
*
60
*
*
*
*
*
*
*
8
9
*
40
20
0
1
2
3
4
5
6
7
10
11
12 13 14 15
16
17
18
19
20 21 R /S a
Q fh s.nd su-3 B S N IL P a ir
F ig u re 1. R esults from one greenhouse point-inoculation disease screening of Q TL-N IL pairs.
F ifteen of the tw enty-one pairs are from unique cross com binations. O pen bars indicate lines w ith
Q fhs.ndsu-3B S alleles; black bars indicate sib lines w ithout Q fhs.ndsu-3B S . a M ean of resistant (R )
parents w ith Q fhs.ndsu-3B S and susceptible (S ) parents. *S ignificant at P < 0.05
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2002 National Fusarium Head Blight Forum Proceedings
ACKNOWLEDGEMENTS
The authors would like to acknowledge R. Dill-Macky, C. K. Evans, G.Thompson, and G.
Linkert for their help in disease screening nurseries and procedures. Funding was provided
by the U.S. Wheat and Barley Scab Initiative, via USDA-ARS.
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 QTLs its two
wheat populations. Theoretical & Applied Genetics 102:1164-1168.
Buerstmayr, H., M. Lemmens, L. Hartl, L. Doldi, B. Steiner, M. Stierschneider, and P. Ruckenbauer. 2002.
Molecular mapping of QTLs for Fusarium head blight resistance in spring wheat. I. Resistance to fungal spread
(type II resistance). Theoretical & Applied Genetics 104:84-91.
Cregan, P., and Q.J. Song. 2002. BARC Primer Pair Annotations [Online]. Available by USWBSI http://
www.scabusa.org/pdfs/BARC_SSRs_011101.html (posted 11-06-01; verified 11-8-02).
Jones, R.K., and C.J. Mirocha. 1999. Quality parameters in small grains from Minnesota affected by Fusarium
head blight. Plant Disease 83:506-511.
Liu, S. and J.A. Anderson. 2002. Marker assisted evaluation of Fusarium head blight resistant wheat
germplasm. Crop Science in press.
Roder, M.S., V. Korzun, K. Wendehake, J. Plaschke, M.H. Tixier, P. Leroy, and M.W. Ganal. 1998. A
microsatellite map of wheat. Genetics 149:2007-2023.
Schroeder, H.W., and J.J. Christensen. 1963. Factors Affecting Resistance of Wheat to Scab Caused by
Gibberella zeae. Phytopathology 53:831-838.
Waldron, B.L., B. Moreno-Sevilla, J.A. Anderson, R.W. Stack, and R.C. Frohberg. 1999. RFLP mapping of QTL
for Fusarium head blight resistance in wheat. Crop Science 39:805-811.
Zhou, W.C., F.L. Kolb, G.H. Bai, G. Shaner, and L.L. Domier. 2002. Genetic analysis of scab resistance QTL in
wheat with microsatellite and AFLP markers. Genome 45:719-727.
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SSR MAPPING OF FUSARIUM HEAD BLIGHT RESISTANCE IN WHEAT
Xiaorong Shen* and Herbert Ohm
Agronomy Department, Purdue University, West Lafayette, IN 47907-1150
*Corresponding Author: PH: (765)494-9138; E-mail: xshen@purdue.edu
ABSTRACT
Fusarium head blight (FHB) of wheat, caused mainly by Fusarium graminearum Schwabe
(telemorph Gibberella zeae), causes reduced yield and lowered grain quality. Identification
of resistance sources and understanding the genetic basis of the resistance is beneficial to
wheat breeding for FHB resistance. Two recombinant inbred wheat populations were developed by single-seed descent from the crosses ‘Ning 894037 × Alondra’ and ‘Patterson ×
F201R’, respectively. The phenotypic evaluation of the RI population Ning 894037 ×
Alondra displayed a continuous distribution with two peaks, suggesting a gene with large
effect controlling the resistance coupled with some genes with relatively small effects. SSR
marker analysis revealed three chromosomal regions associated with FHB resistance in
this population, located on chromosomes 3B, 2D and 6B. The QTL on 3B accounted for
42.5% of the phenotypic variation. The three QTLs collectively explained 51.6% of the
phenotypic variation. SSR marker analysis also provides evidence that the 3BS QTL in
Sumai 3 was derived from Taiwan Wheat instead of the Italian line ‘Funo’, which was
thought to be the donor of FHB resistance from previous pedigree analysis. In the RI population of Patterson × F201R, the phenotypic distribution is bell-shaped, suggesting quantitative inheritance of FHB resistance. Four chromosomal regions associated with resistance to
FHB were identified in this population with SSR markers. The QTLs on chromosomes 1B
and 3A have relatively large effects and accounted for 18.7% and 13.0% of the phenotypic
variation, respectively. The four QTLs jointly accounted for 32.7% of phenotypic variation.
The mapping results showed the genetic diversity of resistance genes in Ning 894037 and
F201R, which represent the Chinese and European resistant sources, respectively. SSR
markers closely linked to FHB resistance QTLs in these two parent lines may be helpful in
breeding programs using marker assisted selection.
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SUMMARY REPORT ON THE 2002 NORTHERN UNIFORM
WINTER WHEAT SCAB NURSERY (NUWWSN)
Clay Sneller1*, Patrick Lipps2, and Larry Herald1
Dept. Horticulture and Crop Science and 2Dept. Plant Pathology,
The Ohio State University, OARDC, Wooster, OH 44691
*Corresponding Author: PH: (330)263-3944; E-mail: sneller.5@osu.edu
1
OBJECTIVE AND INTRODUCTION
This report is a compilation and analysis of data from the cooperative assessment of resistance to
Fusarium head blight (scab) (causal agent Fusarium graminearum (teleomorph: Gibberella zeae
Schwabe.)) in winter wheat germplasm adapted to the northern regions of North America. The
report can be accessed in its entirety on the USWBSI web site.
METHODS
There were 46 lines and four checks in the 2002 trial (Table 1). The entries were successfully
evaluated in 13 field tests and five greenhouse tests. Data was collected on heading date (HD),
height (HGT), disease severity (SEV), disease incidence (INC), disease index (IND), kernel rating
(KR), percent scabby seed (%SS), and DON.
Entry means were analyzed and least square estimate of means over tests were obtained. The
entry x test interaction (ETI) appeared quite large for disease index, incidence, and severity from the
field and greenhouse, so multivariate statistics were used to analyze the interaction and group those
tests that produced similar results for each trait. Entry means were then calculated over the tests
that produced similar rankings. Sets of test that produced similar rankings of entries were called
megaenvironments (ME).
RESULTS
Entry was a significant source of variance for all traits. There was little ETI for heading date, height,
kernel rating, % scabby seed, or DON. Thus, entry means over all tests are appropriate estimators
of genetic value (Table 1). ETI seemed to be an important source of variation of disease severity
from field and greenhouse trials, disease incidence, and disease index.
The ETI for incidence accounted for 30% of the treatment sum of squares. Seven of the nine tests
were place into two ME. One ME consisted of IL+VA and the other consisted of
IN+KY+MO+NY+OH. The correlation of entry means within an ME was generally greater than 0.50.
The NE and ONT tests did not fit in any ME. The correlation between the two MEs was 0.12, suggesting that entry ranking varied between the two MEs. If we were to select the five entries with
lowest incidence in each ME, only one of the selections (IL97-6755) would be the same in both MEs.
Only three (IL97-6755, IL97-1828, and MO981020) of the best 10 selections would be the same in
both MEs. None of five selections for highest incidence would be the same in both MEs. IL97-6755
had the lowest incidence in both MEs, but would be ranked 10th in NE and 28th in ONT. Better
selection concordance would be expected between ONT and NE, or between either outlier test and
either ME. None of the 10 entries selected for low incidence in either ME would be among the five
worst in the other ME. The entry means for incidence in the IL+VA ME were positively correlated
with heading date suggesting the earlier lines may have escaped some affect of the disease. The
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2002 National Fusarium Head Blight Forum Proceedings
opposite trend was present in the other ME (IN+KY+MO+NY+OH) and this may explain why these
MEs gave different entry rankings.
The ETI for field severity accounted for 25% of the treatment sum of squares. Six of nine tests were
places into two MEs (IL+KY+MO+VA and IN+OH). The remaining four tests (NE, NY, ONT, and
SD) were outliers. The correlation of entry means between the two MEs was 0.43, though entry
ranking was different in each ME. Only one (MO980829) of five selections for low severity would be
the same in both ME. There is better concordance if selection pressure is relaxed as six of 10
selections for low severity would be the same in both MEs. None of the five entries selected for high
severity were the same in both MEs. One entry (KY92C-0158-63) among the 10 entries with lowest
severity in the IN+OH ME would be among the five worst in the IL+KY+MO+VA ME.
The ETI interaction for index accounted for 24% of the treatment sum of squares. The ETI pattern
for index was similar to that found for severity as tests that grouped in the same ME for severity
were also grouped together for index. Nine of the 13 tests were placed into two MEs:
IL+KY+MO+VA and AR(2)+IN+KS+OH. The remaining tests (NE, NY, ONT and SD) appeared to
be outliers. The existence of two MEs and four outlier tests show the complex ETI pattern for
disease index. The correlation of entry means between the two MEs was 0.10. Assuming selection
of five entries for low index in each ME, only two entries (IL97-1828 and MO980829) would be
selected in both MEs. Only only four (IL97-1828, MO980829, IL97-6755, and MO981020) would be
selected in both MEs if the ten entries with the lowest index were selected in both MEs. No entry
would be select among the five worst entries in both MEs. None of the 10 entries selected for low
index in either ME would be among the five worst in the other ME. The entry means for index for the
AR(2)+IN+KS+OH ME were negatively correlated to heading date, while entry means for index from
the other ME were positively correlated with heading date. Thus the interaction of heading date with
index may explain why these ME provided different entry rankings.
The ETI for severity in greenhouse assays accounted for 27% of the treatment sum of squares and
three of five tests were placed in a ME (AR+IL+MO). Correlation among these three tests all exceeded 0.55. The IN and KY tests were outliers, though both were more correlated to the ME (r =
0.42) than to each other (r = 0.22). Assuming selection of the best six entries in each AR+IL+MO,
IN, and KY, only about 25% of the selections would be the same between any two tests. Only 20%
of the entries selected for high severity would be the same between two tests.
Using entry means over all tests, heading date and height were not correlated to any disease trait
(exceptions within certain ME are discussed above). There was a high correlation between the
three head disease traits (incidence, severity, and index), and between the head disease traits and
kernel rating and percent scabby seed. Field severity and index were correlated to greenhouse
severity, and this relationship held even when severity and index were averaged within MEs.
Each entry was compared using LSD to the entries with the highest and lowest value for each of the
seven disease traits. Three entries from Missouri and three from Illinois had low values for all seven
traits (Tables 1 and 2). One entry from New York had low scores for six of seven traits while the two
resistant checks (Ernie and Freedom) were low for five of seven traits. All nine of the resistant
entries had low scores for percent scabby seed, DON, and severity in the greenhouse. Twelve
entries had high scores for at least five of seven disease traits. All 12 had high scores for incidence,
field severity, disease index, and kernel rating. Two entries had high scores for all seven traits,
including the susceptible check (Pioneer 2545).
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2002 National Fusarium Head Blight Forum Proceedings
Table 1. Entry means for 2002 NUWWSN. Each entry was compared to the lowest (l) and highest
(h) means in each column using LSD(0.05). “# low scores” is the number of disease traits for which
an entry received a low score, “# high scores” is the times it received a high score.
Trait:
# of tests:
HD
HGT
INC
SEV
IND
KR
%SS
DON
9
7
9
10
13
4
4
2
5
# low
# high
in
%
%
%
0-100
%
PPM
%
scores
scores
54.2 h
1
4
44.8
0
5
Units Days
SEV-GH
1
KY90C-054-6
139
37.3
55 h
34.1 h
23.2
21.4 h
22
16.6 l
2
KY93C-0876-66
140
35.3
64.9 h
40 h
30.1 h
30.9 h
21.5
21 h
3
KY92C-0010-17
140
37
67.2 h
38.5 h
29.5 h
31.1 h
26.7 h
26.3 h
31.2 l
1
6
4
KY92C-0158-63
142
36.3
68.3 h
31.8
27 h
21.6 h
23.6 h
19.8 l
42.8
1
4
5
VA01W447
135
35.6
61.4 h
41.8 h
31.9 h
27.3 h
27.3 h
48.8 h
1
6
6
VA01W461
137
36.6
53.8 h
28.8 l
18.8
24.9 h
14.3 l
15.8 l
40.2
3
2
7
VA01W462
135
34.6
61.8 h
38.4 h
29.4 h
27.5 h
17.5 l
13 l
34.9
2
4
8
VA01W465
139
32.6 l
66.9 h
36 h
28.4 h
20.1 h
22.5 h
28.8 h
40.6
0
6
35.1
62.9 h
36.8 h
30.2 h
29.2 h
24.1 h
14.3 l
55.5 h
1
6
52.9 h
37 h
24.2
25.3 h
17.1 l
20.3
36.5
1
3
46.7
2
4
9
VA01W469
137
10
P97397J1-4-1-4
135
11
P97395B1-4-5-9
133 l
34 l
†
34.1 l
48
41.4 h
18 lh
22.2 h
18 l
12
P97395B1-4-2-7
134 l
34.6
46.9
22.3
13.3 l
14.2 l
14 l
32.7 l
4
0
13
P981128A1-23-1
137
36.3
52.9 h
37.7 h
25.3 h
22.4 h
20.5
15 l
48.9 h
1
5
14
P981238A1-1-11
137
33.6 l
44.3
28.2 l
17.2
15
OH708
140
38.1
54.9 h
41 h
28 h
16
OH712
141
41 h
56.4 h
38.7 h
25.3 h
17
OH719
142
38.9 h
53.4 h
28.8 l
19.7
21.9 h
18
OH720
141
39.9 h
53.2 h
41.2 h
25.6 h
24.7 h
23.5 h
11 l
19
OH685
136
37
54.6 h
45.9 h
28.2 h
26 h
23.3 h
24.8 h
20
IL96-6472
135
36.1
41.9 l
30.9
21
IL97-1828
137
36.3
29.5 l
24.6 l
22
IL97-6755
138
40.6 h
26 l
26.4 l
14.6 l
8.6 l
8.7 l
3 l
19.6 l
7
0
23
IL97-7010
136
39.1 h
38.6 l
29 l
15.7 l
14.4 l
12.7 l
16.3 l
18.6 l
7
0
37.7
24
IL98-6718
135
25
MILLENNIUM
142
26
NE98632
141
27
NE99543
139
38.3
28
NY89052SP-9
143 h
29
NY89086-7120
30
33
31.3 h
12 l
21.8 h
16.5 l
23 h
19 l
18.3 l
15 l
4
1
15.2 l
15 l
48.8 h
3
4
22.5 h
15.3 l
56.4 h
1
6
16.9 l
14.5 l
31.5 l
4
2
19
12.5 l
7.8 l
8 l
13.6 l
7.7 l
11 l
11.4 l
44.8
1
5
63.7 h
0
7
34.1
4
0
32.8 l
7
0
43.6
35.2 h
22.3
10.2 l
14 l
9.5 l
44.9
3
1
39 h
38.6 l
30.2
15.1 l
15.6 l
25.6 h
7.5 l
43.4
4
1
39 h
48.2
31.7
18.8
23.3 h
29.2 h
17.3 l
42.4
1
2
40.7 l
38.1 h
21.6
23.9 h
30.7 h
14.3 l
64.3 h
2
4
38.6
42.6
38.3 h
19.2
11.6 l
17.6 l
22.3 h
53.6 h
2
3
142
38.9 h
48.9
39.6 h
23.6
19.8 h
20.4
37.5 h
40.5
0
3
NY89082-7159
144 h
35.9
48.9
35.5 h
19.7
15.1 l
19.3 l
12.3 l
50.3 h
3
2
31
NY89064SP-7139
143 h
37.6
41.6 l
36.1 h
15.6 l
11.7 l
14.5 l
9.5 l
31.9 l
6
1
32
NY89088-7401
143 h
38.9 h
48.4
32.8
18
18.4 h
20.5
21.5 h
39.8
0
2
33
MDV11-52
136
32.4 l
59.1 h
41.5 h
32.9 h
30.2 h
24.8 h
28 h
46.6
0
6
34
M94*1549-1
137
34.3 l
56.7 h
35 h
26.9 h
22 h
24.6 h
14.5 l
38.6
1
5
35
M95-2994-1
140
35
46
20.9 h
23.1 h
20.3
25.5 l
1
2
36
MO980829
141
39.3 h
25.9 l
17.1 l
8.4 l
5.7 l
12.8 l
6.9 l
16.3 l
7
0
37
MO981020
137
36.7
37.2 l
23.5 l
15.9 l
8.6 l
11 l
18.3 l
19.8 l
7
0
38
MO000925
138
36.1
43.6
28.6 l
20.3
21.8 h
15.2 l
16.8 l
34
3
1
39
MO000926
136
34.4 l
40.3 l
16.9 l
13.8 l
14.3 l
17.8 l
24.7 l
7
0
40
MO000969
137
36.1
46.9
42.8 h
23.1
24.8 h
23.8 h
30.3 l
1
4
41
PATTERSON
136
37.1
50.8
40.1 h
29.6 h
18.1 lh
21.4
11.5 l
60.3 h
2
4
31.3
26 l
20.2
27 h
42
FREEDOM
140
37.6
44.6
22.4 l
15.7 l
20.4 h
17.9 l
13.3 l
16 l
5
1
43
PIONEER 2545
140
36.6
59.1 h
38.3 h
28.4 h
28.4 h
34.2 h
33.3 h
52.1 h
0
7
44
ERNIE
134 l
34.1 l
42.6
23.6 l
20
17 l
16.9 l
13.8 l
24.9 l
5
0
45
D9046-1
136
35.7
41.3 l
31.5
22.9
26.7 h
18.2 l
25.8 h
67.3 h
2
3
46
D9070-1
141
37.7
52
36.7 h
19.7
18.3 lh
13.3 l
17.5 l
33.5 l
4
2
Average
138
37
49.3
34
22.5
19.7
19.5
17.1
38.9
LSD (0.05)
1.8
2.2
16.1
12.7
8.7
13.2
12.2
15
19
†
Indicates a mean that is not different from the lowest (l) or highest (h) mean in the column based on
LSD(0.05)
Variety Development and Uniform Nurseries
263
2002 National Fusarium Head Blight Forum Proceedings
Table 2. Entry means for the most tolerant (top) and susceptible (bottom) entries in the 2002
NUWWSN
Trait:
# of tests:
HD
HGT
INC
SEV
IND
KR
%SS
DON
9
7
9
10
13
4
4
2
5
# low
# high
in
%
%
%
0-100
%
PPM
%
scores
scores
0
Units Days
SEV-GH
21
IL97-1828
137
36.3
29.5 l
24.6 l
13.6 l
7.7 l
11 l
11.4 l
32.8 l
7
22
IL97-6755
138
†
40.6 h
26 l
26.4 l
14.6 l
8.6 l
8.7 l
3 l
19.6 l
7
0
23
IL97-7010
136
39.1 h
38.6 l
29 l
15.7 l
14.4 l
12.7 l
16.3 l
18.6 l
7
0
36
MO980829
141
39.3 h
25.9 l
17.1 l
8.4 l
5.7 l
12.8 l
6.9 l
16.3 l
7
0
37
MO981020
137
36.7
37.2 l
23.5 l
15.9 l
8.6 l
11 l
18.3 l
19.8 l
7
0
39
MO000926
136
34.4
40.3 l
26 l
16.9 l
13.8 l
14.3 l
17.8 l
24.7 l
7
0
31
NY89064SP-7139
1
42
FREEDOM
44
ERNIE
l
143 h
37.6
41.6 l
36.1 h
15.6 l
11.7 l
14.5 l
9.5 l
31.9 l
6
140
37.6
44.6
22.4 l
15.7 l
20.4 h
17.9 l
13.3 l
16 l
5
1
134 l
34.1
42.6
23.6 l
20
17 l
16.9 l
13.8 l
24.9 l
5
0
34.1 h
23.2
21.4 h
22
16.6 l
54.2 h
1
4
21.6 h
23.6 h
19.8 l
42.8
1
4
l
1
KY90C-054-6
139
37.3
55 h
4
KY92C-0158-63
142
36.3
68.3 h
40
MO000969
137
36.1
46.9
42.8 h
23.1
24.8 h
27 h
23.8 h
30.3 l
1
4
27
NE99543
139
38.3
40.7 l
38.1 h
21.6
23.9 h
30.7 h
14.3 l
64.3 h
2
4
15
OH708
140
38.1
11
P97395B1-4-5-9
133 l
34.1
41
PATTERSON
136
7
VA01W462
135
2
KY93C-0876-66
140
35.3
34
M94*1549-1
137
34.3
54.9 h
31.8
27 h
41 h
28 h
16.5 l
15.2 l
15 l
48.8 h
3
4
48
41.4 h
31.3 h
18 lh
22.2 h
18 l
46.7
2
4
37.1
50.8
40.1 h
29.6 h
18.1 lh
21.4
34.6
61.8 h
38.4 h
29.4 h
27.5 h
17.5 l
l
l
64.9 h
40 h
30.1 h
30.9 h
56.7 h
35 h
26.9 h
22 h
21.5
11.5 l
13 l
60.3 h
2
4
34.9
2
4
21 h
44.8
0
5
24.6 h
14.5 l
38.6
1
5
18
OH720
141
39.9 h
53.2 h
41.2 h
25.6 h
24.7 h
23.5 h
11 l
44.8
1
5
13
P981128A1-23-1
137
36.3
52.9 h
37.7 h
25.3 h
22.4 h
20.5
15 l
48.9 h
1
5
3
KY92C-0010-17
140
37
67.2 h
38.5 h
29.5 h
31.1 h
26.7 h
26.3 h
31.2 l
1
6
33
MDV11-52
136
32.4
l
59.1 h
41.5 h
32.9 h
30.2 h
24.8 h
28 h
46.6
0
6
16
OH712
141
41 h
56.4 h
38.7 h
25.3 h
23 h
22.5 h
15.3 l
56.4 h
1
6
5
VA01W447
135
35.6
61.4 h
41.8 h
31.9 h
27.3 h
27.3 h
12 l
48.8 h
1
6
8
VA01W465
139
32.6
66.9 h
36 h
28.4 h
20.1 h
22.5 h
28.8 h
40.6
0
6
9
VA01W469
137
35.1
62.9 h
36.8 h
30.2 h
29.2 h
24.1 h
14.3 l
55.5 h
1
6
19
OH685
136
37
54.6 h
45.9 h
28.2 h
26 h
23.3 h
24.8 h
63.7 h
0
7
43
PIONEER 2545
140
36.6
59.1 h
38.3 h
28.4 h
28.4 h
34.2 h
33.3 h
52.1 h
0
7
Average
138
37
49.3
34
22.5
19.7
19.5
17.1
38.9
LSD (0.05)
1.8
2.2
16.1
12.7
8.7
13.2
12.2
15
19
l
†
Indicates a mean that is not different from the lowest (l) or highest (h) mean in the corresponding column in
Table 1 based on LSD(0.05)
Table 3. Possible sources of resistance for the most resistant entries in Table 2.
Entry
IL97-1828
IL97-6755
IL97-7010
MO980829
Pedigree
Possible source of resistance
P818311-16-2-1-2-3-3/IL90-4813
IL90-4813//IL85-3132-1/NING7840 Ning 7840
IL90-6363//IL90-9464/NING7840
Ning 7840
MO11769/MADISON
MO11769 which is not a descendent of Ernie,
Sumai 3, or Ning 7840
MO981020
MO11769/MADISON
MO11769 which is not a descendent of Ernie,
Sumai 3, or Ning 7840
MO000926
ERNIE/AP HICKORY
Ernie
NY89064SP-7139
88029(84061(6120-15/F29Harus and 6120-15 (Geneva) are moderately
76)/AUGUSTA)/HARUS
resistant
Variety Development and Uniform Nurseries
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2002 National Fusarium Head Blight Forum Proceedings
FUSARIUM HEAD BLIGHT IN HEXAPLOID WHEAT POPULATIONS
DERIVED FROM LINES WITH TYPE I RESISTANCE
R.W. Stack1*, R.C. Frohberg2, and M. Mergoum2
1
Dept of Plant Pathology and 2Dept of Plant Sciences, North Dakota State University, Fargo, ND 58105
*Corresponding Author: PH: 701-231-7077; E-mail: rstack@ndsuext.nodak.edu.
ABSTRACT
Fusarium head blight (FHB) of wheat, caused mainly by Fusarium graminearum Schwabe,
is a serious problem in North Dakota spring wheat.
An adapted, FHB resistant spring wheat, ND2710, was selected from some Sumai 3 derived lines in 1993 and been used in many crosses in the North Dakota breeding program
since that time. It was a parent of the ND cultivar ‘Alsen’, released in 2000, and planted on
over 2 million acres in 2002. The FHB resistance of ND2710 has proven durable over
several years and in a wide range of environments. One flaw of the resistance pattern of
ND2710 is its acceptance of individual primary infections, even though these infections do
not spread to adjacent spikelets. To find a parent which will better resist primary infection
we tested two populations derived from three-way crosses between adapted ND spring
wheat and two lines thought to possess resistance to primary infection (= “Type 1” resistance). One parent was ‘Frontana’, the line in which type 1 resistance was first described;
the other was ‘W9207’, a line derived from intercrossing of 6 of the best Chinese resistance
sources. The two populations were advanced to F-5 by single seed descent and the lines
were tested for FHB in an inoculated, mist-irrigated field nursery in 2001. At 3.5 weeks
post-inoculation, approx. 50-60 spikes in each of two reps were individually scored for FHB
on a 0-100% scale. Grain was harvested from mature spikes and proportion of visually
scabby kernels and level of deoxynivalenol (DON) in the harvested grain determined. The
two populations were similar in mean and distribution for all FHB measures. Overall about
5% of lines had FHB disease measures similar to or better than ND2710, but 22% and 27%
of lines from these populations had FHB incidence values lower than this standard. The
best lines from these populations had incidence values indicating less than half as many
primary infections as ND2710.
(This poster was presented at the Annual Meeting of Amer. Soc. Agron., Indianapolis, IN,
Nov 10-14, 2002)
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2002 National Fusarium Head Blight Forum Proceedings
SCAB SCREENING USING FROZEN SPIKES
A.J. Stewart, B. Kennedy, and D. A. Van Sanford*
Department of Agronomy, University of Kentucky, Lexington, KY40546-0091
*Corresponding Author: PH: 859-257-5811; E-mail: dvs@uky.edu
ABSTRACT
Evaluation of Fusarium head blight (FHB) in the field environment is difficult. The amount of
material in the scab nursery is large and reading the symptoms before the heads begin to
mature can be problematic. To extend the days of reading symptoms a method was suggested in which spikes are harvested 21 days after flowering and frozen (Personal communication, R. Stack via D. Hershman). Spikes from two elite tests, Magnum and Mondo, along
with the checks in the Kentucky variety trial were frozen and data collected from the spikes
in 2002. A sample of approximately 100 spikes per row was harvested with a hand sickle at
two locations, Lexington and Princeton, KY. The spikes were placed into resealable bags
then placed into an ice chest. The samples were transferred into a freezer and were kept
there until needed. The frozen spikes were read at various times. The samples remained
green and symptoms were still visible. Disease incidence was calculated by counting the
number of infected spikes per bag divided by the total number of spikes. Average head
severity was assessed by evaluating 15 infected heads per bag. The preliminary data indicates that freezing infected heads is an effective tool for reading scab symptoms. The data
was analyzed by comparing severity rankings of entries that were frozen in 2002 to field
samples in 2001. Severity values were also compared to greenhouse severity values.
Harvesting wheat at 21 days after flowering does not give peak severity of FHB on the entry
row; however, the severity of the checks were similar between field rankings and frozen
spike rankings in 2002 (r2=0.88, P=0.01). The correlation from the elite test field spikes in
2001 and frozen spikes in 2002 was less encouraging (r2=0.18 P=0.07, both locations).
Further testing will occur during the upcoming year on improving the method into a efficient
tool for screening and selection of resistant FHB genotypes.
Variety Development and Uniform Nurseries
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2002 National Fusarium Head Blight Forum Proceedings
FUSARIUM GRAMINEARUM AND DON IN SINGLE SEEDS
FOLLOWING GREENHOUSE POINT INOCULATION
Dennis M. TeKrony1*, David VanSanford1, Marcy Rucker1,
Cheryl Edge1 and Yanhong Dong2
Department of Agronomy, University of Kentucky, Lexington, KY 40546-0091; and
2
Department of Plant Pathology, University of Minnesota, St Paul, MN 55108
*Corresponding Author: PH: 859-257-3878; E-mail: dtekrony@ca.uky.edu
1
ABSTRACT
The single floret inoculation system is commonly used to screen wheat cultivars and
germplasm for FHB Type II resistance in the greenhouse by a visual rating of the spread of
fungal hyphae in the spike and spikelets. Evaluation of this system in our laboratory across
a wide range of germplasm has shown that the visual ratings of spikelet infection are poorly
associated with the Fusarium graminearum (Schwabe) infection occurring in the seed,
rachis and other floral components the same spikelets. The objective of this research was to
use the single floret inoculation system to relate ratings of visual spikelet infection in the
greenhouse to F. graminearum infection and deoxynivalenol levels in seeds of adjoining
florets in all individual spikelets on each infected spike. The movement of fungal hyphae
and DON into the various components of the spike was evaluated following point inoculation (PI) of a floret at a middle location of the spike for two susceptible (P 2555 and VA 96W326) and three resistant (P 25R18, Roane, Coker 9474) cultivars. Although high levels of
spikelet infection occurred in the susceptible cultivars in the greenhouse, the fungal movement in the spike occurred primarily in two ways; localization around the PI and movement
down the spike from the PI. Thus, severity of greenhouse infection overestimated F.
graminearum seed infection and DON presence in susceptible cultivars and underestimated
fungal infection and DON in resistant cultivars. A close relationship was shown between the
presence of F. graminearum in seed from the right floret with the presence of DON in seed
from the left floret in both susceptible and resistant cultivars. Although DON was present in
seed of resistant cultivars the levels were much lower than susceptible cultivars and often
did not exceed 1 PPM. This investigation should allow us to evaluate the current methods
for screening for Type II resistance to FHB infection in an attempt to develop more accurate
methods. (This research will be presented as a poster at the annual meeting of the US
Barley and Wheat Scab Initiative in Covington, KY on December 7-9, 2002.)
Variety Development and Uniform Nurseries
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2002 National Fusarium Head Blight Forum Proceedings
HOW TO MAKE INTELLIGENT CROSSES TO ACCUMULATE
FUSARIUM HEAD BLIGHT RESISTANCE GENES BASED
ON KNOWLEDGE OF THE UNDERLYING
RESISTANCE MECHANISMS
M. van Ginkel* and L. Gilchrist
CIMMYT, Apdo. Postal 6-641, Mexico, D.F., MEXICO 06600
*Corresponding Author: PH: 52-55-5804-2004; E-mail: m.van-ginkel@cgiar.org
OBJECTIVES
To describe crossing strategies to accumulate mechanisms of resistance to FHB.
INTRODUCTION
A number of mechanisms or Types of resistance, likely coded for by specific genes, underpin wheat’s final response to Fusarium head blight (FHB). Accumulating additively inherited
resistance genes to enhance genetic control of FHB has been proposed (Anonymous, 2001;
Singh and van Ginkel, 1997). But in the absence of specific information on these genes
gene-based breeding is not yet feasible. Until the specific genes that cause these phenotypic response mechanisms are identified, disease evaluation for each mechanism has to
be conducted independently.
However, in the case of FHB, intelligent crosses can be made by applying our knowledge of
the various resistance mechanisms responsible for its genetic control.
Characterizing FHB Resistance
Before applying the mechanisms of resistance present in genetic stocks to a crossing
scheme, they must be carefully characterized, which can be an arduous process.
A description of how the different mechanisms of resistance to FHB are determined at
CIMMYT follows. We base our approach on published literature and local experience. The
Fusarium sp. used mainly in our work is F. graminearum.
Specific inoculation, screening, and evaluation techniques are used for each type of resistance to avoid confounding the disease response observations of the various resistance
mechanisms. Because genotype x environment interactions may affect the ranking of genotypes for any one of the resistance mechanisms, an appropriate number of replications
across and within years is needed. The level of interaction appears to be linked to the level
of resistance: if a genotype’s resistance level is already pretty high and based on the presence of several mechanisms, it will be less affected by the environment and will express
more stable resistance. Resistance based on just one mechanism is more susceptible to
environmental effects.
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2002 National Fusarium Head Blight Forum Proceedings
The targeted germplasm and check cultivars are always inoculated on the same day. Later
upon harvest the response of the tested lines is compared with that of the checks inoculated
on the same day. If this is not done, erroneous and spurious response readings may result. A
broad spectrum of checks is used, ranging from the highest level of susceptibility to the
highest level of resistance, with at least two representatives at each level.
Type I (penetration by the fungus) and II (spread of mycelium throughout the spike) resistances were identified as distinct mechanisms by Schroeder and Christensen in 1963. Two
other mechanisms or types were proposed by Miller and Arnison (1986), Wang and Miller
(1988), and Mezterhazy (1995).
Type I resistance - To avoid interaction with height and maturity, inoculation for Type I
resistance is done by spraying a fusarium spore suspension (50,000 spores per ml) on a
horizontal plane onto labeled spikes at the onset of anthesis. Spikes are evaluated for
resistance a fixed number of days post inoculation, depending on the prevailing conditions
and the disease reaction of the resistant and susceptible checks. The interval (25 to 35
days) between inoculation and evaluation may vary from year to year. However, within the
same year a fixed number of days is used. Thirty days is generally appropriate under Mexican conditions.
Type II resistance - A tuft of cotton gently soaked in inoculum (50,000 spores per ml) is
inserted into a floret in the center of the spike at the onset of anthesis with a pair of tweezers;
each spike is then covered with a glassine pollination bag. The lightweight, narrow bag
prevents additional FHB inoculum from entering the spike through allo-infection and helps
maintain a high level of humidity, which favors disease development. Spikes are evaluated
for resistance 25-35 days post-inoculation.
Type III resistance - Fusarium graminearum produces mycotoxins, especially
trichothecenes, during the infection process. Type III resistance is associated with degradation of toxins in the grain, as described by Miller and Arnison (1986). In preparation for toxin
evaluation and quantification, genotypes are sprayed with an inoculum suspension when
50% of the spikes in a plot have reached anthesis. A 20-g seed sample from the inoculated
spikes is collected at harvest, and resistance is evaluated by quantifying the accumulated
toxin in the laboratory using the FluoroQuant Rommer method.
Resistance types IV and V - Wang and Miller (1988) described Type IV resistance as
tolerance to high DON concentrations. They reported that some cultivars can tolerate high
mycotoxin concentrations with no negative effects on growth. A six-year study led to the
conclusion that this tolerance should be evaluated as a relative parameter of infection, and
that yield response may help describe the disease reaction of the genotypes (Mezterhazy,
1995).
To identify Type IV resistance, a paired plot of each genotype is planted. One plot is treated
as described for Type I, and the other is sprayed with a functional fungicide (e.g., Folicur
Plus) three times during the cycle. The test weight of grain harvested from the fungicidetreated plot is compared with that of grain from the inoculated plot to calculate the percentage loss.
Variety Development and Uniform Nurseries
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2002 National Fusarium Head Blight Forum Proceedings
To evaluate for Type V resistance, grain from the two plots described above is visually
scored (1=very healthy and plump; 5=diseased and shriveled) at the same time to determine
relative grain filling. This parameter has not been described in the literature as a FHB resistance mechanism, but is used at CIMMYT to complement and aid in identifying Type IV
resistance.
EVALUATING PARENTAL STOCKS
All germplasm being considered for crossing is first characterized for FHB resistance, as
described above and as depicted in the Table 1. Commonly these materials include established sources of resistance, promising introductions contributed by colleagues, good
combiners for the desired agronomic traits, and major varieties in the target region. This
information is later used as the basis for making appropriate crosses.
Table 1. Parental characterization for FHB resistance mechanisms. Codings using bold/
Italics/underline/non-underline indicate the relative levels of resistance, with bold and
underlined lettering representing the highest level.
RESISTANCE MECHANISM or TYPE
Entry Cross
I
II
Damage
(%)
Damage
(%)
2.51
2.66
III
IV
V
Toxin
Grain
Grain
(ppm) losses (%) (1-5)
0
21.16
2
6.07
0.14
13.29
2
21.12
0.52
13.18
2
6.43
2.3
2.36
1*
1.49
10.53
0.026
7.68
1
4.90
13.16
0.21
6.62
1
1
GOV/AZ//MUS/3/DODO/4/BOW
2
MILAN/SHA7
0.00
3
ALUCAN/DUCULA
13.73
4
CBRD/KAUZ
3.21
5
R37/GHL121//KAL/BB/3/JUP/MUS/4/2*YMI #6/5/CBRD
6
GUAM92//PSN/BOW
7
NG8675/CBRD
0.26
8.20
0.48
7.67
1
8
ALTAR 84/AE.SQUARROSA (224)//ESDA
4.42
16.89
0.49
1.75
1
9
BCN*2//CROC_1/AE.SQUARROSA (886)
11.56
4.82
0.38
1.68
1*
10
MAYOOR//TK SN1081/AE.SQUARROSA (222)
0.86
7.26
0.49
1.3
1*
11
SABUF/5/BCN/4/RABI//GS/CRA/3/AE.SQUARROSA (190)
1.98
8.46
0.069
8.07
2
12
SHA3/CBRD
3.87
5.99
0
6.94
1
Coding
123
123
123
123
FHB score
very good
good
moderate
poor
CROSSING STRATEGIES
If crossing is to be effective, the mechanisms of resistance should be complementary among
parental stocks, as is evident in Table 1.
Using the information in Table 1, cross combinations can be designed to cross parents that
fully complement one another in the sense that one or the other contributes high levels of
resistance (scored as ‘very good’) for each of the five mechanisms. With luck and properly
xecuted selection, transgressive segregants will subsequently be identified that express
high levels for all five resistance types.
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2002 National Fusarium Head Blight Forum Proceedings
Following parental stock characterization and crossing, the segregating F2 generations are
grown and selected. But also additional crosses can be made on the F1s. We usually opt for
a top cross or, in some cases, a limited backcross, to a line with desirable agronomic type,
high yield potential and yield stability, durable resistance (to other relevant diseases), good
combining ability, and excellent quality. In the case of FHB, we also make doubled haploids
(DH) on a limited number of simple crosses, to enhance our ability to identify homozygous
transgressive progeny in replicated experiments that combine multiple resistance mechanisms.
CONCLUSION
Our approach uses data gathered on the various mechanisms of resistance on relevant
parental stocks to allow more intelligent crossing. Such an approach increases, at least in
theory; the chance of identifying superior progeny carrying accumulated resistance mechanisms against FHB.
EPILOGUE
An improved understanding of the FHB infection process and related resistance mechanisms reveals the potential relationship between FHB and Karnal bunt (KB) resistance. The
infection processes of the two diseases are similar: in both cases, florets are infected during
anthesis, and resistance is very much influenced by environmental fluctuations. Consequently, the same lines may seem resistant in some years but susceptible in others. Alleles
with large effects on resistance have been noted in both diseases (Fuentes-Davila et al.,
1995; Singh et al., 1995), with some lines expressing high levels of resistance following the
introgression of just 2-3 desired alleles. Significant genetic variation for both diseases is
available, and anecdotal evidence suggests that some genetic sources (especially among
the Chinese materials) are resistant to both diseases. If the same genes confer resistance to
the two diseases, this would explain why many Chinese wheats are resistant to KB, though
the disease has not been reported in China. Should this be proven, then these two threats to
wheat production in the USA could be addressed, at least in part, through a concerted
research effort involving groups now independently engaged in research on these two
diseases.
REFERENCES
Anonymous, 2001. National Fusarium Head Blight Forum Proceedings, Holiday Inn Cincinnati-Airport, Erlanger,
Kentucky, USA, December 8-10, 2001.
Fuentes-Davila G., S. Rajaram, and G. Singh. 1995. Inheritance of resistance to Karnal bunt (Tilletia indica
Mitra) in bread wheat (Triticum aestivum L.). Plant Breeding 114: 250-252.
Mezterhazy, A. 1995. Types and components of resistance against FHB o wheat. Plant Breeding 114: 377-386.
Miller, J.D. and Arnison, P.G. 1986. Degradation of deoxinivanenol by suspension cultures of Fusarium head
blight resistant wheat cultivar Frontana. Canadian Journal Plant Pathology 8: 147-150.
Schroeder, H.W. and Christensen, J.J. 1963. Factors affecting the resistance of wheat to scab caused by
Gibberella zeae. Phytopathology 53: 831-838.
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Singh, G., S. Rajaram, J. Montoya, and G. Fuentes-Davila. 1995. Genetic analysis of Karnal bunt resistance in
14 Mexican bread-wheat genotypes. Plant Breeding 114:439-441.
Singh, R.P., M. van Ginkel. 1997. Breeding strategies for introgressing diverse scab resistances into adapted
wheats. Pp. 86-92 in: Fusarium Head Scab: Global Status and Future Prospects. H.J. Dubin, L. Gilchrist, J.
Reeves, and A. McNab (eds.). Mexico, D.F.: CIMMYT.
Wang, Y.Z. and Miller, J.D. 1988. Effects of Fusarium graminearum metabolites on wheat tissue in relation to
Fusarium head blight resistance. J. Phytopathology 122: 118-125.
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APPARENT AND ACTUAL SEED QUALITY IN SOFT RED WINTER
WHEAT INFECTED WITH FUSARIUM GRAMINEARUM
V.L.Verges, B. Kennedy, A.J.Stewart, D. TeKrony and D.A. Van Sanford*
Department of Agronomy, University of Kentucky, Lexington, KY40546-0091
*Corresponding Author: PH: 859-257-5811; E-mail: dvs@uky.edu
ABSTRACT
Head scab caused by Fusarium graminearum (Schwabe) has caused significant losses in
the soft red winter wheat crop in Kentucky and in small grain crops in many regions of North
America. Head scab epidemics not only result in significant yield losses, but also can cause
serious reductions in seed quality. To investigate associations between apparent infection
based on spike symptoms, apparent damage to the kernel and the actual seed infection, we
evaluated fifteen (15) lines of the 2002 Southern Scab Nursery. Seeds of each line were
separated into three categories, depending on the visual aspect of the seed, so as to define
“good quality” seed (without any symptom of infection), “shriveled” seed and “poor” seed
(tombstones).Then, to determine the presence of Fusarium graminearum in these three
classes of seed, seeds were plated, five (5) plates of each class, with ten seeds per plate.
Plates were incubated at 20°C and at seven and fourteen days the presence of F.
graminearum was recorded. Also a DON test was run to test the concentration of DON in
these three categories of seeds. Seeds showed high levels of infection, above 90 % in
seeds of poor quality, between 70-80% in shriveled seed and 40-50 % in seed of good
quality. The good quality seed showed more variation, depending on the line, and conditions
in the field. To evaluate the relation between this actual infection in the seed and field data,
percentage of visually scabby kernels was measured for each line, and then correlated with
the actual presence of F. graminearum in the seed. A better correspondence between apparent infection and actual seed infection could be useful in assessing varieties in the field, and
predicting the real infection in the seed, depending on its quality. Correlations of seed
quality and the presence of F. graminearum with DON will be presented.
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EFFECT OF SUMAI 3 CHROMOSOMES ON TYPE II AND
TYPE V SCAB RESISTANCE IN WHEAT
Wenchun Zhou1, Frederic L. Kolb1*, Larry K. Boze1, Norman J. Smith1,
Guihua Bai2, Leslie L. Domier3 and Jingbao Yao4
Department of Crop Science, University of Illinois, 1102 South Goodwin Avenue, Urbana, IL 61801;
Department of Plant & Soil Sciences, Oklahoma State University, Stillwater, OK 74078; 3Department of Crop
Sciences, USDA-ARS-MWA, 1102 South Goodwin Avenue, Urbana, IL 61801.; and 4Institute of Food Crops,
Jiangsu Academy of Agricultural Sciences, Nanjing 210014, P. R. China
*Corresponding Author: PH: (217) 244-6148 ; E-mail: f-kolb@uiuc.edu
1
2
ABSTRACT
Two sets of substitution lines were developed by crossing individual monosomic lines of
Chinese Spring (recipient) with scab resistant cultivar Sumai 3 (donor) and then using the
monosomics as the recurrent male parent for four backcrosses (without selfing after each
backcross). The disomic substitution lines were separated from selfed BC4F2 plants. Chromosome specific SSR markers were analyzed for polymorphism between Sumai 3 and
Chinese Spring. Polymorphic markers were used to identify substitution lines for specific
chromosomes. Based on the specific SSR markers, chromosome substitutions occurred in
thirty-six lines, and six lines segregated alleles from the two parents or were homozygous
for the allele from Chinese Spring. These substitution lines were used to evaluate Type II
(spread within the head) and Type V (deoxynivalenol accumulation within kernels) scab
resistance. The objective was to use the substitution lines to evaluate the effect of individual
chromosomes of Sumai 3 on Type II and Type V scab resistance in the greenhouse. Significant differences in Type II scab resistance and deoxynivalenol (DON) levels among different
Chinese Spring (Sumai 3) substitution lines were detected. Positive chromosome substitution effects on Type II scab resistance were found on chromosomes 2B, 3B, 6B, and 7A from
Sumai 3. Chromosomes 3B and 7A also reduced DON accumulation within the kernels,
while chromosomes 1B, 2D, and 4D from Sumai 3 increased DON concentration. Chromosome 7A from Sumai 3 had the largest effect on resistance to scab spread and DON accumulation. Additional research is in progress on the scab resistance conferred by chromosome 7A.
Key words: Type II scab resistance, Type V scab resistance, substitution lines, SSR,Triticum
aestivum.
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ESTIMATING THE ECONOMIC IMPACT OF A CROP DISEASE: THE
CASE OF FUSARIUM HEAD BLIGHT IN U.S. WHEAT AND BARLEY
William E. Nganje*, Dean A. Bangsund, F. Larry Leistritz,
William W. Wilson, and Napoleon M. Tiapo
Department of Agribusiness and Applied Economics, North Dakota State University, Fargo, ND 58105
*Corresponding Author: PH: 701-231-7459; E-mail: wnganje@ndsuext.nodak.edu
ABSTRACT
Plant diseases, particularly those affecting major agricultural crops, can have serious economic consequences, both for agricultural producers and for the regional economy. Since
1993, the spring grain producing area in the upper Midwest region of the United States has
experienced a prolonged outbreak of Fusarium head blight (FHB), commonly known as
scab, a fungus disease that affects wheat, barley, and other small grains. The purpose of
this paper is to estimate the direct and secondary economic impacts of FHB infestations of
wheat and barley during the period 1998-2000. The findings indicate that scab continues to
be a major problem for U.S. wheat and barley producers. The cumulative direct economic
losses from FHB in hard red spring (HRS) wheat, soft red winter (SRW) wheat, durum
wheat, and barley is estimated at $870 million from 1998 through 2000. The combined
direct and secondary economic losses for all the crops were estimated at $2.7 billion. Two
states, North Dakota and Minnesota, account for about 55 percent of the total dollar losses.
INTRODUCTION
Plant diseases, particularly those affecting major agricultural crops, can have serious economic consequences, both for agricultural producers and for the regional economy.
Fusarium head blight (FHB), commonly known as scab, is a fungus disease that affects
wheat, barley, and other small grains (McMullen and Stack 1999). FHB results in yield
losses, and infected grain is also subject to price discounts. FHB is recognized as a factor
limiting grain production in many parts of the world. Since 1993, the spring grain producing
area in the upper Midwest region of the United States has experienced a prolonged outbreak of FHB (Stack 1999). Centered on the Red River Valley of North Dakota and Minnesota, the FHB outbreak led to serious losses for farmers (McMullen et al. 1997).
The impact on the U.S. wheat and barley industries has been sufficient to stimulate a national response; a consortium of scientists and agribusiness leaders has developed a “U.S.
Wheat and Barley Scab Initiative” to target research and outreach to solving this disease
problem. Because the Scab Initiative must compete with other agricultural and natural
resource problems to secure funding, the consortium sought estimates of the economic
impact of the scab outbreak. Estimates of economic impact of scab are equally important for
crop quality insurance (USDA, 2000). Traditional estimates focused on lost producer income solely due to reduced yields and abandoned acres, may lead to ineffective design of
quality crop insurance instruments. In the very least, economic impact estimates of a disease must incorporate price impacts in a manner that captures the interaction between yield
reduction and price increases or decreases. The purpose of this paper is to estimate the
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direct and secondary economic impacts of FHB infestations of wheat and barley during the
period 1998-2000.
METHODS AND DATA
Study design required choosing which crops and states should be included. The study
focused on three wheat classes (hard red spring, soft red winter, and durum) and barley. For
hard red spring (HRS) wheat, durum wheat, and barley, the affected states were Minnesota,
North Dakota, and South Dakota. For soft red winter (SRW) wheat, the affected states were
Illinois, Indiana, Kentucky, Michigan, Missouri, and Ohio.
Estimating the direct impacts (first round effects) of FHB for each type of grain entailed
separately estimating (1) the effect on production and (2) the effect on prices received by
producers in each year. These estimates were made for multi-county Crop Reporting Districts (CRDs) in each state. The product of the production and price effects was the estimate
of the direct impact of FHB infestation.
To estimate the economic losses due to FHB in a given CRD, the value of production under
‘normal’ conditions was estimated (i.e., if there had been no outbreak). Normal crop value is
the product of two variables: pn, the price that farmers would have received, and qn, their
expected production in absence of scab. For years of scab outbreak, both variables are
unobserved and must be estimated. The lost crop value is then calculated as the difference
between actual and normal crop value (Johnson et al. 1998, Nganje et al. 2001). Nganje et
al. (2001) provide detail methodology of estimating production loss, price impacts, and
secondary economic impacts.
DATA SOURCES
Data on temperature and precipitation by region were obtained from the National Climatic
Data Center (U.S. Department of Commerce). Data on planted and harvested acres, harvested yield, production, and average prices received by producers were obtained from the
National Agricultural Statistics Service (U.S. Department of Agriculture). Average CBT and
MGE futures prices were derived from a database of weekly quotes collected from Grain
Market News (U.S. Department of Agriculture) and the Wall Street Journal. Basis was
calculated as the difference between average price received in a region and the average
futures price. For North Dakota, prices received were available by crop reporting district; in
other states, prices are based on state averages. Prices for the 2000 marketing year were
based on data available through February, 2001. Data on national wheat and barley supplies were from the Wheat Yearbook published by the Economic Research Service of the
U.S. Department of Agriculture.
RESULTS
Production losses over the three-year period were estimated to total 47.8 million bushels of
wheat (all classes) and 42.8 million bushels of barley. Hard red spring wheat dominated the
wheat losses (27.6 out of 47.8 million bushels). Losses for both wheat and barley were
most severe in 2000, followed by 1998 (data not shown).
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Price effects were substantial in all three years, but generally were greatest in 1998. For
hard red spring wheat, the futures price effect ranged from about 1 cent to 7 cents per
bushel, while the basis effect varied among CRDs and between years from less than 10
cents to 71 cents. For other wheat classes, the futures price effect was always less than 3
cents per bushel, while the basis effect exceeded $1 per bushel in some cases. The total
price effect for barley varied from 13 to 80 cents per bushel (data not shown).
The direct economic impact from FHB was greatest in 1998 ($457 million) and least in 2000
($160 million) (Table 1). Both SRW and HRS wheat producers sustained substantial losses
in 1998 ($235 million and $144 million, respectively). In both 1999 and 2000, HRS wheat
growers had the largest losses. The total loss for the three-year period was estimated at
$871 million, or an average of $290 million annually. Overall, the price effect accounted for
77 percent of the direct impacts of FHB (Table 1).
Table 1. Direct Economic Effects from Fusarium head blight in the United States, by Crop
and State, 1998 through 2000.
Crop
Economic
Effect
1998
1999
2000
Total,
1998-2000
--------------------- $ million ----------------------25.6
19.5
36.9
82.0
118.5
97.2
32.7
248.4
144.1
116.7
69.6
330.5
HRS
Production Loss
Price Effect
Total
Durum
Production Loss
Price Effect
Total
2.0
18.5
20.5
10.1
18.8
28.9
12.5
8.4
20.9
24.6
45.7
70.4
SRW
Production Loss
Price Effect
Total
16.0
219.0
235.0
3.2
77.7
80.9
5.5
11.9
17.5
24.8
308.6
333.4
Barley
Production Loss
Price Effect
Total
28.7
28.8
57.5
17.1
9.8
26.9
27.1
24.8
51.9
72.9
63.4
136.4
All Crops
Total
457.2
253.5
159.9
870.6
Among the affected states, North Dakota had substantially the greatest impacts in each of
the three years (Table 2). Overall, North Dakota accounted for 41 percent of all direct impacts from FHB, followed by Minnesota and Ohio. The losses sustained by North Dakota’s
wheat producers averaged 10.5 percent of the total cash receipts from wheat sales over the
period 1998-2000. For barley growers, the losses were even more severe, averaging 25.7
percent of the value of barley production over the three-year period. In 2000, the losses
associated with FHB represented 35.9 percent of total barley sales (USDA, National Agricultural Statistics Service).
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Table 2. Direct Economic Effects from Fusarium head blight in the United States, by State
and Crop, 1998 through 2000.
T o ta l,
1998
1999
2000 1998 - 2000
------------------------ $ m illio n ------------------------
S ta te
C rop
N o rth
D a ko ta
HRS
D u ru m
B a rle y
T o ta l
8 0 .8
2 0 .4
3 7 .0
1 3 8 .2
5 3 .3
2 8 .7
2 1 .7
1 0 3 .8
4 8 .8
2 0 .9
4 4 .2
1 1 3 .9
1 8 2 .9
7 0 .0
1 0 3 .0
3 5 5 .8
M in n e so ta
HRS
D u ru m
B a rle y
T o ta l
3 8 .6
0 .1
1 9 .9
5 8 .7
3 8 .5
0 .2
5 .1
4 3 .7
1 1 .7
a
7 .6
1 9 .3
8 8 .8
0 .3
3 2 .7
1 2 1 .8
O hio
SR W
7 0 .0
2 7 .4
5 .1
1 0 2 .5
Illin o is
SR W
4 8 .6
1 7 .5
0 .3
6 6 .4
S o u th
D a ko ta
HRS
B a rle y
T o ta l
2 4 .8
0 .6
2 5 .3
2 4 .9
0 .1
2 5 .1
9 .1
a
9 .2
5 8 .9
0 .7
5 9 .6
M isso u ri
SR W
3 8 .8
8 .7
4 .0
5 1 .5
M ich ig an
SR W
3 3 .1
1 3 .8
4 .2
5 1 .0
In d ia n a
SR W
2 3 .8
8 .0
2 .6
3 4 .3
K e n tu cky
SR W
2 0 .7
5 .6
1 .3
2 7 .6
a L ess tha n 0 .1
To estimate the secondary impacts of FHB infestations, the direct effects were assumed to
primarily represent a reduction of producer net revenues (i.e., the activities and expenditures
associated with crop production occur with or without scab infestation). The direct economic
effects were therefore allocated to the Households sector of the input-output model. Over
the three-year study period, the $870.6 million of direct impacts resulted in an additional
$1,809.3 million of secondary impacts, for a total economic impact of almost $2.7 billion
(Table 3). Impacts were greatest in 1998 ($1.4 billion).
The distribution by state of the total economic impacts of FHB infestations was similar to that
of the direct effects (Table 4). North Dakota experienced the largest effects, both on average
and for each year. The total economic impact for North Dakota averaged more than $365
million annually over the study period, almost 41 percent of the total impacts of FHB.
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Table 3. Total (Direct and Secondary) Economic Impacts for Fusarium head blight in All
Crops, by Economic Sector and Year, Northern Great Plains and Central United States,
1998 through 2000.
Economic Sector
1998
1999
2000
Total,
1998-2000
-------------------------------- $ million ----------------------------Agriculture
43.0
23.8
15.0
81.8
Construction
41.2
22.9
14.4
78.5
Communication & Public
Utilities
48.2
26.7
16.9
91.8
340.5
188.8
119.1
648.3
76.9
42.6
26.9
146.3
Households
709.8
393.5
248.2
1351.5
Government
49.4
27.4
17.3
94.0
Other Sectors1
98.5
54.6
34.4
187.5
Total Direct Impacts
457.2
253.5
159.9
870.6
Total Secondary Impacts
950.2
526.8
332.3
1809.3
1407.4
780.3
492.2
2679.9
Retail Trade
Finance, Insurance,
& Real Estate
Total
1
Includes sectors such as business, professional, personal, and social services, transportation, and manufa
DISCUSSION
The purpose of this study was to estimate the economic losses from FHB infestations suffered by U.S. wheat and barley producers during the period 1998 to 2000. The study was
intended to provide an update to earlier work by Johnson et al. (1998), which estimated
losses to wheat producers from 1993 through 1997. The present study was designed to
estimate the price and yield effects of FHB, accounting for reduced yields, higher abandoned acres, and price impacts on wheat futures and basis, as well as malting and feed
barley prices. The goal was to provide policy makers with estimates of the magnitude,
distribution, and trend over time of FHB-related losses, as well as the secondary economic
impacts resulting from these direct effects. These estimates were of special interest in the
context of obtaining continuing support for the U.S. Wheat and Barley Scab Initiative, which
provides funding for research to develop scab resistant varieties, as well as other research
and educational efforts to solve this disease problem.
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Table 4. Total (Direct and Secondary) Economic Impacts from Fusarium head blight, All
Crops, by State, in the Northern Great Plains and Central United States, 1998 through 2000.
S ta te
1998
1999
2000
T o ta l
---------------------------- $ m illio n ----------------------------
B y S ta te
--- % —
ND
4 2 5 .4
3 1 9 .4
3 5 0 .5
1 0 9 5 .4
4 0 .9
M N
1 8 0 .6
1 3 4 .6
5 9 .5
3 7 4 .8
1 4 .0
OH
2 1 5 .6
8 4 .3
1 5 .6
3 1 5 .5
1 1 .8
IL
1 4 9 .7
5 3 .7
0 .9
2 0 4 .3
7 .6
SD
7 8 .0
7 7 .2
2 8 .3
1 8 3 .4
6 .8
M O
1 1 9 .5
2 6 .9
1 2 .3
1 5 8 .7
6 .8
M I
1 0 1 .8
4 2 .3
1 2 .9
1 5 7 .1
5 .9
IN
7 3 .2
2 4 .6
8 .0
1 0 5 .7
3 .9
KY
6 3 .7
1 7 .3
4 .1
8 5 .0
3 .2
1 4 0 7 .4
7 8 0 .3
4 9 2 .2
2 6 7 9 .9
---
T o ta l
The findings indicate that scab continues to be a major problem for U.S. wheat producers.
Scab-related losses for wheat growers were estimated to average $245 million annually
from 1998 through 2000, compared to $261 million annually during 1993-1997 (Johnson et
al. 1998). The scab effects on wheat were substantially less in 2000 than in previous years,
which may reflect the introduction of FHB resistant varieties in North Dakota and Minnesota.
However, it also may reflect weather conditions that were less conducive to scab development.
Although North Dakota had substantially the greatest impacts, scab losses affect producers
over a wide geographic area. Eight of the states included in the study had estimated direct
impacts of at least $10 million per year over the period 1998-2000. The disease also poses
major problems for producers of several classes of wheat, as well as barley.
The scab losses were substantial not only in absolute magnitude but also relative to the
value of affected crops. In North Dakota, scab losses in wheat from 1998 through 2000
averaged more than 10 percent of the value of the wheat crop while barley losses averaged
almost 26 percent of the total crop value over the same period.
Impacts from scab affect not only grain producers but also other sectors of the economy.
Income reductions for farmers lead to reduced revenues for a variety of agricultural supply
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and service businesses, and economic linkages result in subsequent effects on many
sectors of local and state economies. For every dollar in direct scab losses to producers,
more than two dollars in secondary economic effects are incurred.
Overall, the direct and secondary economic impacts of FHB infestations have been found to
be substantial and widely distributed, both geographically and among economic sectors.
Further, these losses dwarf the resources presently committed in combating the problem
(funding for the U.S. Wheat and Barley Scab Initiative was $4.3 million in FY 2000). Continued support for research in this area should be relatively easy to justify.
Estimates of the economic impact of scab also have important implications in developing
third party quality risk management strategies, using insurance instruments. The USDA
Loss Adjustment Manual (LAM) Standards Handbook for 2001 and Succeeding Crop Years
emphasize the importance of estimating the reduction in value due to scab.
REFERENCES
Johnson, D. Demcey, G. K. Flaskerud, R. D. Taylor, and V. Satyanarayana. 1998. Economic Impacts of
Fusarium head blight in Wheat. Agricultural Economics Report No. 396, Department of Agricultural Economics,
North Dakota State University, Fargo.
McMullen, Marcia, Roger Jones, and Dale Gallenberg. 1997. “Scab of Wheat and Barley: A Re-emerging
Disease of Devastating Impact.” Plant Disease Vol. 81: 1340-1348.
McMullen, Marcia P., and Robert W. Stack. 1999. Fusarium head blight (Scab) of Small Grains. PP-804
(revised). NDSU Extension Service, North Dakota State University, Fargo.
Nganje, William E., D. Demcey Johnson, William W. Wilson, F. Larry Leistritz, Dean A. Bangsund, and Napoleon
M. Tiapo. 2001. Economic Impacts of Fusarium head blight in Wheat and Barley: 1998-2000. Agribusiness &
Applied Economics Report No. 464. Department of Agribusiness and Applied Economics, NDSU, Fargo.
U.S. Department of Agriculture. Multiple years. Data diskette. National Agricultural Statistics Service, Washington, DC., 2000.
U.S. Department of Agriculture. Grain Market News, Various issues. Agricultural Marketing Service, Grain and
Feed Division, Washington, DC.
U.S. Department of Agriculture. 2001. State Level Data for Field Crops. National Agricultural Statistics Service, http://www.usda.gov/nass/, Washington, DC.
U.S. Department of Commerce. National Climatic Data Center, Office of Environmental Information Service,
National Oceanic and Atmospheric Admin., Washington, DC.
U.S. General Accounting Office. March 1999. Grain Fungus Creates Financial Distress for North Dakota Barley
Producers. Washington, DC.
Wall Street Journal. Various issues. Dow Jones & Company, Inc., New York, NY.
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