• TABLE OF CONTENTS
HIDE
 Front Cover
 Title Page
 Copyright
 Table of Contents
 Foreword
 Preface
 Acknowledgement
 FHB-QTL consortium articles
 Fusarium consortium articles
 International scab nursery consortium...
 Workshop summary
 Outcomes and strategic action...
 Attachment 1 : Agenda for...
 Attachment 2 : List of participants...
 Group photograph
 Back Cover






Title: Global fusarium initiative for international collaboration
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Title: Global fusarium initiative for international collaboration
Physical Description: Book
Language: English
Creator: Ban, T. ( Editor )
Lewis, J. M ( Editor )
Phipps, E. E. ( Editor )
Publisher: International Maize and Wheat Improvement Center (CIMMYT)
Publication Date: 2006
 Subjects
Subject: Farming   ( lcsh )
Agriculture   ( lcsh )
Farm life   ( lcsh )
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Funding: Electronic resources created as part of a prototype UF Institutional Repository and Faculty Papers project by the University of Florida.
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Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: isbn - 970-648-146-X

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Table of Contents
    Front Cover
        Front cover
    Title Page
        Page i
    Copyright
        Page ii
    Table of Contents
        Page iii
        Page iv
        Page v
        Page vi
    Foreword
        Page vii
    Preface
        Page viii
    Acknowledgement
        Page ix
        Page x
    FHB-QTL consortium articles
        Page 1
        Page 2
        Page 3
        Page 4
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    Fusarium consortium articles
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    International scab nursery consortium articles
        Page 73
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    Workshop summary
        Page 123
    Outcomes and strategic action plans
        Page 124
        Page 125
        Page 126
        Page 127
        Page 128
    Attachment 1 : Agenda for the workshop
        Page 129
        Page 130
        Page 131
    Attachment 2 : List of participants registered
        Page 132
        Page 133
        Page 134
    Group photograph
        Page 135
    Back Cover
        Back cover
Full Text




th i JJJ S .iJJ tJfl uJjjJ JmJJmJsJ i.u/

r uJ f JJja -,jJJ 'J jjii L iJJit i JlujJ

T. Ban, J.M. Lewis, and E.E. Phipps, editors









A Strategic Planning Workshop
held at CININIMT, El Batan, Nlexico,
March 14-17"', 2006,
with support from Government of Japan,
Japan-ClNIN\l T FHB project


.. low









The Global Fusarium Initiative
for International Collaboration



T. Ban, J.M. Lewis, and E.E. Phipps, editors



A Strategic Planning Workshop
held at CIMMYT, El Batan, Mexico,
March 14-17th, 2006,
with support from the Government of Japan,
Japan-CIMMYT FHB project


Official Development Assistance


II CIMMYT.
INTERNATIONAL MAIZE AND WHEAT IMPROVEMENT CENTER












CIMMYT (www.cimmvt.org) is an internationally funded, not-for-profit organization that conducts
research and training related to maize and wheat throughout the developing world. Drawing on strong
science and effective partnerships, CIMMYT works to create, share, and use knowledge and technology
to increase food security, improve the productivity and profitability of farming systems, and sustain
natural resources. Financial support for CIMMYT's work comes from many sources, including the
members of the Consultative Group on International Agricultural Research (CGIAR) (www.cgiar.org),
national governments, foundations, development banks, and other public and private agencies.

International Maize and Wheat Improvement Center (CIMMYT) 2006. All rights reserved. The
designations employed in the presentation of materials in this publication do not imply the expression of
any opinion whatsoever on the part of CIMMYT or its contributory organizations concerning the legal
status of any country, territory, city, or area, or of its authorities, or concerning the delimitation of its
frontiers or boundaries. CIMMYT encourages fair use of this material. Proper citation is requested.

Correct citation: Ban, T., J.M. Lewis, and E.E. Phipps (eds.) 2006. The Global Fusarium Initiative for
International Collaboration: A Strategic Planning Workshop held at CIMMYT, El Batan, Mexico;
March 14 17, 2006. Mexico, D.F.: CIMMYT.

AGROVOC Descriptors: Wheats; Barley; Fusarium; Scabs; Fungal diseases; Genetic resources;
Genetic variation; Breeding methods; Selection; Genetic resistance;
Molecular markers; Disease resistance; Food production; Research projects;
Planning; International cooperation; Japan; Turkey; UK; Latin America;
Mexico

Additional Keywords: Strategic Plan


AGRIS Category Codes: H20 Plant Diseases
F30 Plant Genetics and Breeding


Dewey Decimal Classif.: 632.4


ISBN: 970-648-146-X











Contents


Foreword vii

Preface viii

Acknowledgments ix

Contributed Research Papers

Session 1: FHB-OTL Consortium

Global Fusarium Initiative for International Collaboration in Genetic Studies and Breeding for Fusarium
Head Blight Resistance in W heat and Barley....................................................................................................... 1
T. Ban*

M arker-Assisted Selection for FHB Resistance in W heat ........................................................................................3
J.A. Anderson*, S. Liu, X. Zhang Y. Jin, R. Dill-Macky, and S. Chao

EUREKA CEREQUAL: Research Strategies Towards Improving Wheat Quality by Resistance to Fusarium
H ead Blight (FH B) ........................................................................................................................................................4
V. Korzun*, E. Ebmeyer, F. Wilde, J. Haeberle, M. Schmolke, L. Hart, G. Zimmermann, and T. Miedaner

Molecular Mapping of QTLs for Resistance to Fusarium Head Blight in Asian Wheat ...................................8
G.-H. Bai*, H.-X. Ma, J.-B. Yu, J. Yang, W.-C. Zhou, P.-G. Guo, G.E. Shaner, and F.L. Kolb

QTL for the Resistance to Wheat Fusarium Head Blight and Deoxynivalenol Accumulation in Wangshuibai
under Field Conditions .....................................................................................................................................................12
H. X. Ma*, K. M. Zhang L. Gao, G. H. Bai, H. G. Chen, Z. X. Cai, and W. Z. Lu

Research on Molecular Mapping of Fusarium Head Blight Resistance in Wheat at IFA-Tulln, Austria..........13
H. Buerstmayr*, B. Steiner, and M. Lemmens

Evidence that Resistance to Fusarium Head Blight and Crown Rot are Controlled by Different Genes in
W heat ...........................................................................................................................................................................15
G.Q. Xie, M.C. Zhang, T. Magner, T. Ban, S. Chakraborty, and C.J. Liu*
Fusarium Head Blight Resistance from Wide Crosses in Bread Wheat and Durum........................... ..........20
G. Fedak*, W. Cao, A. Xue, M. Savard, J. Gilbert, J. Clarke, and D. Somers

Utilization of Wild Genetic Resources for the Improvement of FHB resistance in Wheat Breeding ...............24
M. Kishii*, R. Delgado, V Rosas, A. Cortes, S. Cano, J. Sanchez, A. Mujeeb-Kazi, J. Lewis, and T. Ban

Nobeoka Bozu, an Unused Resistance Source and its Utilization in Improving Resistance to FHB...................28
A. Mesterlhzy*

DNA Marker Analysis for FHB-Resistance Pyramiding from Different Germplasms......................................30
J.R. Shi*, H. Yang Q. Lu, D. Xu, and T. Ban










Session 2: Fusarium Consortium


Global Biodiversity in Fusarium graminearum (Gibberella zeae) and F. culmorum populations and
Implications for Breeding Resistance to Fusarium Head Blight .........................................................................31
T. Miedaner*

Diversity of Fungal Populations Associated with Fusarium Head Blight in Uruguay .......................................35
S. A. Pereyra*, S. Vero, G. Garmendia, M. Cabrera, and M. J. Pianzolla

Fusarium Pathogens of W heat in Australia .......................................................................................................42
S. Chakraborty*, J.B. Scott, O.A. Akinsanmi, C.J. Liu, V Mitter, and R. Dill-Macky

Turkish Fusarium Isolates from Wheat Crown and Head Can Cause Severe Crown Rot................................46
B. Tunali, J.M. Nicol*, S. Chakraborty, F.Y. Erol, and G. Altiparmak

Vegetative Compatibility Analysis (VCG) and Sequence Related Amplified Polymorphisms (SRAP) in
Understanding Genetic Diversity of Gibberella zeae Isolates from Two Manitoba Fields .................................52
W.G.D. Fernando*, J. X. Zhang, M. Dusabenyagasani, X. W. Guo, H. Ahmed, and B. McCallum

Present Status of the Fusarium graminearum Clade in Europe and Possible Development Strategies..............53
B. T6thand A. Mesterhzy

Cross Fertility of Lineages in Fusarium graminearum (Gibberella zeae)..........................................................54
R.L. Bowden*, J.F. Leslie, J. Lee, and Y.-W. Lee

Fusarium Chemotypes in the UK and Chemotype-Host Interactions ..............................................................60
P. Nicholson*, P. Jennings, M. Thomsett, A Steed, and N. Gosman

Relative Pathogenicity of 3-ADON and 15-ADON Isolates of Fusarium graminearum from the Prairie
Provinces of Canada .................................................................................................................................. .......... .......61
J. Gilbert*, R.M. Clear, T. Ward, and D. Gaba

Comparison of Inoculum Sources in Development of Fusarium Head Blight and Deoxynivalenol Content in
W heat in a Disease Nursery ................................................................................................................................ .......62
A.G. Xue*, G. Butler, H.D. Voldeng G. Fedak, and M.E. Savard.

Development of New Tools to Dissect Fungal Virulence and Plant Resistance Components in a Project
Funded by the Austrian Genome Programme GEN-AU...................................................................................69
U. Gueldener, R. Mitterbauer, M. Peruci, H. Hellmer, G. Wiesenberger, A. Czifersky, K. Brunner, U. Werner,
M.-T. Hauser, F. Berhiller, R. Schuhmacher, R. Krska, M. Lemmens, H. Buerstmayr, and G. Adam*

Implications of Population Variability on the Management of Fusarium Head Blight .....................................72
R. Dill-Macky*



Session 3: International Scab Nursery Consortium

Fusarium Head Blight in Argentina: A Local Company Approach to Breeding for Scab Tolerance ................73
L.J. GonzAlez F.M. Ayala, and H.T. Buck*

Current Status of FHB Research in Romanian Bread Wheat Breeding Program..................................... ..74
M. Ittu*, N.N. Saulescu, G. Ittu, and M. Ciuca










Advancement in FHB Resistant Winter Wheat Cultivar Development Using Frontana as the Resistance
D onor Parent ............................................................................................................................................................... 77
R. Pandeya* and R. Graf

Progress in Improving Fusarium Head Blight Resistant Wheat in Hokkaido, Japan .......................................82
Y. Yoshimura*, K. Nakamichi, S. Kobayashi, T. Nishimura, M. Ikenaga, N. Sato, M. Sato, T. Suzuki, T.
Takeuchi, and A. Yanagisawa

Fusarium Head Blight (FHB), an Emerging Wheat Disease in Tunisia ...........................................................83
M.R. Hajlaoui*, L. Kammoun, S. Gargouri, and M.Marrakchi

Sources of "Environmental Interactions" in Phenotyping and Resistance Evaluation; Ways to Neutralize
Them .............................................................................................................................................................................84
A. Mesterhizy*, B. T6th, and G. Khszonyi,

Strategies and Considerations for Multi-Location FHB Screening Nurseries.................................................93
J. Gilbert* and S.M. Woods

Germplasm Exchange in the Southern Cone of Latin America ........................................................................... 103
M. Diaz de Ackermann*

Considerations in Designing Nurseries for Screening FHB Response in Wheat and Barley...........................09
R. Dill-Macky*, C.K. Evans, M.D. Culler, A.M. Elakkad and K.J. Wennberg

CIMMYT and Turkey's International Shuttle Breeding Program to Develop Wheat Lines with Fusarium
Crown Rot and Other Soil Borne Pathogen Resistances................................................................................. 110
J.M. Nicol*, N. Bolat, A. Bagci, R.T. Trethowan, M. William, H. Hekimhan, A.F. Yidirim, E. Sahin,
H. Eleckcioglu, H. Toktay, B. Tunali, A. Hede, S. Taner, H.J. Braun, T. Payne, M. van Ginkel, M. Keser,
Z. Arisoy, A. Yorgancilar, A. Tulek, D. Erdurmus, 0. Buyuk, and M. Aydogdu

FHB Screening Nursery in Western Canada: Critical Control Points for Large-Scale Fusarium Head Blight
Field Screening Trials .....................................................................................................................................................117
A.L. Brfil-Babel, W.G.D. Fernando*, J. Gilbert, and R. Larios

Searching for Novel Sources of Resistance to Fusarium Head Blight in Barley.................................... ....118
F. Capettini*, S. Grando, T. Ban, and J. Valkoun

Characterization and Development of Argentinean Wheat Germplasm with Resistance against Fusarium
H ead Blight ................................................................................................................................................................ 121
M.T.V Galich, E. Alberione*, M. Helguera, and C. Bainotti


W workshop Sum m ary ............................................................................................................................................. 123

Outcom es and Strategic Action Plans ...............................................................................................................24
Session 1: FHB-QTL Consortium 124
Session: FusariumFungus Consortium 125
Session 3: International Scab Nursery Consortium 126
Resolutions from Meeting 128

Attachment 1: Agenda for the Workshop.................................................................................................. 129

Attachment 2: List of Participants..................................................................................................................... 132






































































































vi











Foreword


Many institutions around the world have devoted
substantial resources to combat Fusarium diseases on
cereal crops, and have met a measure of success.
However, the global community is facing the threat of
imminent epidemics. Even worse, the mycotoxins
produced by the Fusarium fungus cause acute food
poisoning in people and farm animals that are fed
infected grain The risk to human and animal health
and food security is a problem that concern both
developed and less developed countries. Unless steps
are taken to defeat the disease, this threat will
materialize into a much greater problem and as such
requires a global response.

Fusarium head blight (FHB) is a grave threat that
requires an integrated research approach to overcome.
The International Maize and Wheat Improvement
Center (CIMMYT) is working to facilitate the
development of a global platform for international
collaboration on these Fusarium diseases including
FHB and Fusarium crown rot (FCR). Our vision is that
this platform will serve to streamline and aid the
exchange of information, development of collaborative
projects, germplasm enhancement and germplasm
distribution We recognize the need to enhance
international relationships as a part of each
national/international project and consortium.
CIMMYT is taking a proactive stance to elevate the
work of resistance breeding and to raise the profile of
this global challenge.

The development of this Global Fusarium Initiative has
been supported by the Government of Japan since


2004. The challenges and specific activities are based
on the new paradigm which arose from the JIRCAS
Workshop held in February 2004 in Tsukuba, Japan
The concept of the Global Fusarium Initiative was
proposed and accepted at the 2nd International
Symposium on Fusarium Head Blight, incorporating
the 8th European Fusarium Seminar, 11-15 December
2004, Florida USA. A new global collaboration for
consensus QTL mapping of FHB and FCR resistance
in wheat, involving the world's most advanced FHB
researchers, will be one of the activities. Fusarium, the
pathogen that causes FHB, also causes FCR in
Australia, Turkey and other places, another constraint
on global wheat production We have integrated
research and germplasm enhancement for both FHB
and FCR under the Global Fusarium Initiative. This
initiative will encourage communication and
cooperation among individuals, institutions and
governments focusing on this disease.

It is our vision that this workshop will facilitate the
development of a Global Fusarium Initiative to provide
a platform for international collaboration on Fusarium
research and facilitate information exchange,
germplasm enhancement and the development of
breeding methods and materials worldwide.

We will use the untapped potential of this global
communication to combat these Fusarium diseases and
contribute to international efforts in increasing grain
availability and safety.


Masa Iwanaga
Director General
CIMMYT











Preface


Fusarium head blight (FHB, scab) and Fusarium crown
rot (FCR) are important threats to sustainable wheat
and barley production worldwide. The efforts to
combat these diseases have been increasing around the
world throughout numerous countries and research
communities. The plan of this workshop was to join
together these various research communities so that we,
as a global community, can have greater impact and
efficacy in our efforts against these diseases. With
such a global platform we can support the exchange of
information, collaborative research, development and
exchange of germplasm enhancement, the
development of breeding methods and other activities.
CIMMYT has been conducting a holistic operation to
enhance resistance to Fusarium diseases in wheat and
barley germplasm through systematic and intensive
screening in multiple environments and cutting-edge
genetic research Novel genetic variation for wheat is
found among CIMMYT's genebank accessions and
synthetic wheat derivatives. The FHB research at
CIMMYT has been systemized in a simple workflow
on four levels: evaluation of resistance in the field
(phenotyping); genetic characterization by DNA
markers (genotyping); gene discovery; and
development of DNA marker assisted selection (MAS)
for use in breeding. To raise the profile of and
consolidate these efforts, CIMMYT organized an
international workshop-the first in a series-to
highlight the importance of Fusarium diseases, the
status of collaborative efforts to address these diseases,
and future prospects for international collaboration


The agenda involved discussion, planning, and
prioritization for the most critical research needs,
opportunities for web-based knowledge sharing,
opportunities for international collaboration, and action
plans in the following topics:

1. FHB-QTL consortium Wheat FHB-QTL
comparative study for the deployment of
resistance genes, including the analysis of the
bases of resistance, the development of an
effective MAS system and the pursuit of
germplasm enhancement.

2. Fusarium fungus consortium Global
compilation/monitoring system of genetic
diversity, pathogenicity, and toxigenicity from
studies on Fusarium fungi, to control FHB and
FCR.

3. International scab nursery consortium:
Development of new international interactive
Fusarium resistance screening nurseries for
germplasm enhancement and global
compilation of Genotype x Environment x
Management effects on resistance to
FHB/FCR.

This workshop was an invaluable opportunity to
exchange ideas regarding the current status of
FHB/FCR research around the globe.


Hans J. Braun
Director, Global Wheat Program, CIMMYT











Acknowledgments

The editors and workshop beneficiaries would like to
thank the Government of Japan for its financial support
through the Japan-CIMMYT FHB collaborative
research project, and The Ministry of Agriculture,
Forestry and Fisheries of Japan (MAFF) which made
this strategic planning workshop and attendant
proceedings possible. We would like to acknowledge
the contribution of Japan International Research Center
for Agricultural Sciences (JIRCAS) through the
collaborative projects. We wish to give great thanks


Tomohiro Ban and Richard R. Ward,
Workshop Co-organizers, CIMMYT


for the contributions of all of the participants who
willingly shared data, the latest status of research,
experience and your thoughts on the development of
targeted consortium within the Global Fusarium
Initiative. We learned much from your excellent
presentations and benefited greatly from the many
discussions during the workshop. We are happy to
have achieved fruitful outcomes and action plans to
launch the Global Fusarium Initiative.


































































































X






Session 1: FHB-QTL consortium


GLOBAL FUSARIUM INITIATIVE FOR INTERNATIONAL
COLLABORATION IN GENETIC STUDIES AND BREEDING
FOR FUSARIUM HEAD BLIGHT RESISTANCE
IN WHEAT AND BARLEY


T. Ban1'2*

1CIMMYT, Apdo. Postal 6-641, 06600 Mexico, D.F., Mexico
Japan International Research Center for Agricultural Sciences (JIRCAS),
1-1 Ohwashi, Tsukuba, Ibaraki 305-8686, Japan
*Corresponding author: Phone: +52 (55) 5804-2004, Email: t.ban@cgiar.org



ABSTRACT

Fusarium head blight (FHB) is a grave threat that we will only overcome if we integrate all our wisdom and
expertise. Many institutions around the world have devoted substantial resources to combat FHB, and have met
with a measure of success. However, the global community is facing the threat of imminent epidemics. Unless steps
are taken to strategically combat the disease on a global scale, this threat will materialize into a much greater
problem and as such requires a global response. CIMMYT has adopted a holistic approach to enhance novel FHB
resistance among genebank accessions and synthetic wheat derivatives and combine their resistance using
systematic screening in multiple environments and genetic characterizationby DNA markers.

For this reason, CIMMYT is facilitating global communication through the Japan-CIMMYT FHB project supported
by the Japanese government since 2004. The challenges and specific activities are based on the new paradigmwhich
arose from the JIRCAS Workshop held in February 2004 in Tsukuba (Ban, 2004). The concept of the Global
Fusarium Initiative was proposed and accepted at the 2nd International Symposium on Fusarium head blight,
incorporating the 8th European Fusarium Seminar, 11-15 December 2004, Florida USA (Van Ginkel and Ban, 2004).
A new global collaboration for consensus QTL mapping of FHB resistance in wheat, involving the world's most
advanced FHB researchers, will be one of the activities. Fusarium fungi, the pathogen of FHB, also causes Fusarium
crown rot (FCR) in Australia, Turkey and other places, and is another constraint to global wheat production We
have integrated research and germplasm enhancement for both FHB and FCR under the Global Fusarium Initiative.
This initiative will encourage communication and cooperation among individuals, institutions and governments
focusing on this disease.

CIMMYT's role in the Global Fusarium Initiative is to provide a platform for international collaboration on
Fusarium research, and facilitate information exchange, germplasm enhancement and the development of breeding
methods and materials globally. This Global Fusarium Initiative will encourage communication and cooperation
among individuals, institutions and governments focusing on this disease. Specific activities will be linked using a
web site and on-site forums (http://www.fusarium-net.org). Global Genotype x Environmental meta-data
compilation, updated global information, and the development of a global crop information system on FHB data will
be features of this web site. The Global Fusarium Initiative provides the platform to fight this grave threat. We are
leading a new paradigm for international cooperation and collaborative research to combat the disease, which will
contribute to the reduction of poverty and hunger world-wide.


The challenges of this initiative are:
SIdentification of new sources of resistant germplasm and pre-breeding
SDelineation of the nature of wheat resistance to FHB and host-pathogen interaction
SDevelopment of effective cropping systems adjusting pathogen cycle and wheat growth







Session 1: FHB-QTL consortium


Germplasm sharing and intellectual property rights (IPRs) management
SKnowledge sharing among the global community

The specific activities of this initiative are:
SLinking with relevant Fusarium research activities
Website and e-News, http://www.fusarium-net.org
Global compilation of Genotype x Environment x Management meta-data
interactive screening nursery system
Up to date global information onFHB epidemics, toxins and resistant breeding
Biennial meetings for information sharing and focused discussion
Global Crop Information System on FHB


through new international


We aim to acquire potentially novel sources of resistance from global hotspots through our widespread contacts.
Expectations are high that useful resistance genes may be identified during the screening of germplasm accessions
and that the effects of Genotype x Environment x Management interactions and the distribution of Fusarium isolates
will be better understood. In addition, we are working to develop a compilation/monitoring system for Fusarium
genetic diversity, pathogenicity, and toxigenicity to further our abilities to control FHB.

REFERENCES

Ban, T. 2004. Collaborative Research for Fusarium Head Blight Resistance in Wheat and Barley, compiled by T.
Ban, JIRCAS Working Report No.37, 2004, P.80 (ISSN1341-710X), Japan International Research Center for
Agricultural Sciences, Tsukuba.
Van Ginkel, M. and Ban, T. 2004. Global Progress in Identifying and deploying FHB resistance genes. In: Canty,
S.M., Boring, T., Wardwell, J. and Ward, R.W (Eds.), Proceedings of the 2nd International Symposium on
Fusarium Head Blight; incorporating the 8th European Fusarium Seminar; 2004, 11-15 December; Orlando, FL,
USA. Michigan State University, East Lansing, MI. pp. 203.






Session 1: FHB-QTL consortium


MARKER-ASSISTED SELECTION FOR FHB RESISTANCE
IN WHEAT


J.A. Anderson"1, S. Liu1, X. Zhang1, Y. Jin2, R. Dill-Macky3, and S. Chao4

'Department of Agronomy & Plant Genetics, University of Minnesota, 411 Borlaug Hall, St. Paul, MN, 55108,
USA. 2USDA-ARS Cereal Disease Laboratory, 1551 Lindig St., St. Paul, MN, 55108, USA.
3Department of Plant Pathology, University of Minnesota, 495 Borlaug Hall, St. Paul, MN, 55108, USA. 4USDA-
ARS Biosciences Research Laboratory, 1605 Albrecht Boulevard, Fargo, ND, 58105, USA.
*Corresponding Author: Phone: +1(612) 625-9763; E-mail: ander319(diumn.edu



ABSTRACT

Wheat varieties with a higher level of resistance to Fusarium head blight (FHB) would make a substantial
contribution to reducing the losses from this devastating disease. With the recent establishment of the USDA-ARS
Small Grains Genotyping Centers in the U.S., we have increased our efforts in marker-assisted selection (MAS) for
FHB resistance. During 2005 we screened more than 5,000 F2 or F3 individuals for their genotype at Fhbl, the
major quantitative trait locus (QTL) on chromosome 3BS. This will increase to more than 7,000 individuals in
2006. Because additional loci (e.g high molecular weight glutenins, leaf rust resistance) are also being subjected to
MAS in the same individuals, we are practicing allele enrichment, i.e. selection against the homozygous undesirable
types, as a means of maintaining an adequate population size for subsequent selection In the future, a greater
emphasis will be placed on screening BC1F1 individuals with markers. Enriched populations will undergo
phenotypic selection for FHB resistance, and other yield, disease resistance, and end-use quality testing necessary to
produce FHB resistant germplasm and variety candidates. We believe that substantial efforts in phenotypic
assessments for FHB resistance will still be necessary, even with an increase in MAS for this trait, because there are
likely numerous "minor" effect genes that need to be combined with the major QTLs in order to obtain the desired
level of resistance.

There is a need to characterize additional FHB resistance genes and to identify associated diagnostic markers. Our
germplasm screening and QTL mapping efforts are focused on materials that do not contain the Fhbl QTL. Tightly
linked markers at the Fhbl locus are being used to identify this germplasm This increases our chances of finding
novel, major QTL, versus finding yet another source of resistance containing Fhbl. Although other QTLs for FHB
resistance have been located using DNA markers (e.g. loci on 5AS and 6BS), they are not as suitable for widespread
use in MAS because of lack of polymorphism of the markers and/or insufficient linkage disequilibrium between the
markers(s) and QTLs. A global consortium could assist in the prioritization of germplasm for genetic studies and
development of diagnostic markers for new and existing QTLs.







Session 1: FHB-QTL consortium


EUREKA CEREQUAL: RESEARCH STRATEGIES TOWARDS
IMPROVING WHEAT QUALITY BY RESISTANCE TO
FUSARIUM HEAD BLIGHT (FHB)


V. Korzun 1, E. Ebmeyer1, F. Wilde2, J. Haeberle3, M. Schmolke3, L. Hartl3,
G. Zimmermann3, and T. Miedaner2

1 Lochow-Petkus GmbH, Bollersener Weg 5, 29303 Bergen, Germany
2 State Plant Breeding Institute, University ofHohenheim, 70593 Stuttgart, Germany
3 Bavarian State Research Center for Agriculture, V6ttinger StraBe 38, 85354
Freising Germany
*Corresponding author: Email: korzunilochow-petkus.de


OBJECTIVES

This collaborative project between a private plant
breeding company and two scientific institutions has
the following objectives: (1) Mapping and validation
of quantitative trait loci (QTL) for resistance to
Fusarium head blight (FHB) in two winter wheat and
two spring wheat populations, (2) Enrichment of the
QTL regions with AFLP and SSR markers and
development of appropriate STS markers, (3)
Comparison of phenotypic with marker-based selection
for FHB resistance in one spring- and one winter-
wheat population, (4) Determination of the correlated
response for reduction in deoxynivalenol (DON)
content of the grain

INTRODUCTION

FHB is one of the most destructive diseases in small-
grain cereals. In Europe, the damage is mainly caused
by the pathogens Fusarium graminearum and F.
culmorum. Infections lead to severe yield losses, poor
grain quality, and contamination with mycotoxins in
the grain, especially with DON. Resistance to FHB is
quantitatively inherited. The overall aim of the project
was to improve the breeding progress by using
molecular markers that can be applied to practical
plant breeding.

MATERIALS AND METHODS

Two populations of spring wheat (CM82036 [resistant,
Sumai 3/Thornbird-S] x Remus [susceptible], Frontana
[resistant] x Remus) and two populations of winter
wheat (G16-92 [resistant] x Hussar, Dream [resistant]
x Lynx) have been tested for FHB by artificial


inoculation with Fusarium culmorum in multiple
environments. Genomic constitution was analysed with
microsatellite and AFLP markers. In spring wheat,
three QTL were located on chromosomes 3A, 3B and
5A contributing between 15 and 32% of the
phenotypic variance in the respective mapping
populations (Buerstmayr et al. 2003, Steiner et al.
2004).

For establishing a broad-based selection project, a
spring- and a winter-wheat double cross with the above
mentioned resistance donors, but different susceptible
crossing partners (Nandu, Munk for spring wheat, LP-
strain and Brando for winter wheat) were used as
source materials. In spring and winter wheat, 1,200 and
600 F1 plants, respectively, were tested in multi-
locational infection trials and selected for their FHB
resistance in a two- and one-step procedure,
respectively. The 20 best lines were slightly less
diseased than the resistant parents. Moreover, they
were significantly less contaminated by DON than
moderately and highly susceptible genotypes (Wilde
and Miedaner 2006). They were crossed in a factorial
design and selfed. In parallel, both source populations
were analysed for each of three donor QTL alleles that
have been associated with FHB resistance (6A and 7B
from Dream, 2B from G16-92, 3B and 5A from
CM82036 and 3A from Frontana) by one to two
single-sequence repeat (SSR) marker per QTL. Across
selfings, all eight expected donor QTL alleles and
allele combinations were found. In 2004, the
populations selected by phenotype and markers,
respectively, were grown together with the source
populations and standard varieties at four locations.
Inoculum of two highly aggressive, DON-producing
isolates of Fusarium culmorum (FC 33, FC 46) was







Session 1: FHB-QTL consortium


applied with density of 5 x 105 spores ml '. To
consider the variation in flowering date between
entries, all genotypes including the parents were
inoculated four times. Accordingly, FHB rating was
assessed three times by rating the percentage of
infected spikelets per plot (0-100). For data analysis,
the arithmetic mean of all three ratings was used. In
spring wheat, FHB ratings were adjusted to heading
date by multiple regression analysis (adjusted FHB
rating).

RESULTS AND DISCUSSION

In each of the two winter wheat populations, QTL for
combined resistance to initial infection and spread
within plant tissue were detected (Schmolke et al.
2005). In the Dream/Lynx population, two main FHB
resistance QTL were detected on chromosomes 6AL
and 7BS which explain 19% and 21% of the
phenotypic variation QTL on chromosome 6AL
overlapped with a QTL for plant height. Additionally,
minor QTL were identified on chromosomes 2BL and
lB. In the G16-92/Hussar population, a major QTL
was detected on chromosome 2BL explaining 17% of
the phenotypic variation Another QTL was found on
chromosome 1A explaining 14% of the phenotypic
variation and overlapping with a QTL for plant height.

For validation of the identified resistance QTL, near-
isogenic lines were created by backcrossing and selling
combined with marker-based selection Selected lines
of the BC2S3- and BC2S4- generation were tested for
FHB resistance in field trials at four and three
environments in 2005, respectively. In the
Dream/Lynx population, the main resistance QTL on
chromosomes 6AL and 7BS had significant effects on


FHB severity, between 7% and 10%. Among the
genotypes for the QTL validation are some lines
carrying one or both of the resistance alleles, which are
of agronomic interest combining short plants with a
good to medium FHB resistance. The minor QTL had
no significant effects on FHB severity. In the G16-
92/Hussar population, the detected QTL could not be
validated due to loss of resistance probably during
backcrossing of the lines. A new backcross population
will be developed and investigated following this
project.

Most exotic and adapted donor QTL could be verified
in elite wheat background (Table 1). Even in the case
where the individual QTL allele showed no effect
compared to the class without any donor QTL allele it
had a positive additive effect in combination with other
QTL. Highest effects were found for each combination
with donor QTL 6A or 7B in winter wheat and the
combination of donor QTL 3B and 5A in spring wheat.
Effects were clearly smaller for the adapted winter
wheat QTL than for those originating from CM82036
or Frontana.

Marker-based selection on the donor QTL 3B and 5A
in spring wheat resulted in an indirect selection gain on
reduced DON content in the grain (Figure 1). Donor
QTL 3A from Frontana did not contribute to this
effect. Individual bulks within the respective QTL
class nevertheless showed a large significant (P=0.05)
variation in both traits. This is most likely caused by
additional QTL alleles for FHB resistance in the
donors or the susceptible elite cultivars (for the class
without donor QTL alleles) not followed by molecular
markers.


Table 1. Means and effects of eight QTL classes for adjusted FHB rating after inoculation of 12 to 15 F3 5 bulks per
class by Fusarium culmorum across four locations in 2004
Winter wheat Spring wheat
QTL class FHB rating Effect' QTL class FHB Effect'
rating
% %
2B + 6A + 7B 18.7 a2 10.6 3B + 5A + 3A 14.2 a 17.3
6A + 7B 19.0 a 10.4 3B + 5A 16.2 ab 15.3
2B + 6A 18.0 a 11.3 3B + 3A 20.6 b 10.9
2B + 7B 21.lab 8.2 3A+5A 19.1 ab 12.4
2B 22.4 ab 6.9 3B 21.1 bc 10.4
6A3 23.7 ab 5.6 5A 21.4 bc 10.1
7B 26.6 bc 2.7 3A 26.6 cd 4.9
Susceptible 29.3 c Susceptible 31.5 d
Difference to the susceptible class.
2 Different letters mark significant differences (P=0.05).






Session 1: FHB-QTL consortium


Donor-QTL alleles:

* 3A+3B+5A
O 3B+5A
A None


90

80

70
0)
60
E
'50-

S40
o
C-
o
z 30
0
Q 20

10

0


LSD5


0 5 10 15 20 25 30 35 40

Adjusted FHB rating (%)


Figure 1. Association between adjusted FHB rating and DON content of F3 5 bulks selected for the given donor QTL
alleles by SSR markers and tested phenotypically by inoculation with Fusarium culmorum across three locations in
2004


Substantial gain from phenotypic and marker-based
selection for FHB resistance was found in the spring
and winter wheat population Higher gain from
selection could be achieved with exotic donor QTL
from spring wheat compared to the adapted QTL from
winter wheat. Their introgression also led to an indirect
selection gain for DON content in the grain in both
selectionvariants. Application of DNA markers almost
doubled the realized selection gain per year in spring
wheat, but not in winter wheat. Here, both variants had
a similar selection gain per year. Economically, the
marker-based selection is cheaper than the phenotypic
selection when only the major QTL for FHB resistance
are followed. For introgression of exotic donors,
however, a background selection for the genome of the
elite parent would be highly recommendable. Caused
by the high genetic variation within the best marker-
based selected variants additional phenotypic selection
is useful to achieve the maximum selection gain


ACKNOWLEDGEMENTS

This project was financially supported by the German
Federal Ministry of Education and Research (BMBF,
Bonn, Germany) and the breeding company Lochow-
Petkus GmbH, Bergen, Germany, within the German-
French EUREKA Consortium (Project No. V! 2386).

REFERENCES

Buerstmayr H., Steiner B., Hartl L., Griesser M.,
Angerer N., Lengauer D., Miedaner T., Schneider
B., and M. Lemmens. 2003. Molecular mapping of
QTL for Fusarium head blight resistance in spring
wheat. II. Resistance to fungal penetration and
spread. Theor Appl Genet 107:503-508.
Miedaner, T., F. Wilde, B. Steiner, H. Buerstmayr, V.
Korzun, and E. Ebmeyer. 2006. Stacking
quantitative trait loci (QTL) for Fusarium head
blight resistance from non-adapted sources in an
European elite spring wheat background and


A A


A



AAAA


00


I I I I I


I I







Session 1: FHB-QTL consortium


assessing their effects on deoxynivalenol (DON)
content and disease severity. Theor. Appl. Genet.
112: 562-569.
Schmolke, M., G. Zimmermann, H. Buerstmayr, G.
Schweizer, T. Miedaner, V. Korzun, E. Ebmeyer,
and L. Hartl. 2005. Molecular mapping of
Fusarium head blight resistance in the winter
wheat population Dream/Lynx. Theor. Appl.
Genet. 111: 747-756.
Steiner B., Lemmens M., Griesser M., Scholz U.,
Schondelmaier J., and H. Buerstmayr. 2004.
Molecular mapping of resistance to Fusarium
head blight in the spring wheat cultivar Frontana.
Theor Appl Genet 109:215-224.
Wilde, F. and T. Miedaner. 2006. Selection for
Fusarium head blight resistance in early
generations reduces deoxynivalenol (DON)
content in grain of winter and spring wheat. Plant
Breeding 125: 96-98.







Session 1: FHB-QTL consortium


MOLECULAR MAPPING OF QTLS FOR RESISTANCE TO
FUSARIUM HEAD BLIGHT IN ASIAN WHEAT


G.-H. Bai H.-X. Ma23, J.-B. Yu2, J. Yang2, W.-C. Zhou4'5, P.-G. Guo6,
G.E. Shaner7, and F.L. Kolb6

'USDA-ARS-Plant Science and Entomology Research Unit, Manhattan, KS, USA 66506;
2 Kansas State University, Manhattan, KS, USA 66506; 3Jangsu Academy of Agricultural Sciences, Nanjing, China,
210014; 4Lethbridge Research Centre, Agriculture and Agri-Food Canada, Lethbridge, AB, T1J4B1, Canada;
5University of Illinois, Urbana, IL, USA, 61801; 6College of Life Science, Guangzhou University, Guangzhou
510006, China; Purdue University, West Lafayette, IN, 47907.
*Corresponding author: Phone: +1 (785)-532-1124; E-mail: elali ksu icdI


OBJECTIVES


MATERIALS AND METHODS


1) To identify the quantitative trait loci (QTLs) for
type II FHB resistance in different Asian cultivars.
2) To identify closely linked molecular markers to
these QTLs for marker-assisted breeding.
3) To elucidate genetic relationship between QTLs
from different sources.

INTRODUCTION

Fusarium head blight (FHB), mainly caused by
Fusarium graminearum, is an economically important
disease of cereal crops worldwide. FHB can cause
dramatically reductions in grain yield and grain quality
(Bai and Shaner, 2004). The accumulation of
mycotoxins in the infected grain has become a major
concern for human health and animal production (Bai
and Shaner, 2004).

Using resistant wheat cultivars is the most cost-
effective and environmental safe method to reduce
losses caused by FHB. Different types of FHB
resistance have been described (Bai and Shaner, 2004).
Resistance to initial infection (type I) and to FHB
symptom spread within an infected spike (type II), and
low deoxynivalenol (DON) accumulation in infected
grain (type III) have drawn more breeders' attention
Type II is a more stable type of resistance in most of
the resistant cultivars identified to date (Bai and
Shaner, 2004). Many wheat cultivars or landraces from
Asia have been reported to have reasonable type II
resistance (Yu et al., 2006). However, some of the
QTLs in these cultivars have not been well
characterized.


Plant materials
Four populations of recombinant inbred lines (RILs)
were developed by single seed descent from the
crosses Wangshuibai/Wheaton (Zhou et al, 2004),
Ning 7s4", C lik (Bai et al., 1999), Chokwang/Clark
(Yang et al., 2005) and CS-SM3-7ADS/Annong 8455
(Ma et al., 2006a). Wangshuibai is a Chinese landrace
with a high level of FHB-resistance. CS-SM3-7ADS is
a Chinese breeding line highly resistant to FHB that
was derived by replacing chromosome 7A of Chinese
Spring with the corresponding chromosome from
Sumai 3 (Zhou et al., 2002a). Ning 7840 is a Chinese
FHB-resistant cultivar derived from Sumai 3, while
Chokwang is a moderately FHB-resistant cultivar from
Korea (Yang et al., 2005). Wheaton, Clark and
Annong 8455 are all highly susceptible to FHB.

Evaluation of FHB and DON content
All RILs were evaluated for FHB resistance in several
greenhouse experiments by inoculating with
conidiospores of F. graminearum, GZ 3639, a field
isolate from Kansas. Type II resistance was measured
by injecting 1000 conidiospores of isolate GZ 3639
into a central floret of a spike at anthesis with a
syringe. The RILs were prepared for inoculation as
following: after vernalization at 40C in a growth
chamber for eight weeks, six seedlings were
transplanted into a 5'x 5' tora pot (Hummert Int., St.
Louis, MO) containing Metro-mix 360 (Hummert Int.,
St. Louis, MO), and grownina greenhouse bench with
12-h daylight period. All plants in each pot were
inoculated with a single head per plant, and incubated
in a moist chamber for three days to initiate infection
The inoculated plants were then moved to the original







Session 1: FHB-QTL consortium


bench positions and grown at 250C during the day and
220C during the night. The infected and total spikelets
in a spike were counted at 21st day after inoculation
and the proportion of symptomatic spikelets (PSS) was
calculated for the final disease severity. DON content
was determined by direct quantification of DON in the
harvested kernels of Fusarium inoculated spikes using
high-pressure liquid chromatography (HPLC) and
electrospray ionization mass spectroscopy (ESI-MS)
(Mirocha, et al. 1998).

Marker analysis
DNA was isolated from leaf tissue using CTAB
method (Saghai-Maroof et al. 1984). AFLP and SSR
analyses followed Bai et al. (2003). For SSR detection,
an 18bp M13 tail sequence (5'-
ACGACGTTGTAAAACGAC) was added at 5'-end of
each forward SSR primer and an additional M13
primer was labeled with IRdye-700 or IRdye-800 (Li-
Cor, Inc. Lincoln, NE). To amplify SSRs, a touchdown
PCR profile started at 950C for 5 min, followed by 5
cycles of 45 s at 950C, 5 min at 680C, and 1 min at
720C with the annealing temperature that was lowered
by 20C in each following cycle; for another 5 cycles,
the annealing time was 2 min and the temperature was
lowered 20C in each following cycle; in the last 25
additional cycles, the annealing temperature was
constant at 500C with 5 min at 720C for a final
extension AFLP was analyzed in a Li-Cor 4200 DNA
Sequencer (Li-Cor, Inc. Lincoln, NE) and scored by
visual inspection SSR was analyzed in either a Li-Cor
4200 DNA Sequencer or ABI 3100 DNA Analyzer
(Applied Biosystems, Foster City, CA).

Data analysis
Linkage maps were constructed with JoinMap 3.0 (van
Ooijen & Voorips, 2001) with the Kosambi mapping
function (Kosambi, 1944). A minimum logarithm of
odds (LOD) threshold of 3 was used for determining
linkage groups. Simple interval mapping (SIM) and
composite interval mapping (CIM) were performed
using average values over each individual experiment
and on the overall average across all experiments by
using Qgene (Nelson 1997), MapQTL (van Ooijen &
Voorips, 2004) or Cartographer 2.0
(http://statgen.ncsu.edu/qtlcart/WOTLCart.htm). The
threshold of the LOD score for declaring the
significance of a QTL was determined by a 1,000-
permution test. Determination coefficients (R2) for
each QTL were calculated through multiple linear
regressions of the QTLs on the phenotype data using
SAS REG procedure.


RESULTS AND DISCUSSION

QTLs for type II resistance and low DON content in
Ning 7840
Type II FHB resistance of 133 RILs derived from cross
Ning 7s41 (lark was evaluated in four greenhouse
experiments (Bai et al, 1999). One major QTL and
several minor QTLs were identified (Bai et al 1999,
Zhou et al 2002b). The major QTL on 3BS explained
up to 50% phenotypic variation for type II resistance
(Bai et al 1999, Zhou et al 2002b) and 25% for low
DON (Bai et al, 2000). It is a stable QTL, was detected
in all four experiments and physically mapped between
breakage points 3BS-3 and 3BS-8 (Zhou et al 2002b).
Several AFLP and SSR markers were identified in the
QTL region Markers Xgwm533, i-,,,-'3U and
Xbarc147 are the closest SSR markers for the QTL. In
addition, one closely linked AFLP marker linked to the
major QTL was converted into a sequence tagged site
(STS) marker (Guo et al, 2003). These SSR and STS
markers have been used in marker-assisted selection
(MAS) for the major QTL in many breeding programs.

Beside the major QTL on 3BS, additional QTLs with
minor effects were also identified on chromosome 2BL
and 2AS for type II resistance using SSR markers
(Zhou et al, 2002b). More recently, a number of
resistance gene analog (RGA) markers were screened
and five RGA markers were identified as associated
with FHB resistance in the Ning7Xls4 C li k population
(Guo et al, 2006). Three of them associated with a
QTL on chromosome 1AL that explained 12%
phenotypic variation One of the RGA markers on 1AL
was converted into a STS marker. Significant positive
interaction was detected between QTLs on 3BS and on
1AL. This STS marker can be used in MAS to
pyramid QTL on 1AL with others.

QTLs for type II resistance in Chinese Spring-Sumai
3-7A-disomic substitution line
Chinese Spring is a moderately resistant landrace from
China. Its Sumai 3-7A-disomic substitution line (CS-
SM3-7ADS) showed the same high resistance as
Sumai 3 (Zhou et al, 2002a). A population of 97 RILs
was developed from the cross CS-SM3-7ADS/Annong
8455 and evaluated for type II resistance in the
greenhouse (Ma et al 2006a). The result indicated that
CS-SM3-7ADS carries FHB-resistance alleles at five
QTLs on chromosomes 2D, 3B, 4D and 6A. One QTL
on 3BS had the largest effect, and explained 30.2% of
the phenotypic variance for type II resistance and was
located at the same location as that in Sumai 3 and
Ning 7840. In addition, several susceptible factors
were mapped on chromosomes 1A, ID, 4A and 4B of
CS-SM3-7ADS. No QTL for enhanced FHB resistance
was detected on chromosome 7A of CS-SM3-7ADS,







Session 1: FHB-QTL consortium


suggesting the increased FHB resistance in CS-SM3-
7ADS was not due to any major FHB-resistance QTL
on 7A of Sumai 3, but more likely due to removal of
susceptible factors) on 7A of Chinese Spring. Further
evaluation of a set of ditelosomic lines derived from
Chinese Spring for FHB resistance indicated that
ditelosomic lines DT1AS, DT2AS, DT3AS, DT3BL,
DT6BL, DT1DL and DT1DS had a significant greater
FHB than Chinese Spring; whereas lines, DT7AL,
DT3BS, DT6BS, DT7BL, and DT4DL had a
significantly lower FHB than Chinese Spring (Ma et al
2006b). The results suggested that some wheat
cultivars may have both FHB-resistance QTLs and
susceptible factors. In breeding practice, adding FHB-
resistance-enhancing QTLs or removal of susceptible
factors may both significantly increase the level of
wheat resistance to FHB in a wheat cultivar.

FHB resistance in Wangshuibai
Wangshuibai is a FHB-resistant Chinese landrace
unrelated to 'Sumai 3' based on marker data (Bai et al,
2003). A mapping population of 139 RILs was
developed from the cross Wangshuibai/Wheaton (Zhou
et al 2004). After screening about 1300 simple
sequence repeat (SSR) and amplified fragment length
polymorphism (AFLP) markers, we detected seven
QTLs on chromosome 3BS, 1A, 5AS, 5DL, 7AL, and
3DL for type I resistance, and six of them were also
associated with lower DON content except the one on
3DL (J-B. Yu et al, unpublished data). These QTLs
jointly could explain as much as 63.5% of phenotypic
variation for type II resistance and 48.0% for low
DON. The QTL on 3BS showed major effect on both
type II resistance and low DON and was stable across
all four experiments. However, its effect on type II
resistance appears to be smaller than that in Ning 7840.
This QTL in both Ning 7840 and Wangshuibai was
mapped in the same region of 3BS and is most likely
the different alleles of the 3BS QTL.

A new QTL in Chokwang
Chokwang is a moderate FHB-resistant cultivar from
Korea. A population of 79 RILs were derived from the
cross Chokwang/Clark in Purdue University (Yang et
al, 2005). The population was screened with both SSR
and target-region amplified polymorphism (TRAP)
primers. One major QTL, Q/hb.ksu-5DL1, was
identified on chromosome 5DL. The SSR marker
Xbarc 239 was mapped in the QTL region, and also
physically located to the bin of 5DL1-0.60-0.74 by
using Chinese Spring deletion lines. Major QTL was
detected on 5DL suggested that Chokwang contains a
new QTL for FHB resistance that is different from the
one on 3BS of Sumai 3 or Ning 7840. A second QTL
Qfhb.ksu-4BL1 linked to SSR Xbarc1096 was
tentatively mapped on 4BL. In addition, a minor QTL


(Qfhb.ksu-3BS1) was detected on 3BS with marginal
significance in Wangshuibai and mapped on the same
location as that inNing 7840.

In summary, resistant and moderately resistant
cultivars from Asian sources usually carry one major
QTL and several minor QTLs for type II resistance.
The QTL on 3BS is a consistent QTL for type II
resistance in four FHB resistant cultivars, but variation
in effects on type II resistance was observed among
cultivars: major effects in Wangshuibai, Ning 7840
and Chinese Spring, and minor effect in Chokwang.
This QTL is most likely allelic among cultivars
studied. The chromosome locations of three QTLs
were in common between two cultivars: a QTL on 1A
and another near centromere of 3BS presented in both
Wangshuibai and Ning 7840, and the QTL on 5DL
showed a minor effect in Wangshuibai and a major
effect in Chokwang. Minor QTLs on 2A, 2B and 1A
are unique in Ning 7840; QTLs on 4D, 6A are unique
in Chinese Spring; QTL on 3D, 5A and 7A are unique
for Wangshuibai; and QTL on 7B is unique for
Chokwang. QTLs for low DON content were usually
overlapped with QTLs for FHB resistance. Several
QTLs for FHB susceptibility were identified in
Chinese Spring suggesting that removing susceptible
QTLs can be a useful strategy for improving FHB
resistance in wheat cultivars.

ACKNOWLEDGEMENTS

This research is partially funded by U.S. Wheat and
Barley Scab Initiative. Mention of trade names or
commercial products in this article is solely for the
purpose of providing specific information and does not
imply recommendation or endorsement by the U.S.
Department.

REFERENCES

Bai, G.-H., F. L. Kolb, G. E. Shaner & L. L. Domier.
1999. AFLP markers linked to one major QTL
controlling scab resistance in wheat.
Phytopathology 89:343-348.
Bai, G.-H., R. Plattner, G. E. Shaner & F. L. Kolb.
2000. A QTL for deoxynivalenol tolerance in
wheat. Phytopathology. 90 (6):S4.
Bai, G.-H., P.-G. Guo & F. L. Kolb. 2003. Genetic
relationships among head blight resistant cultivars
of wheat assessed on the basis of molecular
markers. Crop Sci. 43:498-507.
Bai, G.-H. & G. E. Shaner. 2004. Management and
resistance in wheat and barley to Fusarium head
blight. Ann Rev. ofPhytopath 42:135-161.







Session 1: FHB-QTL consortium


Guo, P.-G., G.-H. Bai & G. E. Shaner. 2003. AFLP
and STS tagging of a major QTL for scab
resistance in wheat. Theor. & Appl. Genet.
106:1011-1017.
Guo, P.-G., G.-H. Bai, R.-H. Li, G. E. Shaner & M.
Baum. 2006. Resistance gene analogs associated
with Fusarium head blight resistance in wheat.
Euphytica (In press).
Kosambi DD (1944) The estimation of map distances
from recombination values. Ann Eugen 12: 172-
175
Ma, H.-X., G.-H. Bai, X. Zhang & W.-Z. Lu 2006a.
Main effects, epistasis and environmental
interactions of QTLs for Fusarium head blight
resistance in a recombinant inbred population
Phytopathology 96:534-541.
Ma, H.-X., G.-H. Bai,B. S. Gill & L. P. Hart. 2006b.
Deletion of a chromosome arm altered wheat
resistance to Fusarium head blight and
deoxynivalenol accumulation in Chinese Spring.
Plant Disease (In press).
Mirocha, C. J., E. Kolaczkowski, W.-P. Xie, H. Yu &
H. Jelen 1998. Analysis of deoxynivalenol and its
derivatives (batch and single kernel) using gas
chromatography/mass spectrometry. J. Agric.
Food Chem., 46, 1414-1418.
Nelson, J. C. 1997. QGENE: software for marker-
based genomic analysis and breeding. Mol. Breed.
3:239-245.
Van Ooijen, J.W., and Voorrips, R.E. 2001.
JoinMap3.0, Software for the calculation of
genetic linkage maps. Plant Research
International, Wageningen, the Netherlands.
Van Ooijen, J.W. 2004. MapQTL 5, Software for
mapping of quantitative trait loci in experimental
populations. Kyazma B.V., Wageningen,
Netherlands.
Saghai-Maroof, M. A., K. M. Soliman, R. A.
Jorgensen, & R. W. Allard. 1984. Ribosomal
DNA spacer-length polymorphisms in barley:
Mendelian in heritance, chromosomal location,
and population dynamics. Proc. Natl. Acad. Sci.
USA. 81:8014-8018.
Yang, J, G.-H. Bai, & G. E. Shaner. 2005. Novel QTL
for Fusarium head blight resistance in wheat
cultivar Chokwang. Theor. Appl. Genet.
111:1571-1579.
Yu, J.-B., G.-H. Bai, S.-B. Cai, & T. Ban 2006.
Marker-assisted characterization of Asian wheat
lines for resistance to Fusarium head blight.
Theor. Appl. Genet. Online at
http://dx.doi.org/10.1007/s00122-006-0297-z.


Zhou, W.-C., F. L. Kolb, J.-B. Yu, G.-H. Bai, L. K.
Boze & L. L. Domier. 2004. Molecular
characterization of Fusarium head blight
resistance in Wangshuibai with simple sequence
repeat and amplified fragment length
polymorphism markers. Genome 47: 1137-1143.
Zhou, W.-C., F. L. Kolb, G.-H. Bai, L. L. Domier, &
J.-B. Yao. 2002a. Effect of individual Sumai3
chromosomes on resistance to scab spread within
spikes and deoxynivalenol accumulation within
kernels inwheat. Hereditas 137:81-89.
Zhou, W.-C., F. L. Kolb, G.-H. Bai, G. E. Shaner, & L.
L. Domier. 2002b. Genetic analysis of scab
resistance QTL in wheat with microsatellite and
AFLP markers. Genome 45:719-27.






Session 1: FHB-QTL consortium


QTL FOR THE RESISTANCE TO WHEAT FUSARIUM HEAD
BLIGHT AND DEOXYNIVALENOL ACCUMULATION
IN WANGSHUIBAI UNDER FIELD CONDITIONS


H. X. Ma", K. M. Zhang1, L. Gaol, G. H. Bai2, H. G. Chen1,
Z. X. Cai1, and W. Z. Lu1

'Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China. 2 USDA-ARS-Plant Science
and Entomology Research Unit, Kansas State University, Manhattan, KS 66506, USA
*Corresponding Author: PH: (86) 25-8439-0300; E-mail: mahxlliaas.ac.cn



ABSTRACT

Wheat Fusarium head blight (FHB) may cause serious losses in grain yield and quality. Production of
deoxynivalenol (DON) by Fusarium graminearum in infected grain is detrimental to livestock and is also a safety
concern in human foods. Cultivation of cultivars with resistance to FHB and DON accumulation is the most
effective strategy for disease control. Wangshuibai is a Chinese landrace with a high level of resistance to FHB and
DON accumulation, and an F7 population of recombinant inbred lines (RILs) derived from the cross between
Wangshuibai and susceptible cultivar Annong 8455 was developed for molecular mapping of quantitative trait loci
(QTL) for the resistance to FHB and DON accumulation Proportion of scabbed spikelets (PSS) and DON content
were assessed under the field conditions over two years. Three hundred fifty-three SSR and AFLP markers were
mapped on 38 linkage groups covering a genetic distance of 1594 cM. Composite interval mapping (CIM) revealed
that two and three QTL were significantly associated with low PSS and low DON content, respectively, over two
years. QTL on chromosome 3B and 2A explained 17% and 11.5% of the phenotypic variance for low PSS,
respectively, whereas QTL on chromosome 5A, 2A and 3B explained 12.4, 8.5 and 6.2% of the phenotypic variance
for low DON content, respectively. The 3B QTL appeared to be associated mainly with low PSS, and the 5A QTL
primarily with low DON content in Wangshuibai. The 2A QTL had minor effect to both low PSS and DON content.
The SSR markers linked to these QTL should be useful for marker-assisted selection (MAS) of QTL for low PSS
and low DON content from Wangshuibai.







Session 1: FHB-QTL consortium


RESEARCH ON MOLECULAR MAPPING OF FUSARIUM
HEAD BLIGHT RESISTANCE IN WHEAT AT IFA-TULLN,
AUSTRIA


H. Buerstmayr*, B. Steiner, and M. Lemmens

BOKU University of Natural Resources and Applied Life Sciences Vienna, Department for Agrobiotechnology,
IFA-Tulln, Konrad Lorenz Str. 20, A-3430 Tulln, Austria
*Corresponding author: Phone: +43 (0)2272 66280 201, Email: hermannbuerstmavr(@iboku.ac.at


ABSTRACT

At IFA-Tulln we have been working on searching for
resistance sources, genetic analysis of promising
sources, improvement of phenotypic resistance
evaluation methods, and the role of fungal toxins in the
plant-pathogen interaction for more than 10 years.

We generated several recombinant mapping
populations (DHs or RILs) in order to allow for
replicated resistance testing using artificial inoculation
Possibly the most important factor for successful QTL
mapping is the accurate phenotyping of the lines. We
therefore put much emphasis on this aspect by
performing artificial inoculation in replicated (min 2
seasons) experiments. We usually sow replications
within each experiment in staggered time intervals.
This allows each genotype to reach anthesis and to be
inoculated at slightly different micro-environmental
conditions within the same experiment. Under our
conditions about 2 weeks sowing difference will result
in about 2 days flowering difference for winter wheat
and about 7-9 days sowing difference will result in 2
days flowering difference for spring wheat.

In several of our projects we applied different
inoculation methods to evaluate for components of
resistance, like single-spikelet inoculation to test for
resistance to fungal spread and spray inoculation to test
for 'field' resistance.

For molecular genetic analysis we used SSRs and
AFLPs in the past. SSR markers allowed anchoring of
linkage groups relative to published maps (e.g. in the
graingenes database, http://wheat.pw.usda.gov). With
AFLP markers we could generate of many marker data
in relatively short time and at reasonable cost.

The first population we analysed was derived from the
cross CM-82036/Remus, the second was


Frontana/Remus. CM-82036 (a line selected at
CIMMYT from Sumai-3/Thornbird) appeared to carry
two major QTL for disease severity under field
conditions mapping to chromosomes 3BS (Qfhs.ndsu-
3BS, Fhbl) and 5A (Q/hs.ifa-SA) (Buerstmayr et al.
2002, 2003). The QTL on 3BS of CM-82036 confers
resistance to fungal spread and is the same locus found
in a lot of mapping studies based on Asian resistance
sources including Sumai#3, Ning 7840 and W14 (e.g.
Anderson et al. 2001, Zhou et al. 2002, Chen et al.
2006). The 5A QTL appears primarily involved in
resistance to fungal penetration (Buerstmayr et al.
2003, Chen et al. 2006). In a recent study, Lemmens et
al. (2005) found that the FHB resistance QTL at 3BS
co-localizes with the ability to detoxify the mycotoxin
deoxynivalenol.

Compared to CM-82036, Frontana showed more QTL
with smaller individual effects of which those on 3A
and 5A appeared to be relatively stable (Steiner et al.
2004). In an independent Frontana derived population
(Frontana/Seri82) only the QTL on chromosome 3A
was consistent with the Frontana/Remus population
(Mardi et al. 2006).

In a population from the cross Wangshuibai/Seri82 two
QTL were detected, where the major resistance factor
mapped near Qfhs.ndsu-3BS (Mardi et al. 2005).
Unfortunately, different Wangshuibai derived mapping
studies in the literature showed only moderate
agreement with our results and with each other, apart
from the QTL on 3BS. Reasons for the non-agreement
between independent mapping studies may be
manifold including the use of different seed stocks of
the resistant line, the different susceptible parents used,
and different inoculation and testing methods applied.

In an attempt to develop higher resolution maps for
Q/hs.ifa-5A (Buerstmayr et al. 2003) we found
severely suppressed recombination in the 5A QTL







Session 1: FHB-QTL consortium


region, making fine mapping very difficult. On the
other hand several SSR markers appear tightly linked
to the QTL, which is advantageous for marker assisted
selection (unpublished results).

More recently, we were also involved in QTL analysis
using the European winter wheat cross Dream/Lynx
(Schmolke et al. 2005). The results indicate that
European winter wheat possesses different QTL for
FHB resistance than Asian or South-American sources,
opening the possibility of resistance gene pyramiding.
In a marker assisted selection study for three QTL (3B,
5A, 3A) using spring wheat crosses, all three QTL
showed significant effects in reducing FHB severity
and DON content (Miedaner et al. 2006).

Currently ongoing projects in FHB resistance involve
the use of more distantly related resistance sources like
Triticum macha for bread wheat and T. dicoccoum or
T. dicoccoides for durum wheat by using advanced
back-cross QTL mapping approaches (Tanksely and
Nelson 1996).

In addition we work on a project to identify
differentially expressed genes involved in the disease
response by applying cDNA-AFLP on near isogenic
lines for the FHB resistance QTL on 3BS and 5A.

ACKNOWLEDGEMENTS

Participation in the workshop was supported by a
travel grant from the University of Natural Resources
and Applied Life Sciences Vienna. The research
activities were and are supported by the Austrian
Science Fund (projects number P11884-GEN, P16724-
B05, P17310-B05), the Federal Ministry of Science,
the GEN-AU program, the European Union, and the
Provincial Government of Lower Austria.

REFERENCES

Anderson, J.A., Stack, R.W., Liu, S., Waldron, B.L.,
Fjeld, A.D., Coyne, C., Moreno-Sevilla, B.,
Mitchell Fetch, J., Song, Q.J., Cregan, P.B.,
Frohberg R.C. 2001. DNA markers for Fusarium
head blight resistance QTLs in two wheat
populations. Theor. Appl. Genet. 102:1164-1168
Buerstmayr, H., Lemmens, M., Hartl, L., Doldi, L.,
Steiner, B., Stierschneider, M., Ruckenbauer, P.
2002. Molecular mapping of QTL for Fusarium
head blight resistance in spring wheat I: resistance
to fungal spread (type II resistance). Theor. Appl.
Genet. 104: 84-91.


Buerstmayr, H., Steiner, B. Hartl, L., Griesser, M.,
Angerer, N., Lengauer, D., Miedaner, T.,
Schneider, B., Lemmens, M. 2003. Molecular
mapping of QTLs for Fusarium head blight
resistance in spring wheat II: resistance to fungal
penetration and spread. Theor. Appl. Genet. 107:
503-508.
Lemmens, M., Scholz, U., Berthiller, F., Dall'Asta, C.,
Koutnik, A., Schuhmacher, R., Adam, G.,
Buerstmayr, H., Mesterhazy, A., Krska, R.,
Ruckenbauer, P. 2005. The ability to detoxify the
mycotoxin deoxynivalenol colocalizes with a major
quantitative trait locus for Fusarium head blight
resistance in wheat. Mol. Plant Microbe Int. 18:
1318-1324.
Mardi, M., Buerstmayr, H., Ghareyazie, B., Lemmens,
M., Mohammadi, S.A., Nolz, R., Ruckenbauer, P.
2005. QTL analysis of resistance to Fusarium head
blight in wheat using a 'Wangshuibai'-derived
population Plant Breeding 124: 329-333
Mardi, M., Pazouki, L., Delavar, H., Kazemi, M.B.,
Ghareyazie, B., Steiner, B., Nolz, R., Lemmens,
M., Buerstmayr, H. 2006. QTL analysis of
resistance to Fusarium head blight in wheat using a
'Frontana'-derived population Plant Breed. (in
press).
Miedaner, T., Wilde, F., Steiner, B., Buerstmayr, H.,
Korzun, V., Ebmeyer, E. 2006. Stacking
quantitative trait loci (QTL) for Fusarium head
blight resistance from non-adapted sources in an
European elite spring wheat background and
assessing their effects on deoxynivalenol (DON)
content and disease severity. Theor. Appl. Genet.
112: 562-569.
Schmolke, M., Zimmermann, G., Buerstmayr, H.,
Schweizer, G., Miedaner, T., Korzun, V., Ebmeyer,
E., Hartl, L. 2005. Molecular mapping of Fusarium
head blight resistance in the winter wheat
population Dream/Lynx. Theor. Appl. Genet. 111:
747-756.
Steiner, B., Lemmens, M., Griesser, M., Scholz, U.,
Schondelmaier, J., Buerstmayr, H. 2004. Molecular
mapping of resistance to Fusarium head blight in
the spring wheat cultivar Frontana. Theor. Appl.
Genet. 109: 215-224.
Tanksley, S.D., Nelson, J.C. 1996. Advanced
backcross QTL analysis: a method for the
simultaneous discovery and transfer of valuable
QTLs from unadapted germplasm into elite
breeding lines. Theor. Appl. Genet. 92: 191-203.
Zhou, W.C., Kolb, F.L., Bai, G.H., Shaner, G.,
Domier, L.L. 2002. Genetic analysis of scab
resistance QTL in wheat with microsatellite and
AFLP markers. Genome 45:719-727.







Session 1: FHB-QTL consortium


EVIDENCE THAT RESISTANCE TO FUSARIUM HEAD
BLIGHT AND CROWN ROT ARE CONTROLLED BY
DIFFERENT GENES IN WHEAT


G.Q. Xie1'2, M.C. Zhang1'3, T. Magner1, T. Ban4, S. Chakraborty1, and C.J. Liu'*

1. CSIRO Plant Industry, Queensland Bioscience Precinct, 306 Carmody Road, St Lucia, Brisbane 4074, Australia
2. Feed Science Research Institute of Jiangxi Province, College of Animal Science and Technology, Jiangxi
Agricultural University, Nanchang 330045, China
3. Hebei Food and Oil Crops Institute, 440 Yuhua East Road, Shijiazhuang 050031, China
4. Japan-CIMMYT FHB Project, CIMMYT, Apdo. Postal 6-641, 06600 Mexico, D.F., Mexico
*Corresponding author: Email: chunji.liu@,csiro.au


ABSTRACT

To test whether resistance against the two diseases are
conditioned by the same genes, we carried out two
experiments. Firstly, Fusarium head blight (FHB) and
crown rot (CR) were assessed in a common set of
hexaploid wheat genotypes using one aggressive
isolate each of F. graminearum and F.
pseudograminearum. A clear correlation between CR
and FHB severity was not detected for either isolate. In
the second experiment we created and analyzed
populations segregating for the 3BS allele of Sumai 3,
a major FHB resistant locus. As expected, plants with
the Sumai 3 allele showed significant reduction in
FHB severity. However, the presence of the 3BS locus
showed no effect on CR resistance. These results
provide evidence that, although FHB and CR are
caused by the same pathogens, different host genes
may control resistance to these two diseases that infect
different tissues.

INTRODUCTION

Fusarium pathogens cause two serious diseases in
wheat. Of these, FHB has been intensively studied
internationally, while CR, which is a major constraint
to wheat production in Australia, has received less
attention Some of the outcomes of the international
effort include the screening of many thousands of
wheat genotypes and the identification of several lines
with high levels of resistance (Gilchrist et al. 1997; Lu
et al. 2001). Although F. graminearum predominantly
causes FHB and F. pseudograminearum is the most
common CR pathogen, recent studies show that both
pathogens can cause FHB and CR. This raises the
question whether the same genes confer resistance to
the two diseases. The FHB resistant genotypes could


potential be invaluable in our effort to breed crown rot
resistant cultivars if the same genes are involved in
resistance to these two diseases. This paper reports on
two experiments to address this: in one, a set of
hexaploid wheat genotypes was inoculated separately
using an aggressive isolate each of F. graminearum
and F. pseudograminearum; in the second experiment
the progeny of two populations segregating for a major
FHB resistance locus on the chromosome arm 3BS
were analyzed. Results from these two experiments
were summarized in this paper.

MATERIALS AND METHODS

Plant material
In the first experiment, 24 genotypes were used. FHB
bioassay was carried out in a controlled environment
facility at the CSIRO Plant Industry Brisbane
Laboratories, with 25/150C day/night temperature and
65/95% relative humidity, and a 13 -hour photoperiod.
A F. pseudograminearum (CS3096) and F.
graminearum isolate (CS3255) from CSIRO collection
were used. These two isolates are highly aggressive
according to a screening of over 650 isolates collected
in field surveys from Queensland and NSW
(Akinsanmi et al. 2004). Each replicate consisted of
two plants in two separate pots and two replicates were
used for each isolate. Eight to ten spikes from each of
the two replications were inoculated using a modified
"cotton wool" method developed at CIMMYT. At
anthesis a 3mm filter paper saturated with inoculum
(about 10gl suspension of 105 conidia/mL) was placed
into the fourth spikelet from the tip of a spike. The
inoculated spikes were immediately covered with
moistened polythene bags for 48 hours and then with a
paper bag until disease assessment at 21 days after
inoculation The FHB severity was measured as (a) the







Session 1: FHB-QTL consortium


average number of infected spikelets (NIS) below the
point of inoculation and (b) the proportion of infected
spikelets (PIS) from a counting of infected and total
number of spikelets. As susceptible genotypes often
rapidly develop bleaching of spikelets above the point
of inoculation, we, as suggested by Buerstmayr et al.
('i I',I, excluded these spikelets from data summary
and analysis. The same two isolates were used in a
glasshouse bioassay to determine CR resistance of the
24 genotypes. The glasshouse is maintained at 24/15C
day/night temperature with natural illumination Three
replications with five seedlings each were tested for
each genotype-isolate combination Ten-day-old
seedlings were inoculated by placing a 10l droplet of
inoculum (106 conidia/mL) on the stem, 0.5 to 1cm
from the soil surface. The inoculated seedlings were
kept in a humidity chamber for 48 hours then
transferred back to the glasshouse. CR severity was
assessed as the length of discolored stem at 35 days
after inoculation Data were analyzed with t-tests using
Microsoft Excel and arithmetic means were compared
using Duncan's multiple range test.

In the second experiment, two 4-way F2/3 populations
were used:
(A) Baxter/3/Lang//EGA Wylie/Sumai 3
(B) Drysdale/3/EGA Gregory//EGA Wylie/Sumai 3

In this experiment, FHB and CR assays were carried
out following conditions described above using the F.
pseudograminearum isolate CS3096.

Identification of individuals with or without the FHB
resistant 3BS allele of Sumai 3 by molecular marker
analysis
DNA was isolated from all individuals of the two
populations and their parents. Leaf tissue was collected
during controlled environment facility and glasshouse
trials. For each genotype, a small section of a young
leaf was ground with 200 tl DNA extraction buffer


Crown
14
12
10
8
6
4
2
0


Rot

A
R = 0.0275

**
4^^ ^

-.t *


(100mM Tris-HCl pH8.5, 100 mM NaC1, 50 mM
EDTA pH8.0, and 2% SDS) and incubated at 65C for
1-2 h. The sample was extracted once with
phenol/chloroform and DNA was precipitated with
ethanol and dissolved in 100 tL of 1 x TE. Aliquots of
initial extractions were diluted in water to a final
concentration of-25 ng/tl prior to PCR amplification

Two SSR markers, gwm493 and gwm533, were used
to identify the presence/absence of the 3BS locus of
Sumai 3. These two markers, with a map distance of
about 7 cM between them, are known to flank the
major FHB resistance of Sumai 3 on the short arm of
chromosome 3B (Buerstmayr et al 2003; Liu and
Anderson 2003). PCR reactions for SSR amplification
were performed in a total volume of 10 gL containing
5 nM of each primer, 0.2mM of each deoxynucleotide,
1.5 mM MgC12, 0.25 unit Taq polymerase and 50 ng
template DNA. After an initial denaturing step for 3
min at 94oC, 40 PCR cycles were performed with 30 s
at 940C, 15 s at 58 C, 30 s at 72 C, followed by a final
extension step of 7 min at 72 C. PCR products were
separated on 3% agarose gels and visualized by
staining with ethidium bromide.

RESULTS AND DISCUSSION

1. Correlation between CR and FHB resistance
assessed using different genotypes:
Both isolates used caused CR and FHB. CR severity
caused by the F. graminearum isolate ranged from 0.0
to 12.1 with an average of 3.9, and that caused by the
F. pseudograminearum isolate ranged from0.3 to 14.2,
with an average of 4.4. FHB severity caused by the F.
graminearum isolate ranged from 1 to 8.1 (average
3.3), and 1.8 to 12.0 (average 5.3) for the F.
pseudograminearum isolate. A strong correlation was
not detected between CR and FHB resistance for either
of the two isolates (Figure 1).


Crown Rot
10
8 B
6 R2 = 0.002 *
44
4- ,- e t
2- .
0 *


0 2 4 6 8 10
Fusarium Head Blight


Fusarium Head Blight


Figure 1. Relationship between FHB and CR resistance in 24 wheat genotypes assessed
using F graminearum (A) and F pseudograminearum (B) isolates


, r ..







Session 1: FHB-QTL consortium


2. Identification of individuals with or without the
3BS locus of Sumai 3:
Two SSR markers, gwm493 and gwm533, were used
to identify individuals that inherited the 3BS allele of
Sumai 3. The marker gwm493 detected a single locus
but gwm533 detected two loci, gwm533a and
gwm533b (Figure 2). Both of the gwm533 loci were
segregating in one (B) of the 4-way F2 populations but
only gwm533a was segregating in the other. The single
PCR product amplified from either Baxter or Drysdale
was located between the two products of gwm533 and
was allelic with gwm533a (Figure 2).
To test the associations between the two different loci
detected by gwm533 and FHB resistance, FHB


Population A


severity between individuals with or without the
gwm533 allele of Sumai 3 was compared using
Population B with 388 inoculated spikes. Individuals
with the gwm533a allele of Sumai 3 had significantly
lower FHB severity compared with those without this
allele, but the difference between individuals with or
without the gwm533b allele was not significant (data
not shown). This suggests that the gwm533a locus is
more closely associated with FHB resistance than
gwm533b. Thus, data derived from gwm533a was used
here for inferring the presence of the 3BS allele of
Sumai 3.


Population B


- gwm533a
- gwm533b


Figure 2. Profiles of SSR marker gwm533 in the two populations, showing that the marker detected two segregating
loci in one population (B) but only one in the other (A). Lane 1 is Sumai 3 and lane 2 Baxter.


3. Effect of the 3BS locus of Sumai 3 on FHB
resistance:
Significant differences were detected between
individuals with or without the 3BS allele of Sumai 3.
When only individuals with Sumai 3 alleles of both
markers were considered, there was a 27.0% to 42.0%
(average 32.0%) reduction in the NIS and a 22.4% to
39.0% (average 29.2%) reduction in PIS among
individuals that had the 3BS allele of Sumai 3 (Table
1). Compared with the average severity of the local
cultivars used to produce the two 4-way F2
populations (ignoring their different contributions to
the progeny), individuals with the 3BS allele of Sumai
3 reduced FHB severity by an average of 42.0% and
47.8%, respectively. These results confirm that the
presence/absence of the FHB resistant locus on the
short arm of chromosome 3B was effectively selected
using the two flanking SSR markers, and that the locus
has a significant effect on FHB resistance.
4. Effect of the 3BS locus of Sumai 3 on CR
resistance:


The two measurements of CR resistance produced
slightly different results. When visual rating was used,
the difference between the four families from
population A was highly significant. Individuals with
the 3BS locus of Sumai 3 showed slightly improved
CR resistance (Table 2). When the length of the
discolored leaf sheath was used, however, the
difference among the four families from this
population was not significant. For the four families
from population B, highly significant differences were
detected using either rating or the length of discolored
leaf sheath (Table 2). In contrast to the four population
A families, individuals without the 3BS allele of Sumai
3 showed better CR resistance in the four population B
families. The combined results of the two populations
showed that the presence of the 3BS allele of Sumai 3
had no effect on CR resistance for either measure of
CR severity (Table 2).


WIMM Samoa
saw seem







Session 1: FHB-QTL consortium


Table 1. Effect of the 3BS locus of Sumai 3 onFHB severity
Population A Population B Average
Locus allele
NISb PIS NIS PIS NIS PIS
a 7.3 56.9 8.1 53.8 7.5 57.3
gwm493 A 5.2 40.2 4.7 32.8 5.2 41.8
Difference P<0.01 P=0.01 p<0.01 p<0.01 p<0.01 p<0.01
b 7.3 56.9 8.1 54.1 7.6 59
gwm533 B 5.2 40.2 5 34.2 5.3 41.5
Difference P<0.01 P<0.01 P<0.01 p<0.01 p<0.01 p<0.01

gwm493 ab 7.3 56.9 8.1 53.8 7.5 57.2
and AB 5.2 40.2 4.7 32.8 5.1 40.5
gwm533 Difference P<001 001 0.01 pP0.01 pP0.01 p. p. p<0.01


a. a and b represent Baxter/Drysdale alleles of gwm493 and gwm533,
alleles ofgwm493 and gwm533, respectively
b. NIS = Number of infected spikelets
c. PIS = percentage of infected spikelets


respectively; A and B represent Sumai 3


Table 2. Crown rot severity, assessed by either a visual rating or discolored leaf sheath, of individuals wither
without the Sumai 3 alleges of gwm4 3


Locus Alele Population A Population B Average
Locus Allelea
rating length Rating rating rating length
a 1.82 1.94 0.67 0.93 1.31 1.49
gwm493 A 1.29 1.53 1.09 1.44 1.17 1.47
difference P<0.01 P=0.05 P<0.01 P<0.01 p=0.13 p=0.45
b 1.95 1.99 0.67 0.93 1.27 1.42
gwm533 B 1.39 1.64 1.09 1.44 1.25 1.54
difference p=0.08 p=0.18 P<0.01 P<0.01 p=0.44 p=0.21

m493 ab 2.04 2.12 0.67 0.93 1.25 1.42
and AB 1.18 1.89 1.09 1.44 1.12 1.55
gwm533 difference P<0.01 p=0.23 P<0.01 P<0.01 p=0.18 p=0.20


a. a and b represent Baxter/Drysdale allies of gwm493 and gwm533, respectively: A and B represent E umai
alleges of gwm493 and gwm533, respectively


_







Session 1: FHB-QTL consortium


CONCLUSIONS

This paper describes two experiments aimed at
examining the relationship between FHB and CR
resistance. Results from both experiments suggest that
resistance to FHB and CR are controlled by different
genes, although these two diseases can be caused by
the same Fusarium species. This implies that the
numerous FHB resistant genotypes identified in the
international programs may not be very useful in our
CR research and separate screenings for the two
different diseases seem to be essential. However,
further tests with the use of a much larger number of
genotypes would be needed to confirm these results. It
would also be highly desirable if genotypes showing
high levels of resistance to both FHB and CR could be
identified and used to further investigate the genetic
bases of resistance to these two diseases.

ACKNOWLEDGEMENTS

The authors are grateful to Phil Banks (QDPI,
Australia) for providing some of the parental
genotypes, and to Dr. Vivek Mitter and Mr. Ross
Perrott for preparing the inoculum. We thank the CRC
for Tropical Plant Protection and the Grains Research
and Development Corporation for financial support.

REFERENCES

Akinsanmi O.A., Mitter V, Simpfendorfer S,
Backhouse D, Chakraborty S (2-'14) Identity and
pathogenicity of Fusarium spp. Isolated from wheat
fields in Queensland and northern New South
Wales. Australian Journal of Agricultural Research
55, 97-107.
Buerstmayr H, Lemmens M, Hart L, Doldi L, Steiner
B, Stierschneider M, Ruckenbauer P (2002)
Molecular mapping of QTLs for Fusarium head
blight resistance in spring wheat. I. Resistance to
fungal spread (Type II resistance). Theoretical and
Applied Genetics 104, 84-91.
Buerstmayr H, Steiner B, Hart L, Griesser M, Angerer
N, Lengauer D, Miedaner T, Schneider B,
Lemmens M (2003) Molecular mapping of QTLs
for Fusarium head blight resistance in spring
wheat. II. Resistance to fungal penetration and
spread. Theoretical and Applied Genetics 107, 503-
508.
Gilchrist L, Rajaram S, Mujeeb-Kazi A, van Ginkel M,
Vivar H, Pfeiffer W (1997) Fusarium Scab
screening Program at CIMMYT. In: Dubin H.J.,
Gilchrist L, Reeves J, and McNab A (eds):
Fusarium Head Scab: Global status and future
prospects. Mexico, D.F.: CIMMYT. pp7-12.


Liu S, Anderson J.A. (2003) Marker assisted
evaluation of Fusarium head blight resistant wheat
germplasm. Crop Science 43, 760-766.
Lu WZ, Cheng SH, Wang YZ (2001) Wheat Scab
Research in China. Scientific Publication Ltd,
Beijing pp229. ISBN 7-03-009199-X.







Session 1: FHB-QTL consortium


FUSARIUM HEAD BLIGHT RESISTANCE FROM WIDE
CROSSES IN BREAD WHEAT AND DURUM


G. Fedak1 W. Cao A. Xue M. Savard1, J. Gilbert2, J. Clarke3, and D. Somers2

'Eastern Cereal and Oilseed Research Center, Agriculture and Agri-Food Canada, 960 Carling Ave., Ottawa ON
K1A 0C6, Canada. 2Cereal Research Center, 195 Dafoe Road, Winnipeg MN R3T 2R9.
3Semiarid Prairie Agricultural Research Center, P. O. Box 1030 Swift Current SK S9H 3X2
*Corresponding Author: PH: 613-759-1393; E-mail: fedakgaZ(agr.gc.ca


OBJECTIVES

To identify resistance to Fusarium head blight (FHB)
in wild relatives of wheat, to transfer this resistance
into bread wheat and durum and to enhance the
resistance inherent in those two wheat species.

INTRODUCTION

Fusarium head blight has become a devastating disease
of cereals in temperate climate regions of the world.
There appears to be sufficient inoculum built up so that
the occurrence of rainfall during the flowering period
of the crop is certain to cause extensive infection The
single best source of resistance under our conditions
has been the variety Sumai 3. However, under
intensive infection pressure in our epiphytotic nursery,
Sumai 3 will suffer up to 20% floret infection and
deoxyivalenol (DON) content as high as 5.0 ppm We
have employed two methods of enhancing the FHB
resistance of Sumai 3. The first is by combining the
resistance of Sumai 3 with that of unrelated wheats
such as Nyu Bay, Frontana and other Brazilian and
Chinese wheats. The second method is by screening
large numbers of accessions of alien species, selecting
lines with resistance and introducing the resistance to
bread wheat and durum

MATERIALS AND METHODS

The Triticum timopheevii accessions that were
screened for FHB resistance were those previously
identified as having multiple pest resistance (Brown-
Guidera et al., 1996). Accessions of T. monococcum
were obtained from Maxime Trottet of INRA Le Rhou,
Cedex, France and Ae. speltoides accessions from
Maria Zahrieva of INRA Centre de Montpellier in
France. The T. miguschovae (AGD) accessions were
provided by Tamara Ternovska of the Mohyla
Academy in Kiev, Ukraine and the Ae. cylindrica (CD)


source of resistance was provided by Alexander
Rybalka of Odessa Ukraine.

The accessions of Tritordeum (ABH) that we screened
were either produced by ourselves or supplied by A.
Martin of Cordoba, Spain The T. carthlicum (AB)
accession was obtained from the Vavilov Institute of
St. Petersberg, Russia while the ABE amphiploid was
supplied by Fangpu Han formerly of the Northeast
Normal University at Changchun in China. The
screening methods involved growing the plant
materials in growth rooms, inoculating (point and
spray) spikes at 50% anthesis with a 50,000 spores/ml
suspension of F. graminearum. Plants with inoculated
spikes were "misted" for 48 hours and symptoms
scored at 21 days. Inoculation was repeated on
accessions showing minimal symptoms. Resistant
accessions of T. monococcum, Ae. speltoides and T.
miguschovae were crossed onto the cultivar Superb
and hybrid embryos were cultured on B5 medium The
T. monococcum hybrid was backcrossed to the cultivar
Fukuhokomugi; Ae cylindrica derivative was crossed
to Superb whereas three backcrosses to Superb were
required to restore fertility of the hybrid involving Ae.
speltoides. DT 712 (AC Strongfield) was the recipient
parent in the tetraploid manipulations. The ABE
amphiploid was crossed with the Capelli ph mutant to
enhance recombinations whereas repeated backcrosses
to AC Strongfield were carried out to produce the
addition lines.

The derived lines were seeded as one meter rows in the
FHB Nursery, inoculated with corn spawn and
irrigated twice a day. Symptoms were scored at 21
days after 50% anthesis. Incidence and severity scores
were assigned visually and the percentage of
Fusarium-damaged kernels (FDK) was determined on
threshed samples. Duplicate five-gram samples of seed
were ground. One gram of the ground samples was
randomly taken for DON analysis.







Session 1: FHB-QTL consortium


RESULTS AND DISCUSSION

The first field trial of interspecific derivatives was
conducted in the summer of 2004 and the results are
shown in Table 1. Since the check varieties performed
as expected from previous experiments it was
considered that the values for the test lines should be
relatively accurate. The DON contents for the seven
Ae. speltoides-derivatives ranged from 2.9 to 8.5 ppm.
Three of the lines approximated DON levels observed
in the Sumai 3; the best check variety. The DON levels
in the Ae. speltoides and T. timopheevii derivatives
were somewhat higher than in Sumai 3. However, if
the resistance is contributed by alleles different from
those already present in bread wheat, the new
resistance source may be effective in augmenting the
resistance of the known genes.


The field screening of the T. monococcum and Ae.
speltoides was repeated in 2005 with 22 and 70 lines
respectively. For some reason the DON levels in the
checks were very low in 2005, eg. in Sumai 3 it was at
2.9 ppm compared to 5.5 in 2004. Similarly the DON
levels in Roblin, the susceptible check, were 35.0 in
2004 but only 9.1 in 2005. The DON levels in the
derived lines were very low in 2005. The range for the
T. monococcum derivatives was 0.1-1.7 and 0.3-2.0 for
the Ae. speltoides derivative i.e. they overlapped the
levels observed in Sumai 3.

Although the DON levels were so drastically different
in two years of testing the values obtained for the
derived lines relative to the checks was consistent so
we feel that the resistance is real. Although we do not
present DON data for TC 67 (T. timopheevii-derived)
for 2005, we have carried out sufficient study with this
line to conclude that its FHB resistance is stable.


Table 1. FHB symptoms and DON content inprogenies of interspecific crosses with bread wheat (field data, 2004)


Source of
Resistance


Generation Incidence(%)


Ae. speltoides
Line 1
2
3
4
5
6
7

T. monococcum
Line 1

T. timopheevi
TC67

Checks
Sumai3
Nyu Bay
Fukuhokomugi
Roblin
AC Barrie


BC3F4


BC2F4


F9-SSD


Severity(%)


DON
FDK(%) (ppm)


11.7
7.0
6.3
14.7
6.7
10.0
7.0


19.0


10
33
15
80
45


13.9


9.0
13.2
50.0
90.0
20.3


8.5
3.6
4.3
5.3
3.2
2.9
4.8


8.9


5.7


5.5
3.4
7.7
35.0
16.3







Session 1: FHB-QTL consortium


Table 2. FHB symptoms and DON content of progenies from interspecific crosses with bread wheat (field data,
2005)


Source of
Resistance


T. monococcum
Ae. speltoides

Checks
Roblin
Superb
Fukuho
Sumai3


Number of
Generation lines


BC2F6
BC3F6


In our experience, there was a certain amount of
linkage drag accompanying the transfer of FHB
resistance from exotic sources to bread wheat. Even in
transfer of resistance from exotic wheat cultivars of
Brazilian, Chinese and Japanese origin it was
necessary to grow large populations and select
intensively for earliness, lodging resistance and shorter
plant stature. The same was true in progeny of
interspecific hybrids. The derived lines shown in
Tables 1 and 2 all have reasonable agronomics except
that they are several days later in flowering and
maturity than locally- adapted check cultivars. When
tested against local check varieties AC Brio and
Hoffman, the derived lines had hectolitre and thousand
kernel weights that were equal to the checks. In terms
of crude protein content, the T. monococcum and Ae.
speltoides derivatives had levels of 13.9 and 15.3,
respectively. We have also been transferring FHB
resistance from Ae. cylindrica (CD genomes) into
bread wheat. For the 2006 season we will have in
excess of 100 F6 lines to be tested in the epiphytotic
nursery. These lines were derived from crosses to the
cultivar Superb. This source of resistance has also been
crossed into the Canadian cultivars Teal, Barrie,
Domain, Elsa and HY644. Progenies from the latter
are now at the F4 and F5 generation

We are working on the introgressionof FHB resistance
into durum wheat. We received a sample of tetraploid
wheat from the Vavilov Institute labelled as T.
carthlicum. In our indoor screening facility it showed
reasonable Type I resistance to FHB. This line was
crossed to the durum cultivar Strongfield and a
doubled haploid population of about 150 lines


produced in the laboratory of Julian Thomas at Cereal
Research Center (CRC) of Winnipeg. The population
was phenotyped at the ECORC facility during the
winter of 2004. The marker work conducted by Daryl
Somers of CRC showed two major QTL on
chromosomes 2B and 6B that controlled the resistance.
These markers are now being used for MAS in the
durum program.

Introgression of resistance into durum wheat is also
continuing using Tritordeum (ABH), T. miguschovae
(AGD) and an amphiploid between T. turgidum x Th.
elongatum 2x (ABE) as resistance donors. The latter
has excellent FHB resistance. BC2 F4 progeny from
crosses to Tritordeum were grown in the epiphytotic
nursery in 2005. They appeared to have reasonable
resistance but were quite late maturing under our
conditions. We have produced BC2 F4 progeny from
crosses with T. miguschovae and are now screening
progeny indoors before going to field plots.

With amphiploid 8801 we are in the process of
advancing the progeny in two different streams. BC2
progeny have been produced from hybrids with the
Capelli ph mutant where we hope to isolate
recombinants with resistance. In the other stream the
cross was made with Strongfield and backcrossed with
the idea of producing EE addition lines. We now have
30 chromosomes lines showing resistance. The next
step will involve the induction of recombination
between the resistant addition lines and the durum
chromosomes. Once the resistance from alien sources
has been isolated, the next steps will involve
identifying the resistance QTL with molecular markers


FHB
Index

0.3-3.0
0.3-7.5


76.5
24.0
1.5
1.3


DON
Level (ppm)


0.1-1.7
0.3-2.0


9.1
9.2
1.9
2.9







Session 1: FHB-QTL consortium


and using these markers to "pyramid" the various
sources of resistance into adapted cultivars. A number
of mapping populations have been produced for this
purpose and appropriate crosses and intercrosses have
already been made.

From our experience in screening wild relatives of
wheat for FHB resistance, we find low frequencies of
resistant accessions in many alien species. This
phenomenon is reflected in several efforts worldwide
at introgressing alien resistance into wheat. Major
efforts are underway at the Nanjing Agricultural
University (Chen et al., 2004), Purdue University
(Shen et al, 2006) and North Dakota State University
(Oliver et al., 2004). Each of these groups has found
high levels of resistance in particular species and is
concentrating on those. The species that we are
concentrating on are different from the above (Fedak et
al., 2005). This appears to be a pragmatic approach as
in due time, it should be possible to pyramid resistance
genes from numerous unique sources.

REFERENCES

Brown-Guidera G.L. et al. 1996. Evaluation of a
collection of wild timopheevii wheat for resistance
to disease and arthropod pests. Plant Dis. 80: 928-
933.
Chen, P. et al. 2004. Development and utilization of
alien translocation lines for wheat scab resistance
improvement. Proceedings of the 2nd International
Symposium on Fusarium Head Blight. Orlando ,
Florida, 11-15 Dec. 2004 pp. 33.
Fedak, G. et al. 2005. Enhancement of Fusarium head
blight resistance in bread wheat and durum by
means of wide crosses. Proceedings of 7th
International Wheat Conference. Mar del Plata,
Argentina Nov. 27-Dec. 02, 2005 pp. 11.
Oliver, R.E. et al. 2004. Fusarium head blight
resistance in wheat-alien species derivatives.
Proceedings of the 2nd International Symposium
on Fusarium head blight. Orlando, Florida, 11-15
Dec. 2004 pp. 139.
Shen, X. 2006. Development of chromosome
recombinant lines of wheat-Thinopyrum ponticum
with resistance to Fusarium head blight. Plant and
Animal Genomes XIV Conference. Poster 308.







Session 1: FHB-QTL consortium


UTILIZATION OF WILD GENETIC RESOURCES FOR
THE IMPROVEMENT OF FHB RESISTANCE
IN WHEAT BREEDING


M. Kishii*, R. Delgado, V. Rosas, A. Cortes, S. Cano, J. Sanchez,
A. Mujeeb-Kazi, J. Lewis, and T. Ban

CIMMYT, Apdo. Postal 6-641, 06600 Mexico, D.F., Mexico
*Corresponding Author: PH: (52) 55 5804 2004; E-mail: mkishiiicgiar.org


MATERIALS AND METHODS


While accumulation of resistance sources from known
wheat genetic resources is a major task, we still need to
seek additional and more effective sources that we can
use in breeding programs. This is especially the case
for Type I resistance (against initial infection). A fair
number of bread wheat accessions have been reported
as Type II resistant (against spread of infection), but
none are Type I resistant. The diverse source of genes
provided by wheat relatives and alien species has the
potential to produce wheats resistant to FHB.
Ancestral species would be the first choice to explore
because genetic materials can be introduced by
recombination This includes wild types of hexaploid
and tetraploid wheat as well as diploid ancestors of A,
B, and D genomes. A number of alien species has
been reported as resistant, including Thinopyrum,
Leymus, and Roegneria (Mujeeb-Kazi 1986; Wan
1997; Fedak 2003). Wan et al. (1997) reported genus
Roegneria as hyper-resistant for both Type I and Type
II resistance, while Sumai 3 was regarded as
susceptible in Type I. CIMMYT has been working on
D genome synthetics and demonstrating that these are
useful sources for FHB resistance in bread wheat.
Likewise, synthetic wheat of A and B genomes would
be useful for durum breeding. For pursuing further
resistances, we are now turning more attention on alien
sources. After evaluation of the FHB resistance of
amphiploids and addition lines, we started production
of translocation lines. However, one problem for this
production is that it will require significant investments
of time and labor until these become useful in the
breeding program This is a challenge we need to
overcome.


Plant Materials (Table 1)
All synthetic wheats used have been produced in
CIMMYT by crossing durum and diploid ancestor
species followed embryo rescue and chromosome
doubling. There are about 1,100 lines of D genome
synthetic (AABBDD), 150 lines of A genome
synthetic (AAAABB), and 50 lines of B genome
synthetic (AABBBB). Amphiploid and addition lines
of Leymus and Elytrigia were produced in CIMMYT
or Kihara Institute (Japan). The mapping populations
were obtained after crossing resistance synthetic
derivatives and susceptible wheat variety 'Flycatcher'
(FCT).

Production oftranslocation
Amphiploid of Thinopyrum bessarabicum was crossed
twice with wheat line Chinese Spring (phlb).
Translocations between Th. bessarabicum and wheat
chromosomes were detected by genomic in situ
hybridization (GISH). The plants with translocations
were backcrossed with CIMMYT variety PRINIA
several times to produce a translocation line whose
chromosome constitution is 42. Addition lines of
Leymus racemosus were crossed with monosomic lines
whose homoeology is supposed to matchL. racemosus
chromosomes in the addition lines. Centromeric
translocation was screened in their progenies.
Addition lines of L. racemosus were also crossed with
2C chromosome addition lines. The 2C chromosome
is from Aegilops cylindrica and has been known to
induce chromosome breakage and translocation in its
progeny (Endo 1998). Translocation was screened in
the F2 generation The A and D genome translocation
will be induced in the progenies of the crosses between
durum line 'Capelli (phlc)' and Aegilops tauschii
lines.


INTRODUCTION







Session 1: FHB-QTL consortium


Evaluation of Type II resistance
The evaluation was conducted in early September in
Toluca station in Mexico. Five to ten spikes of each
line were point-inoculated with FHB suspension
(50,000 spores / ml) and then covered with glassine
bags. Spread of infection was measured 30-35 days
after the inoculation

RESULT AND DISCUSSION

Bread Wheat

Synthetic wheat/Aegilops tauschii
After screening of 1,000 synthetic wheats of D
genome, we have identified five lines of FHB
resistance whose Type II resistance are equal to or
higher than Sumai 3. Since all five synthetic wheats
were put into different phylogenic groups (data not
shown), they may contain different resistance sources.
Three lines of synthetic derivatives were employed to
propagate nine sets of mapping populations.
Quantitative trait locus (QTL) analysis has been
conducted on one of the mapping populations, and one
strong QTL was detected in chromosome 2DL (Lewis
et al. 2004). It is important to investigate whether the
other four synthetic wheats have novel sources or not.
Resistant synthetic wheats have been crossed with
several CIMMYT varieties to transfer resistance. Field
testing has been conducted in CIMMYT Toluca station
every year with selection of the lines whose infection
level is less than 15%. The F8 were propagated in
2005, and these materials can be used as alternative
resistance sources inbreeding programs.

Leymus racemosus
We used two different L. racemosus accessions and
obtained two sets of addition lines. The first and the
second sets contain ten and six addition lines,
respectively. The evaluation of these lines showed one
mild resistance in the first set and three lines of
resistance in the second set. The difference between
the sets may indicate that the choice of parents is
important for FHB resistance. To transfer these
resistances to bread wheat, we started the production of
translocation lines using 2C gametocidal systems. In
the last year, we have obtained about 40 translocated
chromosomes. Separately from this work, we are now
propagating centromeric translocations using
monosomic lines. After several backcrossings with
CIMMYT varieties, these translocations will be readily
transferred into CIMMYT breeding varieties to see the
effect of the translocation

Thinopyrum bessarabicum
It was reported that amphiploids and addition lines
between Thinopyrum species and wheat would


increase FHB resistance, including using species Th.
elongatum, Th. intermedium, Th. junceiforme, Th.
ponticum (Jauhar et al. 2001; Fedak et al. 2003; Shen
et al. 2004). Since we had a set of addition lines of Th.
bessarabicum, we focused on this species.
Amphiploids between wheat and Th. bessarabicum
showed FHB resistance higher than Sumai 3 (Table 2),
but we observed that only three addition lines
possessed mild resistance (-15%; Table 2). Because
of complex nature of FHB resistance, the resistance
may become diluted in the addition lines, or the
resistance may not be expressed in the wheat
background. We have already obtained four different
translocation lines and also twenty translocated
chromosomes. We will evaluate resistance of these
lines in the future.

Durum Wheat

Tetraploid and AA and BB genome ancestors
Preliminary screening of wild relatives showed that we
would find resistance sources in Triticum dicoccoides
and T. dicoccum (Table 3). The infection level in
those species ranged from 6% to 100%, comparatively
much lower than the level in durum, which was
between 70 and 100%. CIMMYT has about 50 and
200 lines of A genome (AAAABB) and B genome
(AABBBB) synthetic wheats, respectively. When we
tested the A genome synthetic wheats, there were also
some accessions which showed Type II resistance of
the same level as Sumai 3 (-10%). The resistance
sources in tetraploid relatives and in A or B genome
synthetic wheat can be transferred into durum by
conventional breeding methodology.

Transfer of resistance from D genome
Resistance fromD genome can be also transferred into
durum wheat by induction of translocation between A
and D genome. From the study of synthetic wheats of
D genome, we know which Ae. tauschii accessions
have FHB resistance. In 2005, we obtained two kinds
of amphiploids between Ae. tauschii and Capelli
(ph1c) accessions. We are now screening translocation
between A and D genome by GISH (Figures 1 & 2).

One big problem in the use of alien species
We are now heavily involving the production of
translocation lines for FHB resistance in both of bread
and durum wheat. However, one problem is the time
and labor required to induce translocations. Moreover,
one translocation seems to be insufficient to achieve
the full resistance of alien species (Table 2), even
though the resistance of alien species is superior to that
of wheat. We may need to combine multiple
translocations. The task would be quite a burden if
pursued by one institute. Fortunately, several groups







Session 1: FHB-QTL consortium


across the world seem to be working in different
species, though there may be overlapping to a certain
extent. If we can collaborate and divide our task
efficiently, we believe we can achieve our goal much
more easily.

REFERENCES

Endo TR (1988) Induction of chromosomal structural
changes by a chromosome of Aegilops cylindrica
incommonwheat. J Hered 79:366-370
Fedak G, Han F, Cao W, Burvill M, Kriteno S, Wang
L (2003) Identification and characterization of
novel sources of resistance to Fusarium head
blight. In Proceedings of the 10th International
Wheat Genetics Symposium. Vol. 1. Paestum,
Italy, September 1-6 2003. pp 354-356.
Jauhar PP, Peterson TS (2001) Hybrids between durum
wheat and Thinopyrum junceiforme: Prospects for
breeding for scab resistance. Euphytica 118: 127-
136.


Lewis JM, Suenaga K, Van Ginkel M, Gilchrist L, Shi
JR, Jiang GL, Kravchenko S, Mujeeb-Kazi A,
Ward RW (21'" 4) Identification and mapping of a
QTL for Type II resistance to Fusarium head
blight on chromosome arm 2DL of wheat:
Proceedings of Scab Symposium, Dec. 2004. pp
89-92.
Mujeeb-Kazi A, Bernard M, Bekele GT, Mirand JL
(1983) Incorporation of alien genetic information
fromElymus giganteus into Triticumaestivum. In:
Sakamoto S (ed) Proceedings of the 6th
international wheat genetics symposium. Plant
germ-plasm Institute, Kyoto, Japan pp 223-231.
Shen X, Kong L, Ohm H 2" 114) Fusarium head blight
resistance in hexaploid wheat (Triticum
aestivum)-Lophopyrum genetic lines and tagging
of the alien chromatin by PCR markers. Theor
Appl Genet. 108: 808-13.
Wan YF, Yen C, Yang JL (1997) The diversity of
head-scab resistance in Triticeae and their relation
to ecological conditions. Euphytica 97: 277-281.


Table 1. Plant materials utilized in this study.
Lines Number of lines Origin
Synthetic wheat
D genome synthetic wheat (AABBDD) 1,100 CIMMYT
A genome synthetic wheat (AAAABB) 150 CIMMYT
B genome synthetic wheat (AABBBB) 50 CIMMYT

Amphilpoid
Bread wheat Thinopyrum bessarabicam 1 CIMMYT
Bread wheat Thinopyrum elongata 1 CIMMYT
Bread wheat Elytrigia scythica 1 CIMMYT

Alien chromsome addition lines
Thinopyrum bessarabicum 7 CIMMYT
Leymus racemosus set#1 10 Japan
Leymus racemosus set#2 6 CIMMYT

Mapping population (Doubled haploid)
TURACO/5/CHIR3/4/SIREN//ALTAR 84/Ae. tauschii (205)/3/3*BUC/6/ FCT 128 CIMMYT
MAYOOR/TK SN1081/Ae. tauschii(222)/3/ FCT 171 CIMMYT
SABUF/3/BCN/CETA/Ae. tauschii(895)/4/ FCT 125 CIMMYT







Session 1: FHB-QTL consortium


Table 2. Type II resistance in Thinopyrum bessarabicum chromosome lines.
Lines Damage % Type II
Amphilpoid
Wheat- Thinopyrum bessarabicum 8.2
Addition lines
Thinopyrum bessarabicum 29.1
Thnopyrum bessarabicum
2knopyrum bessarabicum 19.8
1i .' bessarabicum 39.4
4J *' bessarabicum 16.6
Thinopyrum bessarabicum 16.0
iAznopyrum bessarabicum 17.2
7J


Table 3. Type II resistance in the field at Toluca, Mexico 2004.

Damage % Type II
Species Genome Number of lines Min Max Average


Triticum durum AABB 16 69.4 100 84.4
Triticum dicoccum AABB 35 6.9 70.3 32.8
Triticum dicoccoides AABB 46 5.8 100 25.3
Triticum monococcum AA 41 9.4 45.7 26.2
*A genome synthetic wheat AAAABB 194 9.5 100 41.8

The analysis was conducted by Dr Maarten Van Gmkel *The test for synthetic wheat was conducted Dr Muleeb Kazl in 2001


J I


4


4-8


BCapet pfle **i c* *ni ** i *
A* ....f .i .'..;... .;"'i
0 P IMoon-- bi3hw Ih
.' iPO l f ,rcm'D g "nom'.._..--
/ Fenn nnm nnn i
FISH

"IlC 1 B I A MBB(QUO~


[ ]a W duruamn MWh nMlhm: gue
from D genome)


Figure 1. Methodology for transfer of B and D
genomes into A genome


Figure 2. Translocation between B/D genomes and
A genome. The arrows indicate transfer of B and D
genomes into A genome chromosomes.






Session 1: FHB-QTL consortium


NOBEOKA BOZU, AN UNUSED RESISTANCE SOURCE AND
ITS UTILIZATION IN IMPROVING RESISTANCE TO FHB

A. Mesterhazy*

Cereal Research non-profit Co., Szeged, Hungary
*Corresponding Author: PH: 36-62-435-235; E-mail: akos.mesterhazy@gk-szeged.hu


ABSTRACT

I received Nobeoka Bozu (NB), a spring type FHB resistance source from Japan, 20 years ago from Dr. M. Kohli of
CIMMYT. The first tests showed a very high level of resistance in FHB symptoms, kernel infection, yield reduction
and deoxynivalenol (DON) accumulation Several crosses were made with high yielding adapted materials such as
Zugoly (Zu), Ringo Star (RSt), Kincso (Ko), R6ka (RE) (abbreviations in Figure 1) to observe its segregating
populations. NB is an agronomically poor, ill-adapted landrace. The heads and grains are small and 1000 kernel
weight is about 25-27 g. The straw is thin and the color is light green, though the segregating populations also
contained plants with light yellow heads. NB has comparable FHB resistance to Sumai 3 (Sum 3) (Figure 1). Of the
progenies derived from the crosses with NB, several highly resistant lines were identified. All are winter type, and
therefore they have special value for winter wheat programs. In Figure 1, the genotype first from the left is a highly
susceptible genotype: it seemingly did not inherit resistance from NB. Some of these materials have also been tested
in Japan, with encouraging results (Ban, unpublished). The agronomic traits of these lines are significantly better
than those of NB, as are baking quality, yield potential, and resistance to other diseases, whilst the resistance seen in
NB has been successfully retained.

NB has highly effective QTL(s) on 3BS type (Liu et al. 2005, Jian-Bin et al. 2006.), but it does not seem to be
identical with the 3BS QTL allele found in Sumai 3 (unpublished), even though it gives strong Type II resistance
(Jian-Bin et al. 2006). Therefore it is highly important to identify the genetic background of the NB QTL(s). We
now have three mapping populations from NB. Their study began in 2006 in the field in collaboration with the
Agricultural University As, Norway. We hope that QTLs may be identified that will have similar significance to the
Sumai 3 QTL(s) and that the identity or diversity problemcanbe answered.

REFERENCES

Sixin Liu, Xiuling Zhang, Michael O. Pumphrey, Robert W. Stack Bikram S. Gill and James A. Anderson, 2005.
New DNA Markers for the Chromosome 3BS Fusarium Head Blight Resistance QTL in Wheat. 2005 National
Fusarium Head Blight Forum, Milwaukee, p. 55.
Jian-Bin Yu, Gui-Hua Bai, Shi-Bin Cai and T. Ban 2006. Marker assisted characterization of Asian wheat lines for
resistance to Fusarium head blight. Theor. Appl. Genet. 113:308-320.







Session 1: FHB-QTL consortium


Figure 1. FHB resistance of lines derived from Nobeoka Bozu,
2004






7 Yield loss%
a C I/Z / 7/ DON ppm


0(N NEN






Session 1: FHB-QTL consortium


DNA MARKER ANALYSIS FOR FHB-RESISTANCE
PYRAMIDING FROM DIFFERENT GERMPLASMS


J.R. Shi1, H. Yang1, Q. Lu1, D. Xu2, and T. Ban2'3

1Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China
2Japan International Research Center for Agricultural Sciences (JIRCAS), Tsukuba, Ibaraki 305-8686, Japan
3Japan-CIMMYT FHB Project, International Maize and Wheat Improvement Center,
Apdo. Postal 6-641, 06600 Mexico, D.F., Mexico



ABSTRACT

Use of resistant cultivars is an effective way to control Fusarium head blight (FHB), an epidemic wheat disease
around the world. Several resistant germplasms, such as Sumai 3, Wangshuibai, and Nobeokabouzu-komugi, have
been screened and widely used in wheat breeding programs. A number of quantitative trait locus (QTL) analyses
indicated that the resistance genes are not identical among resistant germplasms. Pyramiding of different resistance
genes into one wheat cultivar would be useful for breeders to introgress different resistance genes into their locally-
adapted cultivars. By crossing Sumai 3, Wangshuibai, and Nobeokabouzu and following a high FHB pressure
selection for six generations, a line with pyramided resistance, WSY, has been developed. In the present study, we
analyzed the genetic flow between the three parental cultivars and the pyramided resistance line WSY using DNA
markers with a purpose to clarify how many and which resistance genes were accumulated in the pyramided line.
Two-hundred and eiglty three SSR markers and three STS markers from the 21 wheat chromosomes were analyzed.
Of these markers, 115 are from tenFHB resistance QTL regions. The results disclosed that the pyramided line WSY
included different chromosome regions that harbored putative FHB QTLs from the three parental germplasms.
Haplotypes of DNA markers on these QTL regions demonstrated that the 3BS QTL (the major FHB resistance QTL)
was from Nobeokabouzu; QTLs on 1BL 5A and 2BL were from Sumai 3; QTLs on 2AS, 3AS and 6BS were from
Wangshuibai; and the 3BSc QTL was from Sumai 3 and Wangshuibai. This study showed that different resistance
genes from the different resistant germplasms were indeed accumulated in the pyramided resistance line WSY. The
developed pyramided resistance line might be used as a potential resource for FHB resistance in wheat breeding.







Session 2: Fusarium consortium


GLOBAL BIODIVERSITY IN FUSARIUM GRAMINEARUM
(GIBBERELLA ZEAE) AND F. CULMORUM POPULATIONS
AND IMPLICATIONS FOR BREEDING RESISTANCE TO
FUSARIUM HEAD BLIGHT


T. Miedaner*

University of Hohenheim, State Plant Breeding Institute, D-70593 Stuttgart, Germany
*Corresponding author: Phone: +49-711-459-2690; E-mail: miedaner@iuni-hohenheimde


OBJECTIVES

Genetic diversity of a population is the result of all
evolutionary processes that have affected a population
Recombination, gene flow and mutation increase
genetic variation, selection and genetic drift decrease
it. Knowing the amount of genotypic diversity and its
spatial and temporal distribution within populations,
the level of population subdivision, and its importance
for phenotypic traits like aggressiveness and
deoxynivalenol (DON) production, will allow us to
predict the evolutionary potential of the respective
pathogens and generate important implications for
deployment of resistance. This paper will briefly
review the present knowledge on population-genetic
parameters in individual field populations of F.
graminearum and F. culmorum.

INTRODUCTION

Fusarium graminearum (teleomorph Gibberella zeae)
is the most destructive pathogen causing Fusarium
head blight (FHB) worldwide. In Europe other
Fusarium species may play an important role in this
disease, e.g. F. culmorum F. avenaceum, F. poae. F.
graminearum and F. culmorum both produce similar
mycotoxins: DON, nivalenol (NIV), and zearalenone
(ZEA) are among the most important. F. graminearum
naturally infects wheat during flowering by splash-
dispersed ascospores from soil or infected wheat or
maize stubble. Secondary infections by conidiospores
can additionally occur. Humid weather during
flowering is an important prerequisite for epidemics.
Kernels are totally destroyed when susceptible
varieties are infected early or become discoloured and
shrivelled. Seed-borne inoculum of late-infected
kernels that look healthy might play a role in long-
distance transport of the fungus. Ascospores have been
found to be transported at higher altitudes by air


streams (Fernando et al. 2000). G. zeae is a haploid,
homothallic fungus caused by the presence of two
mating type idiomorphs (MAT1-1 and MAT1-2) in each
isolate. Some outcrossing has been observed in the
laboratory (Bowden and Leslie 1999). Crossings
between isolates can easily be done, and recombinants
have to be identified by use of nitrate-nonutilizing
mutants (Bowden and Leslie 1999) or by use of female
strains with a manipulated MAT locus to achieve
obligatory heterothallism (Lee et al. 2003).
Segregation occurs in a haploid fungus in the Fi
generation and dominant molecular markers have the
same information content as codominant markers. For
F. culmorum no sexual stage is known, but two
separate MAT idiomorphs were found (Toth et al.
2004). The importance of F. graminearum for wheat
crown rot and FHB in Australia is reviewed in another
chapter of this volume (Chakraborty et al., p. 42).

LEVELS OF BIODIVERSITY

Four levels of biodiversity are obvious: Variation
among isolates (1) from different parts of the world,
(2) within geographically defined regions, (3) within
and between individual field populations, (4) within
crossing populations.

Large variation between isolates from different
countries and continents is common with each isolate
displaying a unique haplotype (Miedaner et al. 2001).
O'Donnell et al. (2000) were the first to divide F.
graminearum into seven phylogenetic lineages of
different geographic distribution Recently, the
lineages were extended to nine and given species rank
while the postulated geographical barriers dwindle
(O'Donnell et al. 2004). Bowden et al. (2006) could
achieve fertile crossings between lineage 7, the most
common clade in the Northern hemisphere, and all
other lineages and between selected other lineages as







Session 2: Fusarium consortium


well. Genetic variation within one lineage due to traits
other than sequenced structural genes analysed by
O'Donnell still needs to be evaluated.

More important for assessing the evolutionary
potential of F. graminearum with respect to resistance
breeding is the third and fourth level of biodiversity.
Several studies revealed a high level of genetic
diversity by RAPD, AFLP and SSR markers within
individual field populations or populations sampled
across a small spatial scale (Table 1).

In F. graminearum populations there is unequivocally
a high percentage of unique haplotypes and
consequently a large genotypic and genetic diversity
that have been reported. Populations that were sampled
100 to 200 km apart (Naef 2006, Gale et al. 2002) as
well as populations from about 2.500 km apart (Zeller
et al. 2004) showed no or only a low amount of
subdivision According to these studies, more than
97% of allelic variation is shared among populations.
This might be most likely caused by a high gene flow
among populations that was estimated with 7 to 74
migrants.

The evolutionary forces behind this genetic diversity
might be most likely driven by the large population
size of asexually propagated conidia supporting
variation by mutation Additionally, balancing
selection between the parasitic and saprophytic
subpopulations might contribute to genetic variation A
new study compares genetic diversity within field
populations of saprophytic isolates from maize


stubbles (Table 1, Switzerland) with a re-analysis of a
wheat pathogenic population (Miedaner et al. 2001) by
SSR markers (Naef 2006). Genotypic diversity was
significantly higher on maize stubble, allelic richness
and gene diversity, however, were similar in both
populations. A high extent of asexual dispersal was
found and the populations shared six multilocus
haplotypes (MLHs) across a geographical distance of
about 100 km and a temporal difference between both
collections of 11 years. If the selection forces in both
subpopulations act in at least partially different
directions, a balancing selection could maintain large
genetic variation High gene flow together with the
occurrence of environmentally stable MLHs could be
explained by long-distance transport of ascospores
emerged by selling. Similar results from eight parasitic
wheat populations were previously reported by Zeller
et al. (I "' 4). Little linkage disequilibriumwas detected
either in the population as a whole or in any of the
individual eight subpopulations sampled from seven
US states in three years (Zeller et al. 2004). Similarly,
estimates from the Chinese F. graminearum
populations showed gametic equilibrium in 25 out of
36 locus pairs (Gale et al. 2002). These results are in
accord with the hypothesis of a large, randomly mating
population The role of sexual recombination and
especially that of outcrossing in natural populations of
G. zeae is, however, still under debate (Gale et al.
2002). Population analyses of Schilling (1996) and
Naef (2006) revealed no gametic equilibrium
Population-genetic theory, however, shows that even
rare outcrossings may contribute significantly to
genetic diversity.


Table 1. Important quantitative-genetic parameters of individual Fusarium populations
Country Sum of No. of % unique Genotypic Nei's genetic Population Gene
(No. ofpopul.) isolates poly- haplo- diversity diversity H subdivision flow
morphic types Go/N RST Nm
loci

F. graminearum:
Germany (1)1 70 37 76 0.64 0.69
China (4)2 225 9 64 0.31-0.37 0.01-0.07 7-30
USA (8)3 523 26-30 91-100 0.95-1.00 0.00-0.07 7-74
Switzerland (5)4 395 8 76-98 0.59-0.96 0.67-0.70 < 0.02
F. culmorum:
Collection5 24 20 54 High -
Russia (1)6 41 28 56 0.50
Hungary (1)5 13 20 0 0
Switzerland (1)4 29 8 83 0.74 0.34
Schilling 1996, 2Gale et al. 2002, 'Zeller et al. 2004, 4Naef 2006, 'Toth et al. 2004, bMiedaner et al. 2001.







Session 2: Fusarium consortium


The high genetic variation found in two F. culmorum
populations (Table 1, Miedaner et al. 2001, Naef 2006)
illustrates that in this species, forces on a population
level exist that promote diversity without sexual
recombination In contrast, de Nijs et al. (1997) found
in the Netherlands by RAPD analyses only three very
closely related haplotypes among 18 F. culmorum
isolates as expected from a clonally propagating
species. Thirteen isolates from Hungary also were of
clonal origin whereas the analysis of a collection of
different origins with the same markers revealed large
genotypic diversity (Toth et al. 2004). These differing
results can only be clarified by analysing larger
populations from different countries or even
continents.

ANALYSES OF SEGREGATING POPULATIONS
FROM CROSSINGS AND IMPLICATIONS FOR
RESISTANCE BREEDING

Phenotypic traits, especially aggressiveness measured
by symptom development and host colonization after
inoculation, but also mycotoxin production largely
vary on a quantitative scale among isolates. All isolates
of F. graminearum or F. culmorum produce either
DON or NIV, their respective precursors, and ZEA
(Gang et al. 1998, Miedaner et al. 2000, Toth et al.
2004), i.e. each infection in the field will result in
toxin-contaminated grain

If the amount of genetic variation within populations
reflects the evolutionary potential of a pathogen, this
potential should be rated high for G. zeae. In natural
infections, often multiple infections occur with
different MLHs on the same wheat head (Miedaner et
al. 2001, Naef 2006). Additionally, different isolates
may have a differing competitiveness in the natural
habitat that is not necessarily related to aggressiveness
or the amount of toxin production (Miedaner et al.
2004). It is, therefore, of high interest to study the
inheritance of these traits.

Significant (P<0.01) genetic variation for
aggressiveness, host colonization, and DON content
was found among 155 progeny of a cross between two
medium aggressive isolates of European origin, isolate
x environment interactions also was important
(Cumagun et al. 2004). Several transgressive
segregants towards higher aggressiveness and higher
DON production occurred in this population
illustrating that both parental isolates had different
alleles for these traits that recombined in the progeny.
This illustrates the potential of the pathogento increase
its level of aggressiveness by intermating of isolates of
the same geographic region


In the inter-lineage cross between a Japanese barley
isolate (R-5470) and a US wheat isolate (Z-3639), a
major gene for toxin content (TOX1), female fertility
(PERIl) and colony pigmentation (PIG1) was mapped
for the first time (Jurgenson et al. 2002). In close
vicinity of these loci, a gene for pathogenicity
(PATH1) was mapped for the first time (Cumagun et
al. 2004) and at least one major QTL for
aggressiveness located on a different linkage group but
near the trichothecene cluster that contains the DON
vs. NIV switch. Progeny producing DON were, on
average, twice as aggressive as those producing NIV.
This has been previously found also for isolate
collections (Miedaner et al. 2000).

Resistance to both Fusarium species should not be
endangered by selection within populations in the near
future, because no specific host genotype x fungal
isolate interaction occurs in this pathosystem. If,
however, the same resistance genes with high effects
are used globally and genotypes harbouring them are
grown on large acreages, an unspecific increase in
aggressiveness and mycotoxin production might occur
in natural populations of F. graminearum on the long
run To minimize this risk, several genetically
unrelated resistance sources should be introgressed in
national breeding programmes. Besides the well-
known Chinese and South American resistance
sources, additively inherited resistance genes exist in
other gene pools, e.g. among the Middle and East
European winter wheats. By recurrent selection a
rather high resistance level can be achieved. Mapping
studies in these materials detected a high variation for
QTLs on nearly all wheat chromosomes.

FUTURE OUTLINES

For a profound understanding of the structure of F.
graminearum and F. culmorum populations more data
are necessary on the global diversity. For this, a
common array of selection-neutral markers, e.g. SSR
markers, should be used. This would be especially
important for populations of F. culmorum, because
here substantial data are lacking. Populations from the
center of wheat diversity should be included to
understand phylogeny of both species. Another feature
is monitoring of gene flow between parasitic and
saprophytic populations of the same Fusarium species
in the same fields related to the crop rotation More
populations of F. graminearum that have been
obtained from crosses within the same lineage should
be mapped for phenotypic traits to monitor the amount
of segregation variance that might be available in
future field populations.







Session 2: Fusarium consortium


An important issue for the deployment of resistances is
the adaptation of the pathogen to resistant varieties.
This could be tested in various ways, e.g. by growing
resistant lines in hot-spot areas of epidemics or by
monitoring pathogen populations on hosts differing in
resistance.

REFERENCES

Bowden, R. L. and Leslie, J. F., 1999. Sexual
recombination in Gibberella zeae. Phytopathology
89, 182-188
Bowden, R. L. and Leslie, J. F., 2006. This volume.
Cumagun, C. J. R., Bowden, R. L., Jurgenson, J. E.,
Leslie, J. F., and Miedaner, T., 2004. Genetic
mapping of pathogenicity and aggressiveness of
Gibberella zeae (Fusarium graminearum) toward
wheat. Phytopathology 94, 520-526
de Nijs, M., Larsen, J.S., Gams, Rombouts, W.M.,
Wernars, K, Thrane, U., and Notermans, S.H.W.
1997. Variation in random amplified polymorphic
DNA patterns and secondary metabolite profiles
within Fusarium species from cereals from various
parts of The Netherlands. Food Microbiol. 14, 449-
457.
Fernando, W.G.D., Miller, J.D., Paulitz, T.C., Seaman,
W.L., and Seifert, K. 2000. Daily and seasonal
dynamics of airborne spores of Fusarium
graminearum and other Fusarium species sampled
over wheat fields. Canadian Journal of Botany 78,
497-505
Gale L.R., Chen L.F., Hernick C.A., Takamura K.,
Kistler H.C. 2002. Population analysis of Fusarium
graminearum from wheat fields in eastern China.
Phytopathology 92, 1315-22.
Gang, G., Miedaner, T., Schuhmacher, U.,
Schollenberger, M. and H.H. Geiger. 1998.
Deoxynivalenol and nivalenol production by
Fusarium culmorum isolates differing in
aggressiveness toward winter rye. Phytopathology
88, 879-884.
Jurgenson, J. E., Bowden, R. L., Zeller, K. A., Leslie,
J. F., Alexander, N. J., and Plattner, R. D., 2002. A
genetic map of Gibberella zeae (Fusarium
graminearum). Genetics 160, 1451-1460
Lee, J., Lee, T., Lee, Y. W., Yun, S. H., and Turgeon,
B. G., 2003. Shifting fungal reproductive mode by
manipulation of mating type genes: obligatory
heterothallism of Gibberella zeae. Molecular
Microbiology 50, 145-152
Miedaner, T., Reinbrecht, C., and Schilling A.G.
2000. Association among aggressiveness, fungal
colonization, and mycotoxin production of 26
isolates of Fusarium graminearum in winter rye
head blight. Z. PflKrankh PflSchutz 107, 124-134.


Miedaner, T., Schilling, A.G., and Geiger, H.H. 2001.
Molecular genetic diversity and variation for
aggressiveness in populations of Fusarium
graminearum and Fusarium culmorum sampled
from wheat fields in different countries. J.
Phytopathology 149, 641-648.
Miedaner, T., Schilling, A.G., and Geiger, H.H. 2004.
Competition effects among isolates of Fusarium
culmorum differing in aggressiveness and
mycotoxin production on heads of winter rye.
Europ. J. Plant Path 110, 63-70.
Naef, A. 2006. Survival of Fusarium graminearum on
maize crop residues: competition with Trichoderma
atroviride and impact of maize Bt transformation
PhD dissertation Swiss Federal Institute of
Technology (ETH), Zurich, Switzerland. (Diss.
ETH No. 16459).
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. Proc.
Nat. Academy Sci. USA 97, 7905-7910
O'Donnell, K. Ward, T. J., Geiser, D. M., Kistler, H.
C., and Aoki, T. 2004. Genealogical concordance
between the mating type locus and seven other
nuclear genes supports formal recognition of nine
phylogenetically distinct species within the
Fusarium graminearum clade. Fungal Genetics and
Biology 41, 600-623
Toth, B., Mesterlizy, A., Bart6k, T., and Varga, J.
2004. Mycotoxin production and lineage
distribution in Central European isolates of the
Fusarium graminearum clade. In: Canty, S.M., T.
Boring, J. Wardwell, and R.W. Ward (Eds.), Proc.
2nd Int. Symp. Fusarium Head Blight incorp. 8th
Europ. Fusarium Semn, 2004, 11-15 Dec.,Orlando,
FL, USA. Michigan State Univ., East Lansing, MI,
p. 579.
Zeller, K. A., Bowden, R. L., and Leslie, J. F. 2003.
Diversity of epidemic populations of Gibberella
zeae from small quadrats in Kansas and north
Dakota. Phytopathology 93, 874-880
Zeller, K. A., Bowden, R. L., and Leslie, J. F. 2004.
Population differentiation and recombination in
wheat scab populations of Gibberella zeae from the
United States. Molecular Ecology 13, 563-571.







Session 2: Fusarium consortium


DIVERSITY OF FUNGAL POPULATIONS ASSOCIATED WITH
FUSARIUM HEAD BLIGHT IN URUGUAY


S. A. Pereyra", S. Vero2, G. Garmendia2, M. Cabrera2, and M. J. Pianzolla2

'Plant Pathology, INIA La Estanzuela. Ruta 50 km 11. 70000, Colonia, URUGUAY; 2Faculty of Chemistry,
Universidad de la Republica, Gral. Flores 2124. 11800, Montevideo, URUGUAY.
*Corresponding author: Phone: (598) 574 8000; E-mail: sperevra@,inia.org.uv


OBJECTIVES

(1) To identify and quantify the most prevalent
Fusarium species in wheat and barley grains.
(2) To characterize the diversity within the population
ofFusarium graminearum collected in Uruguay

INTRODUCTION

Fusarium head blight (FHB) is a destructive disease of
wheat and barley in the Southern Cone of South
America. In particular, FHB represents one of the main
constraints for wheat and barley production in Uruguay
where moderate to severe outbreaks have occurred in
one of every two years over the past decade (Diaz de
Ackermann and Kohli, 1997; Perea and Diaz, 1980;
Pereyra and Diaz de Ackerman, 2003). Epidemics have
caused extensive damage through direct losses in grain
yield and quality, particularly because of the presence
of mycotoxins, such as deoxynivalenol (DON), in
harvested grain

In Uruguay, FHB is primarily caused by Fusarium
graminearum (Schw.) [teleomorph Gibberella zeae
(Schwein) Petch] (Boasso, 1961; Boeger, 1928;
Pereyra and Stewart, 2001; Pritsch, 1995). Other
species may also incite FHB such as F. poae and F.
culmorum in wheat (Stagno, 1980) and F. poae in
barley (Pereyra and Stewart, 2001). However, there
has not been a systematic survey of Fusarium species
present in wheat and barley grains harvested from
different cultivars, locations, and years.

Previous studies have indicated a differential response
in aggressiveness among 14 F. graminearum isolates
collected during 1991 to 1993 in western-southwestern
Uruguay (Diaz de Ackermann and Kohli, 1997). These
isolates were tested in the greenhouse against three
wheat sister lines (Catbird) with different degrees of
reaction (moderately resistant, moderately resistant to
moderately susceptible, and susceptible) to FHB, both


in the field and greenhouse. No polymorphism was
detected among isolates through RFLPs (Pritsch,
1995).

From a phylogenetic approach, lineage 7 (F.
graminearum sensu O'Donnell) is dominant in Brazil
and Uruguay, although other lineages like 1, 2, 6, and
8 are present in minor frequencies (Leslie and Bowden,
2005; O'Donnell et al., 2004; Zeller et al., 2002; Zeller
et al., 2003).

Knowledge of Fusarium population diversity in
Uruguay is essential for effective disease management
strategies in the region This information would be
useful for ecological and epidemiological studies, to
develop resistant cultivars through improved screening
procedures, and to optimize chemical and biological
control.

MATERIALS AND METHODS

Fusarium species present in Uruguayan wheat and
barley grains
Wheat and barley grain samples (0.2 kg) were
collected from epidemic years 2001 and 2002. Five
wheat cultivars and five barley cultivars that together
comprise the bulk of the commercial hectarage in
Uruguay were tested each year at La Estanzuela,
Young, and Paysandu and planted at different dates.
One hundred arbitrarily selected kernels per cultivar,
planting date and location, were examined each year.
Surface-disinfected grain samples were plated onto
pentachlonitrobenzene (PCNB) agar medium in Petri
plates. Twenty grains were placed per Petri plate with
five replicates and incubated at 20-220C with 12-hr
light and dark cycles for seven days. Colonies growing
with salmon to pink-white color were recorded as
Fusarium species. The proportion of G. zeae colonies
was determined by transferring 10 arbitrarily selected
Fusarium spp. single conidial colonies to carnation-
leaf agar (CLA) medium and potato-dextrose agar







Session 2: Fusarium consortium


(PDA). Cultures were incubated at 20-220C with 12-hr
light and dark cycles for 15 days. The formation of
bluish to black perithecia in CLA cultures indicated the
presence of G. zeae. Fusarium colonies not forming
perithecia were identified to species, based on
procedures and descriptions outlines by Nelson et al.
(1983) and Burgess et al. (1994).

Pathogenicity tests were conducted with single
conidial isolates from different species and
environments (locations/planting dates) in the
greenhouse with susceptible wheat line LE 2294 and
susceptible barley cultivar Estanzuela Quebracho. One
to two spikes per pot (five pots per isolate per plant
species) were inoculated with a concentration of 2 x
104 conidia per ml at mid-anthesis in wheat and at
heading in barley using an airbrush (model VL3,
Paasche Air Brush). Inoculated plants were incubated
in a dew chamber at 20-220C with 12-hr photoperiod
and 100% relative humidity for 72 h Disease severity
(percentage of symptomatic spikelets per spike) was
evaluated 21 days after inoculation

Fusarium graminearum diversity
Sixty-four F. graminearum isolates were obtained
from wheat grains collected in commercial fields in
western Uruguay in 2001, 2002 and 2003. Isolates
were identified from morphological characters
following the procedures of Nelson et al. (1983) and
confirmed by F. graminearum-specific PCR using
primers FgllF/FgllR (Nicholson et al., 1998).
Furthermore, isolates were sent to Dr. K. O'Donnell
lab (MGBR, NCAUR, USDA, Preoria, IL) to
determine by molecular methods, which of the nine
lineages or species within F. graminearum clade, they
belonged to.

All isolates were tested by a combination of
phenotypic and molecular criteria, including
chemotype, DON production, fungicide resistance,
aggressiveness under greenhouse conditions, and
perithecial production under standardized techniques.

Chemotvpe characterization: PCR assays to determine
the presence oftrichothecene genes Tri 5, Tri 7 and Tri
13 were performed. Primers for analysis of
polymorphisms in Tri 7 and Tri 13 genes were as
described by Lee et al. (2001) and Kim et al. (2003),
respectively. The primers for Tri 7 analysis were
GzTri7F and GzTri7R. Isolates producing DON have
11 base pairs insertions rendering it non-functional.
Primers for Tri 13 analysis were GzTril3F and
GzTril3R. Isolates producing DON have deletions of
181 base pairs, rendering it non-functional.


DON production: Each isolate was separately
cultivated on 10 g of rice grains moistened with 5 ml
of sterile water in a 100-ml Erlenmeyer flask, at 280C
for 30 days. Rice cultures were then removed and the
content of each flask was blended with 50 ml methanol
for one minute. Extracts were filtered through filter
paper. DON was detected by Thin Layer
Chromatography according to AOAC and Elisa test
(Veratox DON 5/5 Quantitative DON Test
Neogen) was used in order to quantify DON
concentration

Assessment of fungicide sensitivity: Minimal
inhibitory concentration (MIC) was determined for
thiabendazole- Tecto 500 SC (Syngenta),
tebuconazole- Folicur 450 (Bayer Crop Science) and
metconazole Caramba (BASF). The last two
fungicides are widely used to control FHB in Uruguay.
PDA (200 [l) amended with different fungicide
concentrations were dispensed into the wells of sterile
disposable microtitre plates. After 72 h of incubation at
250C in the darkness, fungal growth was determined
visually. MIC was defined as the lowest concentration
that inhibited fungal growth Two repetitions per
treatment were performed. Fungicide concentrations
assayed were 0, 2, 4, 8, 16, 32, 64, and 128 ppm
Experiments were repeated at least twice.

Aggressiveness: Wheat genotypes with different
reactions to FHB in the greenhouse and field were
planted in the greenhouse: Frontana (resistant),
Sumai#3 (resistant), Ringo Sztar-Mini Mano/Nobeoka
Bozu, Catbird 1073 (moderately resistant), Onix
(moderately resistant to moderately susceptible), INIA
Churrinche (moderately resistant to moderately
susceptible), INIA Mirlo (susceptible), INIA Boyero
(susceptible), and Buck Guarani (susceptible). The
nine F. graminearum isolates with the highest DON
production were tested in a randomized complete
design with 10 plants per cultivar per isolate. One to
two spikes per pot (five pots per isolate per plant
species) were inoculated with a concentration of 2 x
104 macroconidia/ml at mid-anthesis using an airbrush
(model VL3, Paasche Air Brush Company, Harwood
Heights, IL) at 12 psi to deliver ca. 0.2 ml of inoculum
per spike. Controls were mock-inoculated with sterile
deionized water. Inoculated plants were incubated in a
dew chamber at 20 to 220C with a 12-h photoperiod
and 100% relative humidity for 72 h After incubation,
plants were returned to the greenhouse and grown
under the same light and temperature conditions used
prior to inoculation Disease severity was evaluated 7,
14 and 21 days after inoculation and expressed as the
percentage of symptomatic spikelets per spike. A
repetition of this experiment is underway; therefore,
only preliminary results will be presented.







Session 2: Fusarium consortium


Perithecia production: Residue of the wheat cultivar
INIA Churrinche, collected from a commercial field
and without G. zeae perithecia, was used to determine
perithecia production of 14 F. graminearum isolates.
These isolates were selected for the highest DON
production among the 64 isolates. Residue (1.5-cm
long stem pieces including one node) was autoclaved
at 1200C for 25 minutes. Three grams of residue pieces
were inoculated by submerging for two minutes into an
aqueous macroconidia suspension (1 x 105 spores/ml)
of each F. graminearum isolate. Following inoculation,
residue was placed on sterile sand moistened with
sterile water in Petri plates (one gram of residue per
plate per isolate, three replicates per isolate). To
facilitate the development of mature perithecia, residue
pieces were incubated at 20-220C under 12-hr light and
dark cycles for 21 days. Plates were covered with a
plastic lid for three days and then maintained
uncovered for the rest of the experimental period.
Gross box weight was checked each four to five days
and sterile water was added as necessary to maintain
the original weight. Following the 21-day incubation,
perithecia were counted in a 7 mm diameter field using
a dissecting scope at 30x magnification on each
residue piece.

RESULTS AND DISCUSSION

Fusarium species present in Uruguayan wheat and
barley grains
Fusarium graminearum was the primary species
associated withFHB. It comprised 76% and 60% of all
Fusarium species isolated from wheat grains in 2001
and 2002, respectively (Figure 1). Fusarium
graminearum represented 65% and 56% of all
Fusarium species isolated from barley grains in 2001
and 2002, respectively. The results from isolation and
identification of Fusarium species in wheat and barley
grains clearly confirmed that the main species
associated with FHB in Uruguay is F. graminearum as
suggested by previous studies (Boerger, 1928; Boasso,
1961; Pritsch, 1995; Pereyra and Stewart, 2001).
Similar results are reported for North America (Clear
and Patrick, 2000; McMullen et al., 1997; Salas et al.,
1999), some parts of Europe (Parry et al., 1995) and
other countries in South America (Lori et al., 2003;
Reis, 1988). Fusarium graminearum is widespread in
the southern cone of South America and has been
isolated from a wide range of hosts (Fernandez, 1991;
Pereyra et al., 2004; Reis, 1988).

The frequencies with which Fusarium species other
than F. graminearum were recovered varied depending
on both environment and host cultivar. In general, F.
avenaceum, F. culmorum and F. poae were the
following most common species isolated from wheat


grains. Other species included F. equiseti, F.
acuminatum, and F. trincictum. Fusarium poae and F.
equiseti were the most common species after F.
graminearum isolated from barley grains. Other
Fusarium species recovered in barley grains included
F. avenaceum, F. sambucinum, F. trincictum, F.
semitectum, and F. chlamydosporum (Figure 1).
Fusarium avenaceum was the second most common
species found in wheat grains while either F.
culmorum or F. poae was the third most common
species, depending on the year. These three species are
most often found in cooler production areas
(Backhouse et al., 2001; Bottalico and Perrone, 2002)
and this might explain the higher incidence of these
species at the early planting dates in the southern
region of Uruguay (La Estanzuela) where cooler
conditions than the other sites occur at
heading/flowering. Fusarium poae was the second
most prevalent species in barley grains. Under
Uruguayan conditions, infection by F. poae usually
occurs at boot stage, infecting the spike through the
flag leaf sheath in late-August or early-September
when lower temperatures are generally more favorable
for disease development. The virulence and
increasingly wide distribution of F. poae in barley
should not be overlooked by barley breeding programs
when screening lines for resistance to FHB.

All species were pathogenic on wheat and barley in
inoculation tests in the greenhouse, except F.
semitectum on wheat. Greater FHB severity and FHB
incidence on wheat and barley spikes were obtained
with the F. graminearum isolates, followed by F.
avenaceum and F. poae. Cultivars previously
characterized as moderately resistant to moderately
susceptible showed the lowest FHB incidences,
severities, percentages of Fusarium-infested grains,
and grains infested withF. graminearum.

The results from this study have shown that several
potentially important toxin-producing Fusarium
species are common under natural conditions in wheat
and barley grains in Uruguay.

Fusarium graminearum diversity
Phylogenetic lineage 7 (F. graminearum sensu
O'Donnell) was the most common lineage isolated
from wheat grains in Uruguay during 2001, 2002, and
2003 (Figure 2). Sixty-two of the 64 isolates
corresponded to this lineage, one isolate to lineage 1
(F. austroamericanum) and one to lineage 8 (F.
cortaderiae). Presence of these lineages have already
been cited in the Southern Cone of South America by
several authors (Leslie and Bowden, 2005; O'Donnell
et al., 2004; Zeller et al., 2002; Zeller et al., 2003).







Session 2: Fusarium consortium


Based on the presence of genes Tri7 and Tril3, all
isolates belonged to DON chemotype. Several
amplification sizes were obtained from the different F.
graminearum isolates for Tri7 gene, indicating
considerable diversity among these isolates (Figure 3).

Deoxynivalenol (DON) was produced by all 64
isolates in rice culture. Isolates were classified into
three groups according to levels of DON production
(Figure 4). Lineage 8- isolate produced less than 10
ppm of DON, while lineage 1- isolate produced DON
in the range of 100 to 1000 ppm All isolates were 15-
AcDON producers, except isolate of lineage 1, which
corresponded to the 3-AcDON type.

All isolates had high levels of sensitivity against
thiabendazole and metconazole. However, different
levels of sensitivity were observed against
tebuconazole.

Preliminary results showed differences among isolates
with high DON production for aggressiveness and
perithecia production All isolates tested caused visible
symptoms of FHB, however, some isolates did not
cause detectable disease on some resistant wheat
genotypes. The effect of the isolate on FHB severity
was significant (P=0.0001). The average range of FHB
severities on inoculated spikes 21 days after
inoculation was from 18% to 40%. These results are
currently being checked in a repetition of the
experiment.

All isolates produced perithecia under the temperature,
moisture and light conditions of this study. However,
there were significant differences among isolates for
perithecia production (P=0.005). Average perithecia
per gram of residue ranged from 59 to 521.

Results from this study reveal that the population ofF.
graminearum in Uruguay is diverse for several
characteristics. Further work is ongoing to understand
the genetic and pathogenic diversity of the populations
of F. graminearum in wheat and barley producing
areas. Furthermore, information from these studies will
be added to analyses of diversity of Fusarium
populations in the Southern Cone of South America.

REFERENCES

Backhouse, D., Burgess, L. W., and Summerell, B. A.
2001. Biogeography of Fusarium. p.122-136 In: B.
A. Summerell, J. F. Leslie, D. Backhouse, P.
Bryden, and L. W. Burgess, (eds). 2001 Fusarium
Paul E. Nelson Memorial Symposium The
American Phytopathological Society Press. St.
Paul, MN.


Boasso, C. 1961. Estado fitosanitario de los cultivos de
trigo de la reciente cosecha. [Phytosanitary state of
wheat crops in present growing season] Boletin
Informativo 854:7.
Boerger, A. 1928. Observaciones sobre agriculture,
quince afios de trabajos fitot6cnicos en Uruguay.
[Agricultural notes: Fifteen years of crop breeding
in Uruguay]. Montevideo. 436p.
Bottalico, A. and Perrone, G. 2002. Toxigenic
Fusarium species and mycotoxins associated with
head blight in small-grain cereals in Europe. Eur. J.
Plant Pathol. 108:611-624.
Burgess, L. W., Summerell, B. A., Bullock, S., Gott,
K. P., and Backhouse, D. 1994. Laboratory Manual
for Fusarium Research Third edition University
of Sydney, Sydney, Australia.
Clear, R. M. and Patrick, S. K. 2000. Fusarium head
blight pathogens from fusarium-damaged kernels
of wheat in western Canada, 1993 to 1998. Can J.
Plant Pathol. 22:51-60.
Diaz de Ackermann, M. and Kohli, M. M. 1997.
Research on Fusarium head blight of wheat in
Uruguay. p. 13-18 In: H. J. Dubin, L. Gilchrist, J.
Reeves, and A. McNab, (eds.) Fusarium head scab:
Global status and prospects. CIMMYT, DF,
Mexico.
Fernandez, M. R. 1991. Recovery of Cochliobolus
sativus and Fusarium graminearum from living
and dead wheat and nongramineous winter crops in
southern Brazil. Can J. Bot. 69:1900-1906.
Kim, H. S., Lee, T., Dawlatana, M., Yun, S. H., and
Lee, Y. W. 2003. Polymorphism of trichothecene
biosynthesis genes in deoxynivalenol- and
nivalenol- producing Fusarium graminearum
isolates. Mycological Research 107:190-197.
Lee, T., Oh, D. W., Kim, H. S., Lee, J. K., Kim, Y. H.,
Yun, S. H., and Lee, Y. W. 2001. Identification of
deoxynivalenol- and nivalenol-producing
chemotypes of Gibberella zeae by using PCR.
Applied and Environmental Microbiology
67:2966-2972.
Leslie, J. F. and Bowden, R. L. 2005. Field populations
of Gibberella zeae. p.166. In: Canty, S.M.; Boring,
T.; Wardwell, J., Siler, L. and Ward, R. W. (eds.)
Proceedings of the National Fusarium Head blight
Forum; 2005 Dec. 11-13; Milwakee, WI. Michigan
State University, East Lansing, MI.
Lori, G. A., Sistema, M. N., Haidukowski, M. and
Rizzo, I. 2003. Fusarium graminearum and
deoxynivalenol contamination in the durum wheat
area in Argentina. Microbiol. Res. 158:29-35.
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.







Session 2: Fusarium consortium


Nelson, P. A., Tousson, T. A., and Marasas, W. F. O.
1983. Fusarium species: An Illustrated Manual for
Identification The Pannsylvania State University
Press. University Park 193 p.
Nicholson, P.; Simpson, D. R.; Weston, G; Rezanoor,
H. N.; Lees, A. K.; Parry, D. W. and Joyce, D.
1998. Detection and quantification of Fusarium
culmorum and Fusarum graminearum in cereals
using PCR assays. Physiol. Mol. Plant Pathol.
53:17-37.
O'Donnell, K.; Ward, T. J.; Geiser, D. M.; Kistler, H.
C. and Aoki, T. 2004. Genealogical concordance
between the mating type locus and seven other
nuclear genes supports formal recognition of nine
phylogenetically distinct species within the
Fusarium graminearum clade. Fungal Genet. Biol.
41:600-623.
Parry, D. W., Jenkinson, P., and McLeod, L. 1995.
Fusarium ear blight (scab) in small grain cereals -
a review. Plant Pathology 44:207-238.
Perea, C. and Diaz, M. 1980. Relevamiento de
enfermedades de trigo en el Uruguay 1968-1979.
[Wheat diseases survey in Uruguay 1968-1979].
Investigaciones Agron6micas 2:42-51.
Pereyra, S. and Stewart, S. 2001. Investigaci6n en
fusariosis de la espiga de cebada en Uruguay.
[Research on Fusarium head blight of barley in
Uruguay ]. p. 41-68. In: XXI Reuniao Anual de
Pesquisa de cevada: Anais e ata. E. Minella, ed.
EMBRAPA Trigo, Passo Fundo, Brazil.
Pereyra, S. A., Dill-Macky, R. y Garcia, M. 2004.
Survival of Gibberella zeae and inoculum
contribution of diverse plant species in prevalent
crop rotations in Uruguay. p. 489-492. In: Canty,
S. M., Boring, T. Versdahl, K. Wardwell, J. and
Ward, R. W. (eds).Proceedings of the Second
International Symposium on Fusarium Head
Blight; 2004 Dec. 11-15; Orlando, FL, USA.
Michigan State University, East Lansing, MI.
Pritsch, C. 1995. Variabilidad patog6nica enFusarium
spp. agent causal del golpe blanco del trigo.
[Pathogenic variability of Fusarium spp. causal
agent of wheat scab]. FPTA-INIA. Informe final
79p.
Reis, E. M. 1988. Doencas do trigo III. Giberela.
[Wheat diseases III. Fusarium head blight].
Segunda edicao. Sao Paulo, Brazil.13p.
Salas, B., Steffenson, B. J., Casper, H. H., Tacke, B.,
Prom, L. K., Fetch, T. G., and Schwarz, P. B. 1999.
Fusarium species pathogenic to barley and their
associated mycotoxins. Plant. Dis. 83:667-674.


Stagno, J. P. 1980. Enfermedades transmitidas por la
semilla de trigo. [Seed transmitted diseases in
wheat]. SEMAGRO, Boletin 3:30-33.
Zeller, KA.; Vargas, J.I.; Valdovinos-Ponce, G.;
Leslie, J.F. and Bowden R.L. 2002. Population
genetic differentiation and lineage composition
among Gibberella zeae(Fusarium graminearum) in
North and South America. p. 188 In: Canty, S. M.;
Lewis, J.; Siler, L. and Ward, R. W. (eds.)
Proceedings of the National Fusarium Head blight
Forum; 2002 Dec. 7-9; Erlanger, KY. Michigan
State University, East Lansing, MI..
Zeller, KA.; Vargas, J.I.; Valdovinos-Ponce, G.;
Leslie, J.F. and Bowden R.L. 2003. Population
genetic differentiation and lineage composition
among Gibberella zeae in North and South
America. Fungal Genet. Newsl. 50:446.








Session 2: Fusarium consortium


F. acumnatum
2%0


F. culnmornm


F avenaceum
11%

F. equlsetl
3%


F.poae J
4%


. gram.
76%


B









F gram.
60%









D









F. gram.
56%


F. trincictum F


F. acum.
1%

F. aven.
5%


Figure 1. Frequency of Fusarium species isolated from wheat (A,B) and barley (C,D) grain sampled
from regional field trials in 2001 (A,C) and 2002 (B,D)


E F. graminearum (lineage 7)

0 F. austroamericanum (lineage 1)

B F. cortaderiae (lineage 8)


1.5%


Figure 2. Frequency ofFusarium graminearum phylogenetic lineages and corresponding species sensu
O'Donnell et al. (2" i 14 isolated from wheat grains in 2001, 2002 and 2003.






Session 2: Fusarium consortium


< 500 bp


NIV producer
Lineage 8
(F. cortaderiae)


.m. .
-< *c


UI


Figure 3. PCR products obtained for Tri7 gene amplification of different F. graminearum isolates.
Different sizes correspond to different number of 1 lbp insertions.


50%

40%
> 30%

S20%
LL 10%

0%


10-100 100-1000
DON (ppm)


>1000


Figure 4. Frequency ofFusarium graminearum isolates producing different deoxynivalenol (DON)
concentrations in rice grain cultures.







Session 2: Fusarium consortium


FUSARIUM PATHOGENS OF WHEAT IN AUSTRALIA


S. Chakraborty", J.B. Scott', O.A. Akinsanmi2, C.J. Liu V. Mitter1,
and R. Dill-Macky3

1CSIRO Plant Industry, Queensland Bioscience Precinct Brisbane, QLD 4300, Australia; 2Cooperative Research
Centre for Tropical Plant Protection, Plant Pathology Building, 80 Meiers Road, Australia; 3 University of
Minnesota, Department of Plant Pathology, 495 Borlaug Hall, Saint Paul, MN 55108, USA;
*Corresponding author: E-mail: Sukumar.chakrabortvgicsiro.au


ABSTRACT

This paper deals with a comparative study of Fusarium
head blight (FHB) pathogens including Fusarium
graminearum (FG) and F. pseudograminearum (FP)
from Australia and the USA using aggressiveness,
mycotoxin production and genotypic diversity based
on amplified fragment length polymorphism In
addition, it examines phylogenetic relationships within
FP using genealogical analysis of four nuclear genes.
Both Australian FG and FP are genetically diverse,
although perithecia are rarely seen in nature in the
heterothallic teleomorph of FP. Both species produced
mycotoxins in grain cultures and caused FHB in
infection assays. Although toxin levels and FHB
severity varied with the isolate, there was no
association between these two traits. A multi-locus
sequence analysis of FP isolates from Australia,
Canada, New Zealand, Turkey and the USA
differentiated this species into four well-supported
clades, but these were not linked to their geographic
origin The paper stresses the importance of
international collaboration in sharing information,
tools, and resources in making research outputs
applicable to wide geographical regions.

INTRODUCTION

In Australia, crown rot (CR), predominantly caused by
Fusarium pseudograminearum (FP), and head blight
(FHB), predominantly caused by F. graminearum
(FG), are two important diseases of wheat (Burgess et
al., 2001). CR imposes a recurring cost of >$56
million per year over vast areas. The impact of FHB is
sporadic in Australia and limited to an area in northern
New South Wales (NSW) and southern Queensland
(QLD) and as a result, there has been limited research.
The global significance of FHB has prompted
coordinated research efforts in the USA and Europe,
among others, to generate a wealth of knowledge
(Goswami and Kistler, 2004). In contrast, the majority


of CR research originates from Australia despite its
presence in most cereal-growing regions of the world.
Ongoing Australian research over the past 50 years,
reviewed by Burgess et al. (2001), has been boosted by
recent industry initiatives. New research to improve
host resistance to CR and FHB by applying essential
knowledge of pathogen biology, genetics and
epidemiology started in 2001 with the build up of a
culture collection of well characterized isolates of
Fusarium spp. from Australia and overseas. All 17
Fusarium species collected from Australian field
surveys caused FHB and all 10 of these 17 tested
caused CR in plant infection assays where 20% of FP
and FG isolates were aggressive to highly aggressive
for both diseases (Akinsanmi et al., 2004). New
knowledge of pathogen aggressiveness has led to the
development of a new high throughput seedling assay
for CR (Mitter et al., 2006) to accelerate the search for
host plant resistance.

FHB and CR have linked aetiology and epidemiology
but the relationship among Fusarium species and
strains that cause FHB and CR are far from clear,
except that infected crop residue is the primary
inoculum source for both diseases. The narrow
window of FHB infection and the subsequent removal
of infected heads at harvest raise the possibility that the
bulk of FHB inoculum may originate from stem and/or
crown infection How CR infected crops contribute to
FHB inoculum has never been explored despite
increased prominence of CR in the USA in recent
years (Smiley et al., 2005). Similarly, the wealth of
knowledge from global FHB research has not been
applied to CR. To do this, the information gained on
one location/disease must be relevant to another
location/disease. In the USA ascospores are the main
FHB inoculum but these are less aggressive than
macroconidia in CR infection assays. In Australia
macroconidia are the dominant FHB inoculum and
both spore types are equally aggressive for CR (Mitter
et al., unpublished data). In this paper we continue the







Session 2: Fusarium consortium


comparative study of FG and FP from Australia and
the USA using FHB aggressiveness, toxin production
and genotypic diversity. Additionally, phylogenetic
relationships within FP were examined using
genealogical analysis of four nuclear genes.

MATERIALS AND METHODS

Comparison of F. graminearum from Australia and
the USA as FHB pathogen
A total of 13 F. avenaceum, 6 F. crookwellense, 69 FG
and 75 FP isolates from Australia and 6 F. avenaceum,
5 F. culmorum, 130 FG, 5 F. poae, 5 FP and 7 F.
sporotrichoides isolates from the USA were studied
using the same methodology. FHB aggressiveness of
isolates from the USA was studied at the University of
Minnesota on cultivars Wheaton (susceptible), Pioneer
2375 (moderately susceptible) and Alsen (Sumai3-
derived moderately resistant) by point inoculation of a
central spikelet in each of 10 spikes with 10 l
suspension of 105 macroconidia/ml at anthesis. The
Australian isolates were similarly inoculated on to
Wheaton (susceptible), Pioneer 2375 and Sumai3
(moderately resistant) at CSIRO facilities in Brisbane.
Each isolate was grown on autoclaved wheat grains,
ground and toxins extracted and analyzed following
the procedures of Mirocha et al. (1998). Genotypic
diversity was analyzed using amplified fragment
length polymorphism (AFLP) following methods
outlined previously (Akinsanmi et al. 2006).

Phylogenetics ofF. pseudograminearum
DNA from the translation elongation factor-la,
phosphate permease 1, phosphate permease 2 and the
intergenic region between these two genes, a reductase
gene and the (-tubulin gene were amplified and
sequenced for 54 FP isolates from Australia, Canada,
Turkey, New Zealand and the USA. Data were
analyzed separately and as a multi-locus sequence
using both maximum parsimony and Bayesian
inference methods.

RESULTS AND DISCUSSION

Comparison of F. graminearum from Australia and
the USA as FHB pathogen
Results on the Australian isolates are mainly discussed
here. On sterilized wheat grain the Australian FG
isolates produced between 0 and 113 ppm
deoxynivalenol (DON) (mean 10.3, median 1.9)
compared with to 1587 ppm (mean 21.5, median2.2)
for isolates from the USA. Australian FP isolates
produced 0 45 ppm (mean 1.3) DON compared with
0.1-101 ppm (mean 22.6) for isolates from the USA. In
most isolates 3ADON and/or 15ADON were co-
produced in smaller quantities but none produced


Nivalenol to any significant extent. FG and FP isolates
differed in their aggressiveness on the three wheat
varieties; Sumai 3 was the most resistant, 2375 was
intermediate in resistance and Wheaton the most
susceptible. Toxin production in grain culture was not
associated with aggressiveness on any variety,
indicating that mycotoxins are only one component of
aggressiveness. Data on mean FHB severity on three
wheat varieties and DON are shown for FG and FP
(Figure 1).

The genotypic diversity was high; over 90% of FP and
FG isolates had distinct haplotypes. The polyphyletic
Australian FG was grouped into five AFLP clusters
(Figure 2) with reference isolates of lineages 2, 3, 4
and 6 were distributed within two of the Australian
clusters, whereas isolates of lineages 1, 5 and 7 were
contained in a single cluster with other Australian
isolates. Although this suggests FG lineages 1, 2, 3, 4,
5, 6 and 7 (O'Donnell et al., 2004) in Australia this
must be confirmed by gene sequencing since AFLP
clustering does not accurately correlate with lineages.
So far, sequencing has confirmed lineage 7 (FG) and 8
(F. cortideriae) in Australia. Genotypic diversity and
FHB aggressiveness in FG were not significantly
linked. The 5 clusters for the Australian FP isolates
were not as clearly delineated (Figure 3) and unlike
FG, AFLP clusters were significantly association with
FHB aggressiveness. The population structure of the
heterothallic Gibberella coronicola (teleomorph of FP)
and the homothallic G. zeae (teleomorph of FG) was
not that different and neither species was panmictic or
strictly clonal.

Phylogenetics ofF. pseudograminearum
Evolutionary divergence varied between genes and
there was phylogenetic incongruence between the
reductase gene and the translation elongation factor-
la, phosphate permease and (-tubulin genes. A multi-
locus sequence analysis differentiated FP isolates into
three well-supported clades while a single Turkish
isolate formed a fourth clade. The majority of
Australian, all New Zealand, two Canadian and one
isolate from the USA grouped in one clade; five of
seven Canadian isolates formed another; and four
isolates from the USA and two Australian isolates
formed the third clade. These clades were not
consistent across genes to indicate that FP is a single
phylogenetic species as opposed to FG (O'Donnell et
al. 2004). Evolutionary structure and geographic origin
of isolates were not linked, indicating FP to be a
recombining species. The high level of genetic
variation and sexual recombination in the heterothallic
G. coronicola is consistent with a recent finding that
high levels of genetic variation is generated by single
recombination events and that the MAT1-1 and







Session 2: Fusarium consortium


MAT1-2 idiomorphs of the mating type locus occurs in
1:1 ratio in most field populations within Australia
(Bentley et al. 2005).

In Australia the geographical distribution of FG is far
more restricted than the widespread FP. This is
counter-intuitive, since the long distance dispersal of
airborne G. zeae ascospores should have made this
species more widespread than G. coronicola where
perithecia are not frequent. The level of genotypic
diversity and population differentiation or the range of
aggressiveness for FHB as determined from the current
work do not indicate a superior pathogenic or
saprophytic fitness ofFP over FG either. While climate
(Backhouse and Burgess 2002) can influence the
distribution of some Fusarium species, the temperature
difference between northern NSW and southern QLD
is not large enough to explain the differential
distribution of these two species. There is a history of
research on epidemiology and management of CR in
Australia. New information on the structure, genetics
and evolution of the pathogen population is only just
beginning to emerge. The vast wealth of FHB
knowledge is starting to be examined from a CR
perspective by sharing information, tools, and
resources through international collaboration This
must continue, evolve and expand to encompass other
research groups to make research and development
more cost effective and targeted.

ACKNOWLEDGEMENTS

Funding of this work from the Cooperative Research
Centre for Tropical Plant Protection, The U.S. Wheat
and Barley Scab Initiative, The Australian Grains
Research and Development Corporation and CSIRO
Plant Industry are gratefully acknowledged. Technical
assistance from Ross Perrott and Paul Melloy of
CSIRO Plant Industry is gratefully acknowledged.

REFERENCES

Akinsanmi OA, Backhouse D, Simpfendorfer S,
Chakraborty S (2006) Australian Fusarium
graminearum and F. pseudograminearum are both
genetically diverse. Plant Fi,lh. i. ,-' (inpress).
Akinsanmi OA, Mitter V, Simpfendorfer S, Backhouse
D, Chakraborty S (2" 1 4) Identity and pathogenicity
of Fusarium spp. isolated from wheat fields in
Queensland and northern New South Wales.
Australian Journal of Agricultural Research 55,
97-107.
Backhouse D, Burgess LW, 2002. Climatic analysis of
the distribution of Fusarium graminearum, F.
pseudograminearum and F. culmorum on cereals in
Australia. Australasian Plant F, hi. I.. 31, 321-7.


Bentley AR, Summerell BA, Liew ECY, Burgess LW
(2005) Gibberella coronicola: An imperfectly
understood teleomorph Proceedings of the 15th
Biennial Australasian Plant Pathology Society, 26-
29 September, Deakin University, Geelong, VIC.
Burgess LW, Backhouse D, Summerell BA, Swan LJ
(2001) Crown rot of wheat. In: 'Fusarium: Paul E.
Nelson Memorial Symposium'. (Eds Summerell
BA, Leslie JF, Backhouse D, Bryden WL, Burgess
LW). Pp 271-294. The American
Phytopathological Society, St Paul, Minnesota.
Goswami RS, Kistler HC (i' 1'14) Heading for disaster:
Fusarium graminearum on cereal crops. Molecular
Plant Fihi. i.-, .- 5, 515-525.
Mirocha, C.J., Kolaczkowski, E., Xie, W., Yu, H. and
Jelen, H. 1998. Analysis ofdeoxynivalenol and its
derivatives (batch and single kernel) using gas
chromatography/mass spectrometry. Journal of
Agriculture Food Chemistry 46:1414-1418.
Mitter V, Zhang MC, Liu CJ, Ghosh R, Ghosh M,
Chakraborty S (2006) A high throughput
glasshouse bioassay to detect crown rot resistance
in wheat germplasm. Plant Fi.-'1l (in press)
doi: 10.1111/j.1365-3059.2006.01384.x.
O'Donnell K, Ward TJ, Geiser DM, Kistler HC, Aoki
T, 2004. Genealogical concordance between the
mating type locus and seven other nuclear genes
supports formal recognition of nine
phylogenetically distinct species within the
Fusarium graminearum clade. Fungal Genetics
and Biology 41, 600-23.
Smiley RW, Gourlie JA, Easley SA, Patterson L-M,
Whittaker RG 2005. Crop damage estimates for
crown rot of wheat and barley in the Pacific
Northwest. Plant Disease 89, 595-604.







Session 2: Fusarium consortium


0

*

*.


**
*0 *
** 0
0 0


0 30 60 90 120 v
Deoxynivalenol content (ppm)

Figure 1. Mean Fusarium head blight severity (proportion of spikelet infected) on three wheat varieties and
Deoxynivalenol production on grain culture by isolates of Australian Fusarium graminearum (left) and F.
pseudograminearum (right)


-7-


o. I





1.0 5522316 7 112346
c5 c4 c3 c2 cl
Figure 2. Five genotypic clusters (cl-c5) of Australian Fusarium graminearum isolates based on amplified
fragment length polymorphismwith lineages (small numerals) of reference isolates


0.22


S0.41.


g0.60- I
*I ,


c3 c2 cl


Figure 3. Genotypic clusters (cl-c5) of Australian Fusarium pseudograminearum isolates based on amplified
fragment length polymorphism


I A '1 A ir 7







Session 2: Fusarium consortium


TURKISH FUSARIUM ISOLATES FROM WHEAT CROWN
AND HEAD CAN CAUSE SEVERE CROWN ROT


B. Tunali1, J.M Nicol2*, S. Chakraborty3, F.Y.Erol1 and G. Altiparmak1

'Department of Plant Protection, Faculty of Agriculture, University of Ondokuz Mayis, Kurupelit, Samsun, 55139
Turkey; 2CIMMYT International, PK 39, Emek, Ankara, 06511, Turkey;
3CSIRO Plant Industry, Queensland Bioscience Precinct, Brisbane, Queensland 4067, Australia
*Corresponding Author: PH: (90) 312 2873595; Email: j.nicoliacgiar.org


ABSTRACT

This paper deals with the severity of crown rot caused
by Fusarium acuminatum, F. avenaceum, F.
chlamydosporum, F. crookwellense, F. culmorum, F.
equiseti, F. graminearum, F. oxysporum, F.
pseudograminearum, F. semitectum, F. solani, F.
,Ill1 i, o,,li, and F. verticilloides isolated from wheat
fields in Turkey surveyed for Fusarium head blight in
2003 and crown rot in 2005. In a seedling assay
Fusarium species and isolates within a species
significantly influenced the level of crown
discoloration and biomass of the winter wheat cultivar
'Pehlivan'. There was no significant difference among
the 3 most aggressive species, F. culmorum, F.
graminearum and F. pseudograminearum. Where
isolates of the same Fusarium species were obtained
from head and crown tissue, these did not differ greatly
in their aggressiveness on the crown tissue.

INTRODUCTION

The genus Fusarium is one of the most widespread and
economically important groups of fungi with more
than one hundred species attacking most plant species.
On wheat and barley Fusarium species cause two
diseases: a basal stem and root rot often known as
crown rot (CR) and spike infection commonly known
as Fusarium head blight (FHB). Several Fusarium and
other species including F. pseudograminearum
(teleomorph Gibberella coronicola), F. crookwellense,
F. avenaceum (teleomorph. G. avenacea), F.
culmorum, F. acuminatum and Microdochium nivale
(teleomorph Monographella nivalis) can cause CR
(Cook, 1981, Specht and Rush, 1988). A similar range
of species including F. graminearum, F. culmorum, F.
avenaceum, F. sambucinum var. coeruleum, F.
crookwellense, F. sporotrichoides and M. nivale can
cause FHB (Arsenuik et al. 1991., Miedaner et al.,
1993).


Turkey is among the 10 largest wheat producers
worldwide with 16-21 million tones from 9.35 Mha at
an average yield of 2t/ha (Braun et al., 2001). Over
half of the area is located in Central Turkey where
wheat is grown under rainfed or supplementary
irrigation conditions in cereal fallow rotation In the
Central Anatolia Plateau cereal root rots contribute to
significant yield losses of between 24-36% in the
commonly cultivated winter wheats (Aktag et al. 1999;
Hekimhan et al., 2004).

The distribution of Fusarium species associated with
CR in Turkey varies with the geographical region: F.
pseudograminearum is more widespread than F.
culmorum in the Northeast Marmara coastal region
(Aktag et al., 1996), while in the Central Anatolia
Plateau F. culmorum was the most commonly isolated
species from the crown and sub-crown tissue in three
surveys spanning 1994 to 2004 (Aktag et al., 1999;
Tunali et al., 2006) While most tests show F.
pseudograminearum and F. culmorum as the most
pathogenic for CR in Turkey, some reports indicate F.
avenaceum to be equally or more pathogenic than the
other species (Arsenuik et al., 1993; Jenkins and Parry,
1994).

In Turkey FHB epidemics are restricted to years of
high humidity during anthesis and F. graminearum
was the primary pathogen in the Northwest Anatolia in
2001/02 (Tunali et al., 2006). AlthoughF. culmorum is
more aggressive on wheat spikes inpathogenicity tests,
it is considered a secondary pathogen following F.
graminearum infection

In Australia both F. graminearum and F.
pseudograminearum can cause CR and FHB; but the
former predominantly occurs on flag leaf node and
head tissue while the later is common on crown and
stubble (Akinsanmi et al., 2004). Overall, isolates from
stem base and crown are more aggressive for CR and







Session 2: Fusarium consortium


isolates from flag leaf node and head tissue are more
aggressive for FHB. Although several species
including F. culmorum, F. graminearum and F.
pseudograminearum are commonly associated with
CR and FHB in Turkey, their relative aggressiveness
for CR has not been studied in details. This paper
compares the CR aggressiveness of isolates from
several Fusarium species originating from crown and
head tissue of wheat.

MATERIALS AND METHODS

All isolates of Fusarium species used in this work
came from two surveys around plant maturity (Zodoks
growth stage 92) of wheat growing areas in central and
northern Anatolia: survey for FHB in 2003 and for CR
in 2005 (Figurel). Details are given for the 2005 CR
survey where diseased crown and sub crown tissue was
collected from five plants at each of the 32 sites.
Small pieces of infected tissue were washed in tap
water, surface sterilized with 1% NaOCl solution for 3
minutes and plated on 1/4 strength potato dextrose agar
(PDA, 9g PDA, 10g Bacto agar, 1L distilled water)
amended with streptomycin sulfate (100 mg/L) and
Oxytetracyline (60 mg/L). Plates were incubated for 7
days at 25 C, 15 h photoperiod under cool white
fluorescent light and colonies were transferred to
Carnation leaf agar (CLA). To obtain monoconidial
isolates water agar plates were seeded with a conidial
suspension, incubated (Burgess et al., 1994) and a
single germinated macroconidium on a small square of
agar was transferred to a fresh CLA plate using a
sterile needle. Fusarium species were identified using
morphological and cultural characteristics (Booth,
1977; Gerlach and Nirenberg 1982; Tousson and
Nelson, 1985).

The pathogenicity of 51 isolates from 13 Fusarium
species was tested in a glasshouse assay. Plants were
grown in plastic pots (8 cm diameter, 16cm high) with
a mixture of autoclaved commercial potting soil and
local loam soil. Seeds of the winter wheat cultivar
'Pehlivan' were surface sterilized for 3 minutes in a
1% aqueous solution of NaOC1, rinsed twice in sterile
distilled water and coated separately with
macroconidia and mycelia of each of the 51
monoconidial isolates by shaking on a CLA plate of a
7 day old culture. Additional inoculum was added by
placing a 1 cm diameter agar plug with mycelium and
spores from the periphery of CLA cultures of the
respective isolate at the bottom of each of the three 3-
cm deep holes in each pot (Fernandez and Chen,
2005). One Pehlivan seed coated with the same isolate
was placed on top of the agar plug so that the growing
colony was in contact with the seed and the holes were
covered with 3 cm of potting soil. Sterile agar plugs


were used as a control. Three pots were used for each
fungal isolate and treatments were arranged in a
completely randomized design with each pot treated as
one replicate. Plants were watered as necessary and
grown at 25+50C in a greenhouse under a 16 h
photoperiod using fluorescent lights.

Plant emergence was counted 15 days after planting
and symptoms were assessed at harvest on day 70.
Each plant was rated for discoloration of crown and the
sub coronal internodes using a 0-3 scale, where, 0= no
discoloration, 1= trace to 25% discoloration, 2= 25 to
50%, and 3= 50>%. Plants were dried between 2 layers
of paper towels following disease assessment and the
fresh weight was recorded as an estimate ofbiomass.

Data on percentage discoloration were analyzed using
a nested model in an analysis of variance where
isolates were nested within species using the SAS
software.

RESULTS

Eleven isolates comprising F. culmorum (2), F.
graminearum (4) and F. pseudograminearum (5), from
the 2003 FHB survey were used (Table 1). The
remaining 40 isolates of F. acuminatum (4), F.
avenaceum (2), F. chlamydosporum (2), F.
crookwellense (1), F. culmorum (13), F. equiseti (2),
F. oxysporum (2), F. pseudograminearum (1), F.
semitectum (2), F. solani (2), F. ,ili.jllia' (4) and
F. verticilloides (5) originated from crowns of 160
plants sampled from 32 fields in 2005. All species and
isolates caused discoloration of crown and the sub-
coronal internode. The level of aggressiveness differed
significantly both within (Figure 1) and among
Fusarium species but there was no significant
difference among the 3 most aggressive species, F.
culmorum, F. graminearum and F.
pseudograminearum (Table 1). The aggressive species
also greatly reduced plant emergence and survival
(data not shown). The pathogenicity of each species
and isolate was confirmed by following Koch
postulates where infected tissue from the crown and
sub crown internodes were plated onto CLA medium
and the identity of the Fusarium species was re-
confirmed.

The impact of crown rot was evident from a significant
reduction of plant biomass with the three most
aggressive species, F. culmorum, F.
pseudograminearum and F. graminearum, reducing
growth by up to 85% of the uninoculated control
(Table 1). As expected, crown rot severity and plant
biomass were inversely correlated (Figure 2). All
except F. crookwellense had more than 1 isolate per







Session 2: Fusarium consortium


species, but an overwhelming majority of isolates
originated from the crown tissue and only F. culmorum
and F. pseudograminearum isolates originated from
both spike and crown tissues. Overall, the crown rot
severity of isolates originating from the crown tissue
was not different to that of the isolates originating from
the spike, but the very limited number of isolates from
the spike makes this comparison tentative.

DISCUSSION

We have shown that Fusarium species and isolates
within a species significantly influence crown rot
severity and biomass of winter wheat Pehlivan in a
seedling assay and F. culmorum, F. graminearum and
F. pseudograminearum are the 3 most aggressive
species. Where isolates of the same Fusarium species
were available from head and crown tissue, these did
not differ greatly in their aggressiveness on the crown
tissue. The three highly aggressive species also
affected seedling emergence and growth This is
similar to Australian findings where F. graminearum
from wheat spikes and F. pseudograminearum mostly
from crown tissue were equally aggressive for crown
rot under greenhouse tests (Akinsanmi et al., 2004).
Other findings (Fernandez and Chen, 2005) also show
that Fusarium species derived from infected wheat
heads or sub-crown internode/crown have similar
relative pathogenicity on wheat heads. These data
indicate that the entire wheat plant may play an
important role in the survival of the pathogen and act
as inoculum source for both FHB & CR.

Previous work has failed to separate F. graminearum
and F. pseudograminearum based on their crown rot
aggressiveness in seedling bioassays where the same
20% of isolates from both species were aggressive or
highly aggressive for both CR and FHB (Akinsanmi et
al., 2004). Our current study showing F.
pseudograminearum and F. graminearum among two
of the three most highly aggressive species causing CR
in the seedling test, confirms this earlier finding.
Extensive studies in the USA (Smiley and Patterson,
1996) with more than 1200 Fusarium isolates
representing 19 species confirmed F. graminearum, F.
culmorum and B. sorokiniana to be the major species
associated with root and crown rot of wheat. Recent
work has further highlighted the importance of F.
pseudograminearum (Smiley et al., 2005). Although
many other Fusarium species have been isolated from
the crown tissue in this and previous research (Uoti,
1976; Arsenuik et al., 1993 their importance as a CR
pathogen appears to be limited.

Recent extensive surveys of a major cereal producing
region in Turkey have clearly identified F. culmorum


as the main Fusarium species associated with the
crown tissue (Tunali et al. 2006), while F.
pseudograminearum is the dominant pathogen in some
other areas (Aktas et al., 1996). Both are among the
most aggressive Fusarium species for CR. Fusarium
graminearum, the other most aggressive CR pathogen
is also the dominant FHB pathogen in Turkey (Tunali
et al., 2006). CR is a chronic biotic constraint in
Turkey on dryland wheat while FHB is restricted to
years of high humidity during anthesis. Given the
global efforts in identifying FHB resistance, future
research need to explore whether some of the sources
of FHB resistance to also offer resistance to CR. This
must be combined with field-based research on the
inter-relationships between FHB and CR
epidemiology.

ACNOWLEDGEMENTS

We appreciate statistical assistance from Aysun Peksen
at Ondokuz Mayis University.

REFERENCES

Akinsanmi, O. A., V. Miller, S. Simpfendorfer, D.
Backhouse and S. Chakraborty. 2004. Identity and
pathogenicity of Fusarium spp. Isolated from
wheat fields in Queensland and northern New
South Wales. Australasian J Agric. Res. 55: 97-
107.
Aktas, H., H. Bostancioglu, B. Tunali and E. Bayram,
1996. Determination of the root rots disease
agents, their interference with cultural practices
and evaluation of varieties and lines against to
important ones in Sakarya Region Plant
Protection Bulletin, 36(3-4): 151-167.
Arsenuik, E., T. Gora, and, H.J. Czember, 1993.
Reaction of triticale, wheat and rye accessions to
graminaceous Fusarium spp. Infection at the
seeding and adult plant growth stages. Euphytica
70: 175-183.
Booth, C. 1977. Fusarium laboratory guide to the
identification of the major species.
Commenwealth Mycological Institute, Ferry
Lane,Kew Surrey. 58 p.
Braun, H.J., N. Zencirci, F Altay, et al., 2001. Turkish
Wheat Pool. In 'World Wheat Book A History of
Wheat Breeding'. (Eds AP Bonjean, WP Angus)
Lavoisier Publishing: Paris France, pp.851-879.
Burgess, L.W., B.A. Summerell, S. Bullock, K.P. Gott,
D. Backhouse, 1994. Laboratory manual for
Fusarium research 3rd edition Fusarium Research
Laboratory University of Sydney and Royal
Botanic Gardens: Sydney. 133p.







Session 2: Fusarium consortium


Cook, R.J, 1981. Fusarium diseases of wheat and other
small grains in North America. Pages 30-52 in
Fusarium diseases. Biology and Taxonomy. P.E.
Fernandez, M.R and Y. Chen, 2005. Pathogenicity of
Fusarium species on different plant parts of spring
wheat under controlled conditions. Plant Dis. 89:
164-169.
Gerlach, W and H. Nirenberg, 1982. The genus
Fusarium. A pictorial atlas. Kommissionsverlag
Paul Parey, Berlin und Hamburg.p.406.
Hekimhan, H., A. Bagci, J. Nicol, T. Ansoy and S.
Sahin, 2004. Dryland Root Rot: a major threat to
winter cereal production under sub-optimal
growing conditions. 4th international Crop Science
Congress,26 Sep.- 1 Oct., Brisbane, Australia.
www.regional.org.au/au/cs.
Jenkinson, P and D.W. Parry, 1994. Isolates of
Fusarium species from common broad
leaved weeds and their pathogenicity to winter
wheat. Mycol. Res. 98: 776-780.
Miedaner,T., D.C. Borchardt and H.H. Geiger, 1993.
Genetic analysis of inbred lines and their crosses
for resistance to head blight (Fusarium culmorum
,F. graminearum) in winter wheat rye. Euphytica
65:123-133.


Smiley, R.W., H.P. Collins, and P.E. Rasmussen,
1996. Diseases of wheat in long-term agronomic
experiments at Pendleton, Oregon Plant Dis. 80:
813-820.
Smiley RW, Gourlie JA, Easley SA, Patterson L-M,
Whittaker RG 2005. Crop damage estimates for
crown rot of wheat and barley in the Pacific
Northwest. Plant Dis. 89: 595-604.
Specht, L.D., and C.M. Rush, 1998. Fungi associated
with root and foot rot of winter wheat and
populations of Bipolaris sorokiniana in the Texas
panhandle. Plant Dis. 72: 959- 969.
Tousson,T.A, and Nelson,P.E. 1995. A pictorial guide
to the identification of Fusarium species.
FUSARIUM. The Pennsylvania State University
Press. University Park and London p.43.
Tunali B., Biiytik O.,Erdurmus D., Ozseven I. ve
Demirci A. 2006. Fusarium head blight and
deoxynivalenol accumulation of wheat in
Marmara region and reactions of wheat cultivars
and lines to F. graminearum and F.culmorum.
Pakistan J. Plant Pathol. 5(2): 150-156.
Uoti, J, 1976. The effect of five Fusarium species on
the growth and development of spring wheat and
barley. Ann Agric. Fenniae Ser. Phytopathol.
No.62. 15: 254-262.


Sa 881 -1.238
1-_ ii-- / 7- / 1.239- 2.227
------------ ----------------------
Figure 1. Field sites in Central and Northern part of Anatolia, Turkey sampled for
Fusarium species associated with crown rot (2005) and Fusarium head blight (2003) of







Session 2: Fusarium consortium


Table 1. Crown rot severity and fresh weight of winter wheat 'Pehlivan' inoculated in the greenhouse with isolates
from 13 Fusarium species obtained from different parts of wheat plants growing in farmers' fields in Turkey.
Fusarium species Isolates Isolates Total Mean severity Mean fresh
from crown from spike weight
F. pseudograminearum 1 5 6 91.98 A 0.28 D
F. culmorum 13 2 15 91.57 A 0.35 D
F. graminearum 0 4 4 88.61 A 0.43 D
F. avenaceum 2 0 2 59.28 B 1.57 ABC
F. crookwellense 1 0 1 55.17 BC 0.89 CD
F. semitectum 2 0 2 48.13 BCD 1.23 BC
F. oxysporum 2 0 2 46.27 BCD 1.01 CD
F. solani 2 0 2 44.43 BCD 1.41 ABC
F. verticilloides 5 0 5 37.01 CDE 1.60 ABC
F. acuminatum 4 0 4 36.97 CDE 2.03 AB
F. equiseti 2 0 2 31.45 DE 1.82 AB
F. chlamydosporum 2 0 2 31.45 DE 1.65 ABC
F. subglutinans 4 0 4 22.21 E 2.07 A
Total isolates 40 11 51

Within columns, means followed by a different letter are significantly different (P <0.05) according to Ryan-
Einot-Gabriel-Welsch Multiple Range Test.


Severity (%)


Isolates 11


S erticilloides
subglutinans
solani
semitectum
pseudograminearum
oxysporum
graminearum
equiseti
culmorum
chlamydosporum
avenaceum
acuminatum


Figure 2. Crown rot aggressiveness (severity) of isolates from 13 different Fusarium spp. on winter
wheat 'Pehlivan'.







Session 2: Fusarium consortium


3.50

3.00 y= -0.023x + 2.47
Fs R2 = 0.84
p 2.50 Fa Fa
Fs
2.00 -C Fs FeFa

-1.50- Fv e Fsey Fa FFvo
Fso Fay
T 1.00- Fo Fs@ F Fg
c Pse Fp FcFg
S0.50- Fp F c
O O0 C'
Fc0 F p
0.00 20.00 40.00 60.00 80.00 100.00 120.00
Mean Disease Severity (%)



Figure 3. Relationship between fresh plant weight and crown rot severity caused by isolates of 13
different Fusarium spp. on winter wheat 'Pehlivan' screened under greenhouse conditions.
F.a: F. acuminatum, F.av: F.avenaceum, F.c: F. culmorum, F.ch F. chlamydosporum, F.cr: F
crookwellence, F.e: F. equiseti, F.g: F. graminearum, F.o: F. oxysporum, F.p: F. pseudograminearum,
F.s: F. solani, F.se: F. semitectum, F.s: F. itlljlitwioi,iu F.v: F. verticilloides.






Session 2: Fusarium consortium


VEGETATIVE COMPATIBILITY ANALYSIS (VCG) AND
SEQUENCE RELATED AMPLIFIED POLYMORPHISMS
(SRAP) IN UNDERSTANDING GENETIC DIVERSITY
OF GIBBERELLA ZEAE ISOLATES
FROM TWO MANITOBA FIELDS

W.G.D. Femando1*, J. X. Zhang1, M. Dusabenyagasani1, X. W. Guo1,
H. Ahmed1, and B. McCallum2

'University of Manitoba, Department of Plant Science, Winnipeg, MB R3T 2N2, Canada;
Cereal Research Centre, Agriculture and Agri-Food Canada, Winnipeg, MB R3T 2M9, Canada
*Corresponding Author: PH: (1-204) 474 6072; E-mail: d fernando@iumanitoba.ca



ABSTRACT

Gibberella zeae causes Fusarium head blight of wheat. It is one of the most important diseases of cereals in the
Canadian prairies especially the Red River Valley for the last decade. In 2002, 60 isolates of G. zeae were collected
and single spored from naturally infected spikes of wheat from Carman and Winnipeg in Manitoba. These isolates
were compared using vegetative compatibility analysis and PCR-based sequence related amplified polymorphisms
(SRAP). Sixteen vegetative compatibility groups (VCG) were found among the 50 isolates tested. Eight SRAP
primer pairs identified 59 distinct haplotypes. Principal component analysis and UPGMA separated the dataset into
two main groups, each with isolates from both locations. The analysis of molecular variance also revealed that 75%
and 20% of the variance were associated to differences among individual isolates and varieties sampled respectively.
Geographic location was not a significant source of variation at P=0.05 and accounted for only 4% of total variance.
A low correlation between VCG and SRAP marker data was detected. This study showed that though the genetic
diversity is high among G. zeae isolates, Carman and Winnipeg collections have a similar genetic makeup and are
likely part of the same population A large-scale study has been initiated collecting Fusarium isolates from 15
different farmers' fields located throughout Manitoba. The crop history, environmental conditions, cultivars, and soil
type are different in these fields. The new study will investigate the genotype and chemo-type differences of isolates
frombetween and within fields.






Session 2: Fusarium consortium


PRESENT STATUS OF THE FUSARIUM GRAMINEARUM
CLADE IN EUROPE AND POSSIBLE DEVELOPMENT
STRATEGIES


B. T6th and A. Mesterhazy

Cereal Research Non-profit Company, P.O. Box 391, H-6701 Szeged, Hungary
Author e-mails: beata.tothagk-sze ged. hu akos.mesterhazv Agk-szeged.hu



ABSTRACT

Fusarium head blight caused mainly by Fusarium graminearum and F. culmorum is the most important disease of
wheat in Central Europe. Previous studies clarified that F. graminearum is an assemblage of at least nine
geographically separated species (O'Donnell et al. 2004).

We examined the mycotoxin producing ability, molecular variability and aggressiveness of Fusarium graminearum
isolates originated from Central Europe, and representatives of 8 species identified in the F. graminearum clade.
Mycotoxin producing abilities of the isolates were tested by GC-MS and HPLC analyses. The mycotoxins tested
included type B trichothecenes (deoxynivalenol, 3- and 15-acetyl-deoxynivalenol, nivalenol, 4-acetyl-nivalenol
(fusarenone X)) and zearalenone. All but one of the isolates produced zearalenone. The Central-European isolates
were found to belong to chemotype I (producing deoxynivalenol). Most of them produced more 15-acetyl-
deoxynivalenol than 3-acetyl-deoxynivalenol, indicating that these isolates possibly belong to chemotype lb.

Phylogenetic analysis of random amplified polymorphic DNA (RAPD) profiles of the isolates let us cluster the
Central-European isolates into 10 haplotypes. The three Austrian isolates formed a distinct clade on the tree. We
also examined the variability of the intergenic spacer region (IGS) of the ribosomal RNA gene cluster using IGS-
RFLP analysis. The isolates belonged into 9 haplotypes on the tree based on IGS-RFLP data. Representatives of
species of the F. graminearum clade exhibited unique IGS-RFLP and RAPD profiles. When RAPD and IGS-RFLP
data were combined, almost every single Central-European F. graminearum isolate could be differentiated from
each other (27/30 haplotypes). Such a lack of strict correlation between trees based on different data sets indicates
that recombination took place in the examined population due to frequent outcrossing. Based on RAPD, IGS-RFLP
and sequence data, the majority of the Central-European isolates belong to the F. graminearum sensu strict species
characteristic to the Northern hemisphere, with the exception of a Hungarian isolate, which was closely related to F.
asiaticum based on RAPD and IGS-RFLP data. Further sequence analysis revealed that this isolate belongs to a new
species, which occurs in Hungary and Japan (F. vorosii, species description is in progress; T6th et al. 2005). The
taxonomic assignment of two other Hungarian isolates previously suggested to belong to F. boothii based on
mitochondrial DNA RFLP data were supported by sequence analysis.

Isolates belonging to the F. graminearum species complex exhibited high levels of strain-specific variability in their
aggressiveness and their ability to produce trichothecenes on susceptible cultivars (Goswami et al. 2005). We tested
twenty wheat genotypes in 2003-2004 under field conditions by spraying inocula of isolates of eight species of the
F. graminearum species complex representing geographically isolated populations. The various wheat genotypes
exhibited similar reactions against the different Fusarium isolates, indicating that resistance to F. graminearum
sensu lato was similar to that for the other Fusarium species examined (Mesterihzy et al. 2005). This is an important
message to breeders as the resistance relates not only to any particular isolate ofF. graminearum sensu strict, but
similarly to other isolates of the Fusarium graminearum species complex as well. Based on our results, the new
members of the F. graminearum species complex do not seem to have any importance for breeding. Therefore
resistant genotypes can be grown successfully in areas where different members of the species complex dominate or
occur mixed.







Session 2: Fusarium consortium


CROSS FERTILITY OF LINEAGES IN
FUSARIUM GRAMINEARUM (GIBBERELLA ZEAE)


R.L. Bowden"1, J.F. Leslie2, J. Lee2, and Y.-W. Lee3

'USDA-ARS Plant Science and Entomology Research Unit, Manhattan, KS;
Department of Plant Pathology, Kansas State University, Manhattan, KS;
3School of Agricultural Biotechnology, Seoul National University, Seoul, Korea
*Corresponding Author: E-mail: i bo"\ dcn ii kL.s u d i


INTRODUCTION

Fusarium graminearum Schwabe is the main causal
agent of Fusarium head blight of wheat and barley
around the world. The name Fusarium graminearum
Schwabe is used to designate the anamorph (asexual
conidial stage) of the fungus. The name Gibberella
zeae (Schwein) Petch is used to designate the
teleomorph (sexual ascospore stage) of the fungus.
Both names are commonly used in the literature to
denote this fungus. G. zeae is homothallic, but it can
also outcross in the laboratory (Bowden and Leslie,
1999) and there are several lines of evidence that it
outcrosses in the field (Schmale et al. 2006; Zeller et
al., 2004).

O'Donnell et al. (2000) divided Fusarium
graminearum into seven phylogenetic lineages based
on the partial sequences of six nuclear genes.
Phylogenetic analyses of the sequences for each gene
gave similar patterns, a phenomenon termed
genealogical concordance. The genealogical


concordance indicates that members of the
phylogenetic lineages in F. graminearum have
exchanged little or no genetic material with each other
in recent evolutionary history. O'Donnell et al. (2000)
noted a strong global pattern in the distribution of the
lineages, which may help explain the lack of genetic
exchange. The number of lineages was later extended
to eight and a genealogical discordance involving
genes in the trichothecene cluster was reported (Ward
et al., 2002). The existence of phylogenetic lineages in
this fungus has been independently confirmed and is
generally well accepted (Jeon et al, 2003; Vargas et al,
2001).

Recently, O'Donnell et al. (2"' 14 extended the number
of lineages to nine and gave them species rank (Table
1). The diagnoses for each of the nine species were
based on uniquely fixed single nucleotide
polymorphisms (SNPs). The phylogenetic analyses of
eleven nuclear genes indicated that the nine species
together form a closely related monophyletic group or
clade. Two additional phylogenetic species of the


Table 1. Correspondence of lineage and species designations within the F. graminearum clade.
Lineage Designation Species Designation References
1 F. austroamericanum O'Donnell et al. (2000, 2004)
2 F. meridionale O'Donnell et al. (2000, 2004)
3 F. boothii O'Donnell et al. (2000, 2004)
4 F. mesoamericanum O'Donnell et al. (2000, 2004)
5 F. acaciae-mearnsii O'Donnell et al. (2000, 2004)
6 F. asiaticum O'Donnell et al. (2000, 2004))
7 F. graminearum O'Donnell et al. (2000, 2004)
8 F. cortaderiae Ward et al. (2002); O'Donnell et al. (21" 14)
9a F. brasilicum O'Donnell et al. (21 14)
F.. .. Toth et al. (2005)
F. gerlachiib Ward et al. (2005)
a proposed lineage designation
b species name informally proposed at time of this writing.







Session 2: Fusarium consortium


Fusarium graminearum clade have been proposed
informally (T6th et al., 2005; Ward et al., 2005) and a
total of 13 phylogenetic species have been suggested to
exist (Starkey et al., 2005).

With the exception of SNPs, differences between the
nine phylogenetic species or lineages appear to be few.
Ward et al. (2002) reported that trichothecene
mycotoxin chemotype (nivalenol, 3ADON, or
15ADON) did not correlate well with phylogenetic
lineage. O'Donnell et al. (21"' 4) found overlapping
ranges for conidial characters such as length, width,
and widest region of the conidium. They claimed that a
combination of conidial characters could be used to
recognize three species. Even these modest claims are
questionable due to the very small number of strains
examined.

The pathological uniqueness of these newly described
species also appears to be low. Cumagun et al (21 "14)
tested progeny from a cross between lineage 6 (F.
asiaticum O'Donnell, T. Aoki, Kistler et Geiser) and
lineage 7 (F. graminearum Schwabe sensu strict
O'Donnell et al. 2004) and found no transgressive
segregation for aggressiveness to wheat. This result
suggests that aggressiveness factors were similar in
both parents. Goswami and Kistler (2005) tested nine
phylogenetic species for pathogenicity to wheat and
found that aggressiveness and production of
trichothecene mycotoxins were strain-specific rather
than a species-specific character. T6th et al. (2005)
tested 20 wheat genotypes with differing resistance
against eight members of the F. graminearum species
complex. They found no evidence of differential
resistance reactions.

Despite the phylogenetic evidence for genetic isolation
of the lineages, there is clear evidence that hybrids
between lineages occur in the field. O'Donnell et al.
(2000) reported a naturally occurring hybrid between
lineage 2 and lineage 6 in a collection from Nepal.
Evidence for the hybrid nature of the strain was very
strong, including an intragenic recombination event in
the TRI101 gene. Leslie et al. (2005) reported a few
putative hybrids from Brazil and Uruguay between
lineages 1 and 7 as well as 2 and 7 based on AFLP
markers and sequences of four nuclear genes. One
significant problem with detecting natural hybrids is
the small number of loci for which lineage-specific
alleles have been described. In addition, many of the
characterized loci are closely linked, which reduces
genome coverage. The result is that the power to detect
interlineage hybrids is low using current methods and
the frequency of naturally occurring hybrids is still
largely unknown


The ability of different lineages of F. graminearum to
hybridize in the laboratory is well established. Bowden
and Leslie (1999) described a crossing procedure using
nitrate non-utilizing (nit) mutants to distinguish
recombinants. For additional discussion of genetic
methods in this fungus see Bowden and Leslie (21i '4).
Cross fertility was demonstrated between lineages 3, 6,
and 7 in laboratory studies (Bowden and Leslie, 1999).
Jurgenson et al. (2002) constructed an entire genetic
map based on 99 progeny from a cross between lineage
6 and lineage 7 strains. Commenting on this cross,
O'Donnell et al. (21l 4) suggested that it was not
surprising that morphologically indistinguishable sister
species can form hybrids, albeit with a reduction in
fertility.

Although hybrids among these lineages clearly occur
in the field and the laboratory, our understanding of
potential genetic exchange among lineages is
incomplete. It is unknown whether the expectation of
low fertility in pairings between lineages is correct. It
also is unknown whether cross fertility between
lineages is the exception or the rule in the F.
graminearum clade. We assessed cross fertility
between nine lineages ofF. graminearum in laboratory
crosses using two different methods.

RESULTS

In the first test, three strains ofF. graminearum lineage
7 with an insertion in the mating (MAT) locus that
renders them heterothallic (Lee et al., 2003) were used
as females and standard tester strains of each of the
nine lineages were used as males. Ascospore
production was variable, but depended on particular
combinations of strains rather than on lineage. All
males from all lineages produced viable progeny with
at least two of the lineage 7 female strains. At least one
male of each lineage had high fertility with at least one
female of lineage 7.

In the second test, strains representing nine lineages
were crossed as complementary nit mutants in a diallel
design Every lineage was able to pair with at least two
other lineages (Table 2). Surprisingly, many of the
lineages failed to pair with themselves. These negative
results are most likely due to poor female fertility of
some strains and/or the small number of strains tested.
Therefore, the results in this table probably represent
an underestimate of the amount of potential cross
fertility between lineages. Nevertheless, the results
from this experiment confirmed that lineage 7 is highly
cross fertile with all other lineages.







Session 2: Fusarium consortium


Table 2. Summary of diallel cross fertility test of lineages of F. graminearum using nitrate non-utilizing (nit)
mutants.
(fLineag 1 (1) 2 (2) 3 (3) 4 (2) 5 (2) 6 (3) 7 (5) 8 (4) 9 (2)
(# of strains)
1 (1) ++
2(2) ++
3 (3) + + ++ ++ +
4(2) + + + ++++ + +++
5(2) +++
6 (3) +++ ++ ++++
7 (5) +++ +++ ++ ++ +++ ++++ ++++ ++++ +++
8(4) ++ + + +++ ++
9(2) ++
a _" = no recombinants produced; "+" = 1-5 recombinant colonies per plate; "++" = 6-25 recombinants; "+++"
= 26-100; "++++" = >100 recombinants. The highest rating among the individual lineage pairings is shown


DISCUSSION

Phylogenetic analyses indicate a history of genetic
isolation among the lineages of F. graminearum
(O'Donnell et al., 2000, 2004). This isolation may be
relatively recent, since the phylogenetic lineages
apparently differ very little from each other.
Nevertheless, according to the genealogical
concordance phylogenetic species recognition concept,
these lineages merit species rank (O'Donnell et al.
2004).

Limited field surveys and extensive laboratory crosses
reveal that genetic exchange can occur between these
lineages. Every single lineage was able to cross with
two or more other lineages. In many cases, interlineage
pairings were highly fertile (Table 2). Lineage 7 could
serve as a universally cross-fertile lineage.
Furthermore, many progeny of an interlineage cross (6
x 7) were highly aggressive on wheat heads and were
sexually fertile (Cumagun et al, 2004; Jurgenson et al.,
2002) indicating that interlineage progeny may have
adequate fitness. According to the biological species
concept, these lineages would not merit species rank.

Remarkably, two different ways of looking at genetic
isolation resulted in opposite conclusions about species
limits in the F. graminearum clade. Opinions about
species definitions differ and there is no universally
accepted methodology for recognizing and delimiting
species (Sites and Marshall, 2004). In our opinion,
these morphologically and pathologically
indistinguishable phylogenetic lineages should be
classified as separate species only if we can reasonably
expect them to remain genetically distinct when they


co-occur in the field. Unfortunately, neither species
concept is capable of reliably predicting genetic
exchange in this case.

Phylogenetic analyses provide an objective, but
retrospective, assessment of genetic exchange or
isolation Historical genetic isolation should be
predictive of future genetic isolation for sympatric
(overlapping ranges) lineages. However, it is difficult
to determine if sympatry is an historical or recent
situation as strains from some lineages may have
moved recently in global trade. If sympatry is recent,
there may not have been sufficient time for hybrids to
proliferate. Evidence for historical genetic isolation of
allopatric (non-overlapping ranges) lineages is not
necessarily a good predictor of cross fertility when
geographically isolated lineages are reunited because
the isolation mechanism could be solely geographic.

Laboratory cross fertility studies provide an objective,
but artificial, assessment of genetic exchange or
isolation If laboratory test crosses are consistently
negative while appropriate controls are consistently
positive, then the lab tests should be predictive for
genetic isolation under field conditions. However,
consistently positive laboratory crosses are not
necessarily a good predictor cross-fertility under field
conditions since undiscovered genetic or ecological
isolating mechanisms may be inhibiting genetic
exchange or selecting against hybrids in the field.

The conflicting conclusions about species boundaries
in the F. graminearum clade may indicate that these
lineages are in the early stages of speciation, but we do
not know the mechanisms of genetic isolation that







Session 2: Fusarium consortium


allowed them to diverge. Laboratory crossing results
do not support fertility barriers as a mechanism,
especially not for lineage 7. Neither is there evidence
for ecological separation since all appear to be good
pathogens, at least on wheat (Goswami and Kistler,
2005; T6th et al., 2005). All are presumably good
saprophytes on crop residue, which is where the sexual
stage occurs. A reasonable working hypothesis is that
geographic isolation allowed the lineages to develop
independent evolutionary trajectories. Migration of
strains, probably through the global grain trade, may
have changed the situation The situation may be
complicated because evolutionary trajectories of
lineages may now vary indifferent locations.

The question of genetic exchange among lineages in
this group will probably best be resolved empirically
through intensive surveys of field populations in areas
of geographic overlap among lineages. There are
several places in the world where multiple lineages of
F. graminearum are known to co-occur (Jeon et al,
2003; O'Donnell et al., 2000, 2004; Toth et al., 2005;
Vargas et al, 2001). If significant frequencies of
hybrids are found in the field, then lineage distinctions
may be blurred and species rank may not be justified.
Accurately determining the hybrid frequency is
problematic with current methods, but this may be
solved with new population genomics tools.

Pending resolution of the conflicting conclusions
regarding species boundaries, researchers have a
choice of concepts for F. graminearum. Researchers
should not feel compelled to use one concept or the
other, as long as they are clear which one they are
using. Each choice has advantages and disadvantages.


O'Donnell et al. (21'-i"4 advocated formal recognition
of the lineages in the F.graminearum clade as species.
An advantage of the new species names is that they
promote collection of data that is specific for each
phylogenetic lineage. Currently there are no significant
morphological, sexual, pathological, ecological, or
toxicological phenotypes that are lineage-specific. The
main disadvantage is that assigning a strain to species
requires amplifying and sequencing diagnostic genes
or using primers targeting specific single nucleotide
polymorphisms (SNPs). This method of identification
may be daunting for many applied researchers. A high
throughput multilocus genotyping assay for
identification of species and chemotypes has been
described (Ward et al., 2005), which could help
ameliorate the problem A second disadvantage is that
hybrids between lineages may easily escape detection
because only a few loci can be assayed with current
technology. If hybrids are misidentified as pure
species, then information about the phylogenetic
species may be incorrect. A third disadvantage is that
using species names for phylogenetic lineages could
promote erection of quarantines that serve as non-tariff
trade barriers. At this time, there are no data that
justify erecting quarantines based on these lineages.
Use of a precautionary principle for quarantines of
cryptic species/lineages is ultimately a philosophical,
economic, and political issue.

Leslie and Summerell (2006) recommended using the
single name, Fusarium graminearum, for all of the
phylogenetic lineages/species associated with this
group. The main advantage is simplicity and ease of
identifying strains. This polytypic view of F.
graminearum is justified by the high genetic similarity


Table 3. Correspondence of names for different concepts of Fusarium graminearum.

Phylogenetic lineage view Phylogenetic species view
Narrow F. graminearum Schwabe lineage 7 F. graminearum Schwabe
sense or or
F. graminearum Schwabe lineage 7 sensu F. graminearum Schwabe sensu O'Donnell et al.
O'Donnell et al, 2000 2004
or or
F. graminearum sensu strict F. graminearum sensu strict
or
Gibberella zeae (Schwein)Petch
Broad F. graminearum Schwabe F. graminearum species complex
sense or or
F. graminearum clade F. graminearum clade
or or
F. graminearum sensu lato F. graminearum sensu lato
or
Gibberella zeae (Schwein)Petch
SDesignations that are NOT ambiguous are inbold.







Session 2: Fusarium consortium


and monophylly of the phylogenetic lineages. The
main disadvantage is potential loss of lineage-specific
information For many studies, this may not be critical.
In cases where lineage could be important, such as
resistance screening nurseries, researchers are strongly
encouraged to report the lineage designations. The
second disadvantage is that O'Donnell et al. (2'k14)
abandoned the lineage designations when naming the
new species. There is no problem correlating lineages
and species for the first nine lineages (Table 1).
However, there could be ambiguity for future
lineages/species. Lineages are informal taxa and this
can be easily solved by numbering lineages in the
order in which the corresponding species are
described.

A problem generated by the new species descriptions
is the potential ambiguity of the names Fusarium
graminearum Schwabe and Gibberella zeae
(Schwein) Petch O'Donnell et al (21'1 4) emended the
concept of Fusarium graminearum to encompass only
phylogenetic lineage 7, but changed neither the species
epithet nor authority. Unfortunately, this allows for
ambiguity of the species concept being used. Gale et
al. (2005) suggested that the name Gibberella zeae
(Schwein) Petch was implicitly emended by
O'Donnell et al. (2_,14) to represent only F.
graminearum lineage 7, leaving none of the other
species/lineages with a defined sexual stage name. To
avoid confusion, they recommended that the Fusarium
designations be used rather than Gibberella zeae. Of
course, that does not solve the problem for F.
graminearum.

Accordingly, researchers should strive to be clear
which concept of F. graminearum is being used. A
suggested correspondence of names is given in Table 3
for the phylogenetic lineage view and the phylogenetic
species view of the F. graminearum clade. T6th et al
(2005) used F. graminearum sensu strict and F.
graminearum sensu lato to represent the narrow and
broad interpretations, respectively. Although this lacks
formality, at least for the moment it is unambiguous.
When a formal name is required, the full species
epithet and authority should be used.


ACKNOWLEDGEMENTS

This is contribution number 07-26-A from the Kansas
Agricultural Experiment Station


REFERENCES

Bowden, R. L. and J. F. Leslie. 1999. Sexual
recombination in Gibberella zeae. F/l ,ili. j. .~ '
89: 182-188.
Bowden, R. L., J. F. Leslie, J. E. Jurgenson & J. Lee.
2004. "Genetic mapping in Gibberella zeae." In:
Canty, S.M., Boring, T., Wardwell, J. and Ward,
R.W (Eds.), Proceedings of the 2nd International
Symposium on Fusarium Head Blight;
incorporating the 8th European Fusarium Seminar;
2004, 11-15 December; Orlando, FL, USA.
Michigan State University, East Lansing, MI. pp.
555-556.
Cumagun, C. J. R., R. L. Bowden, J. E. Jurgenson, J.
F. Leslie, and T. Miedaner. 2004. Genetic
mapping of pathogenicity and aggressiveness of
Gibberella zeae (Fusarium graminearum) towards
wheat. hi, i. ./ i. -..-: 94: 520-526.
Gale, L. R., Bryant, J. D., Calvo, S., Giese, H., Katan,
T., O'Donnell, K., Suga, H., Taga, M., Usgaard,
T. R., Ward, T. J., and Kistler, H. C. 2005.
Chromosome complement of the fungal plant
pathogen Fusarium graminearum based on
genetic and physical mapping and cytological
observations. Genetics 171:985-1001.
Goswami, R.S., and Kistler, H. C. 2005. Pathogenicity
and In Planta mycotoxin accumulation among
member of the Fusarium graminearum species
complex on wheat and rice. Phytopathology
95:1397-1404.
Jeon, J.-J., Kin, H., Kim, H.-S., Zeller, K.A., Lee, T.,
Yun, S.-H., Bowden, R.L., Leslie, J.F., Lee, Y.-
W., 2003. Genetic diversity of Fusarium
graminearum from maize in Korea. Fungal Genet.
Newslett. 50 (Suppl.), 142 (Abstract).
Jurgenson, J. E., R. L. Bowden, K. A. Zeller, J. F.
Leslie, N. J. Alexander, and R. D. Plattner. 2002.
A genetic map of Gibberella zeae (Fusarium
graminearum). Genetics 160: 1452-1460.
Lee, J., Lee, T., Lee, Y.-W., Yun, S.-H., Turgeon,
B.G., 2003. Shifting fungal reproductive mode by
manipulation of mating type genes: obligatory
heterothallism of Gibberella zeae. Mol. Microbiol.
50, 145-152.
Leslie, J. F., A. A. Saleh & R. L. Bowden 2005.
Naturally occurring hybrids of Fusarium
graminearum. Fit, I r'o'i'. 'i. \1- 95: s58.
Leslie, J. F. & B. A. Summerell. 2006. Fusarium
Laboratory Manual. Blackwell Professional
Publishing, Ames, Iowa. 385 pp.







Session 2: Fusarium consortium


O'Donnell, K., H. C. Kistler, B. K. Tacke, and H. H.
Casper. 2000. Gene genealogies reveal global
phylogeographic structure and reproductive
isolation among lineages of Fusarium
graminearum, the fungus causing wheat scab.
Proc. Natl. Acad. Sci. (USA) 97: 7905-7910.
O'Donnell, K., T. J. Ward, D. M. Geiser, H. C. Kistler,
and T. Aoki. 2004. Genealogical concordance
between the mating type locus and seven other
nuclear genes supports formal recognition of nine
phylogenetically distinct species within the
Fusarium graminearum clade. Fungal Genet.
Biol. 41: 600-623.
Sites, J. W., Jr., and Marshall, J. C. 2004. Operational
criteria for delimiting species. Annual Review of
Ecology, Evolution, and Systematics 35:199-227.
Schmale, D.G., Leslie, J. F., Zeller, K. A., Saleh, A.
A., Shields, E. J., and Bergstrom, G.C. 2006.
Genetic structure of atmospheric populations of
Gibberellazeae. Phytopathology 96:1021-1026
Starkey, D. E., Ward, T. J., O'Donnell, K. L., Geiser,
D. M., Kuldau, G., Clear, R. M., Gale, L. R.,
Kistler, H. C., and Aoki, T. 2005. Delineation of
species boundaries within Fusarium graminearum,
the causative agent of Fusarium head blight.
Fungal Genetics Newsletter 52 (Suppl.): 71.
T6th, B., Mesterhazy, A., Kdszonyi, G., and Varga, J..
2005. "Common Resistance of Wheat to Members
of the Fusarium graminearum Species Complex
and F. culmorum". IN: Canty, S. M., Boring, T.,
Wardwell, J., Siler, L., and Ward, R. W. (Eds.).
Proceedings of the National Fusarium Head Blight
Forum; 2005 Dec. 11-13; Milwaukee, WI. East
Lansing: Michigan State University. p. 93.


Vargas, J. I., R. L. Bowden, K. A. Zeller, and J. F.
Leslie. 2001. Comparisons of North and South
American populations of Gibberella zeae.
7ith I Ifi., ,, / 1. 91: s91.
Ward, T. J., J. P. Bielawski, H. C. Kistler, E. Sullivan,
and K. O'Donnell. 2002. Ancestral
polymorphism and adaptive evolution in the
trichothecene mycotoxin gene cluster of
phytopathogenic Fusarium. Proc. Natl. Acad. Sci.
(USA) 99: 9278-9283.
Ward, T. J., Starkey, D., O'Donnell, K., Clear, R.,
Gaba, D., Patrick, S., Kistler, H. C., Gale, L.,
Elmer, W. H. 2005. "FHB Species and
Trichothecene Toxin Diversity in North America".
IN: Canty, S. M., Boring T., Wardwell, J., Siler,
L., and Ward, R. W. (Eds.). Proceedings of the
National Fusarium Head Blight Forum; 2005 Dec.
11-13; Milwaukee, WI. East Lansing: Michigan
State University. p. 170.
Ward, T. J., Starkey, D., Page, B., and O'Donnell, K.
2005. "A Multilocus Genotyping Assay for
Identification of Fusarium Head Blight Species
and Trichothecene Toxin Chemotypes". IN:
Canty, S. M., Boring, T., Wardwell, J., Siler, L.,
and Ward, R. W. (Eds.). Proceedings of the
National Fusarium Head Blight Forum; 2005 Dec.
11-13; Milwaukee, WI. East Lansing: Michigan
State University. p. 169.
Vargas, J. I., R. L. Bowden, K. A. Zeller, and J. F.
Leslie. 2001. Comparisons of North and South
American populations of Gibberella zeae.
7Th I,.. ,, ./., I.- 91:s91.
Zeller, K. A., R. L. Bowden, and J. F. Leslie. 2004.
Population differentiation and recombination in
wheat scab populations of Gibberella zeae in the
United States. Molecular Ecology 13:563-571.






Session 2: Fusarium consortium


FUSARIUM CHEMOTYPES IN THE UK AND
CHEMOTYPE-HOST INTERACTIONS

2 1
P. Nicholson P. Jennings2, M. Thomsett A Steed' and N. Gosman

'John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK
2Central Science Laboratory, Sand Hutton, York, Y04 1LZ, UK
*Corresponding Author: PH: (44) 1603 450616; E-mail: paul.nicholsonw&Zbbsrc.ac.uk



ABSTRACT

Prior to 1998, the predominant trichothecene-producing species associated with Fusarium head blight on wheat in
the UK was Fusarium culmorum. Since that time, the occurrence of F. graminearum has increased year-on-year
relative to F. culmorum and it is now the more prevalent. We have developed a series of PCR-based assays to
characterise isolates with respect to trichothecene biosynthetic genes and infer their chemotype. Two chemotypes of
F. culmorum are present in the UK. One type produces predominantly nivalenol (NIV), while the other produces
deoxynivalenol (DON) and 3-acetyl DON (3ADON). The latter is a consequence of the deletion of a region of the
principal trichothecene gene cluster encoding TRI7 and the promoter region of TRI8. Whereas a single chemotype of
F. graminearum predominates in the USA producing DON and 15-acetyl DON (15ADON), three chemotypes are
present inthe UK. These are NIV producers, DON/3ADON producers and DON/15ADON producers. It is unknown
why, despite the very recent appearance of F. graminearum in the UK, there should be more apparent chemotype
diversity here than in the USA.

We have been carrying out a series of experiments to establish whether chemotype affects interaction with host
plants or has other biological consequences. Preliminary results indicate that different chemotypes interact
differently with wheat and other cereal hosts. These results will be presented and discussed in relation to host
resistance and epidemiology.






Session 2: Fusarium consortium


RELATIVE PATHOGENICITY OF 3-ADON AND 15-ADON
ISOLATES OF FUSARIUM GRAMINEARUM FROM THE
PRAIRIE PROVINCES OF CANADA


J. Gilbert*, R.M. Clear, T. Ward, and D. Gaba

Cereal Research Centre, AAFC, 195 Dafoe Road, Winnipeg, Manitoba R3T 3E5; Grain Research Laboratory,
Canadian Grain Commission, 1404-303 Main St. Winnipeg Manitoba R3C3G8; Microbial Genomics Research
Unit, USDA/ARS/NCAUR, 1815 N. University St., Peoria, IL 61604
*Corresponding Author: PH: 204-983-0891; E-mail: jgilberti_,agr. gc.ca



ABSTRACT

Eighteen isolates of Fusarium graminearum Schwabe, 3 producing 15-ADON and 3 producing 3-ADON from each
of the Canadian provinces of Manitoba, Saskatchewan and Alberta, were tested for relative pathogenicity and
consistency of production of toxin, on two Canadian spring wheat cultivars, 'Roblin' (S) and '5602 HR' (MR). The
experimental design was a 3-replicate randomized complete block. Each replicate consisted of a pot containing 2 or
3 plants of one cultivar, and were grown in a cooled greenhouse. At anthesis, 2 to 5 heads per pot were inoculated
with one isolate and heads covered with glassine bags for 48 h to promote a favourable environment for disease
development. Disease was scored at 14 d and 21 d after inoculation and recorded as percentage infected spikelets.
These preliminary results showed no significant differences in pathogenicity among isolates from the three
provinces and producing either 3-ADON or 15-ADON. Toxin analysis by GC/MS of seeds from the inoculated
heads found higher levels of DON in the susceptible (45 ppm) vs resistant (14 ppm) wheat. All isolates formed their
respective 3-ADON or 15-ADON analogs. The 3-ADON isolates formed, on average, 16.1 ppm DON on 5602HR
and 56.9 ppmon Roblin, higher than the 15-ADON isolates which averaged 11.9 ppm and 33.6 ppm respectively.







Session 2: Fusarium consortium


COMPARISON OF INOCULUM SOURCES IN
DEVELOPMENT OF FUSARIUM HEAD BLIGHT AND
DEOXYNIVALENOL CONTENT IN WHEAT
IN A DISEASE NURSERY


A.G. Xue*, G. Butler, H.D. Voldeng, G. Fedak, and M.E. Savard.

Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada, K.W. Neatby Building 960
Carling Avenue, Ottawa, ON K1A 0C6, Canada
*Corresponding author. Telephone: 1-613-759-1513. Email: axue ag.r. c.ca


OBJECTIVES

The objectives of this study were to compare three
inoculum sources for their efficacy in FHB induction
and progression and, in addition, to determine if
disease development and DON accumulation in wheat
grains were affected by the inoculum sources and
inoculation treatments used in a disease nursery.

INTRODUCTION

Fusarium head blight (FHB), caused by Gibberella
zeae (Schwein) Petch (anamorph = Fusarium
graminearum Schwabe) is the most important disease
of wheat (Triticum aestivum L.) in North America
(Gilbert and Tekauz 2000; McMullen et al. 1997).
Infection of wheat by the pathogen results in reduced
grain yield and quality, and kernel contamination with
deoxynivalenol (DON) and other mycotoxins, which
are harmful to livestock and pose a safety concern in
human food (Miller 1995; Placinta et al. 1999).
Currently, there is no satisfactory cultural or chemical
control for FHB (McMullen et al. 1997; Shaner 2003;
Tekauz et al. 2003).

The use of genetic resistance is considered one of the
most practical and environmentally safe measures to
control FHB (Bai and Shaner 1996; Gilbert and
Tekauz 2000). High levels of resistance to FHB have
been identified in wheat germplasm (Fedak et al. 2001;
van Ginkel et al. 1996; Wilcoxson et al. 1992) and
considerable breeding has been conducted to develop
resistant cultivars and lines (Comeau et al. 2003;
Pandeya et al. 2003; van Ginkel et al. 2003).
Resistance to F. graminearum has been characterized
by a typical quantitative reaction (Mesterhazy 1995;
Schroeder and Christensen 1963) and evaluated in
FHB nurseries at multiple locations and years (Dill-


Macky 2003). Since FHB does not always appear in
consecutive years, a disease nursery has become an
essential part of most wheat breeding programs aimed
at FHB resistance across Canada and the United States
(Evans et al. 2003; Humphreys et al. 2001; Pandeya et
al. 2003). However, there has been considerable
variation among breeding programs in the use of
inoculum source, inoculation treatment, and
assessment method (Dill-Macky 2003; Evans et al.
2003). As a result, breeders have frequently
encountered inconsistent disease reactions in their
breeding lines, leading to significant loss of time and
resources (Bockus et al. 2001; Campbell and Lipps
1998). Althoughbothconidial suspension and infested
barley, corn or wheat kernels are commonly used
inoculants in FHB nurseries (Dill-Macky 2003; Evans
et al. 2003; Groth et al. 1999), there has been little
information available on the relative efficacy of these
methods.

MATERIALS AND METHODS

Three single-spore isolates ofF. graminearum, DAOM
178148, DAOM 212678, and AC Taho P2G1, obtained
from the Canadian Collection of Fungal Cultures at the
Eastern Cereal and Oilseed Research Centre (ECORC),
Ottawa, Canada, were used. These isolates were
chosen because they are known to be aggressive (Xue
et al. 2004). Conidial suspensions were made
separately for each isolate. The final spore suspension
used for field inoculation consisted of a 1/3 volume of
each of the three isolates.

Infested kernel inoculum was prepared by soaking
barley and corn kernels in water separately overnight.
Equal volumes of both were mixed into aluminum
trays (21" x 12.5" x 3.5") and autoclaved. Each tray
was then inoculated with 100 ml of a spore suspension







Session 2: Fusarium consortium


at 5 x 104 spores ml-1 under sterile conditions, labeled,
and incubated at ambient room temperature (20-25 oC)
to allow the kernels to be fully covered by mycelium.
The infested kernel inoculum was made separately for
each isolate and equal volumes of the three isolates
were used for field inoculation Inocula were produced
in April each year in preparation for field inoculations
in June.

Wheat debris consisted of FHB-infected wheat spikes
and peduncles collected from the previous years' FHB
nursery, resulting from artificial inoculation with the
infested barley and corn kernels. Debris was air-dried,
cut into 2 cmpieces, and stored at 4 C until used.

Field experiments were conducted at ECORC, Ottawa,
Ontario from 2001 to 2003. Three spring wheat
genotypes, including a FHB susceptible cultivar AC
Foremost and two moderately resistant lines CIMMYT
11 (Cross: SHA4/3/2*CHUM18//JUP/BJY; Selection
History: CMBW91Y02946M-030TOPM-1Y-010M-
010Y-015M-10Y-OM; Origin PTB96E1 #107) and
HY644 (Fedak et al. 2001), were used. Experiments
were arranged in a split plot design with inoculum
source and inoculation frequency assigned to the main
plots and wheat genotype assigned to the subplots.
Plants were grown in two-row plots, 1.5 m long with
18 cm row spacing. There was 20 cm between
subplots and 3.0 m between main plots, with four
replicates in 2001 and 2002 and three in 2003. To
prevent inter-plot interference, a four-row plot of
barley was seeded between the main plots and as a
border around the experiment. Plants were hand-
harvested at maturity, air-dried, and threshed.

Inoculum from each of the three sources was applied
two and three times in each growing season For the
treatment of two applications of conidial suspension,
inoculations were conducted at 50% anthesis (Zadoks
scale 65) and 3-4 days later. Treatment of three
applications of conidial suspension included these two
applications plus an additional application 3 days after
the second application, when plants were at the end of
their flowering stage (Zadoks scale 69). At each
inoculation, approximately 50 ml of the conidial
suspension was sprayed evenly onto the spikes in each
plot with an ultra low-volume Microfit Herbaflex
inoculator (Micron Sprayers Ltd., Bromyard, UK).

For treatments with two applications of infested barley
and corn kernels and two applications of infested
wheat debris, inoculations were conducted about three
and two weeks before anthesis (Zadoks scale 15-30).
Treatments of three applications of the respective
inocula were these two applications and a third
inoculation at one week before anthesis (Zadoks scale


47). At each inoculation, approximately 50 g of the
infested kernels or 45 g of infested debris was scattered
evenly by hand between the two rows of each plot.
Control plots were not inoculated. A non-inoculated
control treatment with two applications of
Folicur432F (tebuconazole, Bayer CropScience Inc.,
Calgary, AB) was added to the experiments in 2002
and 2003 because of the expected high disease
pressure in the FHB nursery. The fungicide was
applied at 50% anthesis and seven days after the first
application, at the recommended rate for cereals using
a polyethylene compressed air sprayer (Chapin
Manufacturing Inc., Batavia, NY). Sprinkle irrigation
was applied each year for about 0.5 h each in the
morning and afternoon (excluding rain days), starting
at the first inoculation with infested kernels and debris
and continuing until about 3 wk after anthesis, when
plants were at the soft dough stage.

The development of FHB was monitored by visually
estimating disease severity on ten randomly selected
plants from each plot using a 0-9 scale described by
Xue et al. ('21 14). The ten plants were labeled at the
heading stage when disease symptoms were absent.
Assessments were carried out six times during each
growing season at 5-7 day intervals starting at 50%
anthesis (first inoculation date of conidial suspension)
and ending at the early maturity stage (3-4 wk after the
final date of inoculation with conidial suspension).
Severity of FHB over time was summarized as area
under the disease progress curve (AUDPC) for each
plot using the formula described by Wilcoxson et al.
(1975). In addition, critical-time disease severity (DS)
and percentage of infected spikelets (IS) of the 10
selected plants were rated at the soft dough stage,
which occurred 21-25 days after the final date of
inoculation with conidial suspension depending on
genotype maturity. At the soft dough stage, plot
disease severity for a population of approximately 200
spikes per plot was estimated for both incidence
(percentage of infected spikes) and severity
(percentage of infected spikelets of the diseased
spikes). An FHB index (incidence x severity)/100 was
derived to give an assessment of plot severity (Groth et
al. 1999).

Percentage of FDK (kernels with a chalky white
appearance) was determined for 300 randomly selected
seeds per plot with a magnifying lamp. Analyses for
DON were carried out at the Mycotoxin Research
Laboratory, ECORC. Each DON analysis was
conducted using a 30-g seed sample from each plot.
The concentration of DON was determined by the
competitive direct enzyme-linked immunosorbent
assay (CD-ELISA) procedure using monoclonal
antibodies (MABs) as described by Sinha et al. (1995).







Session 2: Fusarium consortium


RESULTS AND DISCUSSION

Symptoms of FHB were observed on all wheat
genotypes each year (Figure 1). The level of FHB in
the nursery was considered adequate as demonstrated
by the fast disease progression and severe infection of
the susceptible cultivar 'AC Foremost' in all three
years. Disease development was slower and final
disease severity was notably lower for wheat
genotypes CIMMYT 11 and HY644 than cultivar 'AC
Foremost'. High levels of natural inoculum were
present as indicated by the development of FHB in the
uninoculated and fungicide treated controls. It was not
known whether the infection of uninoculated controls
was from the ground applied inocula or residues of
adjacent fields. Although FHB was observed in the
fungicide protected control treatment in 2002 and
2003, the disease symptoms appeared about 7-10 days
later, had slower disease progression and lower final
disease severity than other treatments inboth years.

Analysis of variance indicated highly significant
differences among the inoculation treatments and
among the genotypes (P < 0.01) for all parameters in
all three years except for FDK in 2003 where the
differences among inoculation treatments were not
significant (Data not shown). Inoculation treatment x
genotype interaction was not significant for any


parameters in 2003, but a significant interaction was
observed for AUDPC and DS in 2001, for FHB index
in 2002, and for percentage of IS in both years. The
combined analyses over years indicated heterogeneity
of variances for all variables, thus a model with
heterogeneous residual errors (a different residual error
for each year) was used for significance testing and
drawing inferences over years. Overall, there were
significant differences among inoculation treatments,
genotypes, and genotype x year interaction (Table 1).
The interaction of inoculation treatment x genotype
was significant only for percentage of IS and that of
inoculation treatment x year for DON only. There
were no significant three-way interactions. This
implies that the results of the experiments in all three
years were consistent with respect to the inoculation
treatments for all but one of the parameters. The lack
of a significant interaction effect of inoculation
treatment x genotype for most of the parameters
indicates that the genotypes responded similarly to the
different inoculation treatments. We conclude that the
experimental protocol and disease assessment
parameters provided conditions allowing the effects of
the treatments to be observed and rated consistently
and satisfactorily.


Table 1. Mean squares from three years of test of fixed effects of inoculation treatment, genotype, and inoculation
x genotype interaction and selected variance components for area under the disease progress curve (AUDPC),
critical-time disease severity (DS), Fusarium head blight (FHB) index, percentage of infected spikes (IS), Fusarium
damaged kernels (FDK), and deoxynivalenol (DON) content in spring wheat.

Source of variance df AUDPC DS FHB index IS FDK DON

Fixed effect F valuesa
Inoculations (I) 6 6.7** 4.0** 5.9** 5.9** 10.1** 3.1*
Genotype (G) 2 16.9* 9.9* 44.2** 59.8** 36.1** 20.4**
IxG 12 1.1 0.8 1.1 3.4** 0.6 1.1

Variance component X2 values
GxY 1 87.6** 88.7** 37.5** 19.3** 11.0"* 431.8**
IxY 1 1.1 1.3 2.1 1.6 1.3 10.7**
GxIxY 1 0.0 2.1 0.0 0.0 0.4 0.1
Note: FHB index, IS and FDK were arcsine square root transformed and DON was log transformed prior to analysis
to stabilize variances. aTests for fixed effects were Type III F tests in a mixed model with random effects of year,
replicate, genotype by year, for log DON only, and inoculation treatment by year. Residual variances were
heterogeneous over years and modeled as such bVariance components were tested with likelihood ratio tests and
where nonsignificant were not included in the final model. *,**significant atP < 0.05 and P < 0.01, respectively.







Session 2: Fusarium consortium


'AC Foremost' CIMMYT 11 HY644
9
8
7
6-
5-
4
3
2



















Julian date 2002
0


Julian date 2001


7 -
6
5 -
4 4-
(D 3
Co 2
I 1 -



Julian date 2002
9
8
7-
6-
5
4
3-
2

0-


Julian date 2003

Conidial suspension -4- 2-applications 3-applications
Infested kernels -m- 2-applications -F- 3-applications
Infested debris -y- 2-applications -v-- 3-appli nations
Non-inoculation -- natural -o- Folicur 432F



Figure 1. Fusarium head blight disease progress curves for seven inoculation treatments in 2001 and eight
treatments in 2002 and 2003 on three wheat genotypes in a disease nursery in Ottawa, Ontario. Each point is the
mean of four replications in 2001 and 2002 and three replications in 2003.







Session 2: Fusarium consortium


All inoculated treatments had significantly greater
AUDPC and DS in 2002, FHB index and percentage of
IS in 2001 and 2002, and FDK in all three years than
the non-inoculated control (Table 2). Overall,
inoculation treatments were highly significantly (P <
0.01) greater than the non-inoculated control in all
parameters except DON in the combined analyses.
The non-inoculated treatment with two applications of
Folicur432F was significantly lower in all parameters
than the mean of the inoculated treatments in 2002 and
2003, when the fungicide treatment was used. There
were significant differences among the inoculation
treatments for all parameters. However, the contrast
comparing the two numbers of applications (two
applications versus three applications) was not
significantly different for any parameter in any year or
in the combined analysis (P > 0.15). This result
indicates that two inoculations produced as much FHB
symptoms (AUDPC of 18.3 versus 18.4 over all
genotypes and years; DS of 5.6 versus 5.6; FHB index
of 48.7 versus 48.0; and, percentage of IS of 32.4
versus 32.0), FDK (36.8 versus 34.5), and DON (25.9
versus 24.8) as three inoculations, regardless of
inoculum source. The means shown in Table 2 have
been averaged over the two numbers of applications
for simplicity and clarity of presentation Infections
following inoculation with conidial suspensions or
infested kernels were significantly greater than
infection following application of the infested debris
for all parameters except FDK, where there was no
difference between infested kernels and infested
debris. Inoculation with conidial suspensions also
produced higher percentages of IS than the infested
kernels. These results indicated that infested debris,
although a natural source of inoculum, is less effective
than the use of conidial suspension or infested kernels
for inoculation in FHB nurseries.

AC Foremost was the most susceptible of the three
wheat genotypes to FHB as measured by the
parameters in all three years (Table 2). All assessment
parameters identified that CIMMYT 11 and HY644
were less susceptible than AC Foremost by any
inoculation treatment. Genotype CIMMYT 11 had
significantly lower FDK than HY644, but there was no
significant difference between the two genotypes for
all other parameters. The genotypical reactions were
in agreement with previous field observations that
CIMMYT 11 and HY644 were moderately resistant
and AC Foremost was susceptible (Fedak et al. 2001).

Although the present study demonstrates that both
conidial suspension and infested kernels are equally
effective inoculants in detecting genotypical
differences in reaction to FHB in a disease nursery,
there are considerable differences in efficacy of the


inoculum production and application between the two
inoculation methods. The approach of spraying
conidial suspension onto flowering spikes requires that
plots must be carefully scouted for anthesis prior to
inoculation and inoculated individually. Since wheat
genotypes vary considerably in anthesis date, and this
critical time lasts for only 3-5 days, inoculation must
be carried out daily for as long as 3 weeks, to screen
the 10,000 to 20,000 lines in a typical wheat breeding
program for FHB resistance (Campbell and Lipps
1998; Evans et al. 2003). The procedure is time-
consuming and labor-intensive. Results sometimes
vary considerably because of different environmental
conditions at the time of inoculation, uneven flowering
as a result of late tillers or late emergence, types of
plant resistance, and a possible variation in the
pathogen spore viability (Dill-Macky 2003). In
contrast, the use of infested barley and corn kernels
approaches the natural situation Under moist
conditions in a nursery, the infested kernels produce
ascospores about 7-10 d after application, releasing
spores over a 2-3 wk period (Paulitz 1996). Infested
barley and corn kernels are simple and inexpensive to
produce and store, require less time for application in
the nursery, and can be applied to all genotypes in a
single application regardless of anthesis date.
Therefore, the infested barley and corn kernel method
is less labor intensive, more economical, and suitable
for screening large numbers of genotypes in a FHB
nursery.

ACKNOWLEDGEMENTS

We thank Y. Chen, F. Sabo, P. Matthew, R. Stanley,
and T. Potter for technical assistance.

REFERENCES

Bai, G.-H., and Shaner, G.E. 1996. Variation in
Fusarium graminearum and cultivar resistance to
wheat scab. Plant Dis. 80: 975-979.
Bockus, W.W., Davis, M.A., and Bowden, R.L. 2001.
Rankings of wheat cultivars after using different
times and methods to rate Fusarium head blight.
In Proceedings of the 2001 National Fusarium
Head Blight Forum 8-10 Dec. 2001, Erlanger,
KY. p. 227.
Campbell, K.A.G., and Lipps, P.E. 1998. Allocation
of resources: Sources of variation in Fusarium
head blight screening nurseries. Phytopathology,
88: 1078-1086.
Comeau, A., Dion, Y., Rioux, S., Butler, G., Langevin,
F., Martin, R.A., Nass, H., Fedak, G., Xue, A.G.,
Voldeng, H., Gilbert, J., and Dubuc, J.P. 2003.
Progress in developing cultivars and germplasm
with FHB resistance in eastern Canada. In







Session 2: Fusarium consortium


Proceedings of the 3rd Canadian Workshop on
Fusarium Head Blight. 9-12 Dec. 2003,
Winnipeg, MB. pp. 140-141.
Dill-Macky, R. 2003. Inoculation methods and
evaluation of Fusarium head blight resistance in
wheat. In Fusarium Head Blight of Wheat and
Barley. Edited by K.J. Leonard and W.R.
Bushnell. American Phytopathological Society
Press, St. Paul, MN. pp. 184-210.
Evans, C.K., Garvin, D.F., and Dill-Macky, R. 2003.
Comparative evaluation of the uniform regional
scab nursery for spring wheat parents under
dryland and mist-irrigated conditions. In
Proceedings of the 2003 National Fusarium Head
Blight Forum. 13-15 Dec. 2003, Bloomington,
Minn pp. 245-249.
Fedak, G., Gilbert, J., Comeau, A., Voldeng, H.D.,
Savard, M., and Butler, G. 2001. Sources of
Fusarium head blight resistance in spring wheat.
In Proceedings of the 2nd Canadian Workshop on
Fusarium Head Blight. 3-5 Nov. 2001, Ottawa,
ON. pp. 30-35.
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.
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.
Humphreys, G., Brown, D., Clarke, J., Depauw, R.,
Fox, S., Nass, H., Shugar, L., and Voldeng, H.D.
2001. Progress towards Fusarium head blight
resistant spring wheat in Canada. In Proceedings
of the 2nd Canadian Workshop on Fusarium Head
Blight. 3-5 Nov. 2001, Ottawa, ON. pp. 5-8.
McMullen, M.P., Jones, R., and Gallenberg, D. 1997.
Scab of wheat and barley: a re-emerging disease
ofdevastating impact. Plant Dis. 81: 1340-1348.
Mesterhazy, A. 1995. Types and components of
resistance to Fusarium head blight of wheat. Plant
Breed. 114: 377-386.
Miller, J.D. 1995. Fungi and mycotoxins in grain
implications for stored product research J. Stored
Prod. Res. 31: 1-16.
Pandeya, R., Graf, R., Etienne, M., Matthew, P.,
Kalikililo, A., and McLean, M. 2003. Progress in
national winter wheat Fuarium research and
development. In Proceedings of the 3rd Canadian
Workshop on Fusarium Head Blight. 9-12 Dec.
2003, Winnipeg, MB. pp. 59-71.
Paulitz, T.C. 1996. Diurnal release of ascospores by
Gibberella zeae in inoculated wheat plots. Plant
Dis. 80: 674-678.


Placinta, C.M., D'Mello, J.P.F., and Macdonald,
A.M.C. 1999. A review of worldwide
contamination of cereal grains and animal feed
with Fusarium mycotoxins. Anim. Feed Sci.
Technol. 78: 21-37.
Schroeder, H.W., and Christensen, J.J. 1963. Factors
affecting ressitance of wheat to scab caused by
Gibberellazeae. Phytopathology 53: 831-838.
Shaner, G.E. 2003. Epidemiology of Fusarium head
blight of small grain cereals in North America. In
Fusarium Head Blight of Wheat and Barley.
Edited by K.J. Leonard and W.R. Bushnell.
American Phytopathological Society Press, St.
Paul, MN. pp. 84-119.
Sinha, R.C., Savard, M.E., and Lau, R. 1995.
Production of monoclonal antibodies for the
specific detection of deoxynivalenol and 15-
acetlydeoxynivalenol by ELISA. J. Agric. Food
Chem. 43: 1740-1744.
Tekauz, A., Hellegards, B., and Savard, M.E. 2003.
Fungicide efficacy for control of FHB in large-
scale wheat plots. In Proceedings of the 3rd
Canadian Workshop on Fusarium Head Blight. 9-
12 Dec. 2003, Winnipeg, MB. p. 156.
Van Ginkel, M., Gilchrist, L., Capettini, F., Kazi, M.,
Pfeiffer, W., William, M., Ban, T., and Lillemo,
M. 2003. International approach to breeding for
Fusarium head blight resistance. In Proceedings
of the 3rd Canadian Workshop on Fusarium Head
Blight. 9-12 Dec. 2003, Winnipeg, MB. p. 122.
Van Ginkel, M., Van der Schaar, W., Zhuping, Y., and
Rajaram S. 1996. Inheritance of resistance to
scab in two cultivars of wheat from Brazil and
China. Plant Dis. 80: 863-867.
Wilcoxson, R.D., Skovmand, B., and Atif, A.H. 1975.
Evaluation of wheat cultivars for ability to retard
development of stem rust. Ann Appl. Biol. 80:
275-281.
Wilcoxson, R.D., Busch, R.H., and Ozmon, E.A.
1992. Fusarium head blight resistance in spring
wheat cultivars. Plant Dis. 76: 658-661.
Xue, A.G., Armstrong, K.C., Voldeng, H.D., Fedak,
G., and Babcock, C. 2004. Comparative
aggressiveness of isolates of Fusarium species
causing head blight on wheat in Canada. Can J.
Plant Pathol. 26: 81-88.










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Session 2: Fusarium consortium


DEVELOPMENT OF NEW TOOLS TO DISSECT FUNGAL
VIRULENCE AND PLANT RESISTANCE COMPONENTS IN
A PROJECT FUNDED BY THE AUSTRIAN GENOME
PROGRAMME GEN-AU


U. Gueldener R. Mitterbauer2, M. Peruci2, H. Hellmer2, G. Wiesenberger2,
A. Czifersky2, K. Brunner2, U. Werner2, M.-T. Hauser2, F. Berhiller3,
R. Schuhmacher3, R. Krska3, M. Lemmens4, H. Buerstmayr4, and G. Adam2*

'Munich Information Center for Protein Sequences (GSF Forschungszentrum fiir Umwelt und Gesundheit, GmbH)
Neuherberg, Germany. 2Institute of Applied Genetics and Cell Biology, Department of Applied Plant Sciences and
Plant Biotechnology, BOKU Vienna, Austria. 3Christian Doppler Laboratory for Mycotoxin Research, Center for
Analytical Chemistry, Department for Agrobiotechnology, IFA-Tulln, BOKU Vienna, Austria. Institute of Plant
Production Biotechnology, Department for Agrobiotechnology, IFA-Tulln, BOKU Vienna, Austria.
*Corresponding Author: PH: 0043-136006-6380; Email: gerhard.adam@boku.ac.at


OBJECTIVES

The progress made in a three-year, interdisciplinary,
nationally-funded project will be reviewed. We suggest
that the most important task for the future is to set up
internationally-coordinated collaborative efforts to
maintain central repositories for Fusarium genomics
data and for mutant strains created invarious genomics
projects, and to establish improved links between the
fungal research community, phytopathologists and
plant breeders.

INTRODUCTION

In recent years enormous progress has been made in
Fusarium genomics. The most significant step was the
generation of a high quality genome sequence of F.
graminearum, funded by the National Research
Initiative of USDA (USDA/NSF Microbial Genome
Sequencing Project. Principal investigators: Corby
Kistler, USDA, ARS Cereal Disease Lab, University
of Minnesota; Dr. Jin-Rong Xu at Purdue University;
Dr. Frances Trail at Michigan State University; Dr.
Bruce Birrene, Whitehead Institute, Boston). The
genome sequence and an automated annotation is now
publicly accessible at the Broad Institute web site
together with sequences of several other fungal species
(Fungal Genome Initiative, see:
http://www.broad.mit.edu/annotation/fgi/, and for the
Fusarium graminearum database see:
http://www.broad.mit.edu/annotation/fungi/fusarium/).


With the help of the genome sequence it should be
possible to identify fungal genes required for full
virulence, to determine the virulence mechanisms, and
to dissect plant resistance components which are at
least partially able to antagonize such fungal virulence
factors and contribute to the complex trait Fusarium
resistance in crop plants. The long term goal is to use
the tools of genomics to dissect plant resistance into
measurable resistance components, improving the
mapping and utilization of such genes quantitatively
increasing resistance of crop plants. This task most
likely requires the interdisciplinary collaboration of
plant breeders, chemists and fungal researchers.

RESULTS AND DISCUSSION

Within the Austrian genome program GEN-AU a
project (coordinated by G. Adam) was funded aiming
to improve the understanding of Fusarium virulence
and plant resistance mechanisms. The first goal was to
establish a user friendly bioinformatics resource to
allow straightforward utilization of the genome
sequence by the international research community.
Consequently MIPS (Munich Information Center for
Protein Sequences) was included as subcontractor to
establish the Fusarium Genome Database (FGDB:
http://mips. sf.de/genre/proi/fusarium/).

As the current computer algorithms used to predict
fungal genes from DNA sequences are still imperfect,
independent automated annotations by Broad and
MIPS using different software frequently led to







Session 2: Fusarium consortium


alternative gene models. Meanwhile a significant
portion of the F. graminearum genome is also
manually annotated. More detailed information on
features of the FGDB is available in Giildener et al.,
2006a. The MIPS gene predictions were also very
important for the design of gene specific oligos for the
now commercially available Affymetrix gene chip for
F. graminearum (Giildener et al., 2006b).


The GEN-AU/MIPS database was also very helpful in
identifying candidate target genes with a putative role
in virulence. We have, for instance, identified
phosphopantetheinyltransferase (PPT1) as a new
virulence factor (Peruci et al., unpublished). Many
fungal secondary metabolites are produced by
polyketide synthases (PKSs) or non-ribosomal peptide
synthases (NRPSs), both of which require
posttranslational attachment of the prosthetic group 4'-
phosphopantetheine to the respective acyl- or peptidyl-
carrier domains. Disruption of the PPT1 gene, which is
equivalent to a loss of all PKSs and NRPSs leads as
expected to a dramatically reduced virulence on wheat,
indicating that potentially new or overlooked Fusarium
metabolites could play an important role in disease
development. The challenge for future work is to
dissect the possibly redundant role of the multiple PKS
and NRPS genes of Fusarium. To this end we
currently develop new tools for Fusarium functional
genomics. The Cre/lox system should allow removal of
the transformation marker and its subsequent reuse.
The Cre recombinase gene was cloned behind the
Trichoderma xylanase promoter and placed together
with a hygromycin B resistance marker between
repeated loxP-sites. This cassette should be stable on
glucose medium, but allow self-excision on xylose
medium.
Another problem is to detect quantitative effects of
gene disruptions on virulence. We are developing
competitive virulence assays, which should reduce the
problem of large variation due to failed inoculations
into wheat heads ("disease escape"). In brief, two
unique 20 bp tags (uptag and downtag sequences) are
introduced during the gene disruption The distribution
of tags is coordinated by MIPS (FGDB). The tag
sequences can be amplified from infected plant tissue
by unique primers, labeled and hybridized to
immobilized tag sequences. Shifts in the relative
amount of tags reamplified by unique primers should
reveal differences in virulence between mutants
present in the mixed inoculum. We are currently
performing reconstitution experiments with tri5 and
other mutants ofF. graminearum.


As biosynthesis of deoxynivalenol (DON) was
previously the best known virulence factor we started
to characterize mechanisms in plants relevant for DON
resistance. By heterologous expression in yeast we
identified a UDP-glucosyltransferase from the model
plant Arabidopsis thaliana (Poppenberger et al., 2003).
The chemists in our team developed reference
materials and analytical techniques based on LC-
MS/MS (Berthiller et al, 2003) and for the first time
identified DON-30-glucoside also in naturally
Fusarium infected wheat (Berthiller et al., 2005).
Meanwhile it was possible to show that DON
resistance co-localizes with the main previously
identified spreading resistance QTL derived from
Sumai-3 (Lemmens et al., 2005). Much higher ability
to form the glucoside-conj ugate of DON was shown to
be present in plants containing the 3BS Fusarium
resistance QTL.

Other lines of research in the GEN-AU project deal
with the possible role of the estrogenic Fusarium
metabolite zearalenone. We have evidence supporting
the hypothesis that zearalenone (ZON) may also be
relevant for virulence on plants by interfering with
ethylene signal transduction (Werner, 2005). On the
other hand, ZON is rapidly metabolized into ZON-40-
glucoside in most plants (Berthiller et al., 2006) and is
therefore most likely a nearly defeated virulence
factor. We also succeeded in cloning a ZON-
glucosyltransferase fromArabidopsis (Poppenberger et
al., 2006).

In summary, the Fusarium graminearum genome
sequence and Fusarium genomics tools, together with
the advances in metabolomics should lead to the
identification of new relevant virulence genes and
mechanisms. A challenge for the future will be to
maintain the MIPS FGDB and support a curator for
community annotation efforts, and to incorporate the
growing set of data from microarray experiments after
the end of the GEN-AU project. Another challenge for
the future is to coordinate the production of Fusarium
knock-out strains and to preserve the generated strains
for easy access by other groups.

Our Austrian project has showed that by bringing
together researchers with complementary expertise
(fungal genetics, chemistry, phytopathology and plant
breeding) significant progress can be made in a short
time. The main challenge for the future will be to
establish ways to fund internationally coordinated
efforts to solve the Fusarium problem







Session 2: Fusarium consortium


REFERENCES

Berthiller, F, Schuhmacher, R, Buttinger, G,
Freudenschuss, M, Adam, G, Krska, R (2003)
Synthesis of deoxynivalenol-glucosides and their
characterization using QTrap LC-MS/MS.
MycotoxinRes. 19: 47-50
Berthiller, F, Dall'Asta, C, Schuhmacher, R, Lemmens,
M, Adam, G, Krska, R (2005) Masked
mycotoxins: determination of a deoxynivalenol
glucoside in artificially and naturally
contaminated wheat by liquid chromatography-
tandem mass spectography. J. Agric. Food Chem.
53: 3421-3425
Berthiller, F, Werner, U, Sulyok, M, Krska, R, Hauser,
MT, Schuhmacher, R (2006) Liquid
chromatography coupled to tandem mass
spectrometry (LC-MS/MS) determination of phase
II metabolites of the mycotoxin zearalenone in the
model plant Arabidopsis thaliana. Food Addit.
Contain. 23: 1194-200.
Giildener, U, Mannhaupt, G, Munsterkotter, M, Haase,
D, Oesterheld, M, Stumpflen, V, Mewes, HW,
Adam, G (2006) FGDB: a comprehensive fungal
genome resource on the plant pathogen Fusarium
graminearum. Nucl. Acids Res. 34: D456-D458
Giildener, U, Seong K-Y, Boddu, J, Cho, S, Trail, F,
Xu, J-R, Adam, G, Mewes, HW, Muehlbauer, GJ,
Kistler, HC (2006) Development of a Fusarium
graminearum Affymetrix GeneChip for profiling
fungal gene expression in vitro and in plant.
Fungal Genetics and Biology, in press.
Lemmens, M, Scholz, U, Berthiller, F, Dall'Asta, C,
Koutnik, A, Schuhmacher, R, Adam, G,
Buerstmayr, H, Mesterhazy, A, Krska, R, and
Ruckenbauer, P (2005). The ability to detoxify the
mycotoxin deoxynivalenol colocalizes with a
major quantitative trait locus for Fusarium head
blight resistance in wheat. Molec. Plant Microbe
Interact. 18: 1318-1324
Poppenberger, B, Berthiller, F, Lucyshyn D, Sieberer,
T, Schuhmacher, R, Krska, R, Kuchler, K, Gldssl,
J, Luschnig, C, Adam, G (2003): Detoxification of
the Fusarium mycotoxin deoxynivalenol by a
UDP-glucosyltransferase from Arabidopsis
thaliana. J. Biol. Chem 278: 47905-14


Poppenberger, B, Berthiller, F, Bachmann, H,
Lucyshyn, D, Peterbauer, C, Mitterbauer, R,
Schuhmacher, R, Krska, R, Gldssl, J, Adam, G
(2006) Heterologous expression of Arabidopsis
UDP-glucosyltransferases in Saccharomyces
cerevisiae for production of zearalenone-4-O-
glucoside. Appl. Environ Microbiol. 72: 4404-
4410
Werner, U. (2005) Characterization of the effect of the
Fusarium toxin zearalenone in Arabidopsis
thaliana. PhD. thesis, BOKU University of
Natural Resources and Applied Life Sciences,
Vienna






Session 2: Fusarium consortium


IMPLICATIONS OF POPULATION VARIABILITY ON THE
MANAGEMENT OF FUSARIUM HEAD BLIGHT


R. Dill-Macky

Department of Plant Pathology, University of Minnesota, 495 Borlaug Hall, 1991 Upper Buford Circle, St. Paul,
MN, 55108
*Corresponding Author: PH: (612) 625-2227; E-mail: ruthdmuiumnedu



ABSTRACT

The principal pathogens associated with Fusarium head blight (FHB or scab) of wheat and barley are Fusarium
graminearum (Schw.) (teleomorph Gibberella zeae (Schw. & Petch) and F. culmorum (W.G. Smith) Sacc.. Other
species including F. avenaceum (Corda: Fr.) Sacc., F. crookwellense Burgess, Nelson & Toussoun, F. equisiti
(Corda) Sacc., F. poae (Peck) Wollenw., F. sporotrichioides Sherb. and Michrodochium nivale (Fr.) Samuels & I.C.
Hallett (teleomorphMonographella nivalis (Schaffnit) E. Miller) may also be associated with FHB. The frequency
of the association of these species with FHB varies among geographical locations over time. The taxonomy of the
genus Fusarium was originally based on morphological traits and has been revised following studies of anamorph-
teleomorphs connections and DNA sequences. These studies have demonstrated the immense diversity within the
taxonomic groupings within the genus Fusarium, and indeed within F. graminearum. Historically scientists
interested in taxonomy have constructed, deconstructed and reconstructed taxonomic groupings. In recent years
there has been much debate over the taxonomic classification of the genus Fusarium. For researchers focused on the
management of FHB the genetic diversity of the pathogens which incite FHB is important only as it effects the
biology of the pathogen and thus impacts the efficacy of disease control practices.

Host genetic resistance is considered the most effective and economic means to control plant diseases. Although
resistance to FHB is obviously highly desirable, immunity to Fusarium has not been identified in wheat or barley.
The development of wheat and barley with improved resistance to FHB currently relies on the introgression of
multiple genes conferring partial resistance. To aid the genetic resistance already deployed and control FHB prior to
the deployment of resistance, fungicides are being used in the United States. It has been demonstrated that fungicide
applications can reduce the severity of epidemics although again disease control is only partial. Cultural control
options appear limited given the current constrains, both agronomic and economic, on crop selection, crop rotation
sequence and tillage practices. It would appear that rather than elimination of this disease that we will likely
manage FHB through the deployment of the best host resistance available, the judicious use of fungicides and the
implementation of available cultural control practices. The use of these disease management options are likely to be
facilitated by forecasting systems being developed for use in the United States.

The use of an integrated approach to FHB management will reduce the chance that any single control option will
influence the genetic structure of Fusarium populations and should be cognizant that other forces will influence the
population structure of Fusarium spp. which spend much of their life as saprophytes on the residues of both
gramineous and non-gramineous plants.






Session 3: International scab nursery consortium


FUSARIUM HEAD BLIGHT IN ARGENTINA: A LOCAL
COMPANY APPROACH TO BREEDING FOR SCAB
TOLERANCE


L.J. Gonzalez, F.M. Ayala, and H.T. Buck*

Buck Semillas S.A., Av. 41 y 28, 7637 La Dulce, Argentina
*Corresponding author: Phone: 54-2262-434061; E-mail: hbuckZAbucksemillas.comar



ABSTRACT

FHB has become a disease of increasing importance in most wheat-producing countries. Annual wheat production in
Argentina may vary between 13 and 16 MMT and nearly 60% is exported. Wheat production is marketed in three
commercial grades. The commercial standard allows up to 3% damaged kernels (which include damage by FHB),
but the Alimentary Codex establishes no regulations on mycotoxin levels in cereal or cereal products. Thus, local
end users such as millers and bakers purchase according to their requirements, usually having to implement prior
testing for DON in years of high scab incidence. Foreign customers may expect variations in grain quality and toxin
levels of shipments according to year and shipment port. Due to lack of segregation into different end use quality
classes, Argentine wheat competes in the international market mostly by price, its buyers often being low resource
countries.

Fusarium resistant wheat should not only prevent farm production losses but also contribute to protect consumers'
health, especially in less developed countries with no regulations on toxin content.

Buck Semillas S.A. is a local private bread and durum wheat breeding company; data on work aiming to improve
Fusarium tolerance will be presented. Crosses between elite lines and scab tolerant materials derived from Chinese
and local germplasm are made regularly. Field evaluations of segregating material and stabilized lines are carried
out in several locations. Natural field infections show wide yearly variations according to weather conditions,
location and tillage system. Promising advanced lines are artificially infected using Fusarium conidia under partially
controlled conditions. Visual notes are taken on artificially infected spikes and also on the development ofFusarium
on their kernels in humid chamber. Results obtained in the last two years-some of them not easy to interpret-will
be discussed and materials showing stable tolerant behavior will be mentioned.

In recent years a joint project between the Instituto de Recursos Biol6gicos at INTA Castelar and three private sector
breeding companies has been implemented, aiming to develop enhanced germplasm for scab resistance using
molecular marker assisted backcross selection

Buck Semillas strongly supports the development of an International Interactive Scab Resistance Screening Nursery
within the framework of the Global Fusarium Initiative and would be willing to collaborate on field screening and/or
germplasm exchange. Given the importance of local private breeders-whose varieties cover around 90% of
Argentina's wheat planted area-their inclusion in this project should eventually be taken into consideration







Session 3: International scab nursery consortium


CURRENT STATUS OF FHB RESEARCH IN ROMANIAN
BREAD WHEAT BREEDING PROGRAM


M. Ittu*, N.N. Saulescu, G. Ittu, and M. Ciuca

Agricultural Research-Development Institute (ARDI) Fundulea, 1 N. Titulescu street, Romania
*Corresponding Author: Phone: (4021) 3150805; E-mail address: ittumiricic.ro / giiuiu p IaIc to


OBJECTIVES

The objectives of this study were to review the latest
results obtained in nationally funded projects on
improvement of resistance to Fusarium head blight
(FHB) in winter bread wheat. Part of our breeding
efforts are devoted to cooperation in international FHB
wheat nurseries.

INTRODUCTION

Winter bread wheat and maize are the main food crops
in Romania. An increasing interest is currently shown
in durum wheat and in some extent in triticale. Under
favourable conditions for epidemical development of
Fusarium diseasess, all these crops may be damaged
in both conventional and organic farming systems by
yield reduction and contamination with mycotoxins
(Ittu, 2001a, Ittuetal., 2004).

The spread of intensive wheat cultivars, not necessarily
very resistant, in parallel with an expansion of
wheat/corn rotation, has emphasized in the past
decades the need for a multidisciplinary approach to
Fusarium research, focused on successful management
of risks.

In wheat, breeding cultivars that combine higher
resistance to FHB (mainly Type II) and low content of
DON, with other desirable agronomic traits, is
considered, the most reliable strategy of control, in
terms of efficiency, costs and protection of
environment (Bai and Shaner, 1996). This is also the
main breeding approach for the development of
resistance to FHB in Romanian winter bread wheat.
We have developed a multi-environment field
procedure (year/location) of phenotypic assessment,
under artificial inoculation (point/single head method)
(Ittu et al, 1992, Ittu et al, 2001c).

As a result of continuous breeding efforts to improve
resistance to FHB in bread winter wheat at ARDI


Fundulea and others of its regional breeding centres,
some valuable genotypes with a high and stable level
of type II resistance and lower DON content have been
obtained. One of these achievements is Fundulea
201R, not related to Chinese sources of FHB
resistance, previously reported (Ittu et al., 1998,
2001b). Four quantitative trait loci (QTLs) for
resistance to FHB were found on chromosomes 1B,
3A, 3D and 5 A (Shen, Ittu & Ohm, 2003). F 201R is
characterized also by a multiple resistance to the main
foliar diseases (powdery mildew, leaf and stripe rusts
and septoriosis) and good winterhardiness.

CURRENT GOALS

Continuous improvement of screening techniques,
selection of genotypes that combine a higher resistance
to FHB than that of Fundulea 201R, with resistance to
other pathogens and better agronomic traits are our
current research goals.

At least two prerequisite selected Fusarium isolates
and combined pre and post-harvest criteria are used per
genotype/year combination for inoculation and
assessment, respectively. For a better interpretation of
experimental field data, we use classification of the
entries according to their inoculation day. Between
groups inoculated on different days the influence of
climatic factors could also be very informative.

Recently, two three-year, multidisciplinary, nationally-
funded projects have been initiated: (1) MAS for FHB
resistance and DON contamination in winter wheat
(BIOTECH-4545/2004) and (2) Neutralization of
harmful effect of Fusarium mycotoxins in the entire
food chain (CEEX-25/2005).

SELECTION FOR RESISTANCE

Trials performed in recent years demonstrated that a
higher level of resistance to FHB than that identified in
Fundulea 201R could be achieved, concomitantly with










a better combination of desirable traits: bread making
quality, preharvest sprouting resistance, Al tolerance,
BYDV resistance etc. The newest Romanian advanced
lines are derivatives of bread wheat/triticale crosses (F
00628G34-1) and of bread wheats with complementary
levels of resistance to FHB.

The use of microsatellite markers associated with
resistance to FHB has been used particularly to
validate the resistance derived from crosses among
Romanian and Asian sources of resistance.

These results represent a good premise for further
progress on improvement of both reliable methods of
assessment and the level of resistance to FHB in
Romanian winter bread wheat durum wheat and
triticale.

INTERNATIONAL COOPERATION

Firmly convinced about the global potential of ring
trials for improved resistance to FHB, in the rational
and accelerated selection of more resistant and adapted
winter wheat genotypes, we have maintained and


Session 3: International scab nursery consortium


developed an active partnership in international/
European/ bilateral trials with CIMMYT, United States
Wheat and Barley Scab Initiative (USWBSI) etc.
Beginning approximately two years ago the European
Fusarium Ringtest (EFR) was founded, and includes,
besides the Czech Republic and Romania (initiating
countries), Germany, Switzerland and France. We are
very interested to cooperate with other countries within
the framework of the Global Fusarium Initiative, for
the creation of an International Scab Nursery
Consortium and the Development of a new
International Interactive Scab Resistance Screening
Nursery (IISRSN) for germplasm enhancement and
global compilation of data on Genotype x Environment
x Management effects on resistance to FHB (Ban,
2005). Although Romania only grows winter wheat,
we can grow spring wheat nurseries. We would,
however, like winter wheats also to be included in this
project.

Possible ways for interfacing CIMMYT's 11th Scab
Resistant Screening Nursery with existing regional
FHB nurseries should be explored.


Table 1. New lines of bread winter wheat with improved resistance to FHB, expressed as area under disease
progress curve (AUDPC) and relative head weight (RHW), as % of control, plus other desirable traits


Polymorphism linked to
Polymorphismlinked to Resistance to FHB Other desirable
Line FHB on chromosomes traits
traits
3A 3B 6B AUDPC RHW(%)
F 00146G3-2
X 135 53
(95584G1/Debut/96831)

F 99051G3-3 INC2 Preharvest
X 139 63
(91375G4-13/135U2-103) sprouting resistance

F 01459G4-1
F G-1o Bm X 146 72 BYDV resistance
(B YD V-Scott2/Boema)

F 00628G34-1
(191Tr2-1221Fuz/Bucur//pol. lib)

F 01096G2-2
(96915G1-1/96869G1-1)
F 99419G4-1All-1
F /99419G4-A X 161 57 Al tolerance
(Colonia Bucur)







Session 3: International scab nursery consortium


REFERENCES

Bai, G.-H., and Shaner, G.E. 1996. Variation in
Fusarium graminearum and cultivar resistance to
wheat scab. Plant. Dis. 80: 975-979.
Ban, T., Kishii, M., Ammar, K., Murakami, J., Lewis,
J., William, M., Pena, R. J., Payne T., Singh R.,
and Trethowan R.. 2005. CIMMYT's challenges
for global communication and germplasm
enhancement for FHB resistance in durum and
bread wheat. In Proceedings of the 2005 National
Fusarium Head Blight Forum, Milwaukee, WI,
December 11-13, 2005: 6-11
Ittu, M., SAulescu, N., N., and Ittu, G. 1992. Cultivar x
isolate x location interactions according to different
criteria of estimation for Fusarium scab resistance
in wheat and triticale, Newsletter on Plant
Breeding for resistance, 1(1): 6.
Ittu, M., SAulescu, N., N., and Ittu, G. 1998. Breeding
for resistance to Fusarium head blight in Romania.
In H. J. Braun et al. (Eds.) Wheat: Prospects for
global improvement, Kluwer Academic Publishers,
The Netherlands: 87-92.
Ittu, M., 2001(a), Occurrence of FHB in Romania and
control strategy. In A. Logrieco (Ed.): Occurrence
of toxigenic fungi and mycotoxins in plants, food
and feed in Europe. Agriculture and biotechnology.
COST Action835: 147-151.
Ittu, M., SAulescu, N., N., and Ittu, G. 2001(b).
Fusarium head blight resistance in doubled-haploid
lines derived from crosses with a resistant winter
wheat parent In: Z. Bed6 and L. Lng (eds.) Wheat
in a global environment. Proceedings of the 6
International Wheat Conference, June 4-9 2000,
Budapest, Hungary. Kluwer Academic Publishers,
The Netherlands: 367-372.
Ittu, M., SAulescu, N., N., and Ittu, G. 2001(c).
Progress in breeding for scab resistance in
Romania on wheat. In Proceedings of the
2001National FHB Forum, Erlanger, KY, USA,
December 8-10 2001, p. 83-88.
Ittu, M., Trif, A., Belc, N., 2004. Toxigenic fungi and
mycotoxins in Romania:challenges and
approaches-An overview. In: A.Logrieco and A.
Visconti (eds.) An overview of Toxigenic fungi
and mycotoxins in Europe. Kluwer Academic
Publishers, The Netherlands: 185-194.
Shen, X., Ittu, M., and. Ohm, H., W. 2003.
Quantitative trait loci conditioning resistance to
Fusarium head blight in wheat, Crop Sci 43: 850-
857.







Session 3: International scab nursery consortium


ADVANCEMENT IN FHB RESISTANT WINTER WHEAT
CULTIVAR DEVELOPMENT USING FRONTANA AS THE
RESISTANCE DONOR PARENT


R. Pandeyal* andR. Graf2

1Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada, Central Experimental Farm,
Ottawa, Ontario K1A 0C6. 2AAFC, Lethbridge Research Centre, Highway #3, East Lethbridge,P.O. Box 3000,
Main, Lethbridge, Alberta T1J 4B1
*Corresponding Author: Phone: (613) 759 1636; E-mail: pandevars(agr. gc.ca


ABSTRACT

Fusarium head blight (FHB) has become a national
problem, and continues to cause economic and product
quality/safety damage to the Canadian wheat crop.
Continued development of FHB resistant winter wheat
is of vital importance to Canada, including Ontario,
Quebec and Atlantic Canada and the prairie provinces.
Despite the likelihood of escape in western Canada,
incorporation of FHB resistance into winter wheat is
important, particularly for the moist eastern prairies.
Furthermore, the incorporation of FHB resistance into
new CWRW cultivars may allow national registration
for all parts of Canada. Food/feed safety and
sustainability of the overall wheat agri-economy in
Canada (and other major wheat producing regions in
USA, Asia, Europe and Australia) have been affected
by mycotoxin residues on grains and grain products
due to the frequently-occurring fungal disease FHB,
caused by Fusarium graminiarum.

Agriculture and Agri-Food Canada has successfully
developed genetic stocks (FHB 143, FHB 147, FHB
148 and FHB 161) from the crosses between Frontana
(Brazilian FHB resistance donor) and standard
commercial cultivars, namely Harus, Augusta and
Frederick winter wheat cultivars. Backcrosses to the
respective FIs were conducted to restore plant types
and winter wheat growth habits. Using these resistant
genetic stocks in crosses with the commercial
cultivars, namely Augusta, Casey, Diana and Augusta,
several thousand doubled haploids (DH) were
generated via wheat-maize pollination and embryo
rescue techniques. All DH lines were screened for
their resistance to FHB in the Fusarium epiphytotic
nursery. Selected DH were evaluated for agronomic,
disease and quality parameters.


We have made progress at AAFC-ECORC, in
cooperation with Hyland Seeds of W.G. Thompson
Limited, and developed and released a soft red winter
wheat in 2001-02. This was FT Wonder, the first FHB
resistant/tolerant wheat in North America that has the
lowest DON and visual symptoms of all winter wheat
cultivars grown in Eastern Canada. Commercial seed
sale and production has already begun In 2005,
AAFC-ECORC registered FT-Action and Ashley, the
two first soft white winter wheat cultivars resistant to
FHB in Canada (and anywhere in the world), in
cooperation with Hyland Seed.

INTRODUCTION

Wheat (of all classes) is a very important crop in the
Canadian agri-economy with a contribution of 12% or
$3-4 billion towards the total Farm Cash Receipt (from
animals and crops) of $36 billion Western Canadian
provinces are the main producing region, accounting
for well over 90% of wheat. Amongst the eastern
provinces, Ontario produced 4.7% of the total wheat
cash receipt, accounting for well over $150 million in
2005. Ontario mainly produces soft white and soft red
winter wheat; lately there has been a surge of interest
in soft red and hard red winter wheat.

Over the past 20 years, Fusarium head blight caused by
Fusarium graminearum, has gained national and
international prominence as one of the most
devastating cereal diseases. FHB affects almost all
wheat growing regions of the world where warm,
moist conditions prevail during the flowering and
grain-filling period (Mesterhazy, 1983). In Canada,
the estimated losses to the cereal grain industry from
1993 to 2000 are in excess of $1 billion (Fernando,
1999) through impacts on every aspect of the grain
industry (Gilbert and Tekauz, 2000):










Fusarium-damaged kernels (FDK) may contain
high concentrations of deoxynivalenol (DON)
which makes the grain unfit for human and
animal consumption (Charmley et al., 1994).
Yields are reduced.
The presence of FDK results in downgrading
and lost quality premiums (McMullen et al.,
1997).
Milling, baking, and pasta-making properties
are altered (Dexter et al., 1996) due to the
destruction of starch granules, cell walls, and
endosperm proteins (Bechtel et al., 1985;
Nightingale et al., 1999).
Seed producers must deal with reduced
germination (Gilbert and Tekauz, 1995).
Lost sales in the domestic and export markets
due to low FDK tolerances (Charmley et al.,
1994).

FHB has moved progressively westward from eastern
Canada since the 1980s, and was detected in Alberta in
1994 (Clear and Patrick, 2000). Major epidemics
occurred in Ontario in 1982 and 1986, causing severe
damage to soft white wheat production and overall
usability. The most recent epidemic in 1996 almost
destroyed the entire wheat crop in Ontario and north-
eastern USA. The main reason for the escape of winter
wheat in western Canada is that the crop usually
reaches anthesis before temperature and humidity
conditions are favourable for infection (Brule-Babel
and Fernando, 2001).

Several types for resistance to FHB have been
characterized: 1) resistance to initial infection, 2)
resistance to spread following infection, 3) resistance
to kernel infection, 4) reduced mycotoxin production,
5) yield maintenance in the presence of disease
(Mesterhazy, 1995; Chen, 1996). These components
have been related to various morphological or physical
factors (Schroeder and Christensen, 1963; Mesterhazy,
1995) including head type, spike density, plant
phenology, trapped anthers, plant height, and rate of
grain fill (Stack, 1999). Based on the complexity of
the resistance mechanisms, it is not surprising that
numerous studies have shown that FHB resistance is
conditioned by multiple genes with dominant or
additive effects. Pandeya (1998) concluded that genes
conferring FHB resistance in wheat were located on
most of the chromosomes of the A, B and D genomes.

Genetic variability is essential for the development of
FHB resistant cultivars. Sources of resistance/
tolerance have been introduced to North America from
China, Brazil, Europe and Japan (Fedak et al., 2001).
Canada's first FHB-resistant wheat cultivar, a soft red


Session 3: International scab nursery consortium


winter wheat named 'FT Wonder' was registered in
2002, through the collaborative efforts of AAFC and
Hyland Seeds. Two new cultivars of soft white winter
wheat were registered in January 2003. These cultivars
derive their resistance from the Brazilian cultivar
'Frontana'. Resistance from other germplasm is in
various stages of development.

Continued development of FHB resistant winter wheat
is of vital importance in Ontario, Quebec and Atlantic
Canada. Despite the likelihood of escape in western
Canada, incorporation of FHB resistance into winter
wheat is important, particularly for the moister eastern
prairies. Furthermore, the incorporation of FHB
resistance into new CWRW cultivars may allow
national registration for all parts of Canada.

MATERIALS AND METHODS

Fusarium resistance breeding in winter wheat began in
the early 1980s. Frontana (a Brazilian spring wheat)
was found to be a tolerant variety, and it had the
tolerance to initial infection (Type I resistance).
Frontana was then crossed with our standard winter
wheat cultivars, such as Fredrick, Harus and Augusta
in 1984. Individual plants in the F1 were back-crossed
to the three commercial cultivars to restore phenotype
and restoration of winter wheat growth habits. Plants
from the F2 generation were planted in the greenhouse
for seed increase, and 625 individual plants were
selected from the BC1-F4 generation in 1986-87.
These 625 plants were grown in the greenhouse and
inoculated with a FHB culture suspension with a
spore-concentration of 50,000 per ml. Individual heads
were rated three weeks after inoculation Head-row
lines of the selected individuals were planted. A
suitable method of inoculation, to assess the tolerance
of different winter wheat lines, was developed.
Injection and spray methods of inoculation were
compared for pedigree derived and doubled haploid
lines. The results are presented for the formative phase
of germplasm development and the actual utilization
for cultivar development as follows:

A pedigree population consisting of 625 lines was
sprayed with Fusarium head blight spores (50,000
spores per ml of suspension) under the field and/or
greenhouse epiphytotic nursery conditions in 1990-91.
Lines were rated visually ona 1-10 scale (where higher
numbers indicate susceptibility). Samples of selected
lines with rating score of less than or equal to four
were analysed for the mycotoxin deoxynivalenol
(DON) by GC-MASS SPEC method.

Based on their visual symptom ratings (VSR), 336
lines (100 with rating zero, 64 with 1, 48 with 2, 56











with 3 and 68 with 4) were advanced for the next cycle
of epiphytotic evaluation and selection in 1991-92.
Further inoculation and selection were conducted and a
total of 176 lines were advanced for the third cycle of
selection in 1992-93. We determined DON in the
1992-93 selections (total 176) by monoclonal antibody
base technique developed at ECORC


RESULTS AND DISCUSSION


The formative phase for germplasm development
Over a three year period, continuous selection pressure
was applied in favour of low visual rating and/or DON.
By the 1992-93 crop season, a large number of lines
were identified, with DON values ranging from 3 ppm
to 43 ppm. We succeeded in transferring genes for
resistance from a spring to winter wheat. The data
distribution for disease rating suggested multigenic
inheritance control. Correlation analysis revealed no
definite physical associations between visual rating
and DON content (Table 1). This indicated that
symptom expression and DON are under separate
genetic controls. Low or no correlation between DON
and VSR appeared to be compatible with a discrete
class distribution (suggesting few major genes with
modifiers maybe controlling the characteristics).

Figures 1 and 2 depict the results of three cycles of
inoculation and selections. One of the main findings
was that the visual symptom rating and DON contents
were not related as presented in the Table 1.

Lines with LOW-LOW, LOW-HIGH, HIGH-LOW
and LOW-LOW combinations of DON and VSR were
identified. The inference that DON and VSR were
independently inherited appeared justified. Based on
low DON and VSR, 27 lines were advanced for final
evaluation in 1993-94 (Table 2).

The lines FHB 143, 147, 148 and 161 were chosen as
winter wheat lines with FRONTANA type resistance
as donor parents in our current FHB resistance
breeding for soft winter pastry wheat. These pedigree
lines exhibited low VSR and low DON. Also, the sub-
lines of the four FHB lines showed stable performance
for VSR (see FHB Index, Figure 3). Doubled haploids
(DH) were generated via maize pollination and embryo
rescue technique from the F1 between the four FHB
lines and current standard pastry wheat. Our primary
goal was to develop a resistant variety.

Cultivar development study
The doubled haploids were evaluated for their
agronomic traits and disease (FHB) resistance. Over
the past three years, we have developed three cultivars
for release.


Session 3: International scab nursery consortium



Table 1. Inoculation procedures and correlations in
doubled haploids population
Correlation
DON vs visual rating 0.39 SPRAY
0.31 INJECTED
VSR spray vs VSR injected 0.92
DON injected vs DON spray 0.67




GENOT'E DISIRIBUION 1993
FIB N ES
MNA MEAN -' SA HARUS MEAN -

23 23
5 a 1 21
2 20| |



0

0 1 2 3 4 5 6 7 8 9
RATING

Figure 1. Third cycle selection for VSR


GENOTYPE DISTRIBUTION 193
HB LINES
INA btAKN -tAi MEbLIAN 21A4






IC *






Figure 2. Third cycle selection for DON


Table 2. Number of entries for VSR and DON:
Independent factors
Visual rating DON Number of
Visual concentration entries
HIGH HIGH 25
HIGH LOW 55
LOW HIGH 41
LOW LOW 53












---*


[ni,, I ,,, | -





S ..-... 1 ... .. II ,' 1


Figure 3. FHB resistance stability of the sub-lines
selected from the four main genetic stocks according to
FHB index (y-axis)


Session 3: International scab nursery consortium


The line OTH 017-033, from the cross FHB 147 and
Casey, was released and registered as FT Wonder. FT
Wonder consistently showed resistance in terms of
VSR and DON (Table 3). The commercialization of
FT Wonder began during the fall of 2005 (Table3).

We have also developed two first soft-white winter
wheat cultivars (FT Action and Ashley, Tables 4 and
5). Seed increase and commercialization process have
begun

Our results clearly indicate that the most suitable
procedure for the inoculation was the spore suspension
spray. The method closely mimics the natural infection
process. The results from the point inoculation were
inconsistent in delineating susceptible and resistant
genetic lines. Fusarium-damaged kernels were much
more closely related to the DON content of the lines.


Table 3. Comparative FHB Index and DON ratings for FT-Wonder (OTH 017-033), a soft red winter wheat cultivar
in inoculated nurseries in Nairn and Ottowa over multiple years.
97-98 98-99 99-00 00-01
Nairn Nairn Ottawa Nairn Nairn Nairn FHB Nairn Ottawa
FHB Index FHB Index FHB Index FHB Index DON (ppm) Index % DON (ppm) FHB Index
% % % % %
AUGUSTA 25.0 62.5 47.5 65.0 6.0 30.0 0.64 30.0
AC Ron 25.0 80.0 54.0 70.0 6.0 40.0 0.47 22.5
Freedom 15.0 52.5 43.7 45.0 4.0 25.0 0.43 25.0
2540 25.0 80.0 44.0 70.0 5.6 32.5 1.60 15.0
OTH017-033 5.0 20.0 10.0 25.0 1.9 7.5 0.23 12.5
Check Mean 15.0 52.5 43.7 45.0 4.0 32.5 0.43 25.0
The columns have values for the FHB indices and DON toxin level under inoculated nursery conditions


Table 4. Comparative FHB performance of OTH 013-081 (FT Action), a soft white winter wheat
(DH from the cross Augusta x FHB 148) in inoculated (Inocu.) and natural conditions over multiple
years.
2000 2001 2002 Overall mean
Ino cu. Ino cu. Natural Inocu. Natural Ino cu. Natural
Station year 3 3 1 6 5 12 6
Varieties
FHBI FHBI 0-9 FHBI 0-9 FHBI
Augusta 52.3 34.2 2.2 24 2.6 36.8 2.4
AC Ron 45.8 35 2.2 26.7 4.7 35.8 3.5
Freedom 26.8 32.5 3 21.3 1.5 26.9 2.3
P2540 17.3 36.7 4.8 19.7 4.2 24.6 4.5
OTF 013-081 12 5.8 0.7 8.5 1.4 8.76 1.1

FHBI is the FHB Index = (% infected head X % infected spikelet)/100. 'Station year' is the number of sites
evaluated under inoculated or natural conditions in the given year. The means in the Table 4 (and 5) are presented as
these were measured. The CV and LSD are not provided, because generally CV for FHB resistance are very high
due to strong environmental and local plot effects. The absolute values are taken as indicator of FHB resistance







Session 3: International scab nursery consortium


Table 5. Comparative FHB performance of Ashley (TWF 020-038), a soft white winter wheat cultivar
(DH from the cross Diana x FHB 147) for FHB Indices under inoculated conditions in Nairn and Ottawa.
2000 2001 2002 Overall mean
Naim Nairn Ottawa Nairn Ottawa Inocu.
Station year 1 1 1 1 1 4
Varieties
Augusta 30 42.5 21 27.5 36.20
AC Ron 68 40 30 3.5 30 42.00
Freedom 35 32.5 32.5 9 25 31.25
P2540 60 45 20 22.5 25 34.00
TWF020-038 30 7.5 22.5 0.3 15 18.75

Mean of check 35 32.5 32.5 9 2025
CV 8.05 19.22 44.1 51.96 29.4
LSD 10.8 13.7 11.83 16.8 8.6
'Station year' is the number of sites evaluated under inoculated or natural conditions in the given year. The means in
the Table 5 are presented as these were measured. TWF 20-38 is approved for registration and named as 'Ashley'.


REFERENCES


Anderson, J.A.., Sorrell, M.E., and Tanksley, S.D.
1993. Crop Sci. 33: 463-459.
Brule-Babel, A.L. and Fernando, D. 2001. Breeding
winter wheat for resistance to Fusarium head
blight in the eastern prairies. In: Fedak, G. and
Choo, A. (compilers) Proc. Second. Canadian
workshop on Fusarium head blight. Ottawa,
Canada. p. 20.
Charmley, L.L., Rosenberg, A. and Trenholm, H.L.
1994. Factors responsible for economic losses to
Fusarium nycotoxin contamination of grains,
foods and feedstuffs. In: Miller, J.D. and
Trenholm, H.L. (eds.) Mycotoxins in grain:
compounds other than aflatoxin Eagan Press, St.
Paul, USA. pp. 471-486.
Clear, R.M. and Patrick, S.K. 2000. Fusarium head
blight pathogens isolated from fusarium-damaged
kernels of wheat inwestern Canada, 1993 to 1998.
Can J. Plant Pathol. 22: 51-60.
Dexter, J.E., Clear, R.M. and Preston, K.R. 1996.
Fusarium head blight: effect on the milling and
baking of some Canadian wheats. Cereal Chem
73: 695-701.
Fedak, G., Gilbert, J., Comeau, A., Voldeng H.,
Savard, M. and Butler, G. 2001. Sources of
Fusarium head blight resistance in spring wheat.
In Fedak, G. and Choo, A. (compilers) Proc.
Second Canadian workshop on Fusarium head
blight. Ottawa, Canada. p. 30.
Fernando, D. 1999. Overview of the Fusarium
situation in Canada. In Clear, R.M. (compiler)
Proc. Canadian workshop on Fusarium head
blight. Winnipeg, Canada. pp. 12-15.


Gilbert, J. and Tekauz, A. 1995. Effects of fusarium
head blight and seed treatment on germination,
emergence, and seedling vigour of spring wheat.
Can J. Plant Pathol. 17: 252-259.
Gilbert, J. and Tekauz, A. 2000. Review: recent
developments in research on fusariuym head
blight of wheat in Canada. Can J. Plant Pathol.
22: 1-8.
Meidaner, T. 1997. Breeding wheat and rye for
resistance to Fusarium head blight of wheat. Plant
Breed. 116: 201-220.
Mesterhazy, A. 1995. Types and components of
resistance to Fusarium head blight of wheat. Plant
Breed. 114: 377-386.
Morrison, W.P. and Peairs, F.B. 1998. Response
model concept and economic impact. In:
Quisenberry, S.S. and Peairs, F.B. (eds.) Response
model for an introduced pest the Russian wheat
aphid. Entomol. Soc. Amer., London, Maryland.
pp. 1-11.
Pandeya, R.S. and Sinha, R.C. 1998. Selection and
breeding for Fusarium Head Blight resistance in
wheat using biotechnology-based diagnostic
procedures. In: Behl, Singh and Lodhi (Ed). Crop
improvement for stress tolerance. Proceedings of
"International conference on sustainable crop
production in fragile environments". Haryana
Agricultural University, Hissaar, India. pp. 182-
201.
Stack, R.W. 1999. Return of an old problem:
Fusarium head blight of small grains. Amer.
Phytopathol. Soc.
http://www.scisoc.org/feature/FHB/Top.html.






Session 3: International scab nursery consortium


PROGRESS IN IMPROVING FUSARIUM HEAD BLIGHT
RESISTANT WHEAT IN HOKKAIDO, JAPAN


Y. Yoshimural K. Nakamichil, S. Kobayashil, T. Nishimural, M. Ikenagal,
N. Satol, M. Sato2, T. Suzuki2, T. Takeuchi2, and A. Yanagisawal

1Hokkaido Prefectural Kitami Agricultural Experiment Station
(Yayoi, Kunneppu-tyou, Hokkaido, 099-1496 Japan)
2Hokkaido Prefectural Central Agricultural Experiment Station
(Naganuma-tyou, Hokkaido, 069-1395 Japan)
*Corresponding Autor: Phone: (81) 157-47-3806; E-mail: vosimuvlgbagri.pref.hokkaido.p



ABSTRACT

Fusarium head blight (FHB) is the most serious disease for wheat production in Hokkaido Island, northern Japan
Wheat production in Hokkaido accounts for about 60% of domestic wheat in Japan and the yield losses caused by
rain damage, pre-harvest sprouting and FHB are estimated at 10-20% of total production Exposure to continuous
rain conditions during the wheat maturing stage has become more frequent in Hokkaido. In 2002 the Japanese
government applied maximum limits of 1.1 mg/kg for deoxynivalenol (DON) in raw cereals for human
consumption and feed material, 1.0 mg/kg in feed for various animal species and 4.0 mg/kg for cattle older than 3
months. The FHB resistance of Hokkaido wheat varieties has not been sufficient to avoid contamination with
mycotoxins produced by some Fusarium pathogens.

Evaluation and selection of FHB resistant wheats are conducted by several methods, such as spray inoculation of
Fusarium spores onto wheat heads or spreading Fusarim inoculum using oat cultures with mist irrigation in the
field nursery, and a single floret injection method in the greenhouse. For developing resistant wheat lines, we use
Asian resources, such as Sumai 3, Japanese landraces, and breeding materials developed at Kyushu Agricultural
Research Center, as well as Brazilian varieties and European materials. These materials have many inferior
characters under Hokkaido conditions, such as low yield potential, low quality, lodging, and susceptibility to cold
and snow mold. Since 2004, we started marker-assisted selection to introduce FHB resistance quantitative trait
loci (QTLs) into spring wheat germplasm. We are actively screening and developing good resistant germplasm
and lines with excellent resistance to FHB in both winter and spring wheat. These lines show low DON
accumulations. There was significant correlation between Fusarium-damaged kernels (FKD) and DON content in
inoculated field experiments at Kitami Agricultural Experiment Station The yield potential of these breeding FHB
resistant lines is lower than Hokushin, a leading variety in Hokkaido and they are still inferior in some traits:
snow mold in winter wheat, lodging and quality.






Session 3: International scab nursery consortium


FUSARIUM HEAD BLIGHT (FHB), AN EMERGING WHEAT
DISEASE IN TUNISIA

M.R. Hajlaoui*, L. Kammoun, S. Gargouri, and M. Marrakchi

Laboratore de protection des v6getaux, INRA Tunisie
*Corresponding Author: Phone: 267 1235317; E-mail : hajlaoui.rabeh@iresa.agrinet.tn


ABSTRACT

Fusarium spp. are ubiquitous plant pathogens causing seedling blight, root and foot rot and head blight in wheat
(Triticum aestivum L.) over a broad range of environmental conditions. The most economically important disease is
head blight (FHB), resulting in yield losses, a severe reduction in grain quality, and mycotoxin contents.

Wheat crops produced in Tunisia are often susceptible to root and foot rot diseases caused by F. culmorum and F.
pseudograminearum. An outbreak of FHB occurred in 2004, localised around regions where higher rainfall occurred
during anthesis in April. A collection of Fusarium isolates obtained from foot and head of wheat from different
locations in Tunisia were identified using a combination of morphological and molecular criteria. Results showed
thatMichrodochium nivale, (syn F. nivale), F. culmorum, F. pseudograminearum and F. avenaceum are a dominant
pathogenic species isolated from both foot and head of durum wheat. FHB is a disease of growing concern since
several species that cause the disease can produce trichothecene mycotoxins such as nivalenol (NIV) and
deoxynivalenol (DON). ELISA analysis of naturally contaminated grains (23%) revealed the presence of 0.053 ppm
of DON mycotoxin

Trichothecenes also are phytotoxic and act as virulence factors on sensitive host plants. Recent reports suggest that
strains of Fusarium that produce DON may be more aggressive toward hosts than NIV-producing strains. A total of
90 isolates of F. culmorum and F. speudograminearum were chemotaxonomically classified into DON and NIV
chemotypes based on specific amplification of Tris7 and Tril3 genes involved in trichothecene biosynthesis. Only
three isolates of F. culmorum were of the NIV chemotype while the other 87 isolates were of the DON chemotype.
Our results suggest that strains of Fusarium prevailing in Tunisian cereal growing regions belong to DON
chemotype and stress the importance of considering FHB as an emerging disease of wheat.







Session 3: International scab nursery consortium


SOURCES OF "ENVIRONMENTAL INTERACTIONS" IN

PHENOTYPING AND RESISTANCE EVALUATION; WAYS TO
NEUTRALIZE THEM


A. Mesterhazy*, B. T6th, and G. Kiszonyi

Cereal Research non-profit Co., Szeged, H-6701 Szeged, P.O.Box 391, Hungary
*Corresponding Author: Phone: +36 (30) 415-9730; Email: akos.mesterhazv@,gk-szeged.hu


OBJECTIVES

Our objective is to give an overview of the
environmental influences in Fusarium resistance
research It is often suggested that inconsistent results
are a consequence of the environment. We will show
that most deviations are not of an environmental
nature: they have genetic and methodic causes, and the
heterogeneity of the population tested and differences
in epidemic severity are also responsible for the
problems. At the end several recommendations are
given to improve phenotyping or resistance evaluation
and research

INTRODUCTION

The high level of environmental influence in Fusarium
research has been often pointed to as the most
important source of the frequently inconsequent results
(Wilde and Miedaner 2006, Chen et al. 2003,
Buerstmayr et al. 2003). The interactions may be so
strong that that genotype ranking may be very different
between tests. The problem is especially great for the
inoculations and spraying tests similar to natural
conditions; whereas the problem is less for the more
stable point inoculation results. It is necessary for us to
determine the content of the environmental interaction
In the past decades we have performed many
experiments with results that support the statement that
the 'environmental effects' contain many very
different influences which are not of environmental
origin We should produce comparable data, but in
most cases data cannot be compared as conditions
producing these data are different. In a quantitative
trait locus (QTL) analysis this is a serious problem
The problem is less pronounced in practical resistance
breeding where highly productive and less exact
methods are used that correspond to the need of
handling thousands of lines. However, when the
amount of resistance is to be determined, for example
for registration of cultivars, the problem is greater.


Therefore, this is also a problem in breeding, though
less central.
The experimental material used in this study originates
from our breeding and research program, the testing of
DH population within FUCOMYR FP5 projects
CM82036/Remus and Frontana/Remus, the screening
nursery from the FUCOMYR project, and fungicide
tests from the Szeged program. Inoculation methods
were described by Buerstmayr et al. (2003) and
Mesterhzy (1995, 1999).

PASSIVE INFLUENCING AGENTS

Morphological traits
Plant height significantly influences disease severity.
In 1985 we had a severe FHB epidemic, where four
replicated trials provided natural FHB data. Parallel
with this about 100 genotypes included in these trials
were tested using the bagging method. The plant height
classes revealed significant differences: when 90 cm is
considered as standard, the dwarfs (60-70 cm) had 20-
30 % more disease severity and the tall genotypes 20-
30 % less FHB. However, the means of the same
groups did not differ significantly under artificial
infection (Mesterhzy 1987). This means that plant
height has nothing to do with FHB resistance; for
further details see Parry et al. (1995), Miedaner (1997),
Mesterhzy et al. (2005) and Steiner et al. (21114).
Therefore plant height is a pseudo resistance factor.
The story is similar for awns: awned lines showed on
average up to 80 % more natural infection than
awnless plants, but means for artificial inoculation data
were the same (Mesterhzy 1987). Higher head density
(compact heads) is also an infection severity increasing
factor, as demonstrated by Steiner et al. (2" 4), but its
influence was moderate.

Longflowering period
In mapping populations or breeding materials
flowering time differences might be two weeks or
more. Ecological conditions over this long period are
never stable. In Table 1 the Frontana/Remus results










show that earlier inoculation dates under bagging and
mist irrigation resulted in significantly higher FDK and
yield loss than later dates. The earliest materials were
exposed to four times as much misting as the latest
Table 2 shows the results of this population from 2004.
Here the FHB AUDPC data show a more than five-
fold difference between inoculation dates, but FDK is
relatively stable. Here the earliest genotypes showed
the least infection In 2004 the warm period, with
higher infection severity, was in June, whereas in 2002
the earliest inoculation was made in warmer weather,
followed by a cooler period. Table 3, with data from a
cultivar resistance test of over 100 genotypes, shows a
similar picture. In each case FDK shows much less
deviation It seems that FDK is much more suitable to
characterize DH populations than FHB AUDPC data,
and that that infections in the palea or glume do not
automatically cause higher grain infection

Leaf diseases
In some cases (Mesterlhzy 1987) leaf diseases
significantly increase FHB severity. We found up to a
40% increase in FHB severity compared with the


Session 3: International scab nursery consortium


severity on healthy plants .We observed this also in
artificial stem rust infected plants compared to plants
that were not infected. For this reason it is reasonable
to control leaf diseases, at the latest when the flag leaf
emerges.

Epidemic severity
Epidemic severity has a significant impact on cultivar
differences. Isolates of low aggressiveness will not
differentiate between medium and highly resistant
genotypes, as both will be nearly without symptoms.
The most aggressive isolate will present differences
between these two groups, but all medium susceptible
to highly susceptible genotypes will show the
maximum level of disease (Figure 1). Therefore a
mean of different epidemics gives more precise
information on the amount of resistance than any
single epidemic. Isolates used in parallel can replace
multilocation tests so that the environmental
interaction is near zero as all epidemics generated by
the different isolates have the same environmental
determination


Table 1. Frontana/Remus population data grouped according to inoculations dates, Szeged, 2002

Important traits grouped according to inoculation dates

Inoculation Plant Leaf FHB FDK Relative
date height Lodging diseases AUDPC % Yield %
May cm % mean Mean Mean
10 82 2.2 22 368.4 34.9 30.8
13 103.4 0.8 19.8 248.8 31.4 48.7
15 109.8 4.3 24.1 303.2 25.8 54.8
21 115.9 10.6 13.8 315.2 19.4 61.6


Mean


102.8


308.9


27.9


Table 2. AUDPC and FDK values for the Frontana/Remus population in 2004, Szeged

Means for genotypes according to inoculation time

Inoculation AUDPC FDK% n
date May/June
19 151 37 29
21 173 40 72
24 169 31 29
26 440 44 49
7 832 35 27







Session 3: International scab nursery consortium


Table 3. Mean AUDPC and FDK of the genotypes of the screening nursery by inoculation time, Szeged
nursery, 2003-2005

2003 AUDPC FDK 2004 AUDPC FDK 2005 AUDPC FDK
21 May 475.25 25.05 25 May 96.3 6.1 20 May 229.8 15
24 May 772.41 30.71 30 May 73.8 6.7 26 May 361 15.5
26 May 885.14 23 6 June 139.29 9.22 28 May 576.9 22.5
2 June 2284.37 47.28 2 June 788.7 23.8
7 June 1710.3 43.18








80
60
40
20" Isolate 3
2 & & r Isolate 2
O- ol IA Isolate 1





Figure 1. FDK data (%) from the 2002 resistance tests with three differently aggressive isolates, i.e.
three different epidemic severities.


METHODOLOGICAL CONSIDERATIONS

The main stream in the methodology is the application
of methods that simulate natural infection In the
simplest case the experimental field is sited where high
humidity often occurs and helps the infection process.

Corn stubble, infected maize or other cereal grains can
serve as inoculum source, and mist irrigation helps to
ensure high humidity to initiate infection As heading
takes about two weeks, several waves of infection
occur. Additional rains initiate further infections. The
early genotypes receive much more inoculum than the
late ones; therefore large differences in infection
severity may develop, even when ecological conditions
are relatively stable. With respect to flowering time,
the duration of humid conditions is much longer for the
early than for the later ripening groups, so an over-
infection of the early materials is a real problem, but it
cannot be solved within this methodological
framework


Spray inoculation regimes
Spray inoculation regimes always involve some means
of securing high humidity. Many authors believe that
with this method both Type I and Type II resistance
canbe tested together; we support this view.

The sprayer goes every 3-4 days through the test field,
inoculating the whole area. After each inoculation mist
irrigation is applied for two days. Misting regimes
differ: it may be continuous for 8-10 hours, or
equipment may be controlled by computer to mist 2-3
times an hour. This is clearly a problem in obtaining
comparable data. However, this method gives higher
disease pressure, so selection for higher resistance is
easier, which works well for breeding purposes.

A more sophisticated version is when the spraying
occurs only once for each plot and the 3-5 inoculations
are made according to flowering. However, mist
irrigation is used after each inoculation; therefore the
humid period will be 3-5 times greater in the earliest










than in the latest group. Previous results showed that a
two-day humid period increased infection severity by
30% over a one-day period, and the increase varied
between 0 to 100% in individual genotypes
(Mesterhzy 1978). The data show that each genotype
may have a distinct optimal humid period. Even where
we cannot secure this, a stable humid period length is
much better than any significant change during the
vegetation period. Using mist irrigation this problem
cannot be solved. Mist irrigation has also a disease
development modifying effect. In cool weather it
significantly cools the temperature of the heads,
especially when the weather is windy, and has a
negative influence on disease development. On hot
days the cooling effect is positive, but the water drops
can function as a glass lens and the plant tissue can be
damaged. The result is that even within one test,
disease development can be modified in both
directions.

A solution is to spray the heads at flowering and
ensure humidity using plastic bags (Mesterhzy 1978,
1983, 1995). At spraying, 15-20 heads are sprayed
from every side, thanbagged for 48 hrs. The bagging
has the advantage that the wet period is the same for all
inoculations. In cool weather, through the
greenhouse effect in the bag, the temperature is
higher than outside, therefore under these
conditions this method provides better disease
development. On hot days the disease
development is slower than optimal, but heat
damage is seldom seen; perhaps 1 in 100
genotypes shows susceptibility. Two assistants
can inoculate 4-500 groups of heads in a
morning.

The results show very similar tendencies using
both methods, single spraying and misting and
single spraying and bagging (Table 4). The
agreement between FDK values is much better,
indicating the less sensitive nature of the FDK
values. AUDPC values seem to be less
informative under mist irrigation However, the
results from bagged tests give AUDPC values
that relate more closely to FDK than in the data
from the mist irrigation regime. Large effect
QTLs are more stable under different inoculation
regimes.

Point inoculation
The method is well known, and tests Type 2
resistance. Most tests are done in a controlled
environment (growth chamber, greenhouse),
therefore the quality of the data is mostly
satisfactory. When it is done in the field, the
changing environment over the inoculation


Session 3: International scab nursery consortium


period can influence disease development differently.

Symptoms to be evaluated
Most papers deal with visual FHB evaluation, and
neglect other traits. Disease evaluation stops when
heads turn yellow, but 3-4 weeks remain from this time
until harvest. In this period significant changes may
occur in FDK and DON contamination These are both
key traits, and the value of the lot depends on the DON
contamination It is important that FDK is much more
stable than FHB (Tables 2, 3, 4). In the
CM82036/Remus population the correlation between
the mist-irrigated and bagged regimes is 0.6352 for
AUDPC and r = 0.8284 for FDK, therefore FDK is
more valuable for important research tasks.

Pure isolates or mixtures
There has long been discussion over whether to use
isolates or their mixtures. Mesterhzy (1977)
compared the effect of pure isolates and their mixtures.
In all cases the mixture had lower aggressiveness than
the arithmetical mean of the participant inocula alone.
As its effect could not be forecasted, we did not use
this approach In the rusts, where different specialized
races exist, a mixture is inevitable when a field test is



Table 4. Comparison of spray inoculation + mist
irrigation and spray inoculation + bagging methodical
regimes according to QTL groups, CM82036/Remus DH
population, 2002-2004

BAGGING AUDPC AUDPC FDK % FDK %
QTL group Check Mean Check Mean
3B/5A 0 44.7 0 2.3
3B 0 153.4 0 8.5
5A 0 164.6 0 10.3
no QTL 0 313.8 0.1 15.4
LSD 5 % 0 79.4 0 2.6
MIST
IRRIGATION AUDPC AUDPC FDK % FDK %
QTL group Check Mean Check Mean
3B/5A 19.7 783.3 0.3 10.1
3B 23.3 932.4 4.2 25.4
5A 32.9 1215.9 1 19.8
no QTL 47.5 1434.5 4.4 35.7


LSD 5 %


13.8 145.3


2 4.4










done. However, in cereal Fusaria no races exist, so
this argument does not apply. The data in figures 1 and
2 data demonstrate that different aggressiveness levels
cause different disease levels, with varying usefulness
in differentiating levels of resistance. A mixture is not
better or worse than a single isolate, and provides only
one epidemic situation For this reason we believe that
the use of separate isolates increases greatly the
exactness of the work without increasing
environmental influence.

Conidium concentration and aggressiveness
The aggressiveness of the isolates is not stable
(Mesterhazy 1977). Tests over a number of years have
demonstrated that inocula from the same isolate may
change their aggressiveness markedly; the isolate/year
interactions show this clearly (Mesterhzy 1987, 1995,
Mesterhzy et al. 1999, 2003). The aggressiveness and
conidium concentration are not closely correlated
when different inocula are compared, since all inocula
react differently to dilution However, excellent
correlations are observed when the same inoculum is
diluted to different concentrations (Mesterazy 1977).
The consequence is that changing the inoculum for
different inoculation dates in an experiment is not
recommended, as it may result in additional variation
Enough inoculum should be produced for all
inoculations. In regulating aggressiveness, the
conidium concentration is of secondary importance.
We use an aggressiveness test to give us direct
information about the aggressiveness of a given
inoculum.

Experimental design
In a number of experiments the number of replicates is
low, sometimes only one. From these tests no correct
estimation of the environmental effect is possible. It is
clear that cost of the experiment should be kept to a
minimum, but 2-3 replicates per treatment per location
or year is unavoidable to achieve a comprehensive data
set and to present fair conclusions.

Variation width of the population in resistance
When the variability in the population is narrow, every
small influence can severely affect the picture. Larger
genetic differences help genotype ranks to remain
more stable. This is one source of the phenotyping
errors in mapping populations where low or medium
effective QTLs are tested. When large effect QTLs are
in the test, the resistance differences are larger and
these can be easier identified than the smaller
deviations.


Session 3: International scab nursery consortium


PHYSIOLOGICAL RESISTANCE TRAITS

The Fusarium QTLs determining different components
or types of resistance are the most important resistance
components we have to deal with A given resistance
level is a combined effect of several QTLs. When this
combined effect results in a high level of resistance,
the disease severity remains low in any situation, as
shown in figure 1. The higher the resistance level, the
greater the stability of the resistance (Mesterhzy
1995). All the influences we have discussed affect the
disease severity in the genotypes. As susceptibility
increases, the greater the potential influence of these
effects on disease development, and so the probability
of possible errors increases sharply. The highly
effective QTLs are more stable (Table 3). The medium
or small effect QTLs can easily be misidentified (Table
5). Some markers are more stable: GWM293 gave log
of odds (LOD) values greater than 2.0 in three of the
four data sets. If the data were more precise, the matrix
would probably be more complete.



Table 5. LOD values for the Frontana/Remus DH
population in different years and traits, 2002-2004,
Szeged.


Szeged genotyping and
phenotyping


Chromo- Marker LOD LOD LOD LOD
some
3A *DuPw227

3A *GWM1121 3.92
3A *GWM1110

3A *GWM720 3.16
5A *BARC197

5A *GWM293 2.19 2.56 2.15
5A *GWM156 2.43
5A *GWM304

2D *GWM261
2D *A66 3.56 5.93

2D *A169
2D *GWM614B 2.47 3.67










INFLUENCE OF ENVIRONMENTAL
CONDITIONS

The influence of warm and rainy weather has been
understood since Atanasoff (1920). They are even now
the most important epidemiological factors; recent
forecasting systems use the functions very effectively.
Many factors that influence these traits, such as
location near a lake or river, soil type, etc., influence
disease development significantly. The influence of
temperature and precipitation on FHB AUDPC, FDK,
DON, and yield loss based on a seven-year fungicide
test is shown in Table 6. The correlations between
traits are very close, indicating that the different traits
are strongly interdependent. High May temperatures
had the strongest effect on FDK; the correlation
between DON and mean temperature was not
significant. Interestingly, the June and July
temperatures were almost neutral in their effect on the
FHB traits. The rains in the inoculation period were
not significant as the 48 hr polyethylene bag coverage
ensured the humidity. However, the rains before
inoculation were important, because after the removal
of the bags the higher soil humidity contributed to dew
development and so indirectly enhanced infection
severity. The June rains had, against expectations, no
significant influence. However, the July rains were


Session 3: International scab nursery consortium


very influential and correlated strongly with DON.
This means the rains before harvest are the most
important for toxin accumulation Harvest time was
normally at the end of July. The columns of the
correlation matrix also show that the different traits are
influenced somewhat differently, and this may be one
cause of the different responses in different
experiments.

HOW LARGE IS THE ENVIRONMENTAL
INTERACTION?

In the CM82036/Remus population the FDK data
present remarkable results (Table 7). The genotype,
isolate, year and location effect are highly significant.
There are large differences in disease severity between
genotypes, isolates, years and locations. However, the
interactions including genotypes (bold printed in Table
7) show significance at 0.001 level, but their value is
very small compared to the main genotype effect and
in each case differ significantly from the main
genotype effect. Therefore, the genotype ranking is
surprisingly stable in different environments. For the
Frontana/Remus population the data are somewhat
different (Table 8). The extent of the test was smaller;
only 16 rather than 64 data points were behind each
genotype. The trend is similar. The main effects are


Table 6. Correlation coefficients between FHB traits and weather parameters, 1998-2004

Trait group Traits DON mgkg-1 FHB % Yield red. % FDK %

FHB traits FHB % 0.8612**
Yield
red. % 0.8368** 0.9802****
FDK. % 0.6943o 0.8807** 0.9030***
Mean May -0.4553 -0.6646o -0.7004o -0.8338***
temperature June -0.4379 -0.1183 -0.1019 -0.3201
July 0.2618 0.2126 0.2013 0.138
Mean -0.4696 -0.493 -0.5141 -0.7174o
May 20-
Rain 31 -0.0094 0.032 -0.0951 -0.2468
May 0.7114o 0.7701* 0.6357 0.5077
June 0.1506 -0.1732 -0.1521 0.1199
July 1-20 0.7322o 0.7224o 0.7177o 0.6856o
July 0.9635**** 0.8525** 0.7858* 0.6899o
Total 0.7854* 0.5327 0.4785 0.5697
**** P = 0.001, *** P = 0.01, ** P = 0.02, P =0.05, o = P = 0.10,

Bold printed: significant correlations




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