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<strong>Septoria</strong> <strong>and</strong><br />
<strong>Stagonospora</strong><br />
<strong>Diseases</strong> <strong>of</strong> <strong>Cereals</strong>:<br />
A Compilation <strong>of</strong> Global Research<br />
M. van Ginkel,<br />
A. McNab,<br />
<strong>and</strong> J. Krupinsky,<br />
editors
<strong>Septoria</strong> <strong>and</strong> <strong>Stagonospora</strong><br />
<strong>Diseases</strong> <strong>of</strong> <strong>Cereals</strong>:<br />
A Compilation<br />
<strong>of</strong> Global Research<br />
Proceedings <strong>of</strong> the Fifth<br />
International <strong>Septoria</strong> Workshop<br />
September 20-24, 1999<br />
<strong>CIMMYT</strong>, Mexico<br />
M. van Ginkel, A. McNab, <strong>and</strong> J. Krupinsky, editors<br />
Dedicated to the memory <strong>of</strong><br />
Dr. Zahir Eyal
ii<br />
The Organizing Committee expresses it sincere thanks to the Workshop Sponsors:<br />
Bayer de México, S.A., <strong>and</strong> Zeneca Mexicana, S.A.<br />
<strong>CIMMYT</strong> (www.cimmyt.mx or www.cimmyt.cgiar.org) is an internationally funded, nonpr<strong>of</strong>it<br />
scientific research <strong>and</strong> training organization. Headquartered in Mexico, the Center works with<br />
agricultural research institutions worldwide to improve the productivity, pr<strong>of</strong>itability, <strong>and</strong><br />
sustainability <strong>of</strong> maize <strong>and</strong> wheat systems for poor farmers in developing countries. It is one <strong>of</strong> 16<br />
similar centers supported by the Consultative Group on International Agricultural Research<br />
(CGIAR). The CGIAR comprises over 55 partner countries, international <strong>and</strong> regional<br />
organizations, <strong>and</strong> private foundations. It is co-sponsored by the Food <strong>and</strong> Agriculture<br />
Organization (FAO) <strong>of</strong> the United Nations, the International Bank for Reconstruction <strong>and</strong><br />
Development (World Bank), the United Nations Development Programme (UNDP), <strong>and</strong> the<br />
United Nations Environment Programme (UNEP). Financial support for <strong>CIMMYT</strong>’s research<br />
agenda also comes from many other sources, including foundations, development banks, <strong>and</strong><br />
public <strong>and</strong> private agencies.<br />
<strong>CIMMYT</strong> supports Future Harvest, a public awareness campaign that builds underst<strong>and</strong>ing about<br />
the importance <strong>of</strong> agricultural issues <strong>and</strong> international agricultural research. Future Harvest links<br />
respected research institutions, influential public figures, <strong>and</strong> leading agricultural scientists to<br />
underscore the wider social benefits <strong>of</strong> improved agriculture—peace, prosperity, environmental<br />
renewal, health, <strong>and</strong> the alleviation <strong>of</strong> human suffering (www.futureharvest.org).<br />
© International Maize <strong>and</strong> Wheat Improvement Center (<strong>CIMMYT</strong>) 1999. Responsibility for this<br />
publication rests solely with <strong>CIMMYT</strong>. The designations employed in the presentation <strong>of</strong> material<br />
in this publication do not imply the expressions <strong>of</strong> any opinion whatsoever on the part <strong>of</strong><br />
<strong>CIMMYT</strong> or contributory organizations concerning the legal status <strong>of</strong> any country, territory, city, or<br />
area, or <strong>of</strong> its authorities, or concerning the delimitation <strong>of</strong> its frontiers or boundaries.<br />
Printed in Mexico.<br />
Correct citation: van Ginkel, M., A. McNab, <strong>and</strong> J. Krupinsky, eds. 1999. <strong>Septoria</strong> <strong>and</strong> <strong>Stagonospora</strong><br />
<strong>Diseases</strong> <strong>of</strong> <strong>Cereals</strong>: A Compilation <strong>of</strong> Global Research. Mexico, D.F.: <strong>CIMMYT</strong>.<br />
ISBN: 970-648-035-8<br />
AGROVOC descriptors: Wheats; Triticum; Triticum aestivum; S<strong>of</strong>t wheat; Triticum durum; Hard<br />
wheat; Winter crops; Plant diseases; Fungal diseases; <strong>Septoria</strong>; <strong>Stagonospora</strong>; Blotches;<br />
Mycosphaerella; Epidemiology; Plant breeding; Selection; Disease resistance; Genetic control; Gene<br />
location; Cultural control; Plant response; Research projects<br />
Additional Keywords: Triticum tauschii<br />
AGRIS category codes: H20 Plant <strong>Diseases</strong><br />
F30 Plant Genetics <strong>and</strong> Breeding<br />
Dewey decimal classification: 632.4<br />
Additional information on <strong>CIMMYT</strong> is available on the WorldWideWeb at: www.cimmyt.cgiar.org.
Table <strong>of</strong> Contents<br />
vi In Memoriam, Dr. Zahir Eyal<br />
vii Foreword<br />
1 Opening remarks<br />
1 Historical Aspects <strong>and</strong> Future Challenges <strong>of</strong> an International Wheat Program<br />
S. Rajaram<br />
19 Session 1: Pathogen Biology<br />
19 Biology <strong>of</strong> the <strong>Septoria</strong>/<strong>Stagonospora</strong> Pathogens: An Overview<br />
A.L. Scharen<br />
23 Molecular Analysis <strong>of</strong> a DNA Fingerprint Probe from Mycosphaerella graminicola<br />
S.B. Goodwin <strong>and</strong> J.R. Cavaletto<br />
26 Characterization <strong>of</strong> <strong>Septoria</strong> tritici Variants <strong>and</strong> PCR Assay for Detecting <strong>Stagonospora</strong> nodorum <strong>and</strong><br />
<strong>Septoria</strong> tritici in Wheat<br />
S. Hamza, M. Medini, T. Sassi, S. Abdennour, M. Rouassi, A.B. Salah, M. Cherif, R. Strange, <strong>and</strong> M.<br />
Harrabi<br />
32 Populations <strong>of</strong> <strong>Septoria</strong> spp. Affecting Winter Wheat in the Forest-Steppe Zone <strong>of</strong> the Ukraine<br />
S. Kolomiets<br />
34 <strong>Septoria</strong> passerinii Closely Related to the Wheat Pathogen Mycosphaerella graminicola<br />
S.B. Goodwin <strong>and</strong> V.L. Zismann<br />
37 <strong>Septoria</strong>/<strong>Stagonospora</strong> Leaf Spot <strong>Diseases</strong> on Barley in North Dakota, USA<br />
J.M. Krupinsky <strong>and</strong> B.J. Steffenson (poster)<br />
39 Interrelations among <strong>Septoria</strong> tritici Isolates <strong>of</strong> Varying Virulence<br />
S. Ezrati, S. Schuster, A. Eshel, <strong>and</strong> Z. Eyal (poster)<br />
41 Session 2: The Infection Process<br />
41 <strong>Stagonospora</strong> <strong>and</strong> <strong>Septoria</strong> Pathogens <strong>of</strong> <strong>Cereals</strong>: The Infection Process<br />
B.M. Cunfer<br />
46 Aggressiveness <strong>of</strong> Phaeosphaeria nodorum Isolates <strong>and</strong> Their In Vitro Secretion <strong>of</strong> Cell-Wall-<br />
Degrading Enzymes<br />
P. Halama, F. Lalaoui, V. Dumortier, <strong>and</strong> B. Paul<br />
50 Growth <strong>of</strong> <strong>Stagonospora</strong> nodorum Lesions<br />
A.M. Djurle (poster)<br />
51 Session 3A: Host-Parasite Interactions<br />
51 Genetic Control <strong>of</strong> Avirulence in Mycosphaerella graminicola (Anamorph <strong>Septoria</strong> tritici)<br />
G.H.J. Kema <strong>and</strong> E.C.P. Verstappen<br />
53 Cytogenetics <strong>of</strong> Resistance <strong>of</strong> Wheat to <strong>Septoria</strong> Tritici Leaf Blotch<br />
L.S. Arraiano, A.J. Worl<strong>and</strong>, <strong>and</strong> J.K.M. Brown<br />
54 A Possible Gene-for-Gene Relationship for <strong>Septoria</strong> Tritici Leaf Blotch Resistance in Wheat<br />
P.A. Brading, G.H.J. Kema, <strong>and</strong> J.K.M. Brown<br />
56 Diallel Analysis <strong>of</strong> <strong>Septoria</strong> Tritici Blotch Resistance in Winter Wheat<br />
X. Zhang, S.D. Haley, <strong>and</strong> Y. Jin<br />
59 Analysis <strong>of</strong> the <strong>Septoria</strong> Monitoring Nursery<br />
L. Gilchrist, C. Velazquez, <strong>and</strong> J. Crossa<br />
63 Session 3B: Host Parasite Interactions<br />
63 Host – Parasite Interactions: <strong>Stagonospora</strong> nodorum<br />
E. Arseniuk <strong>and</strong> P.C. Czembor<br />
71 Identification <strong>of</strong> a Molecular Marker Linked to <strong>Septoria</strong> Nodorum Blotch Resistance in Triticum<br />
tauschii Using F2 Bulked Segregant<br />
N.E.A. Murphy, R. Loughman, R. Wilson, E.S. Lagudah, R. Appels, <strong>and</strong> M.G.K. Jones<br />
74 Inheritance <strong>of</strong> <strong>Septoria</strong> Nodorum Blotch Resistance in a Triticum tauschii Accession Controlled by a<br />
Single Gene<br />
N.E.A. Murphy, R. Loughman, R. Wilson, E.S. Lagudah, R. Appels, <strong>and</strong> M.G.K. Jones<br />
77 Session 4: Population Dynamics<br />
77 Population Genetics <strong>of</strong> Mycosphaerella graminicola <strong>and</strong> Phaeosphaeria nodorum<br />
B.A. McDonald, C.C. Mundt, <strong>and</strong> J. Zhan<br />
83 Characterization <strong>of</strong> Less Aggressive <strong>Stagonospora</strong> nodorum Isolates from Wheat<br />
E. Arseniuk, H.S. Tsang, J.M. Krupinsky, <strong>and</strong> P.P. Ueng<br />
85 A Vertically Resistant Wheat Selects for Specifically Adapted Mycosphaerella graminicola Strains<br />
C. Cowger, C.C. Mundt, <strong>and</strong> M.E. H<strong>of</strong>fer<br />
iii
iv<br />
87 Genetic Variability in a Collection <strong>of</strong> <strong>Stagonospora</strong> nodorum Isolates from Western Australia<br />
N.E.A. Murphy, R. Loughman, E.S. Lagudah, R. Appels, <strong>and</strong> M.G.K. Jones<br />
90 Mating Type-Specific PCR Primers for <strong>Stagonospora</strong> nodorum Field Studies<br />
R.S. Bennett, S.-H. Yun, T.Y. Lee, B.G. Turgeon, B. Cunfer, E. Arseniuk, <strong>and</strong> G.C. Bergstrom (poster)<br />
93 Session 5: Epidemiology<br />
93 Epidemiology <strong>of</strong> Mycosphaerella graminicola <strong>and</strong> Phaeosphaeria nodorum: An Overview<br />
M.W. Shaw<br />
98 Spore Dispersal <strong>of</strong> Leaf Blotch Pathogens <strong>of</strong> Wheat (Mycosphaerella graminicola <strong>and</strong> <strong>Septoria</strong> tritici)<br />
C.A. Cordo, M.R. Simón, A.E. Perelló, <strong>and</strong> H.E. Alippi<br />
102 Epidemiology <strong>of</strong> Seedborne <strong>Stagonospora</strong> nodorum: A Case Study on New York Winter Wheat<br />
D.A. Shah <strong>and</strong> G.C. Bergstrom<br />
108 Sessions 6A <strong>and</strong> 6B: Cultural Practices <strong>and</strong> Disease Management<br />
108 Influence <strong>of</strong> Cultural Practices on <strong>Septoria</strong>/<strong>Stagonospora</strong> <strong>Diseases</strong><br />
J.M. Krupinsky<br />
111 Disease Management Using Varietal Mixtures<br />
C.C. Mundt, C. Cowger, <strong>and</strong> M.E. H<strong>of</strong>fer<br />
117 Session 6C: Breeding for Disease Resistance<br />
117 Breeding for Resistance to the <strong>Septoria</strong>/<strong>Stagonospora</strong> Blights <strong>of</strong> Wheat<br />
M. van Ginkel <strong>and</strong> S. Rajaram<br />
127 Breeding for Resistance to <strong>Septoria</strong> <strong>and</strong> <strong>Stagonospora</strong> Blotches in Winter Wheat in the United States<br />
G. Shaner<br />
131 <strong>Septoria</strong> tritici Resistance <strong>of</strong> Wheat Cultivars at Different Growth Stages<br />
M. Díaz de Ackermann, M.M. Kohli, <strong>and</strong> V. Ibañez<br />
134 <strong>Septoria</strong> tritici Resistance Sources <strong>and</strong> Breeding Progress at <strong>CIMMYT</strong>, 1970-99<br />
L. Gilchrist, B. Gomez, R. Gonzalez, S. Fuentes, A. Mujeeb-Kazi, W. Pfeiffer, S. Rajaram, R.<br />
Rodriguez, B. Skovm<strong>and</strong>, M. van Ginkel, <strong>and</strong> C. Velazquez (Field presentation)<br />
140 Selecting Wheat for Resistance to <strong>Septoria</strong>/<strong>Stagonospora</strong> in Patzcuaro, Michoacan, Mexico<br />
R.M. Gonzalez I., S. Rajaram, <strong>and</strong> M. van Ginkel<br />
145 Varieties <strong>and</strong> Advanced Lines Resistant to <strong>Septoria</strong> <strong>Diseases</strong> <strong>of</strong> Wheat in Western Australia<br />
R. Loughman, R.E. Wilson, I.M. Goss, D.T. Foster, <strong>and</strong> N.E.A. Murphy<br />
148 Field Resistance <strong>of</strong> Wheat to <strong>Septoria</strong> Tritici Leaf Blotch, <strong>and</strong> Interactions with Mycosphaerella<br />
graminicola Isolates<br />
J.K.M. Brown, G.H.J. Kema, H.-R. Forrer, E.C.P. Verstappen, L.S. Arraiano, P.A. Brading, E.M. Foster,<br />
A. Hecker, <strong>and</strong> E. Jenny<br />
150 Using Precise Genetic Stocks to Investigate the Control <strong>of</strong> <strong>Stagonospora</strong> nodorum Resistance in Wheat<br />
C.M. Ellerbrook, V. Korzun, <strong>and</strong> A.J. Worl<strong>and</strong> (poster)<br />
154 Evaluating Triticum durum x Triticum tauschii Germplasm for Resistance to <strong>Stagonospora</strong> nodorum<br />
L.R. Nelson <strong>and</strong> M.E. Sorrells (poster)<br />
156 Sources <strong>of</strong> Resistance to <strong>Septoria</strong> passerinii in Hordeum vulgare <strong>and</strong> H. vulgare subsp. spontaneum<br />
H. Toubia-Rahme <strong>and</strong> B.J. Steffenson (poster)<br />
159 S<strong>of</strong>t Red Winter Wheat with Resistance to <strong>Stagonospora</strong> nodorum <strong>and</strong> Other Foliar Pathogens<br />
B.M. Cunfer <strong>and</strong> J.W. Johnson (poster)<br />
160 Partial Resistance to <strong>Stagonospora</strong> nodorum in Wheat<br />
C.G. Du, L.R. Nelson, <strong>and</strong> M.E. McDaniel (poster)<br />
163 Comparison <strong>of</strong> Methods <strong>of</strong> Screening for <strong>Stagonospora</strong> nodorum Resistance in Winter Wheat<br />
D.E. Fraser, J.P. Murphy, <strong>and</strong> S. Leath (poster)<br />
167 Response <strong>of</strong> Winter Wheat Genotypes to Artificial Inoculation with Several <strong>Septoria</strong> tritici<br />
Populations<br />
M. Mincu (poster)<br />
170 Comparison <strong>of</strong> Greenhouse <strong>and</strong> Field Levels <strong>of</strong> Resistance to <strong>Stagonospora</strong> nodorum<br />
S.L. Walker, S. Leath, <strong>and</strong> J.P. Murphy (poster)<br />
173 Session 6D: Chemical Control<br />
173 Adjusting Thresholds for <strong>Septoria</strong> Control in Winter Wheat Using Strobilurins<br />
L.N. Jørgensen, K.E. Henriksen, <strong>and</strong> G.C. Nielsen<br />
177 Concluding Remarks<br />
177 The <strong>Septoria</strong>/<strong>Stagonospora</strong> Blotch <strong>Diseases</strong> <strong>of</strong> Wheat: Past, Present, <strong>and</strong> Future<br />
Z. Eyal (paper presented by A.L. Scharen)<br />
183 List <strong>of</strong> Participants
In Memoriam, Dr. Zahir Eyal<br />
Our friend <strong>and</strong> colleague, Pr<strong>of</strong>essor Zahir Eyal, died Friday, July 30, 1999. Zahir was<br />
intimately involved in all <strong>of</strong> the International <strong>Septoria</strong> Workshops, from the first in 1976 held in<br />
Griffin, Georgia, USA, until the fifth, <strong>and</strong> present, one held in <strong>CIMMYT</strong>, Mexico. He put forward<br />
his many ideas for program <strong>and</strong> participants in a forceful, but thoughtful way, <strong>and</strong> was able to<br />
settle disputes with good humor <strong>and</strong> a smile. Until the last few days <strong>of</strong> his life, Dr. Eyal<br />
continued to work on plans for this <strong>Septoria</strong>/<strong>Stagonospora</strong> workshop.<br />
After finishing agricultural high school in Israel, Zahir went to the USA, where he earned his<br />
B.Sc. degree from Oklahoma State University <strong>and</strong> his Ph.D. from Rutgers. This was followed by a<br />
post-doctoral term at Purdue, where he studied with Jack Schafer <strong>and</strong> the late Ralph Caldwell.<br />
His work on septoria <strong>of</strong> cereals began when he joined the Department <strong>of</strong> Botany at Tel Aviv<br />
University in 1967. He developed an integrated program <strong>of</strong> fundamental <strong>and</strong> applied research<br />
aimed at minimizing the economic impact <strong>of</strong> cereal pathogens, particularly <strong>Septoria</strong> tritici, on<br />
production. He reached out to colleagues in many countries <strong>and</strong> to international organizations,<br />
most especially <strong>CIMMYT</strong>, for cooperation. Over the years, Zahir contributed greatly to those<br />
programs. During his tenure at Tel Aviv University, Pr<strong>of</strong>essor Eyal served two terms as Head <strong>of</strong><br />
the Department <strong>of</strong> Botany (1984-87 <strong>and</strong> 1992-94).<br />
Pr<strong>of</strong>essor Eyal was an enthusiastic teacher, well-loved by students, both undergraduate <strong>and</strong><br />
graduate. He taught in English or in Hebrew with equal facility, sharing his knowledge <strong>and</strong><br />
insights with students <strong>and</strong> faculty during two sabbaticals at Montana State University <strong>and</strong> at<br />
several other institutions. The numerous publications he authored with his students <strong>and</strong> the<br />
important posts those students occupy today attest to the excellence <strong>of</strong> his teaching abilities.<br />
Zahir’s research <strong>and</strong> outreach programs incorporated ideas that were new to his country;<br />
they were solidly anchored in basic science <strong>and</strong> innovative to the end. These programs not only<br />
improved wheat production in Israel but had positive effects on cereal improvement programs<br />
throughout the world. At the time <strong>of</strong> his death, Pr<strong>of</strong>essor Eyal was Director <strong>of</strong> the Institute for<br />
Cereal Crop Improvement at Tel Aviv University, where germplasm <strong>of</strong> wild ancestors <strong>of</strong><br />
cultivated small grains are being preserved, characterized, <strong>and</strong> utilized in breeding improved<br />
cultivars.<br />
Dr. Eyal’s contributions to research, teaching, university administration, <strong>and</strong> international<br />
agriculture are many <strong>and</strong> far reaching. He received the Hazera Seed Co. Melamed Award in 1968,<br />
the A.C. Cohen Award in 1978, <strong>and</strong> in 1995 was made a Fellow <strong>of</strong> the American<br />
Phytopathological Society. Pr<strong>of</strong>essor Eyal served as President <strong>of</strong> the Israeli Phytopathological<br />
Society from 1979 to 1982. He will be fondly remembered <strong>and</strong> sadly missed by his multitude <strong>of</strong><br />
friends, colleagues, <strong>and</strong> students throughout the world.<br />
v
vi<br />
Foreword<br />
In the mid-1970s the idea <strong>of</strong> holding a septoria workshop began to take hold among a small<br />
group <strong>of</strong> scientists in the USA. They were interested in exchanging ideas <strong>and</strong> finding ways to<br />
manage the septoria diseases that affect wheat <strong>and</strong> other cereals all over the world. The first<br />
workshop was organized in a matter <strong>of</strong> a few months <strong>and</strong> held in Griffin, Georgia, in 1976.<br />
Among the 50 scientists who attended were a few researchers from outside the US. The<br />
enthusiasm <strong>of</strong> that first workshop led to the development <strong>of</strong> the second, which was a truly<br />
international meeting attended by more than 100 scientists from many countries, held in<br />
Bozeman, Montana, in 1983.<br />
Since then, international septoria workshops have been held about every five years: in<br />
Zurich, Switzerl<strong>and</strong>, in 1989; in Radzikow, Pol<strong>and</strong>, in 1994; <strong>and</strong> this year at <strong>CIMMYT</strong> in El<br />
Batan, Mexico. Each workshop has exp<strong>and</strong>ed the network <strong>of</strong> scientists who share their<br />
knowledge <strong>and</strong> pose the many questions that remain to be solved about these diseases <strong>and</strong> their<br />
management.<br />
The Zurich workshop had increased participation by workers from Europe <strong>and</strong> Africa. The<br />
Radzikow workshop brought increased participation from scientists in eastern Europe. The early<br />
workshops focused on the biology <strong>of</strong> the pathogens <strong>and</strong> breeding strategies, subjects in which<br />
there remain many unanswered questions. The 1994 workshop <strong>and</strong> the current one emphasize<br />
molecular approaches to the genetics <strong>of</strong> the pathogens.<br />
The Fifth International Workshop provides another opportunity to focus on the <strong>Septoria</strong>/<br />
<strong>Stagonospora</strong> diseases, but also to see them in the context <strong>of</strong> the worldwide programs <strong>of</strong><br />
<strong>CIMMYT</strong>, which emphasize collaboration with developing countries with the aim <strong>of</strong> developing<br />
stable high yielding wheat varieties that possess durable resistance to the diseases.<br />
This workshop also gives us the opportunity to remember our friend <strong>and</strong> colleague, Zahir<br />
Eyal, who passed away not long ago. An integral part <strong>of</strong> the program development process <strong>and</strong><br />
the discussions at each workshop, he organized the scientific program for this workshop as well.<br />
Zahir Eyal was an enthusiastic supporter <strong>of</strong> the septoria workshops <strong>and</strong> the international<br />
exchange <strong>of</strong> ideas. He will be missed.<br />
We would like to express our appreciation for the efforts <strong>of</strong> Ravi Singh, Maarten van Ginkel,<br />
<strong>and</strong> Linda Ainsworth, who organized the workshop. We wish to thank Diana Godínez, María<br />
Luisa Varela, <strong>and</strong> Laura Rodríguez for managing the logistical support. We also recognize the<br />
efforts <strong>of</strong> Arnoldo Amaya, María Garay, Lucy Gilchrist, Monique Henry, Gilberto Hernández,<br />
Reynaldo Villareal, Juan José Joven, Marcelo Ortíz, Eliot Sánchez, Kelly Cassaday, Miguel<br />
Mellado, Wenceslao Almazán, <strong>and</strong> Antonio Luna, as well as many other members <strong>of</strong> <strong>CIMMYT</strong><br />
staff who contributed to the success <strong>of</strong> this event.<br />
The International Organizing Committee<br />
September 21, 1999
Opening Remarks<br />
Historical Aspects <strong>and</strong> Future Challenges <strong>of</strong> an<br />
International Wheat Program<br />
S. Rajaram<br />
Wheat Program, <strong>CIMMYT</strong>, El Batan, Mexico<br />
I am immensely honored <strong>and</strong> grateful to the organizing committee <strong>of</strong> the 5 th International <strong>Septoria</strong> Workshop for asking<br />
me to deliver this lecture in the opening session. Even though my presentation is very broad <strong>and</strong> covers many issues, I assure<br />
you that I have been involved in breeding for resistance to septoria tritici blotch for at least 25 years, with some remarkable<br />
success. In this attempt, I would like to recognize the contribution <strong>of</strong> Pr<strong>of</strong>. Zahir Eyal, who served as a consultant on septoria<br />
issues to <strong>CIMMYT</strong> in the 1970s <strong>and</strong> 1980s. Indeed, he <strong>and</strong> I have some common intellectual roots through Pr<strong>of</strong>. Ralph<br />
Caldwell <strong>of</strong> Purdue University. Pr<strong>of</strong>. Eyal’s untimely death <strong>and</strong> departure from the scientific community is a loss to us all. I<br />
dedicate this opening lecture to him.<br />
Wheat is the most widely<br />
grown <strong>and</strong> consumed food crop in<br />
the world. It is the staple food <strong>of</strong><br />
nearly 35% <strong>of</strong> the world<br />
population, <strong>and</strong> dem<strong>and</strong> for wheat<br />
will grow faster than for any other<br />
major crop. The forecasted global<br />
dem<strong>and</strong> for wheat in the year 2020<br />
varies between 840 (Rosegrant et<br />
al., 1995) to 1050 million tons<br />
(Kronstad, 1998). To reach this<br />
target, global production will need<br />
to increase 1.6 to 2.6% annually<br />
from the present production level<br />
<strong>of</strong> 560 million tons. Increases in<br />
realized grain yield have provided<br />
about 90% <strong>of</strong> the growth in world<br />
cereal production since 1950<br />
(Mitchell et al., 1997) <strong>and</strong> by the<br />
first decade <strong>of</strong> the next century<br />
most <strong>of</strong> the increase needed in<br />
world food production must come<br />
from higher absolute yields<br />
(Ruttan, 1993). For wheat, the<br />
global average grain yield must<br />
increase from the current 2.5 t/ha<br />
to 3.8 t/ha. In 1995, only 18<br />
countries world worldwide had<br />
average wheat grain yields <strong>of</strong> more<br />
than 3.8 t/ha, the majority located<br />
in Northern Europe (<strong>CIMMYT</strong>,<br />
1996).<br />
The formidable challenge to<br />
meet this dem<strong>and</strong> is not new to<br />
agricultural scientists who have<br />
been involved in the development<br />
<strong>of</strong> improved wheat production<br />
technologies for the past half<br />
century. For all developing<br />
countries, wheat yields have grown<br />
at an average annual rate <strong>of</strong> over<br />
2% between 1961 <strong>and</strong> 1994<br />
(<strong>CIMMYT</strong>, 1996). In Western<br />
Europe <strong>and</strong> North America the<br />
annual rate <strong>of</strong> growth for wheat<br />
yield was 2.7% from 1977 to 1985,<br />
falling to 1.5% from 1986 to 1995.<br />
Recent data have indicated a<br />
decrease in the productivity gains<br />
being achieved by major wheat<br />
producing countries (Brown, 1997).<br />
In Western Europe, where the<br />
highest average wheat grain yield<br />
is obtained in the Netherl<strong>and</strong>s<br />
(8.6 t/ha), yield increased from 5 to<br />
6 t/ha in five years, but it took<br />
more than a decade to raise yields<br />
from 6 to 7 t/ha. Worldwide,<br />
annual wheat grain yield growth<br />
decreased from 3.0% between 1977-<br />
1985, to 1.6% from 1986-1995,<br />
excluding the USSR (<strong>CIMMYT</strong>,<br />
1996). Degradation <strong>of</strong> the l<strong>and</strong><br />
resource base, together with a<br />
slackening <strong>of</strong> research investment<br />
<strong>and</strong> infrastructure, have<br />
1<br />
contributed to this decrease<br />
(Pingali <strong>and</strong> Heisey, 1997). Whether<br />
production constraints are affected<br />
by physiological or genetic limits is<br />
hotly debated, but future increases<br />
in food productivity will require<br />
substantial research <strong>and</strong><br />
development investment to<br />
improve the pr<strong>of</strong>itability <strong>of</strong> wheat<br />
production systems through<br />
enhancing input efficiencies. Due to<br />
a continuing necessity for multidisciplinary<br />
team efforts in plant<br />
breeding, <strong>and</strong> the rapidly changing<br />
development <strong>of</strong> technologies, three<br />
overlapping avenues can be<br />
considered for raising the yield<br />
frontier in wheat: continued<br />
investments in “conventional<br />
breeding” methods; use <strong>of</strong> current<br />
<strong>and</strong> exp<strong>and</strong>ed genetic diversity;<br />
<strong>and</strong> investigation <strong>and</strong><br />
implementation <strong>of</strong> biotechnology<br />
assisted plant breeding.<br />
Conventional Wheat<br />
Breeding<br />
It is likely that gains to be<br />
achieved from conventional<br />
breeding will continue to be<br />
significant for the next two decades<br />
or more (Duvick, 1996), but these
2 Opening Remarks — S. Rajaram<br />
are likely to come at a higher<br />
research cost than in the past. In<br />
recent surveys <strong>of</strong> wheat breeders<br />
(Braun et al., 1998; Rejesus et al.,<br />
1996), more than 80% <strong>of</strong><br />
respondents expressed concern that<br />
plant variety protection (PVP) <strong>and</strong><br />
plant or gene patents will restrict<br />
access to germplasm. This may<br />
have deleterious consequences for<br />
future breeding success.<br />
Rasmusson (1996) has stated that<br />
nearly half <strong>of</strong> the progress made by<br />
breeders in the past can be<br />
attributed to germplasm exchange.<br />
Regional <strong>and</strong> international<br />
nurseries have been an efficient<br />
means <strong>of</strong> gathering data from<br />
varied environments <strong>and</strong> exposing<br />
germplasm to diverse pathogen<br />
selection pressures, while<br />
providing access <strong>and</strong> exchange <strong>of</strong><br />
germplasm. Breeders utilize these<br />
cooperative nurseries extensively<br />
in their crossing programs (Braun<br />
et al., 1998). However, the number<br />
<strong>of</strong> cooperatively distributed wheat<br />
yield <strong>and</strong> screening nurseries has<br />
been greatly reduced during the<br />
past decade.<br />
Investments needed for<br />
breeding efforts will increase with<br />
increasing yield levels. Further,<br />
progress to develop higher yielding<br />
cultivars is reduced with every<br />
objective added to a breeding<br />
program. Though the list <strong>of</strong><br />
important traits may get longer <strong>and</strong><br />
longer, little if any assistance has<br />
been provided by economists to<br />
prioritize breeding objectives.<br />
Considering that a wheat breeding<br />
program like <strong>CIMMYT</strong> allocates<br />
around 60% <strong>of</strong> its resources to<br />
durable resistance breeding, the<br />
need for research in this field is<br />
obvious. Due to high costs, we see<br />
durable resistance breeding as one<br />
<strong>of</strong> the first fields where<br />
transformation should be applied<br />
by breeders through introgression<br />
<strong>of</strong> one or more genes controlling<br />
disease resistance.<br />
Adoption <strong>of</strong> <strong>CIMMYT</strong>-<br />
Based Germplasm<br />
<strong>CIMMYT</strong>’s breeding<br />
methodology is tailored to develop<br />
widely adapted, disease resistant<br />
germplasm with high <strong>and</strong> stable<br />
yield across a wide range <strong>of</strong><br />
environments. The impact <strong>of</strong> this<br />
approach has been significant.<br />
The total spring bread wheat<br />
(Triticum aestivum L.) area in<br />
developing countries, excluding<br />
China, is around 63 million ha, <strong>of</strong><br />
which 36 million ha or 58% are<br />
planted to varieties derived from<br />
<strong>CIMMYT</strong> germplasm (Table 1)<br />
(Byerlee <strong>and</strong> Moya, 1993;<br />
Rajaram, 1995). During the 1966-90<br />
period, 1317 bread wheat cultivars<br />
were released by developing<br />
countries, <strong>of</strong> which 70% were either<br />
direct releases from <strong>CIMMYT</strong><br />
advanced lines or had at least one<br />
<strong>CIMMYT</strong> parent (Byerlee <strong>and</strong><br />
Moya, 1993). For the 1986-90<br />
period, 84% <strong>of</strong> all bread wheat<br />
cultivars released in developing<br />
countries had <strong>CIMMYT</strong> germplasm<br />
in their pedigrees. Simultaneously<br />
the use <strong>of</strong> dwarfing genes has<br />
continued to increase over time.<br />
Today, regardless <strong>of</strong> the type <strong>of</strong><br />
wheat, more than 90% <strong>of</strong> all wheat<br />
varieties released in developing<br />
countries are semidwarfs, which<br />
covered 70% <strong>of</strong> the total wheat area<br />
in developing countries by the end<br />
<strong>of</strong> 1990 (Byerlee <strong>and</strong> Moya, 1993).<br />
The continuous adoption <strong>of</strong><br />
semidwarf spring wheat cultivars<br />
in the post-Green Revolution<br />
period (1977-90) resulted in about<br />
15.5 million tons <strong>of</strong> additional<br />
wheat production in 1990, valued<br />
at about US$ 3 billion, <strong>of</strong> which<br />
50%, or US$ 1.5 billion, is attributed<br />
to the adoption <strong>of</strong> new Mexican<br />
semidwarf wheat cultivars (Byerlee<br />
<strong>and</strong> Moya, 1993). In 1990, an<br />
estimated 93% <strong>of</strong> the total spring<br />
bread wheat production in<br />
developing countries, excluding<br />
China, came from semidwarf<br />
spring wheats, which covered<br />
about 83% <strong>of</strong> the total spring bread<br />
wheat area in developing countries<br />
(Byerlee <strong>and</strong> Moya, 1993).<br />
Table 1. Origin <strong>of</strong> spring bread wheat varieties in<br />
developing countries.<br />
<strong>CIMMYT</strong> <strong>CIMMYT</strong><br />
NARS cross<br />
<strong>CIMMYT</strong> No<br />
cross parents ancestor <strong>CIMMYT</strong><br />
1966-90 45% 28% 3% 24% *<br />
1991-97 58% 30% 3% 9%<br />
* Estimated.<br />
Note: Excluding China. NARS=national agricultural<br />
research system.<br />
The cornerstone <strong>of</strong> <strong>CIMMYT</strong>’s<br />
breeding methodology is targeted<br />
breeding for the megaenvironments,<br />
the use <strong>of</strong> a diverse<br />
gene pool for crossing, shuttle<br />
breeding, selection for yield under<br />
optimum conditions, <strong>and</strong> multilocational<br />
testing to identify<br />
superior germplasm with good<br />
disease resistance. In this paper we<br />
would like to present some recent<br />
developments in <strong>CIMMYT</strong>’s Wheat<br />
Program.<br />
Targeted breeding: The<br />
mega-environment concept<br />
To address the needs <strong>of</strong> diverse<br />
wheat growing areas, <strong>CIMMYT</strong><br />
introduced in 1988 the concept <strong>of</strong><br />
mega-environments (ME) (Rajaram<br />
et al., 1994). Mega-environments<br />
are defined as a broad, not<br />
necessarily contiguous areas,<br />
occurring in more than one country<br />
<strong>and</strong> frequently transcontinental,<br />
defined by similar biotic <strong>and</strong><br />
abiotic stresses, cropping system<br />
requirements, consumer<br />
preferences, <strong>and</strong>, for convenience,
y volume <strong>of</strong> production.<br />
Germplasm generated for a given<br />
ME is useful throughout that<br />
environment, accommodating<br />
major stresses but perhaps not all<br />
the significant secondary stresses.<br />
Within an ME, millions <strong>of</strong> hectares<br />
are addressed with a certain degree<br />
<strong>of</strong> homogeneity as relates to wheat.<br />
By 1993, 12 ME had been defined, 6<br />
for spring wheats (ME1-ME6), 3 for<br />
facultative wheats (ME7-ME9), <strong>and</strong><br />
3 for winter wheats (ME9-ME12).<br />
Details <strong>of</strong> each ME are given in<br />
Table 2.<br />
Table 2. Classification <strong>of</strong> megaenvironments (MEs) used by the <strong>CIMMYT</strong> Wheat Program.<br />
Historical Aspects <strong>and</strong> Future Challenges <strong>of</strong> an International Wheat Program 3<br />
Use <strong>of</strong> diverse genepools to<br />
maintain genetic diversity<br />
Recent surveys conducted by<br />
the <strong>CIMMYT</strong> Economics Program<br />
have found that 58% <strong>of</strong> all wheat<br />
varieties in developing countries<br />
derive from <strong>CIMMYT</strong> germplasm;<br />
this percentage rises to more than<br />
80%, if varieties with parents <strong>of</strong><br />
<strong>CIMMYT</strong> origin are also included<br />
(Table 1). This spectacular success<br />
puts an enormous burden on<br />
<strong>CIMMYT</strong> to continually diversify<br />
its germplasm base for resistance<br />
<strong>and</strong> stability parameters.<br />
Broad-based plant germplasm<br />
resources are imperative for a<br />
sound <strong>and</strong> successful breeding<br />
program. Utmost attention is given<br />
to the genetic diversity within<br />
<strong>CIMMYT</strong> germplasm to minimize<br />
the risk <strong>of</strong> genetic vulnerability,<br />
since it is grown on large areas <strong>and</strong><br />
is widely used by national<br />
programs. I believe that the use <strong>of</strong><br />
genetically diverse material is<br />
m<strong>and</strong>atory to increase yield<br />
potential <strong>and</strong> yield stability in the<br />
future. In any year 500-800 parental<br />
lines are considered for crossing.<br />
Major Representative<br />
Year<br />
breeding<br />
Area Moisture Temperature Growth breeding locations/ began at<br />
ME Latitudea (m ha) b regimec regimed habit Sowne objectivesf, g regions <strong>CIMMYT</strong><br />
SPRING WHEAT<br />
1 Low 32.0 Low rainfall, Temperate Spring A Resistance to Yaqui Valley, 1945<br />
irrigated lodging, SR, LR, YR Mexico; Indus<br />
Valley, Pakistan;<br />
Gangetic Valley,<br />
India; Nile Valley,<br />
Egypt<br />
2 Low 10.0 High rainfall Temperate Spring A As for ME1 + North African 1972<br />
resistance to Coast, Highl<strong>and</strong>s <strong>of</strong><br />
YR, <strong>Septoria</strong> East Africa, Andes,<br />
spp., sprouting <strong>and</strong> Mexico<br />
3 Low 1.7 High rainfall Temperate Spring A As for ME2 + Passo Fundo, 1974<br />
acid soil<br />
tolerance<br />
Brazil<br />
4A Low 10.0 Low rainfall, Temperate Spring A Resistance to Aleppo, Syria; 1974<br />
winter drought, Settat, Morocco<br />
dominant <strong>Septoria</strong> spp., YR<br />
4B Low 5.8 Low rainfall, Temperate Spring A Resistance to Marcos Juárez, 1974<br />
summer drought, Argentina<br />
dominant <strong>Septoria</strong> spp.,<br />
Fusarium spp.,<br />
LR, SR<br />
4C Low 5.8 Mostly Hot Spring A Resistance to Indore, India 1974<br />
residual drought, <strong>and</strong><br />
moisture heat in seedling<br />
stage<br />
5A Low 3.9 High rainfall Hot Spring A Resistance to Joydepur, 1981<br />
/ irrigated, heat, Helmin- Bangladesh;<br />
humid thosporium<br />
spp., Fusarium<br />
spp., sprouting<br />
Londrina, Brazil<br />
5B Low 3.2 Irrigated , Hot Spring A Resistance to Gezira, Sudan; 1975<br />
low humidity heat <strong>and</strong> SR Kano, Nigeria<br />
6 High 5.4 Moderate Temperate Spring S Resistance to Harbin, China 1989<br />
rainfall/ SR, LR, Helminsummer<br />
thosporium spp.,<br />
dominant Fusarium spp.,<br />
sprouting,<br />
photoperiod<br />
sensitivity<br />
(cont’d.)
4 Opening Remarks — S. Rajaram<br />
Table 2. Continued.<br />
Major Representative<br />
Year<br />
breeding<br />
Area Moisture Temperature Growth breeding locations/ began at<br />
ME Latitudea (m ha) b regimec regimed habit Sowne objectivesf, g regions <strong>CIMMYT</strong><br />
WINTER/FACULTATIVE WHEAT<br />
7 High – Irrigated Moderate Facultative A Rapid grain Zhenzhou, China 1986<br />
cold fill, resistance<br />
to cold, YR, PM,<br />
BYD<br />
8A High – High rainfall Moderate Facultative A Resistance to Chillan, Chile 1986<br />
/ irrigated, cold cold, YR, <strong>Septoria</strong><br />
long season spp.<br />
8B High – High rainfall Moderate Facultative A Resistance to Edirne, Turkey 1986<br />
/ irrigated, cold <strong>Septoria</strong> spp.,<br />
short season YR, PM, Fusarium<br />
spp., sprouting<br />
9 High – Low rainfall Moderate Facultative A Resistance to Diyarbakir, Turkey 1986<br />
cold cold, drought<br />
10 High – Irrigated Severe cold Winter A Resistance to<br />
winterkill, YR,<br />
LR, PM, BYD<br />
Beijing, China 1986<br />
11A High – High rainfall Moderate Winter A Resistance to Temuco, Chile 1986<br />
/irrigated, cold <strong>Septoria</strong> spp.,<br />
long season Fusarium spp.,<br />
YR, LR, PM<br />
11B High – High rainfall Severe cold Winter A Resistance to Lovrin, Romania 1986<br />
/ irrigated, LR, SR, PM,<br />
short season winterkill,<br />
sprouting<br />
12 High – Low rainfall Severe cold Winter A Resistance to<br />
winterkill,<br />
drought, YR,<br />
bunts<br />
Ankara, Turkey 1986<br />
Source: Adapted from Rajaram et. al. (1995).<br />
a Low = less than about 35-40 degrees.<br />
b Exact area distribution for winter/facultative wheat is not available.<br />
c Refers to rainfall just before <strong>and</strong> during the crop cycle. High = >500mm; low = 17.5°C; cold =
wheat to increase grain size. The<br />
six highest yielding lines derived<br />
from this program outyielded their<br />
breadwheat parent by 5-20% in<br />
yield trials in Cd. Obregon, Mexico.<br />
Shuttle breeding within<br />
Mexico<br />
Young <strong>and</strong> Frey (1994) provide<br />
two factors that influence the<br />
success <strong>of</strong> a shuttle program: a) the<br />
use <strong>of</strong> a germplasm pool<br />
encompassing genotypes with<br />
broad adaptation, <strong>and</strong> b) the use <strong>of</strong><br />
selection environments eliciting<br />
different responses from plant<br />
types. They also state that the<br />
wheat breeding program <strong>of</strong> N.E.<br />
Borlaug met these conditions.<br />
When Borlaug started the shuttle<br />
breeding approach in 1945, his only<br />
objective was to speed up breeding<br />
for stem rust resistance. Since then,<br />
segregating populations have been<br />
shuttled 100 times between the two<br />
environmentally contrasting sites<br />
in Mexico, Cd. Obregon <strong>and</strong> Toluca<br />
(Braun et al., 1992).<br />
Some <strong>of</strong> the salient points <strong>of</strong><br />
this shuttle breeding program are:<br />
• Cd. Obregon is situated at 28 o N<br />
at 40 masl, in the sunny, fertile,<br />
<strong>and</strong> irrigated Yaqui Valley <strong>of</strong><br />
Sonora. Wheats are planted in<br />
November when temperatures<br />
are low <strong>and</strong> harvested in April/<br />
May when temperatures are<br />
high. The yield potential <strong>of</strong><br />
location is high (±10 t/ha);<br />
wheat diseases are limited to<br />
only leaf rust, Karnal bunt, <strong>and</strong><br />
black point.<br />
• The Toluca location is<br />
characterized by high humidity<br />
(precipitation: ±1000 mm). The<br />
nursery is planted in May/June<br />
when temperatures are high <strong>and</strong><br />
harvested in October when they<br />
are low. High humidity causes<br />
Historical Aspects <strong>and</strong> Future Challenges <strong>of</strong> an International Wheat Program 5<br />
incidence <strong>of</strong> many diseases<br />
including rust, septorias, BYD,<br />
<strong>and</strong> fusarium.<br />
An important result <strong>of</strong> shuttle<br />
breeding was the selection <strong>of</strong><br />
photo-insensitive wheat genotypes.<br />
Initially, selection for photoperiod<br />
insensitivity was unconscious, but<br />
only this trait permitted the wide<br />
spread <strong>of</strong> the Mexican semidwarfs<br />
(Borlaug, 1995). Today, this trait has<br />
been incorporated into basically all<br />
spring wheat cultivars grown<br />
below 48 o latitude <strong>and</strong> is now also<br />
spreading to wheat areas above 48 o<br />
N (Worl<strong>and</strong> et al., 1994).<br />
Multi-locational testing<br />
<strong>and</strong> wide adaptation<br />
About 1500 sets <strong>of</strong> yield trials<br />
<strong>and</strong> screening nurseries consisting<br />
<strong>of</strong> around 4000 advanced bread<br />
wheat lines are annually sent to<br />
more than 200 locations. Multilocational<br />
testing plays a key role in<br />
identifying the best performing<br />
entries for crossing. Since the<br />
shuttle program permits two full<br />
breeding cycles a year, it takes<br />
around five to six years from<br />
crossing to international<br />
distribution <strong>of</strong> advanced lines to<br />
cooperators. This “recurrent<br />
selection program” ensures<br />
continuous <strong>and</strong> rapid pyramiding<br />
<strong>of</strong> desirable genes.<br />
Ceccarelli (1989) pointed out<br />
that the widespread cultivation <strong>of</strong><br />
some wheat cultivars should not be<br />
taken as a demonstration <strong>of</strong> wide<br />
adaptation, since a large fraction <strong>of</strong><br />
these areas are similar or made<br />
similar by use <strong>of</strong> irrigation <strong>and</strong>/or<br />
fertilizer. Therefore, the term wide<br />
adaptation has been used mainly to<br />
describe geographical rather than<br />
environmental differences. If this is<br />
true, the genotypic variation<br />
should be considerably higher than<br />
GxE interaction in ANOVAs <strong>of</strong><br />
<strong>CIMMYT</strong> trials. Braun et al. (1992)<br />
showed that this is not the case.<br />
When subsets <strong>of</strong> locations were<br />
grouped by geographical <strong>and</strong>/or<br />
environmental similarities, GxE<br />
interaction was mostly greater than<br />
the genotypic variance. The<br />
environmental diversity <strong>of</strong> sites<br />
where <strong>CIMMYT</strong>’s 21st<br />
International Bread Wheat<br />
Screening Nursery was grown <strong>and</strong><br />
the diversity among genotypes in<br />
this nursery were demonstrated by<br />
Bull et al. (1994). They classified<br />
similarities among environments<br />
by forming subsets <strong>of</strong> genotypes<br />
from the total dataset <strong>and</strong><br />
comparing them with the<br />
classification based on the<br />
remaining genotypes. Using this<br />
procedure they concluded that it<br />
was not possible to form a stable<br />
grouping <strong>of</strong> environments, because<br />
little or no relationship existed<br />
among them.<br />
Conclusions drawn from trials<br />
carried out on research stations are<br />
always open to critics who argue<br />
that these results do not necessarily<br />
reflect conditions in farmers’ fields.<br />
However, the wide acceptance <strong>of</strong><br />
<strong>CIMMYT</strong> germplasm by farmers in<br />
MEs 1-5 does not support the view<br />
that the wide adaptation <strong>of</strong><br />
<strong>CIMMYT</strong> germplasm is based on<br />
geographical rather than<br />
environmental differences.<br />
Breeding for High Yield<br />
Potential <strong>and</strong> Enhanced<br />
Stability<br />
Selection <strong>of</strong> segregating<br />
populations <strong>and</strong> consequent yield<br />
testing <strong>of</strong> advanced lines are<br />
paramount for identifying high<br />
yielding <strong>and</strong> input responsive<br />
wheat genotypes. The increase in<br />
yield potential <strong>of</strong> <strong>CIMMYT</strong>
6 Opening Remarks — S. Rajaram<br />
cultivars developed since the 1960s<br />
is shown in Figure 1 (Rees et al.,<br />
1993). The average increase per<br />
year was 0.9%, <strong>and</strong> there is no<br />
evidence that a yield plateau has<br />
been reached. This progress in<br />
increasing genetic yield potential is<br />
closely associated with an increase<br />
in photosynthetic activity (Rees et<br />
al., 1993.). Both photosynthetic<br />
activity <strong>and</strong> yield potential<br />
increased over the 30-year period<br />
by some 25%. These findings may<br />
have major implications for<br />
<strong>CIMMYT</strong>’s future selection strategy<br />
since there is evidence that wheat<br />
genotypes with a higher<br />
photosynthetic rate have lower<br />
canopy temperature, which can be<br />
easily, quickly, <strong>and</strong> cheaply<br />
measured using a h<strong>and</strong>-held<br />
thermometer. If this is verified in<br />
future trials, this trait may be used<br />
by breeders to increase selection<br />
efficiency for yield potential. This<br />
technique may be particularly<br />
useful in selecting wheat genotypes<br />
adapted to environments where<br />
heat is a production constraint.<br />
Yield per se is closely associated<br />
with input responsiveness.<br />
Increasing the input efficiency at<br />
low production levels can shift<br />
crossover points, provided they<br />
exist, <strong>and</strong> enhance residual effects<br />
<strong>of</strong> high genetic yield potential.<br />
Furthermore, combining input<br />
efficiency with high yield potential<br />
will allow farmers to benefit from<br />
such cultivars over a wide range <strong>of</strong><br />
input levels. The increase in<br />
nitrogen use efficiency is shown in<br />
Figure 2 (Ortiz-Monasterio et al.,<br />
1995).<br />
<strong>CIMMYT</strong>’s breeding strategy<br />
has resulted in the development <strong>of</strong><br />
widely grown varieties, such as<br />
Siete Cerros, Anza, Sonalika, <strong>and</strong><br />
Seri 82, which at their peak were<br />
grown on several million<br />
hectares. Seri 82 was released for<br />
irrigated as well as rainfed<br />
environments. Reynolds et al.<br />
(1994) reported that Seri 82 was<br />
the highest yielding entry in the<br />
1st <strong>and</strong> 2nd International Heat<br />
Stress Genotype Experiment.<br />
Seri 82 can be considered as the<br />
first wheat genotype truly<br />
adapted to several MEs,<br />
particularly to ME1, ME2, ME4,<br />
<strong>and</strong> ME5. A comparison between<br />
Seri 82 <strong>and</strong> Pastor, a recently<br />
developed <strong>CIMMYT</strong> cultivar,<br />
demonstrates the progress made<br />
in widening adaptation during<br />
the last ten years. Figure 3 shows<br />
the performance <strong>of</strong> Pastor<br />
(Pfau/Seri//Bow) in <strong>CIMMYT</strong>’s<br />
13th Elite Spring Wheat Yield<br />
Nursery. In 50 trials grown in all<br />
6 MEs, Pastor yielded<br />
significantly (P=0.01) lower than<br />
the highest yielding entry only<br />
in eight trials. This figure also<br />
demonstrates that Pastor has no<br />
tendency to crossover at any<br />
yield level. While we do not<br />
reject that crossover may exist<br />
for some cultivars, Pastor <strong>and</strong><br />
Seri 82 are clear examples that it<br />
is possible to combine abiotic<br />
stress tolerance with high yield<br />
potential. Figure 4 shows the<br />
yield difference between Seri 82<br />
<strong>and</strong> Pastor. Only in 16 out <strong>of</strong> 50<br />
trials did Seri outyield Pastor.<br />
The latter cultivar proves that<br />
breeding for wide adaptation<br />
has not yet reached its limit.<br />
Apart from the physiological<br />
basis <strong>of</strong> yield potential, the yield<br />
gains in <strong>CIMMYT</strong> wheats are<br />
due to the utilization <strong>of</strong> certain<br />
genetic resources. The<br />
germplasm has been paramount<br />
to increase yield in <strong>CIMMYT</strong>’s<br />
Wheat Program <strong>and</strong> in<br />
Yield in kg/ha<br />
8000<br />
Y=-104607+56.56x<br />
7500<br />
7000<br />
R=0.96***<br />
Gain/year 0.9%<br />
6500<br />
6000<br />
1960<br />
1965 1970 1975 1980 1985 1990<br />
Variety year <strong>of</strong> release<br />
Figure 1. Mean grain yields for the historical series<br />
<strong>of</strong> bread wheat varieties for the year 1990-93 at Cd.<br />
Obregon, Mexico (data from Rees et al., 1993).<br />
Grain yield in kg/ha<br />
8000<br />
7000<br />
6000<br />
5000<br />
4000<br />
3000<br />
2000<br />
50<br />
300 kg N<br />
ON<br />
55 60 65 70 75 80 85<br />
Genotype year <strong>of</strong> release<br />
Figure 2. Grain yield <strong>of</strong> the historical series <strong>of</strong> bread<br />
wheats at Cd. Obregon, Mexico, at 0 <strong>and</strong> 300 kg/ha N<br />
application (data from J.I. Ortiz-Monasterio et al.,<br />
1995).<br />
Yield (kg/ha)<br />
12000<br />
10000<br />
8000<br />
6000<br />
4000<br />
2000<br />
0<br />
0<br />
2000 4000 6000 8000 10000<br />
Location mean yield (kg/ha)<br />
Maximum yield<br />
Pastor significantly different<br />
from highest yield in entry<br />
Mean<br />
Figure 3. Yield <strong>of</strong> Pastor at 50 locations <strong>of</strong> the 13th<br />
ESWYT.<br />
Yield (kg/ha)<br />
4000<br />
3000 Pastor<br />
2000<br />
1000<br />
0<br />
-1000<br />
Seri 82<br />
-2000<br />
-3000<br />
600<br />
1000<br />
2000<br />
3000<br />
4000<br />
5000<br />
6000<br />
7000<br />
8000<br />
Location mean yield (kg/ha)<br />
9000<br />
10000<br />
Figure 4. Yield difference between Pastor <strong>and</strong> Seri 82<br />
at 50 locations <strong>of</strong> the 13th ESWYT.
Minnesota’s barley program<br />
(Rasmusson, 1996). Some examples<br />
are listed next.<br />
• The incorporation <strong>of</strong> Norin 10 x<br />
Brevor germplasm not only<br />
produced dwarf wheats, but also<br />
simultaneously gave high yield.<br />
• Spring <strong>and</strong> winter crosses<br />
involving the variety Kavkaz<br />
resulted in Veerys, representing<br />
high yield potential <strong>and</strong><br />
enhanced yield stability<br />
(Figure 5).<br />
• The incorporation <strong>of</strong> the Lr19<br />
gene <strong>and</strong> Aegilops squarrosaderived<br />
synthetic wheats has<br />
further increased yield potential.<br />
The variety Super Seri has the<br />
Lr19 gene (Figure 6) <strong>and</strong> a<br />
derivative <strong>of</strong> Ae. squarrosa is<br />
given in Table 3.<br />
Breeding for durable disease<br />
resistance<br />
From the beginning,<br />
incorporating durable, non-specific<br />
disease resistance into <strong>CIMMYT</strong><br />
germplasm was a high priority,<br />
since breeding widely adapted<br />
germplasm with stable yields<br />
without adequate resistance against<br />
the major diseases would be<br />
impossible. The concept goes back<br />
to Niederhauser et al. (1954),<br />
Borlaug (1966), <strong>and</strong> Caldwell<br />
(1968), who advocated developing<br />
general resistance in the <strong>CIMMYT</strong><br />
Yield (t/ha)<br />
10<br />
8<br />
6<br />
4<br />
2<br />
ISWYN 15<br />
2 3 5<br />
Environments (t/ha)<br />
7 9<br />
Figure 5. Performance <strong>of</strong> Veery in 73 global<br />
environments (ISWYN 15).<br />
Historical Aspects <strong>and</strong> Future Challenges <strong>of</strong> an International Wheat Program 7<br />
program versus the specific or<br />
hypersensitive type. Very diverse<br />
sources <strong>of</strong> resistance for rusts <strong>and</strong><br />
other diseases are intentionally<br />
used in the crossing program. The<br />
major sources are germplasm from<br />
national programs, advanced<br />
<strong>CIMMYT</strong> lines, germplasm<br />
received from the <strong>CIMMYT</strong> or<br />
other genebanks, <strong>and</strong> <strong>CIMMYT</strong>’s<br />
wide crossing program.<br />
<strong>CIMMYT</strong>’s strategy in the case<br />
<strong>of</strong> cereal rusts is to breed for<br />
general resistance (slow rusting)<br />
based on historically proven stable<br />
genes. This non-specific resistance<br />
can be further diversified by<br />
accumulating several minor genes<br />
<strong>and</strong> combining them with different<br />
specific genes to provide a certain<br />
degree <strong>of</strong> additional genetic<br />
diversity. This concept is also<br />
applied to other diseases like<br />
septoria leaf blotch,<br />
helminthosporium spot blotch, <strong>and</strong><br />
fusarium head scab. The present<br />
situation <strong>of</strong> <strong>CIMMYT</strong> germplasm<br />
regarding resistance to major<br />
diseases may be summarized as<br />
follows:<br />
• Stem rust (Puccinia graminis f.sp.<br />
tritici) resistance has been stable<br />
after 40 years <strong>of</strong> utilization <strong>of</strong> the<br />
genes derived from the variety<br />
Hope. Losses due to stem rust<br />
Veery<br />
Average<br />
yield<br />
have been negligible since the<br />
late 1960s. The resistance is<br />
based on the gene complex Sr2,<br />
which actually consists <strong>of</strong> Sr2<br />
plus 4-5 minor genes pyramided<br />
into three to four gene<br />
combinations (Rajaram et al.,<br />
1988). Sr 2 alone behaves as a<br />
slow rusting gene. Since there<br />
has been no major stem rust<br />
epidemic in areas where<br />
<strong>CIMMYT</strong> germplasm is grown,<br />
the resistance seems to be<br />
durable.<br />
• Leaf rust (Puccinia recondita f.sp.<br />
tritici) resistance has been<br />
stabilized by using genes<br />
derived from many sources, in<br />
particular the Brazilian cultivar<br />
Frontana (Singh <strong>and</strong> Rajaram,<br />
1992). No major epidemic has<br />
been observed in almost 20<br />
years. Four partial resistance<br />
genes, including Lr 34, give a<br />
slow rusting response <strong>and</strong> have<br />
been the reason for the<br />
containment <strong>of</strong> leaf rust<br />
epidemics in the developing<br />
world during the last 15 years.<br />
About 60% <strong>of</strong> <strong>CIMMYT</strong><br />
germplasm carries one to four <strong>of</strong><br />
these partial resistance genes.<br />
Lr34 is linked to Yr18 as well as<br />
to a morphological marker (leaf<br />
tip necrosis) that makes the gene<br />
particularly attractive for<br />
8500<br />
Super Seri<br />
8000<br />
Bacanora 88<br />
7500<br />
Seri 82 Oasis 86<br />
7000 Yecora 70<br />
Ciano 79<br />
Nacozari 76<br />
6500<br />
6000<br />
1960<br />
Siete Cerros 66<br />
Pitic 62<br />
1970 1980<br />
Year <strong>of</strong> release<br />
1990 2000<br />
Figure 6. Increase in grain yield potential <strong>of</strong><br />
<strong>CIMMYT</strong>-derived wheats as a function <strong>of</strong> year <strong>of</strong><br />
release.<br />
Grain yield in kg/ha
8 Opening Remarks — S. Rajaram<br />
breeders (Singh, 1992a, b).<br />
<strong>CIMMYT</strong> continues to look for<br />
new sources <strong>of</strong> partial resistance.<br />
• Stripe rust (Puccinia striiformis):<br />
Slow rusting genes like Yr18<br />
have been identified (Singh,<br />
1992b); however, their<br />
interaction is less additive than<br />
for leaf <strong>and</strong> stem rust. More<br />
basic research is needed to<br />
underst<strong>and</strong> the status <strong>of</strong> durable<br />
resistance in high yielding<br />
germplasm. The breakdown <strong>of</strong><br />
Yr9 in West Asia <strong>and</strong> North<br />
Africa <strong>and</strong> the present yellow<br />
rust epidemics underline the<br />
need for the release <strong>of</strong> cultivars<br />
with accumulated durable<br />
resistance.<br />
• <strong>Septoria</strong> tritici: Initially all<br />
semidwarf cultivars developed<br />
for irrigated conditions were<br />
susceptible. Today more than<br />
eight genes have been identified<br />
in <strong>CIMMYT</strong> germplasm <strong>and</strong> two<br />
to three genes in combination<br />
provide acceptable resistance.<br />
Future activities will concentrate<br />
on pyramiding these genes <strong>and</strong><br />
spreading them more widely<br />
within <strong>CIMMYT</strong> germplasm<br />
(Jlibene, 1992; Matus-Tejos,<br />
1993).<br />
• Karnal bunt (Tilletia indica):<br />
More than five genes have been<br />
identified <strong>and</strong> most <strong>of</strong> them are<br />
partially dominant. Genes<br />
providing resistance to Karnal<br />
bunt have been incorporated<br />
into high yielding lines (Singh et<br />
al., 1995).<br />
• Powdery mildew (Erysiphe<br />
graminis f.sp. tritici): <strong>CIMMYT</strong><br />
germplasm is considered<br />
vulnerable to this disease. The<br />
disease is absent in Mexico <strong>and</strong><br />
the responsibility to transfer<br />
resistance genes has been<br />
delegated to <strong>CIMMYT</strong>’s regional<br />
breeder in South America.<br />
Breeding for<br />
Drought Tolerance<br />
There has been a large<br />
transformation in the productivity<br />
<strong>of</strong> wheat due to the application <strong>of</strong><br />
Green Revolution technology. This<br />
has resulted in a doubling <strong>and</strong><br />
tripling <strong>of</strong> wheat production in<br />
many environments, but especially<br />
in irrigated areas. High yielding<br />
semidwarf wheats have<br />
continuously replaced the older tall<br />
types at a rate <strong>of</strong> 2 million ha per<br />
year since 1977 (Byerlee <strong>and</strong> Moya,<br />
1993).<br />
There is a growing recognition<br />
that the dissemination, application,<br />
<strong>and</strong> adoption <strong>of</strong> this technology<br />
has, however, been slower in<br />
marginal environments, especially<br />
in the semiarid environments<br />
affected by poor distribution <strong>of</strong><br />
water <strong>and</strong> drought. The annual<br />
gain in genetic yield potential in<br />
drought environments is only<br />
about half that obtained in<br />
irrigated, optimum conditions.<br />
Many investigators have attempted<br />
to produce wheat varieties adapted<br />
to these semiarid environments<br />
with limited success. Others have<br />
criticized the Green Revolution<br />
technology (Ceccarelli et al., 1987)<br />
for failing to adequately address<br />
productivity constraints in<br />
semiarid environments, although<br />
their own recommended<br />
technology has had limited impact,<br />
in particular in farmers’ fields. This<br />
criticism is in clear contrast to the<br />
actual acceptance <strong>of</strong> semidwarf<br />
wheat cultivars in rainfed areas,<br />
since most <strong>of</strong> the 16 million ha<br />
increase in the area sown to<br />
Mexican semidwarf wheats in the<br />
mid-1980s occurred in rainfed<br />
areas; in 1990, more than 60% <strong>of</strong> the<br />
dryl<strong>and</strong> area in developing<br />
countries were planted with<br />
semidwarfs (Byerlee <strong>and</strong> Moya,<br />
1993).<br />
Definition <strong>of</strong> semiarid<br />
environments <strong>and</strong><br />
description<br />
<strong>of</strong> drought patterns<br />
In Table 2, the major global<br />
drought patterns observed in<br />
wheat production are presented<br />
(Rajaram et al., 1994, Edmeades et<br />
al., 1989). Through respectively<br />
dealing with spring (ME4A),<br />
facultative (ME9), <strong>and</strong> winter<br />
wheat (ME12), these three MEs are<br />
characterized by sufficient rainfall<br />
prior to anthesis, followed by<br />
drought during the grain-filling<br />
period. In South America, the<br />
Southern Cone type <strong>of</strong> drought<br />
(ME4B) is characterized by<br />
moisture stress early in the crop<br />
season, with rainfall occurring<br />
during the post-anthesis phase. In<br />
the Indian Subcontinent type <strong>of</strong><br />
drought stress (ME4C), the wheat<br />
crop utilizes water reserves left<br />
from the monsoon rains during the<br />
previous summer season. In the<br />
Subcontinent the irrigated wheat<br />
crop (ME1) may also suffer drought<br />
due to a reduced or less than<br />
optimum number <strong>of</strong> irrigations.<br />
The traditional methodology<br />
for breeding for drought stress is<br />
typified by h<strong>and</strong>ling all segregating<br />
populations under target<br />
conditions <strong>of</strong> drought, <strong>and</strong> the use<br />
<strong>of</strong> local l<strong>and</strong>races is recommended<br />
in the breeding process (Ceccarelli<br />
et al., 1987). The methodology rests
on the assumption that the agroecological<br />
situation facing the<br />
farmer does not vary in its<br />
expression over time <strong>and</strong> that<br />
responsiveness <strong>of</strong> varieties to<br />
improved growing conditions will<br />
not be needed. It also assumes that<br />
crossover will always occur below<br />
a certain yield level under dry<br />
conditions, where modern high<br />
yielding varieties <strong>of</strong> a responsive<br />
nature would always yield less<br />
than traditional l<strong>and</strong>race-based<br />
genotypes. Such crossovers may<br />
occur for selected genotypes, <strong>and</strong><br />
one should always be open to the<br />
possibility that there are real<br />
“drought tolerance” traits<br />
operating at the 1 t/ha <strong>and</strong> below<br />
yield level that adversely affect<br />
high yield potential at the 4 t/ha<br />
<strong>and</strong> higher yield levels. So far such<br />
traits have not been identified at<br />
<strong>CIMMYT</strong>. In any case, crossover<br />
would be restricted to such harsh<br />
conditions, where in fact farmers<br />
choose—rightfully so—not to grow<br />
wheat at all, but to produce other,<br />
more drought tolerant crops such<br />
as barley or sorghum, or resort to<br />
grazing (van Ginkel et al., 1998).<br />
At <strong>CIMMYT</strong> we advocate an<br />
“open-ended system” <strong>of</strong> breeding<br />
in which yield responsiveness is<br />
combined with adaptation to<br />
drought conditions. Most semiarid<br />
environments differ significantly<br />
across years in their water<br />
availability <strong>and</strong> distribution<br />
pattern. Hence it is prudent to<br />
construct a genetic system in which<br />
plant responsiveness provides a<br />
bonus whenever conditions<br />
improve due to higher rainfall.<br />
With such a system, improved<br />
moisture conditions immediately<br />
translate into greater gains for the<br />
farmer.<br />
Historical Aspects <strong>and</strong> Future Challenges <strong>of</strong> an International Wheat Program 9<br />
The Veerys<br />
In the early 1980s, when<br />
advanced lines derived from the<br />
spring x winter cross Kavkaz/<br />
Buho//KAL/BB (CM33027) were<br />
tested in 73 global environments <strong>of</strong><br />
the 15th International Wheat Yield<br />
Nursery (15th ISWYN) (Figure 5),<br />
their performance was quite<br />
untypical compared to any<br />
previously known high yielding<br />
varieties. In later tests, we found<br />
that these lines, called Veerys, carry<br />
the 1B.1R translocation from rye<br />
<strong>and</strong> that the general performance<br />
<strong>of</strong> such germplasm was superior<br />
not only in high yielding<br />
environments but particularly<br />
under drought conditions (Villareal<br />
et al., 1995; Table 4). From the Veery<br />
cross 43 varieties were released,<br />
excluding those released in Europe.<br />
The Veerys represent a genetic<br />
system in which high yield<br />
performance in favorable<br />
environments <strong>and</strong> adaptation to<br />
drought could be combined in one<br />
genotype. The two genetic systems<br />
are apparently not always<br />
incompatible, although others have<br />
claimed that their combination<br />
would not be possible. However, it<br />
is possible to hypothesize a plant<br />
system in which efficient input use<br />
<strong>and</strong> responsiveness to improved<br />
levels <strong>of</strong> external inputs (in this<br />
case, available water) can be<br />
combined to produce germplasm<br />
for marginal (in this case, semiarid)<br />
environments that at least<br />
maintains minimum traditional<br />
yields <strong>and</strong> expresses dramatic<br />
increases whenever conditions<br />
improve. The impacts described<br />
below support the utilization <strong>of</strong><br />
this methodology.<br />
• By the mid-1980s <strong>CIMMYT</strong><br />
germplasm occupied 45% <strong>of</strong> the<br />
semiarid wheat areas with 300-<br />
500 mm <strong>of</strong> rainfall, <strong>and</strong> 21% <strong>of</strong><br />
the area less than 300 mm<br />
(Morris et al., 1991), including<br />
large tracts in West Asia/North<br />
Africa (WANA). By 1990 63% <strong>of</strong><br />
the dryl<strong>and</strong> areas, especially in<br />
ME4A <strong>and</strong> ME4B, was planted<br />
with semidwarf wheats (Byerlee<br />
<strong>and</strong> Moya, 1993), many carrying<br />
the 1B/1R translocation.<br />
• To support the above<br />
assumptions, an experiment was<br />
conducted (Calhoun et al., 1994;<br />
Tables 5 <strong>and</strong> 6) to determine<br />
how the most modern <strong>and</strong><br />
widely (spatially) adapted<br />
germplasm compared to<br />
commercial germplasm from<br />
countries representing the<br />
Mediterranean region (ME4A),<br />
the Southern Cone <strong>of</strong> South<br />
America (ME4B), <strong>and</strong> the Indian<br />
Table 4. Effect <strong>of</strong> the 1BL.1RS translocation on yield characteristics <strong>of</strong> 28 r<strong>and</strong>om F2-derived F6<br />
lines from the cross Nacozari 76/Seri 82 under reduced irrigated conditions.<br />
Plant characteristics 1BL.1RS 1B Mean diff.<br />
Grain yield 4945 4743 202 *<br />
Above-ground biomass at maturity (t/ha) 12600 12100 500 *<br />
Grains/m2 14074 13922 152NS<br />
Grains/spike 43.5 40.6 2.9 *<br />
1000-grain weight (g) 37.1 36.5 0.5 *<br />
Source: Villareal et al. (1995).<br />
Note: NS: not significatnt, * : significant at the 0.05 level.
10 Opening Remarks — S. Rajaram<br />
Table 5. Wheat genotypes representing adaptation to different moisture environments.<br />
ME1 ME4A ME4B ME4 Irrigation<br />
(Mediterranean)<br />
(Southern Cone)<br />
(Subcontinent)<br />
Super Kauz, Pavon 76, Genaro 81, Opata 85<br />
Almansor, Nesser, Sitta, Siete Cerros<br />
Cruz Alta, Prointa Don Alberto, LAP1376, PSN/BOW CM69560<br />
C306, Sonalika, Punjab 81, Barani<br />
Source: Calhoun et al. (1994).<br />
Table 6. Grain yields <strong>of</strong> selected wheat genotypes grouped by adaptation <strong>and</strong> tested under<br />
moisture regimes in the Yaqui Valley, Mexico, 1989-90 <strong>and</strong> 1990-91<br />
Full Late<br />
Adaptation group<br />
Early Residual<br />
irrigation1 drought2 drought3 moisture4 ME1 Irrigation 6636 a * ME4C ME4B ME4C Mediterranean<br />
Southern Cone<br />
Subcontinent<br />
6342 b<br />
5028 c<br />
4778 c<br />
4198 a<br />
3990 ab<br />
3148 bc<br />
3245 bc<br />
4576 a<br />
4390 b<br />
4224 b<br />
3657 c<br />
3032 a<br />
2883 b<br />
2359 c<br />
2704 b<br />
Source: Calhoun, et al. 1994<br />
1 Received 5 irrigations; 2 received 2 irrigations early before heading; 3 received one irrigation for<br />
germination <strong>and</strong> two post heading; 4 received one irrigation for germination only.<br />
* Means in the same column followed by the same letter are not significantly different at P=0.05.<br />
Subcontinent (ME4C), under<br />
conditions artificially simulating<br />
those three MEs. The most<br />
widely adapted <strong>CIMMYT</strong> lines<br />
outyielded the commercial<br />
varieties in all artificially<br />
simulated environments.<br />
• Nesser is an advanced line with<br />
superior performance in<br />
drought conditions bred at<br />
<strong>CIMMYT</strong>/Mexico <strong>and</strong> identified<br />
at ICARDA/Syria. The cross<br />
combines a high yielding<br />
<strong>CIMMYT</strong> variety Jupateco <strong>and</strong> a<br />
drought tolerant Australian<br />
variety W3918A. The<br />
performance <strong>of</strong> Nesser in<br />
WANA’s ME4A environments<br />
has been widely publicized<br />
(ICARDA, 1993), <strong>and</strong> the line is<br />
considered by ICARDA to<br />
represent a uniquely drought<br />
tolerant genotype. However, it<br />
was selected at <strong>CIMMYT</strong>/<br />
Mexico under favorable<br />
environments, <strong>and</strong> carries a<br />
combination <strong>of</strong> input efficiency<br />
<strong>and</strong> high yield responsiveness. It<br />
performs similarly to the Veery<br />
lines in the absence <strong>of</strong> rust.<br />
The breeding scheme<br />
The breeding scheme<br />
described below is used to<br />
combine the two genetic systems.<br />
Two contrasting selection<br />
environments are alternated,<br />
allowing alternate selection for<br />
input efficiency <strong>and</strong> input<br />
responsiveness.<br />
F1 Crosses involving spatially<br />
widely adapted germplasm<br />
representing yield stability <strong>and</strong><br />
yield potential, with lines with<br />
proven drought tolerance in<br />
the specific setting <strong>of</strong> either<br />
ME4A, ME4B or ME4C. Winter<br />
wheats <strong>and</strong> synthetic<br />
germplasm are emphasized.<br />
F2 Individual plants are raised<br />
under irrigated <strong>and</strong> optimally<br />
fertilized conditions <strong>and</strong><br />
inoculated with a wide<br />
spectrum <strong>of</strong> rust virulence.<br />
Only robust <strong>and</strong> (horizontally)<br />
resistant plants are selected.<br />
These may represent<br />
adaptation to favorable<br />
environments.<br />
F3, F4 The selected F2 plants are<br />
evaluated using a modified<br />
pedigree/bulk breeding system<br />
(Rajaram <strong>and</strong> van Ginkel, 1995)<br />
under rainfed conditions or very<br />
low water availability. The<br />
selection is based on individual<br />
lines rather than individual<br />
plants. The progenies are<br />
selected based on such criteria as<br />
spike density, biomass/vigor,<br />
grains/m 2 , <strong>and</strong> others (van<br />
Ginkel et al., 1998) (Table 7). This<br />
index helps identify lines which<br />
may adapt to low water<br />
situations.<br />
F5, F6 The selected lines from F4<br />
are further evaluated under<br />
optimum conditions.<br />
Table 7. Genotypic correlation (rg) between<br />
agronomic traits <strong>and</strong> final grain yield, for<br />
optimum environment (full irrigations) <strong>and</strong><br />
reduced water regime (late drought,<br />
Mediterranean type) in wheat.<br />
Moisture regime<br />
Full Late<br />
Trait irrigation drought<br />
Days to heading 0.40 0.19<br />
Days to maturity 0.29 0.27<br />
Grain fill period -0.32 0.36<br />
Height -0.39 0.05<br />
Peduncle length<br />
Relative peduncle<br />
-0.46 0.22<br />
extrusion -0.51* 0.25<br />
Spike length -0.28 -0.50*<br />
Spike/m2 -0.12 0.64**<br />
Grains/spike 0.62* -0.42<br />
Grains/m2 0.74** 0.68**<br />
Yield/spike 0.55* -0.64**<br />
1000 grain weight 0.08 -0.45<br />
Test weight 0.13 0.05<br />
Harvest index 0.83** -0.39<br />
Biomass 0.90** 0.94**<br />
Straw yield 0.52* 0.86**<br />
Yield / day (planting) 0.99** 0.57*<br />
Yield / day (heading) 0.94** 0.44<br />
Biomass / day (planting) 0.86** 0.69**<br />
Biomass / day (heading) 0.74** 0.63**<br />
Vegetative growth rate 0.32 0.63**<br />
Spike growth rate 0.62** -0.58*<br />
Grain growth rate 0.17 -0.44<br />
*, ** indicate significance at the 0.05 <strong>and</strong><br />
0.01probability level, respectively.<br />
Source: van Ginkel et al. (1998).
F7, F8 Simultaneous evaluations<br />
under optimum <strong>and</strong> low water<br />
environments. Selection <strong>of</strong> lines<br />
showing outst<strong>and</strong>ing<br />
performance under both<br />
conditions. Further evaluation in<br />
international environments is<br />
carried out for purposes <strong>of</strong><br />
verification.<br />
The proposed breeding<br />
methodology is supported by<br />
research published in recent years<br />
by others, not only on wheat<br />
(Bramel-Cox et al., 1991; Cooper et<br />
al., 1994; Duvick, 1990, 1992;<br />
Ehdaie et al., 1988; Uddin et al.,<br />
1992; Zavala-Garcia et al., 1992),<br />
where the importance <strong>of</strong> testing<br />
<strong>and</strong> selecting in a range <strong>of</strong><br />
environments, including wellirrigated<br />
ones, has shown to<br />
identify superior genotypes for<br />
stressed conditions. The<br />
methodology aims at combining<br />
input efficiency with input<br />
responsiveness by alternating<br />
selection environments during the<br />
breeding process.<br />
Future Research<br />
Directions<br />
Yield stability <strong>and</strong> yield<br />
potential<br />
Traxler et al. (1995) analyzed<br />
grain yield increases <strong>and</strong> yield<br />
stability <strong>of</strong> bread wheat cultivars<br />
released during the last 45 years. In<br />
the early period <strong>of</strong> the Green<br />
Revolution, when rapid yield<br />
increases occurred, variance for<br />
yield concomitantly increased.<br />
Since the early 1970s, yield stability<br />
has increased at the cost <strong>of</strong><br />
increases in yield. However, steady<br />
progress was made in developing<br />
varieties with improved stability,<br />
grain yield or both. For the<br />
developing world, yield stability<br />
Historical Aspects <strong>and</strong> Future Challenges <strong>of</strong> an International Wheat Program 11<br />
increased since the beginning <strong>of</strong> the<br />
Green Revolution (Smale <strong>and</strong><br />
McBride, 1996). While price policy,<br />
input supplies, <strong>and</strong> environmental<br />
variation contribute more to yield<br />
stability than the genotype, the<br />
increasing yield stability reflects<br />
the emphasis given by breeders to<br />
develop germplasm with tolerance<br />
to a wider range <strong>of</strong> diseases <strong>and</strong><br />
abiotic stresses. Sayre et al. (1997)<br />
concluded that from 1964 to 1990,<br />
yield potential in <strong>CIMMYT</strong>-derived<br />
cultivars increased at a rate <strong>of</strong> 67<br />
kg/ha/yr or 0.88% per year. The<br />
data did not suggest that a yield<br />
plateau had been reached <strong>and</strong> the<br />
performance <strong>of</strong> recently released<br />
lines, such as Attilla (pb343) <strong>and</strong><br />
Babax (Baviacora M92) indicates<br />
that yield potential has been<br />
further enhanced. Improvements<br />
made by breeding for yield stability<br />
<strong>and</strong> adaptation may be illustrated<br />
by data for the advanced line<br />
Pastor, which outyielded the<br />
hallmark check cultivar Seri 82 in<br />
34 out <strong>of</strong> 50 locations where the 13 th<br />
Elite Spring Wheat Yield Nursery<br />
was grown (Figure 4). Discussion<br />
on how to increase the yield<br />
potential <strong>of</strong> wheat <strong>of</strong>ten still centers<br />
around traits that contributed to<br />
the success <strong>of</strong> the Green Revolution<br />
varieties more than 30 years ago,<br />
e.g., photoperiod <strong>and</strong> dwarfing<br />
genes (Worl<strong>and</strong> et al., 1998; Sears,<br />
1998).<br />
<strong>CIMMYT</strong> has made a modest<br />
investment in restructuring <strong>and</strong><br />
creation <strong>of</strong> a new plant type<br />
characterized by robust stem, broad<br />
leaf, long spike (30 cm), <strong>and</strong> large<br />
numbers <strong>of</strong> grain per spike. The<br />
new plant type still suffers due to<br />
diseases <strong>and</strong> is deficient in quality<br />
<strong>and</strong> certain agronomic<br />
characteristics. In 1994, we<br />
launched a dynamic breeding<br />
program to correct these<br />
deficiencies.<br />
Plant nutrition<br />
Selecting for yield potential <strong>and</strong><br />
yield stability under medium to<br />
high levels <strong>of</strong> nitrogen has<br />
indirectly increased efficiency for<br />
nutrient uptake. Recently released<br />
<strong>CIMMYT</strong> bread wheat cultivars<br />
require less nitrogen to produce a<br />
unit amount <strong>of</strong> grain than cultivars<br />
released in the previous decades<br />
(Ortiz Monasterio et al., 1997).<br />
Under low N levels in the soil, N<br />
use efficiency increased mainly due<br />
to a higher N uptake efficiency—<br />
the ability <strong>of</strong> plants to absorb N<br />
from the soil—whereas under high<br />
N levels, the N utilization<br />
efficiency—the capacity <strong>of</strong> plants to<br />
convert the absorbed N into grain<br />
yield—increased. In spite <strong>of</strong> the<br />
increased N use efficiency <strong>of</strong><br />
recently released wheat cultivars,<br />
the response to nitrogen <strong>of</strong> wheat<br />
production systems has been<br />
observed to be declining in many<br />
areas <strong>of</strong> Southeast Asia. In Turkey,<br />
where zinc deficient soils are<br />
common, recently released winter<br />
bread wheat cultivars have<br />
improved Zn uptake <strong>and</strong><br />
consequently higher grain yield<br />
than local l<strong>and</strong>races (M. Kalayci,<br />
pers. comm.).<br />
Physiology<br />
A recent survey <strong>of</strong> wheat<br />
breeders suggested that research in<br />
plant physiology has had a limited<br />
impact on wheat improvement<br />
(Jackson et al., 1996). A strong body<br />
<strong>of</strong> evidence now indicates that<br />
physiological traits may have real<br />
potential for complementing early<br />
generation phenotypic selection in<br />
wheat. One <strong>of</strong> the more promising<br />
traits identified is canopy
12 Opening Remarks — S. Rajaram<br />
temperature depression (CTD).<br />
CTD refers to the cooling effect<br />
exhibited by a leaf as transpiration<br />
occurs. While soil water status has<br />
a major influence on CTD, there are<br />
strong genotypic effects under<br />
well-watered, heat-stressed, or<br />
drought-stressed conditions. CTD<br />
gives an indirect estimate <strong>of</strong><br />
stomatal conductance <strong>and</strong> is a<br />
highly integrative trait affected by<br />
several major physiological<br />
processes, including photosynthetic<br />
metabolism, evapotranspiration,<br />
<strong>and</strong> plant nutrition. CTD <strong>and</strong><br />
stomatal conductance, measured<br />
on sunny days during grain filling,<br />
showed a strong association with<br />
the yield <strong>of</strong> semidwarf wheat lines<br />
grown under irrigation, in both<br />
temperate (Fischer et al., 1998) <strong>and</strong><br />
subtropical environments<br />
(Reynolds et al., 1994). In addition,<br />
CTD, as measured on large<br />
numbers <strong>of</strong> advanced breeding<br />
lines in irrigated yield trials, was a<br />
powerful predictor <strong>of</strong> performance<br />
not only at the selection site but<br />
also for yield averaged across 15<br />
international sites. CTD has been<br />
shown to be associated with yield<br />
differences between homozygous<br />
lines, indicating a potential for<br />
genetic gains in yield, in response<br />
to selection for CTD (Reynolds et<br />
al., 1998).<br />
Germplasm is paramount<br />
Three-quarters <strong>of</strong> recently<br />
surveyed wheat breeders felt that a<br />
lack <strong>of</strong> genetic diversity would<br />
limit future breeding advances<br />
(Rejesus et al., 1996), though<br />
genetic diversity was not<br />
considered an immediately limiting<br />
factor in most programs. This<br />
concern was greater from breeders<br />
in developing <strong>and</strong> former USSR<br />
countries (>80%) than from higher<br />
income countries (59%).<br />
Furthermore, in countries where<br />
privatization <strong>of</strong> wheat breeding<br />
programs has occurred,<br />
investments in strategic germplasm<br />
development that may be risky or<br />
important only in the long term<br />
have declined (McGuire, 1997).<br />
A wide range <strong>of</strong> opinion has<br />
been expressed concerning the<br />
abundance or availability <strong>of</strong><br />
usefully exploitable genetic<br />
variability. Allard (1996)<br />
emphasized that the most readily<br />
useful genetic resources were<br />
modern elite cultivars, since these<br />
lines possessed relatively high<br />
frequencies <strong>of</strong> favorable alleles.<br />
Rasmusson <strong>and</strong> Phillips (1997)<br />
have shown that the assumption<br />
that all genetic variability is a result<br />
<strong>of</strong> the inherent exclusive<br />
contribution <strong>of</strong> two parents, per se,<br />
is not necessarily true, considering<br />
results from molecular analysis.<br />
They discuss mechanisms by which<br />
induction <strong>of</strong> genetic variability may<br />
involve altering the expression <strong>of</strong><br />
genes, the possible mechanisms <strong>of</strong><br />
single allele change, intragenic<br />
recombination, unequal crossover,<br />
element transpositions, DNA<br />
methylation, paramutation, or gene<br />
amplification. They also stressed<br />
the possible importance <strong>of</strong> epistasis<br />
effects which may have been<br />
underestimated in the past.<br />
Introduction <strong>of</strong> genetic<br />
variability from distantly related<br />
wheat cultivars, or related or alien<br />
species, has <strong>of</strong>ten been specifically<br />
aimed at the introduction <strong>of</strong> simply<br />
inherited traits (e.g., genes for<br />
disease resistance), but it has<br />
appeared to be <strong>of</strong> limited value in<br />
quantitative trait improvement<br />
(Cox et al., 1997). incorporated<br />
genes for leaf rust resistance from<br />
T. tauschii into bread wheat. With<br />
two backcrosses to the recurrent<br />
wheat parent, leaf rust resistant<br />
winter wheat advanced lines with<br />
acceptable quality <strong>and</strong> equal in<br />
yield to the highest yielding<br />
commercially grown cultivars were<br />
identified. In addition, it has been<br />
postulated that since recombination<br />
between the D genomes <strong>of</strong> T.<br />
aestivum <strong>and</strong> T. tauschii occurred at<br />
a level similar to that in an<br />
intraspecific cross (Fritz et al.,<br />
1995), T. tauschii could be<br />
considered another primary source<br />
<strong>of</strong> genes for wheat improvement.<br />
The number <strong>of</strong> wheat/rye<br />
translocations that have had a<br />
significant impact on wheat<br />
improvement are actually few in<br />
number. The majority <strong>of</strong> the<br />
1BL.1RS translocations occurring in<br />
more than 300 cultivars worldwide<br />
can be traced to one German source<br />
<strong>and</strong> all 1AL.1RS translocations,<br />
widely present in bread wheat<br />
cultivars grown in the Great Plains<br />
<strong>of</strong> the US, trace to one source,<br />
“Amigo” (Schlegel, 1997a,b;<br />
Rabinovich, 1998). Other<br />
translocations carry genes for<br />
copper efficiency (4BL.5R) <strong>and</strong><br />
Hessian fly resistance (2RL.2BS,<br />
6RL.6B, 6RL.4B, 6RL.4A; McIntosh,<br />
1993). Chromosomes 2R <strong>and</strong> 7R<br />
enhance zinc efficiency in wheatrye<br />
addition lines (Cakmak <strong>and</strong><br />
Braun, unpublished). Considering<br />
the impacts which have come from<br />
the use <strong>of</strong> wheat/rye<br />
translocations, further exploitation<br />
<strong>of</strong> these translocations may be<br />
warranted.<br />
While there have been reports<br />
indicating a positive effect <strong>of</strong><br />
1BL.1RS translocations on yield<br />
performance <strong>and</strong> adaptation<br />
(Rajaram et al., 1990), Singh et al.<br />
(1998) determined that with Seri 82,<br />
replacing the translocation with<br />
1BL from cv. Oasis resulted in a<br />
yield increase <strong>of</strong> 3.4 <strong>and</strong> 5.0% in
irrigated <strong>and</strong> moisture stress<br />
conditions, respectively. A further<br />
increase in grain yield in disease<br />
free conditions <strong>of</strong> about 5% was<br />
observed in the irrigated trials<br />
through the introgression <strong>of</strong><br />
7DL.7Ag translocation carrying the<br />
Lr19 gene (from Agropyron<br />
elongatum). This yield increase was<br />
attributed to higher rate <strong>of</strong> biomass<br />
production in the 7DL.7Ag lines.<br />
However, under moisture stress<br />
conditions 7DL.7Ag lines were<br />
associated with a 16% yield<br />
reduction, possibly due to<br />
excessive biomass production in<br />
early growth stages. This would<br />
suggest that the effect <strong>of</strong> the<br />
1BL.1RS translocation is genotype<br />
specific <strong>and</strong> that 7DL.7Ag could be<br />
a useful translocation for<br />
enhancing yield potential at least in<br />
irrigated conditions.<br />
Recent efforts to generate newly<br />
accessible genetic diversity have<br />
involved the reconstitution <strong>of</strong><br />
hexaploid wheat by producing<br />
“synthetic wheat” by crossing<br />
durum wheat (T. turgidum), the<br />
donor <strong>of</strong> the A <strong>and</strong> B genomes,<br />
with T. tauschii, the donor <strong>of</strong> the D<br />
genome (Mujeeb-Kazi et al., 1996).<br />
Villareal (1995) <strong>and</strong> Villareal et al.<br />
(1997) showed that lines derived<br />
after two backcrosses to T. aestivum<br />
showed increased morphoagronomic<br />
variation, <strong>and</strong> resistance<br />
to Karnal bunt <strong>and</strong> head scab.<br />
Under full irrigation in<br />
northwestern Mexico, the yield <strong>of</strong><br />
this material was nearly 8 t/ha.<br />
When tested under drought<br />
conditions for two years, nearly all<br />
<strong>of</strong> the synthetic derivatives had<br />
significantly higher 1000-kernel<br />
weight, with grain yield varying<br />
between 84 to 114%, when<br />
compared with the bread wheat<br />
checks.<br />
Historical Aspects <strong>and</strong> Future Challenges <strong>of</strong> an International Wheat Program 13<br />
The more focused the breeding<br />
objective, the more restricted a<br />
breeder is in the choice <strong>of</strong> suitable<br />
parents. The use <strong>of</strong> genetically<br />
diverse material will continue to be<br />
a prime genetic source for<br />
increasing yield potential, a<br />
complex trait still not well<br />
understood genetically or<br />
physiologically. As long as breeders<br />
have no other readily accessible<br />
tools, genetic diversity <strong>and</strong> the<br />
opportunity for its recombination<br />
through crossing will be important<br />
to break undesired linkages <strong>and</strong><br />
increase the frequency <strong>of</strong> desirable<br />
alleles. Future breakthroughs in<br />
yield potential will likely come<br />
from such genetically diverse<br />
crosses.<br />
Hybrid wheat<br />
Pickett (1993) <strong>and</strong> Picket <strong>and</strong><br />
Galwey (1997) evaluated 40 years<br />
<strong>of</strong> wheat hybrid development <strong>and</strong><br />
concluded that hybrid wheat<br />
production is not economically<br />
feasible because <strong>of</strong> a) limited<br />
heterotic advantage; b) lack <strong>of</strong><br />
advantage in terms <strong>of</strong> agronomic,<br />
quality, or disease resistance traits;<br />
c) higher seed costs; <strong>and</strong> d)<br />
probably most importantly,<br />
heterosis could be “fixed” in<br />
polyploid plants <strong>and</strong> consequently<br />
hybrids would have no advantage<br />
over inbred lines.<br />
The use <strong>of</strong> hybrid crops is<br />
usually targeted to higher yield<br />
potential environments. Results<br />
from South Africa (Jordaan, 1996),<br />
however, show that hybrids<br />
outyield inbred lines by 15% at a 2<br />
t/ha mean production potential<br />
when narrow row spacing <strong>and</strong> low<br />
seeding rates (
14 Opening Remarks — S. Rajaram<br />
Conventional plant breeders<br />
adopt breeding methods that<br />
increase their breeding efficiency<br />
but are conservative when making<br />
methodological changes. A small<br />
survey <strong>of</strong> wheat programs having<br />
unrestricted access to new<br />
biotechnological methods found<br />
that few research programs, <strong>and</strong> no<br />
main-line wheat breeding<br />
programs, routinely used MAS or<br />
quantitatively inherited trait loci<br />
(QTL). Limitation in use is caused<br />
by lack <strong>of</strong> markers for traits <strong>of</strong><br />
interest, population specificity <strong>of</strong> a<br />
given marker, or markers’relatively<br />
high costs when compared with<br />
conventional selection techniques.<br />
These limitations may lessen in the<br />
next decade.<br />
Modern cultivars are the<br />
product <strong>of</strong> recombinations among<br />
the high number <strong>of</strong> l<strong>and</strong>races in<br />
their pedigrees (Smale <strong>and</strong><br />
McBride, 1996). In contemporary<br />
breeding programs, however,<br />
l<strong>and</strong>races are used directly only as<br />
a source <strong>of</strong> qualitatively inherited<br />
traits. Tanksley <strong>and</strong> McCouch<br />
(1997) argued that the lack <strong>of</strong><br />
success from crosses involving<br />
l<strong>and</strong>races for the improvement <strong>of</strong><br />
grain yield was mainly due to<br />
evaluation on a phenotypic basis,<br />
an imprecise indicator <strong>of</strong> genetic<br />
potential. Analyses <strong>of</strong> QTLs have<br />
revealed that loci controlling a<br />
quantitatively inherited trait do not<br />
contribute equally to the observed<br />
variation for the trait, <strong>and</strong> <strong>of</strong>ten a<br />
few QTLs explain most <strong>of</strong> the<br />
observed variation. In rice, QTLs<br />
for yield were identified in a wild,<br />
low yielding relative. After<br />
introgression into modern hybrid<br />
rice cultivars, yield increases <strong>of</strong><br />
17% compared to the original<br />
hybrid were observed. Based on<br />
the observed gains, Tanksley <strong>and</strong><br />
McCouch (1997) identified the need<br />
to more thoroughly evaluate exotic<br />
germplasm. Those accessions most<br />
distinct from modern cultivars may<br />
contain the highest number <strong>of</strong><br />
unexploited, potentially useful<br />
alleles.<br />
The comparative genetic<br />
mapping <strong>of</strong> cereal genomes has<br />
identified a vast amount <strong>of</strong><br />
conserved linearity <strong>of</strong> gene order<br />
(Devos <strong>and</strong> Gale, 1997). This<br />
observation will likely accelerate<br />
the application <strong>of</strong> QTLs in wheat,<br />
as well as aid in the identification<br />
<strong>of</strong> genes required for introgression<br />
from alien species. Considering the<br />
low number <strong>of</strong> loci tagged today in<br />
wheat, the problems related to<br />
developing a high density map for<br />
wheat (Snape, 1998), <strong>and</strong><br />
consequently the limited progress<br />
to identify QTLs for yield in wheat,<br />
we believe that the impact from<br />
this linearity on wheat<br />
improvement will be significant.<br />
Wheat has been successfully<br />
transformed for herbicide<br />
resistance <strong>and</strong> high molecular<br />
weight (HMW) glutenins, using<br />
both the ballistic <strong>and</strong> Agrobacterium<br />
tumefaciens systems (Cheng et al.,<br />
1997). Barro et al. (1997) inserted<br />
two additional HMW glutenin<br />
subunits, 1Ax1 <strong>and</strong> 1Dx5, <strong>and</strong><br />
observed a stepwise improvement<br />
<strong>of</strong> dough strength. Altpeter et al.<br />
(1996) introduced 1Ax1 into<br />
Bobwhite <strong>and</strong> increased total<br />
HMW glutenin subunit protein by<br />
71% over Bobwhite. However, the<br />
effects <strong>of</strong> transformation are not<br />
necessary additive as was shown<br />
by Blechl et al. (1998), who<br />
identified trangenics for HMW<br />
glutenins that also exhibited<br />
decreased accumulation due to<br />
transgene-mediated suppression.<br />
Conclusions<br />
The challenge to annually<br />
produce one billion tons <strong>of</strong> wheat<br />
within the next 25 years is<br />
formidable <strong>and</strong> can be met only by<br />
a concerted action <strong>of</strong> scientists<br />
involved in diverse disciplines<br />
(agronomy, pathology, physiology,<br />
biotechnology, breeding), as well as<br />
economics <strong>and</strong> politics. I am<br />
optimistic that this target will be<br />
met. Today, funds are directed from<br />
breeding towards biotechnology,<br />
<strong>of</strong>ten due simply to the novelty<br />
required for publication.<br />
Eventually, transformation may be<br />
a valuable technique to alter the<br />
performance <strong>of</strong> a genotype;<br />
however, at least during the next<br />
decade, the simple decision <strong>of</strong> a<br />
breeder in the field to “keep or<br />
discard” will contribute more to<br />
yield increase than any other<br />
approach. In conclusion, I agree<br />
with Ruttan (1993) who stated that<br />
“at least for the next two decades to<br />
come, progress through<br />
conventional breeding will remain<br />
the primary source <strong>of</strong> growth in<br />
crop <strong>and</strong> animal production.”<br />
References<br />
Allard, R.W. 1996. Genetic basis <strong>of</strong> the<br />
evolution <strong>of</strong> adaptedness in plants.<br />
Euphytica 92: 1-11.<br />
Altpeter, F.V., V. Vasil, V. Srivastava,<br />
<strong>and</strong> I.K. Vasil. 1996. Integration<br />
<strong>and</strong> expression <strong>of</strong> the highmolecular-weight<br />
glutenin subunit<br />
1Ax1 gene into wheat. Nature<br />
Biotechnology 14: 1155-1159.<br />
Barro, F., L. Rooke, F. Bekes, P. Gras,<br />
A.S. Tatham, R. Fido, P.A. Lazzeri,<br />
P.R. Shewry, <strong>and</strong> P. Barcelo. 1997.<br />
Transformation <strong>of</strong> wheat with high<br />
molecular weight subunit genes<br />
results in improved functional<br />
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15: 1295-1299.
Blechl, A.E., B.S.B. Altenbach, H.Q.<br />
Le, P.W. Gras, F. Bekes <strong>and</strong> O. D.<br />
Anderson. 1998. Genetic<br />
transformation can be used to<br />
either increase or decrease levels <strong>of</strong><br />
wheat HMW-glutenin subunits.<br />
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28-30, Denver, CO. USDA-ARS,<br />
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Bramel-Cox, P.J., T. Barker, F. Zavala-<br />
Garcia, <strong>and</strong> J.D. Eastin. 1991.<br />
Selection <strong>and</strong> testing environments<br />
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Special Publication No. 18. CSSA<br />
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surviving uniform <strong>and</strong> shuttle<br />
selection strategies. Euphytica<br />
76:63-71.<br />
Zavala-Garcia, F., P.J. Bramel-Cox,<br />
J.D.Eastin, M.D. Witt, <strong>and</strong> D.J.<br />
Andrews. 1992. Increasing the<br />
efficiency <strong>of</strong> crop selection for<br />
unpredictable environments. Crop<br />
Sci. 32:51-57.
Session 1: Pathogen Biology<br />
Biology <strong>of</strong> the <strong>Septoria</strong>/<strong>Stagonospora</strong> Pathogens: An<br />
Overview<br />
A.L. Scharen<br />
Department <strong>of</strong> Plant Sciences, Montana State University, Bozeman, Montana, USA<br />
Abstract<br />
More than 2000 form-species <strong>of</strong> fungi, mostly plant parasites, comprise the genera <strong>Septoria</strong> <strong>and</strong> <strong>Stagonospora</strong>. The two<br />
most important pathogens in wheat production are Mycosphaerella graminicola (<strong>Septoria</strong> tritici) <strong>and</strong> Phaeosphaeria<br />
nodorum (<strong>Stagonospora</strong> nodorum). Primary inoculum for the wheat diseases caused by these pathogens is most <strong>of</strong>ten airborne<br />
ascospores, but may also be wind- <strong>and</strong> rain-borne conidia. Field diagnoses may be augmented <strong>and</strong> made more exact by<br />
use <strong>of</strong> rapid immunological tests <strong>and</strong> molecular genetic methods. Infection processes <strong>of</strong> S. tritici <strong>and</strong> S. nodorum are similar,<br />
but penetration by S. tritici is known only via stomata. Patterns <strong>of</strong> occurrence <strong>of</strong> the two pathogens have changed<br />
dramatically in recent years. <strong>Septoria</strong> tritici has become more important in northern Europe, <strong>and</strong> S. nodorum incidence has<br />
increased in parts <strong>of</strong> North Africa. Changes in cultivars <strong>and</strong> cultural practices are thought to be responsible for the shifts in<br />
pathogens <strong>and</strong> diseases. Genetics <strong>of</strong> resistance in the wheat host <strong>and</strong> virulence in the pathogen populations continues to be<br />
unclear. Some gene-for-gene interactions have been shown, but in field situations resistance is generally observed as nonspecific<br />
<strong>and</strong> pathogen populations vary most in aggressiveness.<br />
“Scientists build on foundations<br />
laid by their predecessors…, but<br />
they show great reluctance to<br />
inspect those foundations”<br />
(Ainsworth, 1965). Such reluctance<br />
was evident for many years in the<br />
case <strong>of</strong> the <strong>Septoria</strong>/<strong>Stagonospora</strong><br />
diseases <strong>of</strong> cereal grains.<br />
Nineteenth <strong>and</strong> early twentieth<br />
century workers in Europe <strong>and</strong><br />
North America described the<br />
pathogens <strong>and</strong> the diseases they<br />
cause in considerable detail. But,<br />
only in the years since the advent<br />
<strong>of</strong> cultivars bred for dwarf stature<br />
<strong>and</strong> high yield under conditions <strong>of</strong><br />
intensive culture have the <strong>Septoria</strong>/<br />
<strong>Stagonospora</strong> diseases been<br />
recognized as having major<br />
impacts upon yield <strong>and</strong> quality.<br />
The causal organisms are still<br />
called by several names, as are the<br />
diseases. A concise summary <strong>of</strong> the<br />
latest information on taxonomy<br />
<strong>and</strong> nomenclature was published<br />
in 1997 (Cunfer, 1997) <strong>and</strong> 1999<br />
(Cunfer <strong>and</strong> Ueng, 1999). I believe<br />
that we should urge from this<br />
venue, as did those attending the<br />
Fourth International Workshop on<br />
<strong>Septoria</strong> <strong>of</strong> <strong>Cereals</strong> (July 4-7, 1994,<br />
IHAR, Radzikow, Pol<strong>and</strong>), that all<br />
workers accept <strong>and</strong> use the latest<br />
nomenclature.<br />
More than 2000 form-species <strong>of</strong><br />
fungi, most <strong>of</strong> them plant parasites,<br />
have been assigned to the genera<br />
<strong>Septoria</strong> <strong>and</strong> <strong>Stagonospora</strong>. Two <strong>of</strong><br />
these that attack wheat are <strong>of</strong> most<br />
concern to us. Losses <strong>of</strong> potential<br />
yield world-wide from just these<br />
two are estimated in the millions <strong>of</strong><br />
metric tons <strong>of</strong> grain <strong>and</strong> billions <strong>of</strong><br />
U.S. dollars each year. The common<br />
names <strong>of</strong> the diseases are septoria<br />
tritici blotch <strong>and</strong> stagonospora<br />
nodorum blotch. Classification <strong>and</strong><br />
19<br />
nomenclature <strong>of</strong> these <strong>and</strong> other<br />
pathogens <strong>of</strong> cereals are given in<br />
Table 1 (Eyal, 1999). The <strong>Septoria</strong><br />
anamorphic states have a<br />
teleomorphic state assigned to the<br />
genus Mycosphaerella, while the<br />
<strong>Stagonospora</strong> states have<br />
teleomorphic forms in the genus<br />
Phaeosphaeria. The teleomorphs are<br />
very important because they<br />
furnish the primary inoculum for<br />
disease development under specific<br />
environmental conditions.<br />
Several periods <strong>of</strong> ascospore<br />
deposition (both Mycosphaerella <strong>and</strong><br />
Phaeosphaeria) from the atmosphere<br />
during the months <strong>of</strong> August to<br />
October in the northern<br />
hemisphere <strong>and</strong> February to April<br />
in the southern hemisphere have<br />
recently been shown to have critical<br />
importance in epidemic<br />
establishment (Arseniuk et al.,
20<br />
Session 1 — A.L. Scharen<br />
Table 1. Classification <strong>and</strong> nomenclature <strong>of</strong> the <strong>Septoria</strong> spp. <strong>and</strong> <strong>Stagonospora</strong> spp. fungi on small grain cereals. a<br />
Genus Teleomorph Anamorph Common name Host<br />
<strong>Septoria</strong> spp. Mycosphaerella <strong>Septoria</strong> tritici <strong>Septoria</strong> tritici Wheat<br />
graminicola blotch <strong>of</strong> wheat<br />
1998; Shaw <strong>and</strong> Royle, 1989). The<br />
anamorphic conidia, also called<br />
pycnidiospores, are most important<br />
as secondary inoculum locally as<br />
the crop is growing <strong>and</strong> are<br />
disseminated mainly by rain<br />
splash.<br />
Sources <strong>of</strong> primary inoculum in<br />
areas where the teleomorph is not<br />
known remain a matter <strong>of</strong><br />
controversy <strong>and</strong> speculation,<br />
particularly in the case <strong>of</strong> septoria<br />
tritici blotch. Both S. nodorum <strong>and</strong><br />
S. tritici are found parasitizing a<br />
wide range <strong>of</strong> graminaceous hosts.<br />
(Krupinsky, 1994; Sprague, 1950).<br />
Several species <strong>of</strong> grasses have<br />
been suspected as alternative hosts<br />
<strong>and</strong> inoculum sources, but the<br />
question is yet unresolved. Conidia<br />
from plant debris may act as<br />
primary inoculum for disease<br />
development in some cases. The<br />
fact remains to puzzle us that no<br />
case has been reported in which<br />
septoria tritici blotch <strong>and</strong>/or<br />
stagonospora nodorum blotch<br />
failed to appear because <strong>of</strong> a lack <strong>of</strong><br />
primary inoculum when a<br />
- b <strong>Septoria</strong> tritici f. avenae Oats<br />
- <strong>Septoria</strong> tritici f. holci Holcus<br />
- <strong>Septoria</strong> tritici f. lolicola Lolium<br />
- <strong>Septoria</strong> passerinii Speckled leaf blotch<br />
<strong>of</strong> barley<br />
Barley<br />
- <strong>Septoria</strong> secalis Leaf spot <strong>of</strong> rye Rye<br />
<strong>Stagonospora</strong> spp. Phaeosphaeria <strong>Stagonospora</strong> nodorum <strong>Stagonospora</strong> nodorum Wheat<br />
nodorum blotch <strong>of</strong> wheat<br />
Phaeosphaeria <strong>Stagonospora</strong> avenae Oats<br />
avenaria f. sp. avenae<br />
Phaeosphaeria <strong>Stagonospora</strong> avenae Oats, wheat<br />
a (7)<br />
avenaria<br />
f. sp. triticea<br />
f. sp. triticea <strong>and</strong> triticale<br />
b Teleomorphic stages not found.<br />
susceptible crop <strong>and</strong> favorable<br />
environmental conditions<br />
prevailed.<br />
As late as the 1960s, diagnosis<br />
<strong>of</strong> <strong>Septoria</strong> diseases on wheat was<br />
not well developed even among<br />
plant pathologists <strong>and</strong> plant<br />
breeders. Leaf chlorosis <strong>and</strong><br />
necrosis was <strong>of</strong>ten viewed as part<br />
<strong>of</strong> the natural process <strong>of</strong><br />
maturation. Any <strong>of</strong> several leaf<br />
spotting pathogens could have<br />
been present <strong>and</strong> contributing to<br />
leaf death. When <strong>Septoria</strong> was<br />
recognized, it was <strong>of</strong>ten called<br />
“head septoria” which we know<br />
now as stagonospora nodorum<br />
blotch <strong>and</strong> “leaf septoria” which<br />
we know as septoria tritici blotch.<br />
Both <strong>of</strong>ten occur together <strong>and</strong> with<br />
other pathogens, <strong>and</strong> both can<br />
infect <strong>and</strong> cause symptoms on all<br />
parts <strong>of</strong> the wheat plant.<br />
Field diagnosis on the basis <strong>of</strong><br />
symptomology is regularly done,<br />
but <strong>of</strong>ten laboratory backup, with<br />
microscopic examination <strong>of</strong> spores,<br />
is necessary for accurate diagnosis.<br />
As can be seen in Table 2 (Eyal,<br />
1997), spore size <strong>and</strong> appearance<br />
may overlap, so some doubts may<br />
remain even after microscopic<br />
examination. Rapid tests have been<br />
developed that use immunological<br />
techniques mainly for early<br />
diagnosis in intensive production<br />
areas where chemical control is<br />
commonly used. Molecular genetic<br />
methods have been used recently<br />
to show similarities <strong>and</strong> differences<br />
between species <strong>and</strong> forma speciales<br />
(Arseniuk et al., 1997; Ueng et al.,<br />
1998.). The karyotype <strong>of</strong> S. nodorum<br />
suggests 14-19 chromosomes<br />
having approximately 0.5-3.5<br />
megabase pairs (Cooley <strong>and</strong> Caten,<br />
1991). McDonald <strong>and</strong> Martinez<br />
(1991) determined 14-16 b<strong>and</strong>s<br />
believed to correspond to<br />
chromosomes <strong>of</strong> 0.33-3.5 megabase<br />
pairs in S. tritici.<br />
Regardless <strong>of</strong> the fact that<br />
morphologic <strong>and</strong> genetic<br />
differences <strong>of</strong> considerable<br />
magnitude exist between S.<br />
nodorum <strong>and</strong> S. tritici, histological<br />
studies have shown that the
Table 2. The <strong>Septoria</strong> tritici/<strong>Stagonospora</strong> nodorum pathogens <strong>of</strong> wheat.<br />
Pycnidium Pycnidiospore Number Chromosome<br />
Asexual state (µm) (µm) <strong>of</strong> septa number Lesion<br />
<strong>Septoria</strong> tritici 60-200 35-98 x 1-3 3-5 14-19 a Irregular to<br />
rectangular,<br />
elongated<br />
between veins<br />
<strong>Stagonospora</strong> 160-210 15-32 x 2-4 0-3 14-16b Lens shaped,<br />
nodorum with chlorotic<br />
border<br />
Pseudothecium Ascospore Number<br />
Sexual state (µm) (µm) <strong>of</strong> cells<br />
Mycosphaerella<br />
graminicola 70-100 10-15 x 2-3 2<br />
Phaeosphaeria<br />
nodorum 120-200 23-32 x 4-6 4<br />
a McDonald <strong>and</strong> Martinez (1991).<br />
b Cooley <strong>and</strong> Caten (1991).<br />
infection process <strong>and</strong> the<br />
production <strong>of</strong> pycnidia in the host<br />
leaf bear some remarkable<br />
similarities (Karjalainen <strong>and</strong><br />
Lounatmaa, 1986; Kema et al.,<br />
1996b; Straley, 1979). Usually all<br />
observed conidia <strong>of</strong> S. tritici on leaf<br />
surfaces <strong>of</strong> either resistant or<br />
susceptible cultivars germinated<br />
<strong>and</strong> produced germ tubes. The<br />
same can be said in the case <strong>of</strong> S.<br />
nodorum, but in one case (T.<br />
aestivum Manitou, CI 13775<br />
resistant spring wheat), significant<br />
differences were found in<br />
germination <strong>of</strong> conidia on leaves <strong>of</strong><br />
susceptible <strong>and</strong> resistant cultivars<br />
(Straley, 1979). Hyphae <strong>of</strong> both<br />
species grow extensively over leaf<br />
surfaces, branching <strong>and</strong> <strong>of</strong>ten<br />
crossing stomata <strong>and</strong> guard cells<br />
without entering them.<br />
According to Kema et al.<br />
(1996b), penetrations <strong>of</strong> S. tritici<br />
were strictly stomatal <strong>and</strong> no direct<br />
penetrations <strong>of</strong> the cuticle were<br />
observed. Appressorial structures<br />
were seen in both pathogens, not<br />
associated with particular<br />
anatomical features, but most <strong>of</strong>ten<br />
in epidermal cracks. In the case <strong>of</strong><br />
S. nodorum, direct epidermal cell<br />
penetrations under appressorial<br />
structures could not be observed<br />
with the light microscope, but were<br />
confirmed with the scanning<br />
electron microscope (SEM).<br />
Stomatal penetration by S. nodorum<br />
was readily observed 72 h after<br />
inoculation. Stomata were<br />
penetrated in either the open or<br />
closed condition, with or without<br />
appressoria (Harrower, 1978;<br />
Straley, 1979).<br />
Reported patterns <strong>of</strong> occurrence<br />
<strong>of</strong> S. nodorum <strong>and</strong> S. tritici have<br />
changed dramatically during the<br />
past 15 years. In the early 1980s, S.<br />
nodorum was considered most<br />
important in northern Europe,<br />
eastern USA <strong>and</strong> Western Australia,<br />
while S. tritici was most prevalent<br />
in Mediterranean climates <strong>and</strong> the<br />
Great Plains <strong>of</strong> North America.<br />
Shifts to a greater importance <strong>of</strong> S.<br />
tritici have occurred in the UK <strong>and</strong><br />
are continuing to progress in<br />
central Europe. Some <strong>of</strong> the<br />
Biology <strong>of</strong> the <strong>Septoria</strong>/<strong>Stagonospora</strong> Pathogens: An Overview 21<br />
contributing factors in the UK may<br />
have been host susceptibility,<br />
earlier sowing, increased N<br />
fertilization <strong>and</strong> high summer<br />
rainfall as well as resistance to<br />
certain fungicides (Bayles, 1991).<br />
An interesting situation that<br />
could benefit from a systematic<br />
region-wide study has been<br />
reported from the Maghreb in<br />
North Africa. Populations <strong>of</strong> S.<br />
tritici particularly adapted to durum<br />
wheats have evolved, along with<br />
populations <strong>of</strong> S. nodorum more<br />
adapted to aestivum wheats. This<br />
phenomonon has become<br />
particularly obvious in Morocco,<br />
where a decided increase in<br />
plantings <strong>of</strong> bread wheats has<br />
occurred in recent years (Jlibene et<br />
al., 1995; Saadoui, 1987).<br />
Where sexual reproduction<br />
plays a role in epidemics, new<br />
virulence combinations can be<br />
selected by host virulence genes,<br />
<strong>and</strong> recombination <strong>of</strong> virulence<br />
genes will overcome “pyramids” <strong>of</strong><br />
resistance genes (McDonald, 1997).<br />
Ahmed et al. (1996) concluded that<br />
S. tritici populations adapt to host<br />
cultivars <strong>and</strong> that susceptible<br />
cultivars tend to select higher levels<br />
<strong>of</strong> pathogen aggressiveness in the<br />
field. Although evidence has been<br />
accumulated over a long period for<br />
specific gene-for-gene relationships<br />
in the S. tritici – wheat system (Eyal<br />
et al., 1985; Kema et al., 1996a), the<br />
question remains whether<br />
distinctive races can be identified<br />
with specific virulence for<br />
particular cultivars <strong>and</strong> whether<br />
the interaction indicates a gene-forgene<br />
system in the S. tritici — <strong>and</strong><br />
S. nodorum – wheat pathosystem<br />
(Johnson, 1992).
22<br />
Session 1 — A.L. Scharen<br />
The bulk <strong>of</strong> evidence continues<br />
to indicate that in S. nodorum —<br />
<strong>and</strong> S. tritici – wheat systems, genefor-gene<br />
interactions probably<br />
occur when selected cultures are<br />
inoculated on specific host plants<br />
under controlled conditions, but<br />
when population confronts<br />
population in the field, resistance<br />
to infection in wheat is generally<br />
non-specific <strong>and</strong> aggressiveness is<br />
the principal attribute <strong>of</strong> pathogen<br />
populations.<br />
References<br />
Ahmed, H.U., Mundt, C.C., H<strong>of</strong>fer,<br />
M.E., <strong>and</strong> Coakley, S.M. 1996.<br />
Selective influence <strong>of</strong> wheat<br />
cultivars on pathogenicity <strong>of</strong><br />
Mycosphaerella graminicola<br />
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Phytopathology 86:454-458.<br />
Ainsworth, G.C. 1965. Historical<br />
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Arseniuk, E., Cunfer, B.M., Mitchell,<br />
S., <strong>and</strong> Kresovich, S. 1997.<br />
Characterization <strong>of</strong> genetic<br />
similarities among isolates <strong>of</strong><br />
<strong>Stagonospora</strong> spp. <strong>and</strong> <strong>Septoria</strong><br />
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Radzikow, Pol<strong>and</strong>. pp. 123-126.<br />
McDonald, B.A., <strong>and</strong> Martinez, J.P.<br />
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Saadoui, E.M. 1987. Physiologic<br />
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Shaw, M.W., <strong>and</strong> Royle, D.J. 1989.<br />
Airborne inoculum as a major<br />
source <strong>of</strong> <strong>Septoria</strong> tritici<br />
(Mycosphaerella graminicola)<br />
infections in winter wheat crops in<br />
the UK. Plant Pathology 38:35-43.<br />
Sprague, R. 1950. <strong>Diseases</strong> <strong>of</strong> cereals<br />
<strong>and</strong> grasses in North America.<br />
Ronald Press, New York. 538 pp.<br />
Straley, M.L. 1979. Pathogenesis <strong>of</strong><br />
<strong>Septoria</strong> nodorum (Berk.) Berk. on<br />
wheat cultivars varying in<br />
resistance to glume blotch. Ph.D.<br />
Thesis, Montana State University,<br />
Bozeman.<br />
Ueng, P.P., Subramaniam, K., Chen,<br />
W., Arseniuk, E., Lixin, W.,<br />
Cheung, A.M., H<strong>of</strong>fmann, G.M.,<br />
<strong>and</strong> Bergstrom, G.C. 1998.<br />
Intraspecific genetic variation <strong>of</strong><br />
<strong>Stagonospora</strong> avenae <strong>and</strong> its<br />
differentiation from S. nodorum.<br />
Mycol. Res. 102(5):607-614.
Molecular Analysis <strong>of</strong> a DNA Fingerprint Probe from<br />
Mycosphaerella graminicola<br />
S.B. Goodwin <strong>and</strong> J.R. Cavaletto<br />
USDA-ARS, Department <strong>of</strong> Botany <strong>and</strong> Plant Pathology, Purdue University, West Lafayette, IN, USA<br />
Abstract<br />
Clones hybridizing to the Mycosphaerella graminicola DNA fingerprint probe pSTL70 were identified from<br />
subgenomic libraries <strong>and</strong> sequenced. Analyses <strong>of</strong> the DNA sequences <strong>of</strong> these clones plus the original pSTL70 clone revealed<br />
that pSTL70 contains part <strong>of</strong> the open reading frame for a probable homologue <strong>of</strong> an osmosensing histidine kinase gene from<br />
yeast. The remaining portion <strong>of</strong> the clone contained a partial reverse transcriptase gene sequence <strong>and</strong> a 29 base pair direct<br />
repeat, which could mean that the clone is a transposable element. Methods for converting transposable elements into<br />
improved DNA fingerprinting techniques are discussed.<br />
DNA fingerprinting is a<br />
powerful tool for analyzing the<br />
genetic structure <strong>of</strong> fungal<br />
populations. Several fingerprinting<br />
strategies have been employed,<br />
including those based on the<br />
polymerase chain reaction (PCR)<br />
such as r<strong>and</strong>om amplified<br />
polymorphic DNA (RAPD),<br />
amplified fragment length<br />
polymorphism (AFLP) <strong>and</strong> DNA<br />
amplification fingerprinting (DAF).<br />
These techniques can provide<br />
information on hundreds <strong>of</strong><br />
potential genetic loci in a very short<br />
time. However, each method has<br />
problems that can limit its utility.<br />
The RAPD technique relies on<br />
annealing <strong>of</strong> short (only 10 base)<br />
primers at low temperatures. This<br />
leads to high variability <strong>and</strong> low<br />
transportability to other labs.<br />
AFLPs require a reasonably high<br />
degree <strong>of</strong> sophistication in<br />
expertise <strong>and</strong> facilities, <strong>and</strong> also can<br />
suffer from problems with<br />
repeatability. DAF is operationally<br />
simple but the large number <strong>of</strong><br />
b<strong>and</strong>s produced can be difficult to<br />
separate <strong>and</strong> interpret.<br />
The most widely used DNA<br />
fingerprint technique is restriction<br />
fragment length polymorphism<br />
analysis using small pieces <strong>of</strong><br />
repetitive genomic DNA as probes.<br />
This technique has been used<br />
extensively to analyze the<br />
population biology <strong>of</strong> the<br />
ascomycetes Magnaporthe grisea<br />
(Hamer et al., 1989) <strong>and</strong><br />
Mycosphaerella graminicola<br />
(McDonald <strong>and</strong> Martinez, 1991),<br />
<strong>and</strong> the oomycete Phytophthora<br />
infestans (Goodwin et al., 1992).<br />
Thous<strong>and</strong>s <strong>of</strong> isolates <strong>of</strong> each<br />
species have been analyzed. The M.<br />
grisea repeat (MGR) 586 probe<br />
contains part <strong>of</strong> an inverted repeat<br />
transposon (Farman et al., 1996).<br />
However, the nature <strong>of</strong> the<br />
repeating elements in the M.<br />
graminicola pSTL70 <strong>and</strong> P. infestans<br />
RG57 probes has not been<br />
determined.<br />
This study was initiated to test<br />
whether the M. graminicola pSTL70<br />
probe was part <strong>of</strong> a transposable<br />
element. The long-term goal is to<br />
clone individual DNA fingerprint<br />
loci <strong>and</strong> convert them to a PCR-<br />
23<br />
based system by designing specific<br />
primers to unique regions at each<br />
genetic locus.<br />
Materials <strong>and</strong> Methods<br />
Subgenomic libraries were<br />
constructed from isolates IPO 323<br />
<strong>and</strong> 94269. These are the parent<br />
isolates <strong>of</strong> the M. graminicola<br />
mapping population (Kema et al.,<br />
1996). Approximately 2 µg <strong>of</strong><br />
genomic DNA from each isolate<br />
was digested to completion with<br />
the restriction enzyme Pst I. DNA<br />
fragments from 0.5-3 kb <strong>and</strong> from<br />
3-9 kb for each isolate were excised<br />
from gels <strong>and</strong> purified using<br />
Wizard PCR Prep (Promega,<br />
Madison, WI). The DNA fragments<br />
were then ligated into pBluescript<br />
vector <strong>and</strong> transformed into<br />
competent cells <strong>of</strong> E. coli strain<br />
INValphaF’. White colonies were<br />
transferred into 200 µL LB+amp<br />
medium in 96-well Microtest tissue<br />
culture plates <strong>and</strong> grown at 37°C<br />
overnight. The 96 cultures from<br />
each plate were transferred onto<br />
large (150 x 15 mm) LB+amp agar
24<br />
Session 1 — S.B. Goodwin <strong>and</strong> J.R. Cavaletto<br />
plates using a replica plater, <strong>and</strong><br />
incubated upside down at 37°C<br />
overnight.<br />
Colony lifts were made onto 8 x<br />
12 cm pieces <strong>of</strong> Zeta Probe<br />
(BioRad) membranes by briefly<br />
laying the membrane pieces over<br />
the 96 colonies <strong>and</strong> lifting to pick<br />
up the bacteria. Membranes were<br />
placed colony side up onto blotting<br />
paper soaked with 10% SDS for 3<br />
min, then 0.5 M NaOH/1.5 M NaCl<br />
for 5 min, 0.5 M Tris, pH 8.0/1.5 M<br />
NaCl for 5 min, <strong>and</strong> 6x SSC for 5<br />
min. Finally, a UV Stratalinker<br />
(Stratagene) was used to crosslink<br />
the plasmid DNA to the membrane.<br />
For Southern analysis, pSTL70<br />
DNA was labeled using the<br />
R<strong>and</strong>om Primer Fluorescein<br />
labeling kit with antifluorescein-<br />
HRP (DuPont NEN) <strong>and</strong><br />
hybridized according to the<br />
manufacturer’s instructions.<br />
Approximately 4,000 clones (2,000<br />
from each isolate) were screened.<br />
Positive hybridizations were<br />
verified by digesting each positive<br />
clone with Pst I to release the insert,<br />
separating the fragments on<br />
agarose gels, blotting <strong>and</strong> probing<br />
as described above. Clones that<br />
hybridized after two rounds <strong>of</strong><br />
screening were sequenced using a<br />
Pharmacia ALF automated DNA<br />
sequencer.<br />
Results<br />
Among 4,000 clones screened,<br />
five hybridized strongly to pSTL70.<br />
Complete sequences have been<br />
obtained for the original pSTL70<br />
clone <strong>and</strong> three others. Clones<br />
2E11, 9A5 <strong>and</strong> 11E8 each had large<br />
regions <strong>of</strong> near identity with<br />
pSTL70 (Table 1). However, the<br />
three clones shared no similarity<br />
with each other. BLASTX searches<br />
<strong>of</strong> the putative translation products<br />
identified a number <strong>of</strong> GenBank<br />
accessions with similarity to clones<br />
pSTL70, 2E11 <strong>and</strong> 11E8 (Table 1).<br />
The original pSTL70 clone had high<br />
similarity to a two-component<br />
regulator gene from yeast, Sln1,<br />
<strong>and</strong> a similar gene from C<strong>and</strong>ida<br />
albicans.<br />
Clone 2E11 had even higher<br />
similarity to the same genes. This<br />
clone corresponded to <strong>and</strong><br />
extended the 5’ end <strong>of</strong> pSTL70<br />
(Figure 1). Clone 11E8<br />
corresponded to <strong>and</strong> extended the<br />
3’ end <strong>of</strong> pSTL70 (Figure 1). This<br />
sequence may code for a reverse<br />
transcriptase. The final 239 bases <strong>of</strong><br />
pSTL70 contained part <strong>of</strong> the<br />
putative reverse transcriptase<br />
coding sequence, but not enough <strong>of</strong><br />
the gene to obtain positive BLAST<br />
hits in GenBank.<br />
Table 1. Analysis <strong>of</strong> the Mycosphaerella graminicola DNA fingerprint clone pSTL70 <strong>and</strong> three<br />
additional clones that hybridized to pSTL70 in Southern analysis.<br />
Size Overlap Blastx E<br />
Clone (bp) with pSTL70 results value Comments<br />
pSTL70 2860 N/A Sln1 8e-14 29 bp repeat<br />
2E11 3073 1278 Sln1 2e-15 Gene sequence<br />
9A5 2640 1103 No hits N/A 29 bp repeat<br />
11E8 1636 417 bp Reverse 0.006 Transposable<br />
transcriptase element (?)<br />
pSTL70<br />
2E11<br />
9A5<br />
11E8<br />
The first 917 bases <strong>of</strong> 11E8 are<br />
not related to the sequence <strong>of</strong><br />
pSTL70. Clone 9A5 had no<br />
similarity to any sequences in<br />
GenBank. The first 1103 bases <strong>of</strong><br />
this clone were identical to pSTL70,<br />
but the final 1537 bases were<br />
unrelated (Figure 1). Clones<br />
pSTL70 <strong>and</strong> 9A5 both contained a<br />
29 bp sequence that was t<strong>and</strong>emly<br />
repeated approximately 3.5 times<br />
(Figure 1). Southern analysis <strong>of</strong><br />
genomic DNA from the parents<br />
<strong>and</strong> several progeny isolates <strong>of</strong> the<br />
M. graminicola mapping population<br />
revealed that 9A5 also gave a DNA<br />
fingerprint pattern, while 2E11 <strong>and</strong><br />
16D7 did not (data not shown).<br />
Discussion<br />
The M. graminicola fingerprint<br />
probe pSTL70 (McDonald <strong>and</strong><br />
Martinez, 1991) contains part <strong>of</strong> the<br />
open reading frame for the yeast<br />
two-component regulator gene<br />
Sln1. This gene has been shown to<br />
function as an osmosensing<br />
Sln 1 R<br />
RT<br />
Figure 1. Relationships among the Mycosphaerella graminicola DNA fingerprint probe pSTL70<br />
<strong>and</strong> related clones. Regions <strong>of</strong> overlap are indicated by open boxes, unique regions by single<br />
lines. Clones are labeled on the left. Approximate locations <strong>of</strong> the Sln1 <strong>and</strong> reverse<br />
transcriptase (RT) open reading frames are indicated by arrows. The t<strong>and</strong>em repeats are<br />
indicated by R.
histidine kinase in yeast, although<br />
its function in M. graminicola<br />
remains unknown. Clone 2E11<br />
includes an even larger portion <strong>of</strong><br />
this gene containing the first 500<br />
amino acids <strong>of</strong> the putative yeast<br />
homologue.<br />
Other parts <strong>of</strong> the pSTL70 clone<br />
contain a 29 bp t<strong>and</strong>em repeat <strong>and</strong><br />
a partial coding sequence for a<br />
reverse transcriptase. These<br />
indicate that pSTL70 may contain<br />
part <strong>of</strong> a transposable element.<br />
Hybridization analysis confirmed<br />
that this region <strong>of</strong> the clone was<br />
responsible for the DNA<br />
fingerprint pattern, which<br />
strengthens the transposable<br />
element hypothesis. The MGR586<br />
probe <strong>of</strong> M. grisea also has been<br />
shown to contain a transposable<br />
element flanked by 16 bp t<strong>and</strong>em<br />
repeats <strong>and</strong> 9 bp inverted repeat<br />
sequences (Farman et al., 1996). If<br />
pSTL70 is a transposable element, it<br />
is truncated partway through the<br />
reverse-transcriptase sequence.<br />
Cloning <strong>and</strong> analysis <strong>of</strong> the<br />
remaining portion <strong>of</strong> the reversetranscriptase<br />
gene will be necessary<br />
to test whether it is flanked by<br />
direct <strong>and</strong>/or inverted repeats.<br />
It is surprising that the three<br />
additional clones identified using<br />
pSTL70 as a probe did not overlap<br />
with each other. Two <strong>of</strong> the clones,<br />
9A5 <strong>and</strong> 11E8, contained unique<br />
regions that did not overlap with<br />
pSTL70. There are two likely<br />
explanations for this result. One is<br />
that these clones were from<br />
different loci in the M. graminicola<br />
Molecular Analysis <strong>of</strong> a DNA Fingerprint Probe from Mycosphaerella graminicola 25<br />
genome that shared segments due<br />
to independent movements <strong>of</strong> the<br />
putative transposable element. The<br />
other possibility is that one or more<br />
<strong>of</strong> these clones is a chimera with<br />
multiple inserts. The chimera<br />
hypothesis is being tested by<br />
additional analyses <strong>of</strong> the cloned<br />
sequences.<br />
If the DNA fingerprint pattern<br />
<strong>of</strong> pSTL70 is due to a transposable<br />
element, it may be possible to make<br />
specific PCR primers to amplify<br />
single DNA fingerprint loci. This<br />
could be accomplished using two<br />
different strategies. If the element is<br />
relatively small, primers could be<br />
made in the single-copy regions<br />
flanking the element. These should<br />
give a large amplification product<br />
when the element is present <strong>and</strong> a<br />
small one when it is not. Such<br />
differences could be resolved easily<br />
on agarose gels without the need<br />
for autoradiography, <strong>and</strong> should be<br />
easily transportable to other<br />
laboratories.<br />
The other approach would be to<br />
design one primer within the<br />
transposable element <strong>and</strong> the other<br />
in the flanking region. This would<br />
give a plus/minus polymorphism<br />
at each locus: plus when the<br />
transposable element is present <strong>and</strong><br />
minus when it is absent. The<br />
advantage <strong>of</strong> this approach is that<br />
the primers could be designed so<br />
that the sizes <strong>of</strong> the PCR products<br />
would not overlap. It then may be<br />
possible to use multiplex PCR to<br />
perform DNA fingerprinting <strong>of</strong><br />
individual genetic loci from small<br />
quantities <strong>of</strong> starting genomic DNA<br />
in single PCR reactions. This would<br />
be much faster <strong>and</strong> simpler than<br />
Southern analysis <strong>and</strong> would avoid<br />
the repeatability, transportability,<br />
<strong>and</strong> interpretation problems <strong>of</strong><br />
other PCR-based methods for<br />
fungal population genetic analyses.<br />
Acknowledgments<br />
We thank Bruce McDonald for<br />
providing the original pSTL70<br />
DNA fingerprint clone <strong>and</strong> for<br />
encouragement during the course<br />
<strong>of</strong> this project.<br />
References<br />
Farman, M.L., S. Taura, <strong>and</strong> S.A.<br />
Leong. 1996. The Magnaporthe<br />
grisea DNA fingerprinting probe<br />
MGR586 contains the 3’ end <strong>of</strong> an<br />
inverted repeat transposon. Mol.<br />
Gen. Genet. 251:675-681.<br />
Goodwin, S.B., A. Drenth, <strong>and</strong> W.E.<br />
Fry. 1992. Cloning <strong>and</strong> genetic<br />
analyses <strong>of</strong> two highly<br />
polymorphic, moderately<br />
repetitive nuclear DNAs from<br />
Phytophthora infestans. Curr. Genet.<br />
22:107–115.<br />
Hamer, J.E., L. Farrall, M.J. Orbach, B.<br />
Valent, <strong>and</strong> F.G. Chumley. 1989.<br />
Host species-specific conservation<br />
<strong>of</strong> a family <strong>of</strong> repeated DNA<br />
sequences in the genome <strong>of</strong> a<br />
fungal plant pathogen. Proc. Natl.<br />
Acad. Sci., USA 86:9981-9985.<br />
Kema, G.H.J., E.C.P. Verstappen, M.<br />
Todorova, <strong>and</strong> C. Waalwijk. 1996.<br />
Successful crosses <strong>and</strong> molecular<br />
tetrad <strong>and</strong> progeny analyses<br />
demonstrate heterothallism in<br />
Mycosphaerella graminicola. Curr.<br />
Genet. 30:251-258.<br />
McDonald, B.A., <strong>and</strong> J.P. Martinez.<br />
1991. DNA fingerprinting <strong>of</strong> the<br />
plant pathogenic fungus<br />
Mycosphaerella graminicola<br />
(anamorph <strong>Septoria</strong> tritici). Exp.<br />
Mycol. 15:146-158.
26<br />
Characterization <strong>of</strong> <strong>Septoria</strong> tritici Variants <strong>and</strong> PCR<br />
Assay for Detecting <strong>Stagonospora</strong> nodorum <strong>and</strong> <strong>Septoria</strong><br />
tritici in Wheat<br />
S. Hamza, 1 M. Medini, 1 T. Sassi, 1 S. Abdennour, 1 M. Rouassi, 1<br />
A.B. Salah, 1 M. Cherif, 1 R. Strange, 2 <strong>and</strong> M. Harrabi1 1 Laboratoire de Génétique, Institut National Agronomique de Tunisie, Tunisia<br />
2 Plant Pathology, UCL London, United Kingdom<br />
Abstract<br />
Pathogenic specialization <strong>of</strong> <strong>Septoria</strong> tritici was studied by inoculating 14 isolates <strong>of</strong> the fungus, <strong>of</strong> which seven were<br />
obtained from durum wheat <strong>and</strong> seven were isolated from bread wheat. Isolates obtained from durum wheat were more<br />
virulent on durum wheat, while those isolated from bread wheat were more severe on bread wheat, which revealed<br />
physiologic specialization <strong>of</strong> S. tritici to either bread or durum wheat. The sequence coding for the nuclear 5.8S rDNA <strong>and</strong><br />
the internal transcribed spacer (ITS1 <strong>and</strong> ITS2) were amplified by polymerase chain reaction <strong>and</strong> sequenced for five isolates<br />
adapted to bread wheat <strong>and</strong> five isolates adapted to durum wheat. These sequences were identical between both variants,<br />
resulting in the absence <strong>of</strong> divergence inside tritici species. <strong>Septoria</strong> tritici <strong>and</strong> <strong>Stagonospora</strong> nodorum isolates collected<br />
from Tunisia were tested for amplification with specific primers to these pathogens. This revealed the primer’s efficiency to<br />
distinguish <strong>Septoria</strong> species <strong>and</strong> detect <strong>Stagonospora</strong> nodorum DNA with as little as 2 pg <strong>of</strong> DNA. Time course<br />
quantification <strong>of</strong> S. tritici mycelia after inoculation <strong>of</strong> resistant <strong>and</strong> susceptible cultivars distinguished those cultivars by the<br />
earliest date <strong>of</strong> apparition <strong>of</strong> a PCR product.<br />
Extensive genetic variation for<br />
virulence in <strong>Septoria</strong> tritici or its<br />
telomorph Mycosphaerella<br />
graminicola, characterized by<br />
differential interaction between<br />
host <strong>and</strong> pathogen genotypes,<br />
suggested the involvement <strong>of</strong><br />
specific factors for virulence <strong>and</strong><br />
resistance in the pathosystem<br />
(Kema et al., 1996a). Specific<br />
interaction between M. graminicola<br />
<strong>and</strong> wheat was well demonstrated<br />
by statistical evidence (Kema et al.,<br />
1996b), which suggested gene-forgene<br />
interaction between resistance<br />
<strong>and</strong> virulence in host <strong>and</strong><br />
pathogen, respectively.<br />
Mycosphaerella graminicola<br />
specialization is much more<br />
pronounced on bread wheat <strong>and</strong><br />
durum wheat than differential<br />
specificity on a particular cultivar.<br />
In particular when considering the<br />
presence <strong>of</strong> pycnidia as a disease<br />
parameter, bread wheat <strong>and</strong> durum<br />
wheat isolates were particularly<br />
virulent on bread <strong>and</strong> durum<br />
wheat, respectively. Therefore<br />
bread wheat <strong>and</strong> durum wheat<br />
variants in M. graminicola were<br />
considered. The identical sequence<br />
<strong>of</strong> the internal transcribed spacer<br />
ribosomal DNA (ITS rDNA)<br />
observed by Kema et al. (1996a)<br />
showed that both variants could<br />
not be distinguished using their<br />
ribosomal DNA <strong>and</strong> that both were<br />
therefore from a similar taxonomic<br />
rank.<br />
PCR has been shown to be a<br />
powerful technique to detect small<br />
amounts <strong>of</strong> fungal plant pathogens.<br />
The technique is now commonly<br />
used by field pathologists <strong>and</strong><br />
growers for diagnosing several<br />
plant pathogens. The early<br />
detection <strong>of</strong> plant disease allows<br />
the judicious use <strong>of</strong> agricultural<br />
fungicides; early treatment is <strong>of</strong>ten<br />
more effective because the<br />
pathogen is less well established in<br />
its host. PCR amplification using<br />
specific primers for S. tritici <strong>and</strong><br />
<strong>Stagonospora</strong> nodorum or <strong>Septoria</strong><br />
nodorum was able to distinguish<br />
these pathogens <strong>and</strong> detect a few<br />
fungal cells in infested wheat<br />
tissues well before disease<br />
symptoms were apparent (Beck<br />
<strong>and</strong> Ligon, 1995). Since both<br />
pathogens may infect the same<br />
plant <strong>and</strong> visually distinguishing<br />
them is difficult, the specific<br />
detection <strong>of</strong> S. tritici <strong>and</strong> S. nodorum<br />
is well circumvented using the PCR<br />
technique <strong>and</strong> facilitates the<br />
decision <strong>of</strong> whether or not to treat<br />
with fungicides <strong>and</strong> which<br />
fungicides to use.
According to the previous<br />
observations, this paper describes<br />
physiologic specialization <strong>of</strong> bread<br />
<strong>and</strong> durum wheat isolates collected<br />
from Morocco <strong>and</strong> Tunisia,<br />
respectively, <strong>and</strong> the sequence <strong>of</strong><br />
their ITS rDNA. Specific primers<br />
determined by Beck <strong>and</strong> Ligon<br />
(1995) for PCR detection <strong>of</strong> S. tritici<br />
<strong>and</strong> S. nodorum were used to<br />
differentiate those pathogens from<br />
other fungal pathogens <strong>of</strong> wheat<br />
<strong>and</strong> to detect their presence in<br />
asymptomatic wheat plants. Time<br />
course PCR quantification <strong>of</strong> S.<br />
tritici in infected wheat was<br />
assessed to analyze the evolution <strong>of</strong><br />
mycelia in resistant <strong>and</strong> susceptible<br />
cultivars.<br />
Materials <strong>and</strong> Methods<br />
Plant material <strong>and</strong><br />
experimental design<br />
Thirty-six durum<br />
wheat <strong>and</strong> eight bread<br />
wheat cultivars<br />
(Table 1) were<br />
employed to study<br />
genetic variation for<br />
virulence. Seeds <strong>of</strong> the<br />
cultivars were sown in<br />
trays with 228 alveolus<br />
with 3 cm x 3 cm<br />
surface <strong>and</strong> 5 cm<br />
height. Each alveolus<br />
contained four seeds.<br />
The experiment was<br />
conducted in an<br />
arbitrary complete<br />
block design with three<br />
replicates for each<br />
isolate. Replicates were<br />
blocked in the same<br />
tray, separated by two<br />
rows <strong>of</strong> alveolus.<br />
Characterization <strong>of</strong> <strong>Septoria</strong> tritici Variants <strong>and</strong> PCR Assay for Detecting <strong>Stagonospora</strong> nodorum <strong>and</strong> <strong>Septoria</strong> tritici in Wheat 27<br />
Experimental procedure<br />
Seven isolates <strong>of</strong> S. tritici taken<br />
from durum wheat <strong>and</strong> seven<br />
isolates from bread wheat collected<br />
in different regions <strong>of</strong> Tunisia <strong>and</strong><br />
Morocco, respectively, were used to<br />
inoculate durum <strong>and</strong> bread wheat<br />
cultivars (Table 2). Eleven-day-old<br />
seedlings with emerging second<br />
leaves were inoculated with a<br />
monosporal suspension till run-<strong>of</strong>f.<br />
The inoculum was prepared from<br />
monosporal culture cultivated on<br />
potato dextrose agar (PDA)<br />
medium. The spores were scraped<br />
from the agar, re-suspended in<br />
distilled water, filtered, <strong>and</strong><br />
adjusted to 106-107 spores/ml. After<br />
inoculation the trays were<br />
incubated in a humid chamber for<br />
72 hours <strong>and</strong> returned to a growth<br />
chamber at temperature 20-25°C<br />
during the day, 17°C during the<br />
night, <strong>and</strong> 12 hours photoperiod.<br />
Table 1. List <strong>of</strong> durum <strong>and</strong> bread wheat cultivars used to study<br />
genetic variation for virulence in Mycosphaerella<br />
graminicola.<br />
Code Cultivars Code Cultivars<br />
01 Florence aurore* 23 Medea AC3<br />
02 Florence Aurore x PUSA* 24 Medea AC4<br />
03 Derbessi x Biskri 25 Medea AP1<br />
04 Guelma* 26 Medea AP2<br />
05 EAPC6326127* 27 Allorea*<br />
06 Tunis9* 28 Richelle*<br />
07 Tunis23* 29 Sebei glabre<br />
08 Abdelkader 30 Sebei pubescent<br />
09 Agili pubescent AC1 31 Souri AC8<br />
10 Bidi AP4 32 Souri AC9<br />
11 Bidi AP1 33 Medea AC1<br />
12 Bidi AP3 34 Medea AC2<br />
13 Bidi AP 35 Medea AP6<br />
14 Biskri glabre 36 Medea AP10<br />
15 Biskri glabre AP3 37 Medea RP1<br />
16 Derbessi AC1 38 Biancullida ICM27<br />
17 Derbessi AP1 39 Mahmoudi ICM28<br />
18 Derbessi AP2 40 Mahmoudi ICM67<br />
19 Hamira AC2 41 Khotifa x ICM75<br />
20 Hamira AC3 42 ICM313<br />
21 Hamira AC4 43 ICM314<br />
22 Hamira AC5 44 Hamira<br />
* Bread wheat cultivars.<br />
Disease evaluation<br />
<strong>and</strong> statistical analysis<br />
Disease severity was evaluated<br />
at 21 days after inoculation on the<br />
second leaf <strong>of</strong> the plant <strong>and</strong> using<br />
the presence <strong>of</strong> pycnidia as the<br />
disease parameter (Kema et al.,<br />
1996a). The collected data were<br />
treated with SAS s<strong>of</strong>tware (1993<br />
version). The presence <strong>of</strong> pycnidia<br />
was subjected to analysis <strong>of</strong><br />
variance. The tables <strong>of</strong> means <strong>of</strong> the<br />
14 isolates were subjected to<br />
hierarchical clustering using the<br />
statistical analysis s<strong>of</strong>tware CSTAT.<br />
DNA extraction from fungal<br />
spores <strong>and</strong> infected plants<br />
Spores <strong>of</strong> S. tritici <strong>and</strong> S. nodorum<br />
cultivated on PDA medium were<br />
subcultured in 100 ml flasks <strong>of</strong> yeast<br />
extract <strong>and</strong> glucose liquid medium.<br />
Five-day spore cultures were<br />
centrifuged <strong>and</strong> DNA extraction <strong>of</strong><br />
fungal spores was performed using<br />
CTAB extraction buffer according to<br />
the protocol described by Morjane et<br />
al. (1995). DNA extraction from three<br />
infected leaves was performed<br />
according to the protocol described<br />
by Möller et al. (1992). DNA pellet<br />
after extraction was re-suspended in<br />
50 µl Tris-HCl; 10 mM EDTA; 1 mM<br />
(TE) buffer.<br />
PCR amplification<br />
<strong>and</strong> sequencing<br />
The sequence <strong>of</strong> ITS rDNA<br />
concerned 5 isolates (MAR 1, 2, 3, 4,<br />
5) collected from Morocco (Table 2)<br />
<strong>and</strong> 5 isolates (TUN 1, 2, 3, 4, 5)<br />
collected from Tunisia (Table 2). ITS<br />
amplification was performed with<br />
ITS1 (5’-<br />
TCCGTAGGTGAACCTGCGG-3’)<br />
<strong>and</strong> ITS 4 (5’-<br />
TCCTCCGCTTATTGATATGC-3’)<br />
universal primers. PCR reactions
28<br />
Session 1 — S. Hamza, M. Medini, T. Sassi, S. Abdennour, M. Rouassi, A.B. Salah, M. Cherif, R. Strange, <strong>and</strong> M. Harrabi<br />
Table 2. Experimental code <strong>and</strong> origin <strong>of</strong> Mycosphaerella graminicola collected from Morocco<br />
<strong>and</strong> Tunisia to study genetic variation for virulence.<br />
Isolates collected from Tunisia Isolates collected from Morocco<br />
Origin Origin<br />
Code Location Wheat species Code Location Wheat species<br />
TUN1 Beja DW MAR1 Jemaa Sham BT<br />
TUN2 Mateur DW MAR2 Douyet BT<br />
TUN3 Mornag DW MAR3 Agadir BT<br />
TUN4 El Agba DW MAR4 Ain Orma BT<br />
TUN5 Zaghouane DW MAR5 Azrou BT<br />
TUN6 Siliana DW MAR6 Essaouira BT<br />
TUN7 Mjez El Bab DW MAR7 Tetouan BT<br />
were performed in a volume <strong>of</strong> 50<br />
µl containing 50 mM KCl, 10 mM<br />
Tris-HCl, pH8.3, 1.5 mM MgCl2,<br />
200 µM <strong>of</strong> dNTP, 50 pmole <strong>of</strong><br />
primer, 2.5 units <strong>of</strong> Taq<br />
polymerase (Boerhringer-<br />
Manheim) <strong>and</strong> 25 ng <strong>of</strong> genomic<br />
DNA. Reactions were run in a<br />
Thermolyne, Temptronic model<br />
thermal cycler for 30 cycles, each<br />
consisting <strong>of</strong> 15s at 94°C, 15 s at<br />
50°C, <strong>and</strong> 45 s at 72°C. An<br />
additional 5 min polymerization<br />
step at 72°C ended the<br />
amplification. The products were<br />
analyzed by electrophoresis <strong>of</strong> 20<br />
µl aliquot <strong>of</strong> each PCR sample on<br />
0.8% agarose gel.<br />
Specific DNA amplifications<br />
were performed as determined by<br />
Beck <strong>and</strong> Ligon (1995). ITS1 <strong>and</strong><br />
JB446 (5’-<br />
CGAGGCTGGAGTGGTGT-3’)<br />
primers were used for specific<br />
amplification <strong>of</strong> S. tritici DNA <strong>and</strong><br />
JB433 (5’-<br />
ACACTCAGTAGTTTACTACT-3’)<br />
<strong>and</strong> JB434 (5’-<br />
TGTGCTGCGTTCAATA-3’) were<br />
used to amplify S. nodorum DNA.<br />
PCR was performed as described<br />
above, except the annealing<br />
temperature was 57°C.<br />
ITS sequencing was performed<br />
using 90 ng <strong>of</strong> 500 bp amplified<br />
product with ITS1 <strong>and</strong> ITS4. The<br />
sequence was determined by the<br />
dideoxynucleotide chain<br />
termination method using an<br />
automated sequencer (Perkin<br />
Elmer) at the Darwin Building <strong>of</strong><br />
University College London<br />
(London, United Kingdom).<br />
Fungal DNA amplification in<br />
resistant <strong>and</strong> susceptible<br />
cultivars<br />
To evaluate the competition <strong>of</strong><br />
plant DNA with fungal DNA using<br />
ITS1 <strong>and</strong> ITS4 primers,<br />
amplification was performed using<br />
1 µl <strong>and</strong> 5 µl <strong>of</strong> plant DNA extract<br />
mixed with several amounts (<br />
0.001, 0.01; 0.1; 1; 10; 100 <strong>and</strong> 1000<br />
ng) <strong>of</strong> fungal DNA. The PCR<br />
conditions were the same as<br />
described before with a 57°C<br />
annealing temperature.<br />
The time course amplification<br />
using ITS1 <strong>and</strong> ITS4 primers was<br />
realized using 2 µl <strong>of</strong> DNA extract<br />
from one infected leaf at 3, 6, 9, 14,<br />
18, <strong>and</strong> 22 days after inoculation.<br />
The PCR conditions were the same<br />
as described before, with a 57°C<br />
annealing temperature.<br />
Results <strong>and</strong> Discussion<br />
Bread wheat derived isolates<br />
almost exclusively produced<br />
pycnidia in the bread cultivars,<br />
whereas pycnidial production by<br />
durum wheat derived isolates was<br />
almost entirely restricted to durum<br />
wheat isolates. The analysis <strong>of</strong><br />
variance (Table 3) shows highly<br />
significant differences (p
cultivars. The presence <strong>of</strong> pycnidia<br />
<strong>of</strong> bread wheat derived isolates<br />
does not exceed 15% <strong>of</strong> the leaf<br />
surface (data not shown). An<br />
explanation <strong>of</strong> the observed weak<br />
virulence would be long time<br />
conservation <strong>of</strong> these isolates in<br />
glycerol. Aggressivity <strong>of</strong> these<br />
isolates would be recovered after<br />
their direct isolation from infected<br />
leaves <strong>and</strong> inoculation from fresh<br />
spore culture.<br />
Cluster analysis <strong>of</strong> the 14<br />
isolates according to the presence<br />
<strong>of</strong> pycnidia resulted in nine<br />
different clusters. Bread wheat<br />
derived isolates are clearly<br />
separated from durum wheat<br />
derived isolates (Figure 1). Durum<br />
wheat isolates are clustered into six<br />
groups. Two isolates (TUN7 <strong>and</strong><br />
TUN6) originated from distant<br />
regions (Mjez El Bab <strong>and</strong> Siliana,<br />
respectively) belong to the same<br />
group, whereas isolates from the<br />
same regions belong to different<br />
216.7<br />
150<br />
100<br />
Figure 1. Hierarchical classification <strong>of</strong> 14<br />
<strong>Septoria</strong> tritici isolates as determined by<br />
CSTAT s<strong>of</strong>tware.<br />
Characterization <strong>of</strong> <strong>Septoria</strong> tritici Variants <strong>and</strong> PCR Assay for Detecting <strong>Stagonospora</strong> nodorum <strong>and</strong> <strong>Septoria</strong> tritici in Wheat 29<br />
50<br />
MAR1<br />
MAR2<br />
MAR3<br />
MAR5<br />
MAR4<br />
MAR6<br />
MAR7<br />
TUN7<br />
TUN6<br />
TUN4<br />
TUN5<br />
TUN3<br />
TUN2<br />
TUN1<br />
0.0<br />
groups, indicating genetic variation<br />
for virulence within local<br />
populations.<br />
ITS sequence analysis<br />
Physiological specialization <strong>of</strong><br />
S. tritici to bread wheat <strong>and</strong> durum<br />
wheat led to the hypothesis <strong>of</strong> the<br />
existence <strong>of</strong> two subspecies inside<br />
tritici. For that purpose sequencing<br />
ITS rDNA would provide evidence<br />
about the taxonomy <strong>of</strong> those<br />
variants. The comparison <strong>of</strong> the<br />
consensus sequence <strong>of</strong> the 550 pb<br />
ITS region fragments obtained from<br />
five bread wheat derived isolates<br />
<strong>and</strong> five durum wheat derived<br />
isolates shows 100% homology<br />
(Figure 2). Therefore S. tritici<br />
variants belong to the same<br />
taxonomic rank <strong>and</strong> cannot be<br />
considered as two different<br />
subspecies <strong>of</strong> tritici. Since both<br />
variants coexist in the same field<br />
(McDonald, personal<br />
communication), permanent<br />
Figure 2. Nucleotide sequence <strong>of</strong> the internal transcribed spacer region derived from bread<br />
wheat (MAR) <strong>and</strong> durum wheat (TUN) derived isolates <strong>of</strong> <strong>Septoria</strong> tritici. The sequence includes<br />
the 5.8S rDNA gene <strong>and</strong> the internal transcribed spacer (ITS1 <strong>and</strong> ITS2). The consensus<br />
sequences were deduced from bread wheat <strong>and</strong> durum wheat derived isolates each.
30<br />
Session 1 — S. Hamza, M. Medini, T. Sassi, S. Abdennour, M. Rouassi, A.B. Salah, M. Cherif, R. Strange, <strong>and</strong> M. Harrabi<br />
Figure 2. cont.<br />
conversion <strong>of</strong> one variant to the<br />
other by genetic exchange <strong>of</strong><br />
virulence genes may occur through<br />
sexual reproduction.<br />
Diagnosis <strong>of</strong> S. tritici<br />
<strong>and</strong> S. nodorum<br />
In order to investigate the<br />
specificity <strong>of</strong> S. tritici <strong>and</strong> S.<br />
nodorum specific primers<br />
determined by Beck <strong>and</strong> Ligon<br />
(1995), we examined whether they<br />
produce PCR products from other<br />
fungal pathogens <strong>of</strong> wheat, such as<br />
Fusarium graminearum, Ustilago<br />
maydis, <strong>and</strong> Puccinia graminis. The<br />
S. tritici specific primers JB446 <strong>and</strong><br />
ITS1, <strong>and</strong> S. nodorum specific<br />
primers JB433 <strong>and</strong> JB434 did<br />
produce PCR products only<br />
from those pathogens (Figure<br />
3) revealing that these<br />
primers can be used to<br />
diagnose S. tritici <strong>and</strong> S.<br />
nodorum isolates collected<br />
from Tunisia.<br />
Amounts ranging from 2<br />
µg to 2 pg <strong>of</strong> S. nodorum were<br />
tested by PCR amplification with<br />
their respective specific primers<br />
(JB433, JB434) to determine the<br />
DNA threshold that can be<br />
detected. PCR amplification with<br />
550 bp<br />
JB446 <strong>and</strong> ITS1 JB433 <strong>and</strong> JB434<br />
Labda HindIII<br />
Healthy wheat<br />
<strong>Septoria</strong> tritici<br />
<strong>Stagonospora</strong> nodorum<br />
Fusarium graminearum<br />
Puccinia graminis<br />
Ustilago maydis<br />
Lambda HindIII<br />
Healty wheat<br />
<strong>Septoria</strong> tritici<br />
<strong>Stagonospora</strong> nodorum<br />
Fusarium graminearum<br />
Puccinia graminis<br />
Ustilago maydis<br />
Lambda HindIII<br />
Figure 3. Ethidium bromide stained agarose gel <strong>of</strong><br />
polymerase chain reaction amplification product<br />
using <strong>Septoria</strong> tritici specific primers (JB446 <strong>and</strong><br />
ITS1) <strong>and</strong> <strong>Stagonospora</strong> nodorum specific primers<br />
(JB433 <strong>and</strong> JB434) with healthy wheat DNA <strong>and</strong><br />
fungal DNA <strong>of</strong> wheat pathogen.<br />
Lambda DNA HindIII<br />
2 µg<br />
200 ng<br />
20 ng<br />
2 ng<br />
200 pg<br />
20 pg<br />
2 pg<br />
Lambda DNA HindIII<br />
Figure 4. Ethidium bromide stained agarose gel <strong>of</strong><br />
polymerase chain reaction products from<br />
amplification <strong>of</strong> 0.2 pg to 2 µg <strong>of</strong> genomic DNA <strong>of</strong><br />
<strong>Stagonospora</strong> nodorum DNA using S. nodorum<br />
specific primers JB433 <strong>and</strong> JB434.<br />
JB433 <strong>and</strong> JB434 was able to detect<br />
2 pg <strong>of</strong> S. nodorum DNA (Figure 4),<br />
which is almost the same amount<br />
detected by Beck <strong>and</strong> Ligon (1995).
Provided that S. nodorum fungal<br />
genomes do not exceed 100 Mbp in<br />
those conditions, PCR is able to<br />
detect as little as 20 fungal cells.<br />
Fungal DNA amplification in<br />
resistant <strong>and</strong> susceptible<br />
cultivars<br />
Different fragment sizes 600 bp<br />
<strong>and</strong> 550 bp amplification products<br />
were obtained from plant <strong>and</strong><br />
fungal DNA, respectively, using<br />
ITS1 <strong>and</strong> ITS4 primers (Figure 5).<br />
PCR amplification with ITS primers<br />
<strong>and</strong> using a mixture <strong>of</strong> both fungal<br />
<strong>and</strong> plant DNA templates resulted<br />
in the amplification <strong>of</strong> both<br />
fragments. However, the intensity<br />
<strong>of</strong> the fungal PCR product is<br />
relative to the amount <strong>of</strong> fungal<br />
DNA (Figure 5). These result<br />
suggest that plant DNA could be<br />
used as competitor <strong>of</strong> fungal DNA<br />
amplification with ITS primers.<br />
Time course PCR amplification<br />
using ITS1 <strong>and</strong> ITS4 primers at 0, 3,<br />
6, 9, 14, 18, <strong>and</strong> 22 days after<br />
inoculation <strong>of</strong> susceptible (Karim)<br />
<strong>and</strong> resistant (Jennah Khotifa)<br />
cultivars with S. tritici is shown in<br />
Figure 6. ITS rDNA amplification<br />
products from fungal DNA (550 bp)<br />
appeared at 3 <strong>and</strong> 18 days from<br />
infected susceptible <strong>and</strong> resistant<br />
cultivars, respectively.<br />
This result showed that the<br />
multiplication <strong>of</strong> S. tritici inside the<br />
plant is delayed in the resistant<br />
cultivar. Therefore, the time<br />
interval corresponding to the<br />
beginning <strong>of</strong> PCR product<br />
apparition may be an indicator <strong>of</strong><br />
resistance. Furthermore, the<br />
intensity <strong>of</strong> the PCR product<br />
increased with the days postinoculation<br />
(Figure 6), indicating a<br />
quantitative response <strong>of</strong> the PCR<br />
Characterization <strong>of</strong> <strong>Septoria</strong> tritici Variants <strong>and</strong> PCR Assay for Detecting <strong>Stagonospora</strong> nodorum <strong>and</strong> <strong>Septoria</strong> tritici in Wheat 31<br />
600 bp<br />
550 bp<br />
1000 ng<br />
100 ng<br />
10 ng<br />
1 ng<br />
0.1 ng<br />
0.01 ng<br />
0.001 ng<br />
Figure 5. Ethidium bromide stained agarose<br />
gel <strong>of</strong> polymerase chain reaction products<br />
using ITS1 <strong>and</strong> ITS4 primers with 0.0001 to<br />
1000 ng fungal DNA <strong>of</strong> <strong>Septoria</strong> tritici <strong>and</strong> 1 µl<br />
plant DNA extract from healthy wheat leaves.<br />
550 bp <strong>and</strong> 600 bp are ITS rDNA amplification<br />
products from <strong>Septoria</strong> tritici <strong>and</strong> wheat,<br />
respectively.<br />
Days after inoculation<br />
0 3 6 9 14 18 22<br />
600 bp<br />
550 bp<br />
Karim<br />
600 bp Jennah<br />
550 bp Khotifa<br />
Figure 6. Ethidium bromide stained agarose<br />
gel <strong>of</strong> time course PCR amplification<br />
products using ITS1 <strong>and</strong> ITS4 primers with<br />
<strong>Septoria</strong> tritici infected wheat (susceptible;<br />
Karim <strong>and</strong> resistant; Jennah Khotifa) DNA.<br />
550 bp <strong>and</strong> 600 bp are ITS rDNA amplification<br />
products from S. tritici <strong>and</strong> wheat,<br />
respectively.<br />
assay. These observations are<br />
preliminary results towards a<br />
quantification <strong>of</strong> fungal<br />
proliferation within tissue to<br />
rapidly characterize varying levels<br />
<strong>of</strong> host resistance. Characterization<br />
<strong>of</strong> host resistance by measuring the<br />
fungal biomass inside wheat leaves<br />
was tested using ELISA (Kema et<br />
al., 1996c). This technique did not<br />
detect the fungal antigen within the<br />
48 h-8 dpi interval, although a<br />
slight increase <strong>of</strong> the fungal tissue<br />
was observed microscopically during<br />
that interval. Thus the ability <strong>of</strong> PCR<br />
to detect fungal mycelia in a<br />
susceptible cultivar at 3 dpi better<br />
reflects the colonization <strong>of</strong> wheat<br />
cells by the fungus. These<br />
preliminary results will also help<br />
establish an efficient protocol for<br />
fungicide utilization. Effective<br />
utilization <strong>of</strong> fungicides would<br />
decrease or maintain the intensity <strong>of</strong><br />
the PCR product a long time after its<br />
application.<br />
References<br />
Beck, J.J., <strong>and</strong> J.M. Ligon. 1995.<br />
Polymerase chain reaction assays for<br />
the detection <strong>of</strong> <strong>Stagonospora</strong><br />
nodorum <strong>and</strong> <strong>Septoria</strong> tritici in wheat.<br />
Phytopathology 85:319-324.<br />
Kema, G.H.J., J.G. Annone, R. Sayoud,<br />
C.H. van Silfhout, M. van Ginkel,<br />
<strong>and</strong> J. Bree. 1996a. Genetic variation<br />
for virulence <strong>and</strong> resistance in the<br />
wheat-Mycosphaerella graminicola<br />
pathosystem I. Interaction between<br />
pathogen isolates <strong>and</strong> host cultivars.<br />
Phytopathology 86:200-211.<br />
Kema, G.H.J., R. Sayoud, J.G. Annone,<br />
<strong>and</strong> C.H. van Silfhout. 1996b.<br />
Genetic variation for virulence <strong>and</strong><br />
resistance in the wheat-<br />
Mycosphaerella graminicola<br />
pathosystem II. Analysis <strong>of</strong><br />
interactions between isolates <strong>and</strong><br />
host cultivars. Phytopathology<br />
86:213-219.<br />
Kema, G.H.J., D. Yu, F.H.J. Rijkenberg,<br />
M.W. Shaw, <strong>and</strong> R.P. Baayen. 1996c.<br />
Histology <strong>of</strong> the pathogenesis <strong>of</strong><br />
Mycosphaerella graminicola in wheat.<br />
Phytopathology 86:777-786.<br />
Morjane, H., J. Geistlinger, M. Harrabi,<br />
K. Weising, <strong>and</strong> G. Kahl. 1995.<br />
Oligonucleotide fingerprinting<br />
detects genetic diversity among<br />
Ascochyta rabiei isolates from a single<br />
chickpea field in Tunisia. Curr.<br />
Genet. 26:191-197.<br />
Möller, E.M., G. Bahnweg, H.<br />
S<strong>and</strong>erman, <strong>and</strong> H.H. Geiger. 1992.<br />
A simple <strong>and</strong> efficient protocol for<br />
solation <strong>of</strong> high molecular weight<br />
DNA from filamentous fungi, fruit<br />
bodies <strong>and</strong> infected plant tissue.<br />
Nucleic Acids Res. 20:6115-6116.
32<br />
Populations <strong>of</strong> <strong>Septoria</strong> spp. Affecting Winter Wheat in<br />
the Forest-Steppe Zone <strong>of</strong> the Ukraine<br />
S. Kolomiets*<br />
Institute <strong>of</strong> Plant Protection, Ukrainian Academy <strong>of</strong> Agrarian Sciences, Kiev, Ukraine<br />
Abstract<br />
Data in the literature <strong>and</strong> results <strong>of</strong> our investigations indicate that septoria leaf blotch <strong>of</strong> winter wheat has been reported<br />
annually in the forest-steppe zone <strong>of</strong> the Ukraine. <strong>Septoria</strong> tritici is the predominant species among the causal agents <strong>of</strong><br />
septoria leaf blotch <strong>of</strong> winter wheat. However, the portion caused by <strong>Stagonospora</strong> nodorum increased to 23-44% in recent<br />
years.<br />
<strong>Septoria</strong> tritici, the pathogen that<br />
causes leaf blotch, <strong>and</strong> <strong>Stagonospora</strong><br />
nodorum, the pathogen that induces<br />
spike <strong>and</strong> leaf blotch, are the most<br />
widespread pathogens <strong>of</strong> winter<br />
wheat in the forest-steppe zone <strong>of</strong><br />
the Ukraine.<br />
<strong>Septoria</strong> spp. induce a decrease<br />
in assimilation surface,<br />
developmental retardation,<br />
premature leaf desiccation, <strong>and</strong><br />
1000-grain weight. In epidemic<br />
years, yield losses may reach 30–<br />
50%. Total yield losses caused by<br />
these pathogens all over the world<br />
are estimated at 9 million tons.<br />
Developing cultivars resistant<br />
to the pathogens <strong>and</strong> establishing<br />
their cultivation is impossible<br />
without investigating the<br />
composition <strong>of</strong> pathogenic species<br />
in a given area <strong>and</strong> systematically<br />
recording its changes. Climatic<br />
conditions, the composition <strong>of</strong><br />
biocenoses, <strong>and</strong> the substrate<br />
where pathogens develop<br />
significantly affect the ratio<br />
between species. In the literature<br />
data on the areas occupied by the<br />
pathogens are quite limited, but<br />
can be found in articles published by<br />
Kovalenko (1975), Vasetskaja et al.<br />
(1983), Dyak (1990), <strong>and</strong> Sanina <strong>and</strong><br />
Antsiferova (1991).<br />
In 1995–97, we investigated the<br />
composition <strong>of</strong> <strong>Septoria</strong> pathogens in<br />
the forest-steppe zone <strong>of</strong> the Ukraine<br />
(Kyiv, Cherkasy, Vinnytsya,<br />
Khmelnytskyy, Ternopil, Zhytomyr,<br />
<strong>and</strong> Poltava regions). Studies were<br />
conducted on promising <strong>and</strong><br />
cultivated cultivars <strong>of</strong> winter wheat<br />
using routine methodologies. The<br />
species were identified via<br />
evaluation <strong>of</strong> stable traits: form,<br />
length, <strong>and</strong> width <strong>of</strong> conidia <strong>and</strong><br />
ends.<br />
Our research demonstrated that<br />
the forest-steppe zone <strong>of</strong> the Ukraine<br />
is occupied by both <strong>Septoria</strong><br />
pathogens, but S. tritici<br />
predominated in the 1970s, while S.<br />
nodorum was found only in certain<br />
years (Kovalenko, 1975). In the 1980s<br />
S. nodorum was reported in Kyiv (30-<br />
36%) <strong>and</strong> Ternopil (13-23%) regions<br />
(Dyak, 1990). In Cherkasy region<br />
(6.6%) the pathogen was reported<br />
only in certain years <strong>and</strong> was not<br />
detected at all in Vinnytsya region.<br />
* Author prevented from attending workshop by unforeseen travel problems.<br />
In the mid 1990s, the proportion<br />
<strong>of</strong> S. nodorum increased among the<br />
species. <strong>Septoria</strong> tritici dominated in<br />
all investigated regions: the highest<br />
percentage was reported in<br />
Vinnytsya region (76%), while the<br />
lowest was reported in Ternopil<br />
region (53%). In Kyiv, Cherkasy,<br />
Poltava, Khmelnytskyy, <strong>and</strong><br />
Zhytomyr regions, it reached 68,<br />
65, 69, 72, <strong>and</strong> 72%, respectively<br />
(Figure 1). Data from the literature<br />
<strong>and</strong> our own research results<br />
indicate an increase in the area<br />
occupied by S. nodorum, as well as<br />
in its ratio <strong>of</strong> <strong>Septoria</strong> pathogens<br />
present.<br />
Changes in the ratio <strong>of</strong> the<br />
pathogens may be explained by<br />
changes in climatic conditions, the<br />
range <strong>of</strong> cultivars used <strong>and</strong>,<br />
possibly, by the spread <strong>of</strong> S.<br />
nodorum infection through seeds,<br />
especially in recent years, when<br />
seeds were not properly treated.<br />
Thus the spread <strong>of</strong> S. nodorum–the<br />
most aggressive <strong>and</strong> damaging<br />
<strong>Septoria</strong> pathogen–increased under<br />
the above conditions.
;; yy<br />
;; yy<br />
6<br />
28%<br />
1<br />
32%<br />
;y; ; y y ; ; y y 28%<br />
31%<br />
68%<br />
72%<br />
2<br />
72%<br />
; y 35% 69% ;y<br />
44% 23% 65%<br />
3<br />
77% ;y ;y 4;<br />
y<br />
56%<br />
5<br />
<strong>Stagonospora</strong> nodorum<br />
<strong>Septoria</strong> tritici<br />
7<br />
Figure 1. The proportion between leaf septoriose pathogens <strong>of</strong> winter wheats in the foreststeppe<br />
zone <strong>of</strong> the Ukraine.<br />
1-Kyiv, 2-Poltava, 3-Cherkasy, 4-Vinnytsya, 5-Ternopil, 6-Khmelnytskyy, 7-Zhytomyr regions.<br />
Populations <strong>of</strong> <strong>Septoria</strong> spp. Affecting Winter Wheat in the Forest-Steppe Zone <strong>of</strong> the Ukraine 33<br />
References<br />
Dyak, U.P. 1990. Distribution <strong>of</strong> the<br />
basic inducers <strong>of</strong> septoriosis on a<br />
winter wheat in Ukrainian SSR.<br />
Zaschitz rasteniy 3:7-9.<br />
Kovalenko, S.N. 1975. <strong>Septoria</strong> leaf<br />
blotch <strong>of</strong> a winter wheat in<br />
requirements forest-steppe zone <strong>of</strong><br />
Ukraine SSR. Avtoreferat<br />
diss…k<strong>and</strong>. biol. nauk. Kiev, 21 p.<br />
Sanina, A.A., <strong>and</strong> Antsiferova, L.V.<br />
1991. <strong>Septoria</strong> Sacc. species on<br />
wheat in the European part <strong>of</strong> the<br />
USSR. Mikilogia i fitopatologia<br />
25(3):250-252.<br />
Vasetskaja, M.N., Kulikova, G.N., <strong>and</strong><br />
Borzionova, T.I. 1983. <strong>Septoria</strong><br />
species <strong>of</strong> fungi distributed on<br />
grades <strong>of</strong> wheat in the USSR.<br />
Mykologia i phytopatologia<br />
17(3):210-213.
34<br />
<strong>Septoria</strong> passerinii Closely Related to the Wheat<br />
Pathogen Mycosphaerella graminicola<br />
S.B. Goodwin <strong>and</strong> V.L. Zismann<br />
USDA-ARS, Department <strong>of</strong> Botany <strong>and</strong> Plant Pathology, Purdue University, West Lafayette, IN, USA<br />
Abstract<br />
<strong>Septoria</strong> passerinii is known only from its anamorphic <strong>Septoria</strong> state; no teleomorph has been identified. In culture, S.<br />
passerinii looks very similar to Mycosphaerella graminicola from wheat. Comparisons <strong>of</strong> the nucleotide sequences <strong>of</strong> the<br />
internal transcribed spacer (ITS) regions <strong>of</strong> the ribosomal DNA <strong>of</strong> both species <strong>and</strong> <strong>of</strong> many other fungi in the Dothideales<br />
revealed that the ITS regions <strong>of</strong> S. passerinii <strong>and</strong> M. graminicola differed by only 10 bases out <strong>of</strong> 571 total. Therefore, these<br />
species are very closely related. Phylogenetic analysis showed that S. passerinii <strong>and</strong> M. graminicola grouped together within<br />
a large cluster <strong>of</strong> Mycosphaerella species. Thus, S. passerinii almost certainly has a Mycosphaerella teleomorph.<br />
<strong>Septoria</strong> passerinii causes<br />
speckled leaf blotch on barley. The<br />
colony <strong>and</strong> conidial morphologies<br />
<strong>of</strong> S. passerinii <strong>and</strong> the wheat<br />
pathogen Mycosphaerella graminicola<br />
are very similar. Both are<br />
pathogens <strong>of</strong> cereals, <strong>and</strong> they may<br />
be closely related. However,<br />
nothing is known about the<br />
evolutionary relationships <strong>of</strong> S.<br />
passerinii (Cunfer <strong>and</strong> Ueng, 1999);<br />
no fruiting structures have been<br />
found <strong>and</strong> its teleomorph remains<br />
unknown.<br />
Analyses <strong>of</strong> the internal<br />
transcribed spacer (ITS) region <strong>of</strong><br />
ribosomal DNA have been very<br />
useful for elucidating the<br />
phylogenetic relationships <strong>of</strong> fungi.<br />
This region contains the highly<br />
variable ITS1 <strong>and</strong> ITS2, separated<br />
by the conserved 5.8S ribosomal<br />
RNA gene, <strong>and</strong> is bounded by the<br />
highly conserved 18 <strong>and</strong> 28S<br />
ribosomal RNA genes. This greatly<br />
facilitates analysis, because<br />
polymerase chain reaction (PCR)<br />
primers within the conserved<br />
regions <strong>of</strong> the 18 <strong>and</strong> 28S genes<br />
amplify the intervening ITS region<br />
<strong>of</strong> approximately 600 bp.<br />
Furthermore, many fungal ITS<br />
sequences are available in<br />
GenBank, which exp<strong>and</strong>s the<br />
number <strong>of</strong> species available for<br />
comparison.<br />
The objective <strong>of</strong> this research<br />
was to assemble a database <strong>of</strong> ITS<br />
sequences to test the hypothesis<br />
that S. passerinii is a close relative <strong>of</strong><br />
M. graminicola. The alternative<br />
hypothesis is that S. passerinii could<br />
be more closely related to other<br />
cereal pathogens, such as the<br />
septoria nodorum pathogen <strong>of</strong><br />
wheat <strong>and</strong> barley, Phaeosphaeria<br />
nodorum.<br />
Materials <strong>and</strong> Methods<br />
Isolates <strong>of</strong> species <strong>of</strong><br />
Mycosphaerella, Leptosphaeria, <strong>and</strong><br />
Phaeosphaeria were obtained from<br />
various sources (Table 1). DNA was<br />
extracted from lyophilized tissue<br />
<strong>and</strong> the ITS regions were amplified<br />
using primers ITS4 <strong>and</strong> ITS5.<br />
Amplification was with the<br />
following cycling parameters: 94 C<br />
for 2 min, 30 cycles <strong>of</strong> 93 C for 30 s,<br />
53 C for 2 min, 72 C for 2 min, <strong>and</strong><br />
a final extension <strong>of</strong> 10 min at 72 C.<br />
Amplification products were<br />
purified <strong>and</strong> cloned. Each clone<br />
was sequenced in both directions<br />
on an automated DNA sequencer<br />
<strong>and</strong> several clones were sequenced<br />
per isolate.<br />
DNA sequence alignment,<br />
genetic distance calculation,<br />
bootstrap analysis (1000<br />
replications), <strong>and</strong> a neighborjoining<br />
tree was prepared using<br />
Clustal X (http://www-igbmc.ustrasbg.fr/BioInfo/ClustalX/<br />
Top.html). The final tree was<br />
printed using NJplot.<br />
Results<br />
The ITS regions <strong>of</strong> all S.<br />
passerinii isolates were 571 bases<br />
long, including the primer regions,<br />
<strong>and</strong> were essentially identical. A<br />
BLAST search on GenBank<br />
identified M. graminicola as the<br />
closest match to S. passerinii.<br />
Sequences <strong>of</strong> other species showing<br />
good matches with S. passerinii in<br />
the BLAST search were<br />
downloaded from GenBank, along<br />
with those <strong>of</strong> species <strong>of</strong><br />
Leptosphaeria, Phaeosphaeria, <strong>and</strong><br />
other fungi in the Dothideales<br />
(Table 1). A multiple alignment<br />
revealed that the ITS sequences <strong>of</strong><br />
S. passerinii <strong>and</strong> M. graminicola<br />
differed by only 10 bases (data not
Table 1. Sources <strong>of</strong> isolates or DNA sequences for the internal transcribed spacer database <strong>of</strong><br />
<strong>Septoria</strong> passerinii, Mycosphaerella graminicola, <strong>and</strong> other fungi in the Dothideales.<br />
Species Isolate Growth medium Source<br />
Cladosporium caryigenum LCF — GenBank<br />
Cladosporium herbarum MZKI B-1002 — GenBank<br />
Cladosporium sphaerospermum MZKI B-1005 — GenBank<br />
Dothidea hippophaeos CBS 186.58 — GenBank<br />
Dothidea insculpta CBS 189.58 — GenBank<br />
Hormonema dematioides — — GenBank<br />
Leptosphaeria bicolor — — GenBank<br />
Leptosphaeria conteca — Malt broth ATCC<br />
Leptosphaeria doliolum — — Genbank<br />
Leptosphaeria maculans — — Genbank<br />
Leptosphaeria microscopica — — Genbank<br />
Mycosphaerella citri — Yeast malt broth Tobin Peever<br />
Mycosphaerella fijiensis — PDB ATCC<br />
Mycosphaerella graminicola — — GenBank<br />
Mycosphaerella graminicola IPO323 Yeast malt broth Gert Kema<br />
Mycosphaerella graminicola T1 Yeast malt broth Minnesota<br />
Mycosphaerella graminicola T48 Yeast malt broth Indiana<br />
Mycosphaerella musicola — PDB ATCC<br />
Mycosphaerella pini — — GenBank<br />
Mycosphaerella tassiana CBS 111.82 — GenBank<br />
Ophiosphaerella herpotricha — — GenBank<br />
Ophiosphaerella korrae — — GenBank<br />
Phaeosphaeria avenaria — — GenBank<br />
Phaeosphaeria halima — Emerson YpSs ATCC<br />
Phaeosphaeria nodorum — — GenBank<br />
Phaeosphaeria spartinae — Emerson YpSs ATCC<br />
Phaeosphaeria typharum — Emerson YpSs ATCC<br />
Phaeotheca triangularis MZKI B-950 — GenBank<br />
Rhizopycnis vagum — — GenBank<br />
<strong>Septoria</strong> passerinii 22585 Yeast malt broth ATCC<br />
<strong>Septoria</strong> passerinii 26516 Yeast malt broth ATCC<br />
<strong>Septoria</strong> passerinii P70 Yeast malt broth North Dakota<br />
<strong>Septoria</strong> passerinii P71 Yeast malt broth North Dakota<br />
<strong>Septoria</strong> passerinii P78 Yeast malt broth Minnesota<br />
<strong>Septoria</strong> passerinii P83 Yeast malt broth North Dakota<br />
Trimmatostroma salinum MZKI B-962 — GenBank<br />
shown). The neighbor-joining tree<br />
showed that S. passerinii <strong>and</strong> M.<br />
graminicola clustered together along<br />
with most <strong>of</strong> the other<br />
Mycosphaerella species (Figure 1).<br />
Phaeosphaeria nodorum was in a very<br />
different cluster that mostly<br />
contained species <strong>of</strong> Phaeosphaeria,<br />
Leptosphaeria, <strong>and</strong> Ophiosphaerella<br />
(Figure 1). Bootstrap analysis<br />
indicated strong support for all <strong>of</strong><br />
the major clusters (Figure 1).<br />
Discussion<br />
These data clearly indicate that<br />
S. passerinii <strong>and</strong> M. graminicola are<br />
very closely related. Because these<br />
two taxa were deeply embedded<br />
within a large cluster <strong>of</strong><br />
<strong>Septoria</strong> passerinii Closely Related to the Wheat Pathogen Mycosphaerella graminicola 35<br />
Mycosphaerella species, it seems<br />
certain that S. passerinii has, or was<br />
derived from a progenitor that had,<br />
a Mycosphaerella teleomorph. This<br />
must be confirmed by searching for<br />
the Mycosphaerella stage on infected<br />
barley tissue.<br />
In addition to elucidating the<br />
evolutionary relationships <strong>of</strong> S.<br />
passerinii, the cluster analysis raises<br />
a number <strong>of</strong> interesting questions.<br />
For example, Phaeosphaeria,<br />
Leptosphaeria, <strong>and</strong> Ophiosphaerella<br />
form a large cluster. However, it is<br />
not clear whether Phaeosphaeria<br />
warrants a separate generic<br />
designation. Clarifying the<br />
relationships among the mitosporic<br />
Dothideales (those with no known<br />
sexual stage) will require a larger<br />
number <strong>of</strong> species <strong>and</strong> a more<br />
comprehensive analysis. Aligning<br />
the 5.8S <strong>and</strong> ITS2 regions was fairly<br />
straightforward. However, ITS1<br />
varied greatly in length, which<br />
made alignment difficult. The<br />
alignment used for the cluster<br />
analysis presented here has not<br />
been optimized <strong>and</strong>, thus, should<br />
be considered preliminary.<br />
However, the close relationship<br />
between S. passerinii <strong>and</strong> M.<br />
graminicola is certain.<br />
Reference<br />
Cunfer, B.M., <strong>and</strong> Ueng, P.P. 1999.<br />
Taxonomy <strong>and</strong> identification <strong>of</strong><br />
<strong>Septoria</strong> <strong>and</strong> <strong>Stagonospora</strong> on small<br />
grain cereals. Annual Review <strong>of</strong><br />
Phytopathology (in press).
36<br />
Session 1 — S.B. Goodwin <strong>and</strong> V.L. Zismann<br />
0.05<br />
100<br />
96<br />
100<br />
100<br />
97.4<br />
100<br />
100<br />
100<br />
100<br />
100<br />
100<br />
100<br />
100<br />
99.9<br />
99.6<br />
Leptosphaeria bicolor<br />
Leptosphaeria conteca<br />
Ophiosphaerella korrae<br />
Ophiosphaerellaherpotricha<br />
Phaeosphaeria typharum<br />
Phaeosphaeria halima<br />
Phaeosphaeria avenaria<br />
Phaeosphaeria nodorum<br />
Phaeosphaeria spartinae<br />
Leptosphaeria microscopica<br />
Leptosphaeria maculans<br />
Leptosphaeria doliolum<br />
Rhizopycnis vagum<br />
<strong>Septoria</strong> passerinii P78<br />
<strong>Septoria</strong> passerinii P26516<br />
<strong>Septoria</strong> passerinii P71<br />
<strong>Septoria</strong> passerinii P83<br />
<strong>Septoria</strong> passerinii P22585<br />
<strong>Septoria</strong> passerinii P70<br />
Mycosphaerella graminicola T1<br />
Mycosphaerella graminicola<br />
Mycosphaerella graminicola IPO<br />
Mycosphaerella graminicola T48<br />
Mycosphaerella citri<br />
Mycosphaerella musicola<br />
Mycosphaerella fijiensis<br />
Mycosphaerella pini<br />
100 Dothidea hippophaeos<br />
Dothidea insculpta<br />
Hormonema dematioides<br />
Mycosphaerella tassiana<br />
Cladosporium herbarum<br />
Cladosporium sphaerospermum<br />
Trimmatostroma salinum<br />
Phaeotheca triangularis<br />
Cladosporium caryigenum<br />
Figure 1. Phylogenetic analysis <strong>of</strong> sequences <strong>of</strong> the internal transcribed spacer region <strong>of</strong> the ribosomal DNA <strong>of</strong><br />
<strong>Septoria</strong> passerinii, Mycosphaerella graminicola <strong>and</strong> other fungi in the Dothideales. All bootstrap values<br />
above 90% are indicated.
<strong>Septoria</strong>/<strong>Stagonospora</strong> Leaf Spot <strong>Diseases</strong> on Barley in<br />
North Dakota, USA<br />
J.M. Krupinsky1 <strong>and</strong> B.J. Steffenson2 (Poster)<br />
1 USDA, Agricultural Research Service, Northern Great Plains Research Laboratory, M<strong>and</strong>an, ND, USA<br />
2 Dept. <strong>of</strong> Plant Pathology, North Dakota State Univ., Fargo, ND, USA<br />
Abstract<br />
Diseased barley leaves were collected from fields in North Dakota in 1998. The most common <strong>Septoria</strong>/<strong>Stagonospora</strong><br />
diseases were septoria speckled leaf blotch (<strong>Septoria</strong> passerinii) <strong>and</strong> stagonospora avenae leaf blotch (<strong>Stagonospora</strong><br />
avenae f. sp. triticea). Net blotch (Drechslera teres) <strong>and</strong> spot blotch (Bipolaris sorokiniana) were also commonly<br />
present. <strong>Stagonospora</strong> nodorum blotch (<strong>Stagonospora</strong> nodorum) <strong>and</strong> tan spot (Drechslera tritici-repentis), which are<br />
major diseases on wheat in the area, were detected on barley but at rather low levels. When isolates <strong>of</strong> S. nodorum from<br />
barley were tested on wheat <strong>and</strong> barley in greenhouse inoculations, higher symptom severity ratings were obtained on wheat<br />
compared to barley, indicating that the isolates obtained from barley were probably wheat-type isolates. Because <strong>of</strong> the<br />
importance <strong>of</strong> both <strong>Septoria</strong> passerinii <strong>and</strong> <strong>Stagonospora</strong> avenae f. sp. triticea on barley, selecting barley for <strong>Septoria</strong>/<br />
<strong>Stagonospora</strong> resistance will require screening with both organisms.<br />
Barley (Hordeum vulgare L.) can<br />
be affected by a number <strong>of</strong> plant<br />
diseases that can cause economic<br />
losses in yield <strong>and</strong> quality. The<br />
<strong>Septoria</strong>/<strong>Stagonospora</strong> diseases<br />
common on barley are septoria<br />
speckled leaf blotch (<strong>Septoria</strong><br />
passerinii Sacc.), stagonospora<br />
avenae leaf blotch (<strong>Stagonospora</strong><br />
avenae Bissett f. sp. triticea T.<br />
Johnson [syn. <strong>Septoria</strong> avenae A.B.<br />
Frank f. sp. triticea T. Johnson]), <strong>and</strong><br />
stagonospora nodorum blotch<br />
(<strong>Stagonospora</strong> nodorum [Berk.]<br />
Castellani & E.G. Germano [syn.<br />
<strong>Septoria</strong> nodorum {Berk.} Berk. in<br />
Berk & Broome]) (Kiesling, 1985;<br />
Mathre, 1997). Barley-type isolates<br />
<strong>of</strong> S. nodorum have also been<br />
identified (Cunfer <strong>and</strong> Youmans,<br />
1983; Smedegard-Peterson, 1974). A<br />
cooperative survey was undertaken<br />
to determine the importance <strong>of</strong><br />
<strong>Septoria</strong>/<strong>Stagonospora</strong> leaf spot<br />
diseases common on barley in<br />
North Dakota.<br />
Materials <strong>and</strong> Methods<br />
Diseased barley leaves (green<br />
leaves with leaf spots) were<br />
gathered from naturally-infected<br />
barley plants in the field in 1998.<br />
Leaves were collected from 70<br />
fields located in the southwestern,<br />
central, northeastern, <strong>and</strong> eastern<br />
areas <strong>of</strong> North Dakota. Collected<br />
leaves were dried <strong>and</strong> stored dry at<br />
4C in a refrigerator until processed.<br />
Leaf sections, 2 cm long, from<br />
approximately 8 leaves per<br />
collection, were processed. Leaf<br />
sections were surface-sterilized for<br />
3 min in a 1% sodium hypochlorite<br />
solution containing a surfactant,<br />
rinsed in sterile distilled water,<br />
plated on 2% water agar in plastic<br />
Petri dishes, <strong>and</strong> incubated under a<br />
12-h photoperiod (cool-white<br />
fluorescent tubes) at 21C. After 7<br />
days, leaf sections were examined<br />
for fungi. Pycnidiospores from<br />
pycnidia on the leaf sections were<br />
identified microscopically. The<br />
presence <strong>of</strong> Drechslera teres (Sacc.)<br />
Shoemaker, Bipolaris sorokiniana<br />
(Sacc.) Shoemaker, <strong>and</strong> D. tritici-<br />
37<br />
repentis (Died.) Shoemaker was also<br />
noted. Two to four fungal species<br />
were present on some leaf sections.<br />
Isolates were obtained, grown,<br />
stored, <strong>and</strong> inoculum was prepared<br />
as described by Krupinsky (1997).<br />
Nine isolates <strong>of</strong> S. nodorum<br />
obtained from barley were<br />
compared in glasshouse<br />
inoculations <strong>of</strong> wheat <strong>and</strong> barley<br />
seedling plants. Two wheat<br />
cultivars, Eureka <strong>and</strong> Fortuna, <strong>and</strong><br />
two barley cultivars, Bowman <strong>and</strong><br />
Hector, were used. In a glasshouse,<br />
seedlings were planted, grown,<br />
fertilized, inoculated, incubated,<br />
<strong>and</strong> assessed for percentage<br />
necrosis <strong>of</strong> the first leaf as<br />
previously reported (Krupinsky<br />
1997).<br />
Results <strong>and</strong> Discussion<br />
Of the 531 leaf sections<br />
processed, 45% were infected with<br />
S. passerinii, 37% with S. avenae f.<br />
sp. triticea, <strong>and</strong> 14% with S.<br />
nodorum. Drechslera teres, B.<br />
sorokiniana, <strong>and</strong> D. tritici-repentis
38<br />
Session 1 — J.M. Krupinsky <strong>and</strong> B.J. Steffenson<br />
were identified on 55, 51, <strong>and</strong> 10%<br />
<strong>of</strong> the leaf sections, respectively.<br />
Using the number <strong>of</strong> leaf sections<br />
infected with a particular fungus as<br />
an indicator <strong>of</strong> the relative<br />
importance <strong>of</strong> a fungus, the most<br />
common diseases making up the<br />
leaf-spot disease complex on barley<br />
in North Dakota were in<br />
descending order <strong>of</strong> importance net<br />
blotch, spot blotch, septoria<br />
speckled leaf blotch, <strong>and</strong><br />
stagonospora avenae leaf blotch.<br />
This ranking was similar to those in<br />
Saskatchewan, Canada, north <strong>of</strong><br />
western North Dakota, where D.<br />
teres was considered the most<br />
common leaf spotting pathogen,<br />
followed by B. sorokiniana, which<br />
was more common than <strong>Septoria</strong><br />
spp. (Fern<strong>and</strong>ez et al., 1999). In<br />
Manitoba, Canada, north <strong>of</strong> eastern<br />
North Dakota, leaf spot diseases<br />
were considered to cause minimal<br />
damage in 1998. Drechslera teres <strong>and</strong><br />
B. sorokiniana were found in 90-93%<br />
<strong>of</strong> the barley fields <strong>and</strong> S. passerinii<br />
was recovered in 22% <strong>of</strong> the fields<br />
(Tekauz et al., 1999).<br />
Two <strong>of</strong> these diseases, spot<br />
blotch <strong>and</strong> stagonospora avenae<br />
leaf blotch, are usually minor<br />
diseases on wheat in central North<br />
Dakota. <strong>Stagonospora</strong> nodorum<br />
blotch <strong>and</strong> tan spot, although major<br />
diseases on wheat in North Dakota,<br />
were only isolated at low levels<br />
from barley <strong>and</strong> are not considered<br />
to be serious problems on barley at<br />
the present time. Because <strong>of</strong> the<br />
importance <strong>of</strong> both S. passerinii <strong>and</strong><br />
S. avenae f. sp. triticea on barley in<br />
North Dakota, selecting barley for<br />
<strong>Septoria</strong>/<strong>Stagonospora</strong> resistance will<br />
require screening with both<br />
pathogens.<br />
The nine isolates <strong>of</strong> S. nodorum<br />
obtained from barley consistently<br />
produced higher symptom severity,<br />
as measured by percentage<br />
necrosis, on wheat than on barley.<br />
Overall, Fortuna averaged 28%<br />
necrosis <strong>and</strong> Eureka averaged 29%<br />
necrosis, compared to less than 1%<br />
for Bowman <strong>and</strong> Hector.<br />
Acknowledgments<br />
The authors thank D. Wetch for<br />
technical assistance, as well as D.E.<br />
Mathre <strong>and</strong> M. Babadoost for their<br />
reviews <strong>and</strong> constructive<br />
comments.<br />
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USDA-ARS, Northern Plains Area,<br />
is an equal opportunity/<br />
affirmative action employer <strong>and</strong> all<br />
agency services are available<br />
without discrimination.<br />
References<br />
Cunfer, B.M., <strong>and</strong> Youmans, J. 1983.<br />
<strong>Septoria</strong> nodorum on barley <strong>and</strong><br />
relationships among isolates from<br />
several hosts. Phytopathology<br />
73:911-914.<br />
Fern<strong>and</strong>ez, M.R., Celetti, M.J., <strong>and</strong><br />
Hughes, G. 1999. Leaf diseases <strong>of</strong><br />
barley <strong>and</strong> oat in Saskatchewan in<br />
1998. Can. Plant Dis. Survey 79:75-<br />
77.<br />
Kiesling, R.L. 1985. The diseases <strong>of</strong><br />
barley. Chapter 10, pages 269-312.<br />
In: Barley. D.C. Rasmusson, ed.<br />
American Soc. <strong>of</strong> Agronomy.<br />
Madison, WI. 522 pages.<br />
Krupinsky, J.M. 1997. Aggressiveness<br />
<strong>of</strong> <strong>Stagonospora</strong> nodorum isolates<br />
obtained from wheat in the<br />
Northern Great Plains. Plant<br />
Disease 81:1027-1031.<br />
Mathre, D.E., 1997. Compendium <strong>of</strong><br />
Barley <strong>Diseases</strong>. Second edition.<br />
The American Phytopathological<br />
Society, APS Press, St. Paul, MN.<br />
90 pages.<br />
Smedegard-Peterson, V. 1974.<br />
Leptosphaeria nodorum (<strong>Septoria</strong><br />
nodorum), a new pathogen on<br />
barley in Denmark, <strong>and</strong> its<br />
physiologic specialization on<br />
barley <strong>and</strong> wheat. Friesia 10:251-<br />
264.<br />
Tekauz, A., McCallum, B., Gilbert, J.,<br />
Mueller, E., Idris, M., Stulzer, M.,<br />
Beyene, M., <strong>and</strong> Parmentier, M.<br />
1999. Foliar diseases <strong>of</strong> barley in<br />
Manitoba in 1998. Can. Plant Dis.<br />
Survey 79:74.
Interrelations among <strong>Septoria</strong> tritici Isolates <strong>of</strong><br />
Varying Virulence<br />
S. Ezrati, S. Schuster, A. Eshel, <strong>and</strong> Z. Eyal (Poster)<br />
Department <strong>of</strong> Plant Science, Tel Aviv University, Israel<br />
Reduction in pycnidial<br />
coverage on Seri 82 was found by<br />
Halperin et al. (1996) after<br />
inoculation with two <strong>Septoria</strong> tritici<br />
isolates: ISR398 (avirulent) <strong>and</strong><br />
ISR8036 (virulent). Cross protection<br />
was suggested as a mechanism for<br />
the suppression phenomenon,<br />
triggered by the avirulent isolate.<br />
The aim <strong>of</strong> the current study was to<br />
investigate possible interactions<br />
between new isolates <strong>of</strong> S. tritici on<br />
Seri 82 <strong>and</strong> other wheat cultivars.<br />
Co-inoculations with mixtures<br />
<strong>of</strong> isolates were conducted on<br />
seedlings <strong>and</strong> maturing plants <strong>of</strong><br />
various cultivars. The following S.<br />
tritici isolates <strong>and</strong> wheat cultivars<br />
were used:<br />
• ISR398 - avirulent on Seri 82,<br />
KK, Bobwhite “S” <strong>and</strong> IAS20;<br />
virulent on Shafir.<br />
• ISR8036 - avirulent on KK,<br />
Bobwhite “S” <strong>and</strong> IAS20;<br />
virulent on Shafir <strong>and</strong> Seri 82.<br />
• ISR9812 - avirulent on IAS20;<br />
virulent on Shafir, Seri 82, KK <strong>and</strong><br />
Bobwhite “S”.<br />
• ISR9840 - avirulent on KK <strong>and</strong><br />
Bobwhite “S”; virulent on Shafir,<br />
Seri 82 <strong>and</strong> IAS20.<br />
Pairs <strong>of</strong> isolates were used for<br />
inoculation with 1:1 mixtures on<br />
each <strong>of</strong> the five cultivars. Pycnidial<br />
coverage by the virulent isolate <strong>of</strong><br />
each pair was used as a reference.<br />
The results are shown in Table 1.<br />
Significant suppression <strong>of</strong> pycnidial<br />
coverage was observed when<br />
seedlings were inoculated with<br />
mixtures composed <strong>of</strong> a virulent <strong>and</strong><br />
an avirulent isolate. However, no<br />
suppression was found when isolate<br />
ISR9840 was used as the virulent<br />
component. Since all isolates were<br />
virulent on Shafir, no suppression<br />
was found on this cultivar.<br />
Suppression <strong>of</strong> pycnidial<br />
coverage was observed on maturing<br />
plants <strong>of</strong> Seri 82 in the field<br />
following co-inoculation at GS 37<br />
with a 1:1 isolate mixture <strong>of</strong><br />
Table 1. Reduction in pycnidial coverage on seedlings <strong>of</strong> the wheat cultivars Shafir (SHF), Seri<br />
82 (SER), IAS 20, Bobwhite “S” (BOW) <strong>and</strong> Kavkaz/K4500 (KK), inoculated with 1:1 mixture <strong>of</strong><br />
<strong>Septoria</strong> tritici. Data are percent reduction as compared to pycnidial coverage by the more<br />
virulent isolate in each case.<br />
Wheat cultivars<br />
Isolate mixture SHF SER IAS20 BOW KK<br />
ISR398 + ISR8036 52.2 98.7 - a - -<br />
ISR398 + ISR9812 38.3 78.0 - 99.5 99.7<br />
ISR398 + ISR9840 11.0 7.0 21.6 21.9 -<br />
ISR8036 + ISR9812 16.3 28.9 - - 71.8<br />
ISR8036 + ISR9840 26.7 24.3 33.8 27.4 -<br />
ISR9812 + ISR9840 26.1 24.3 32.8 63.4 100.0<br />
a Cultivar resistant to both isolates.<br />
39<br />
ISR398+ISR8036. The other five<br />
isolate mixtures on all wheat<br />
cultivar combinations are currently<br />
being tested in field trials.<br />
The identity <strong>of</strong> pycnidia<br />
developed on leaves <strong>of</strong> Seri 82 <strong>and</strong><br />
Shafir, co-inoculated with<br />
ISR398+ISR8036, was verified using<br />
specific PCR primers. Isolate<br />
ISR8036 dominated (>70%) the<br />
pycnidial population on seedlings<br />
<strong>and</strong> from the onset <strong>of</strong> the epidemics<br />
in field plots <strong>of</strong> both Shafir <strong>and</strong> Seri<br />
82. The low fraction <strong>of</strong> ISR398 on<br />
the susceptible cultivar Shafir is<br />
explained by competition. In the<br />
mixture ISR398+ISR8036, isolate<br />
ISR8036 has a competitive<br />
advantage over ISR398, attributed<br />
to its higher aggressiveness. The<br />
same experimental approach will be<br />
adapted to the other isolate<br />
mixtures under investigation, once<br />
specific PCR primers are obtained<br />
for ISR9812 <strong>and</strong> ISR9840.<br />
The structure <strong>of</strong> natural<br />
populations <strong>of</strong> this pathogen is<br />
determined by both cultivar-isolate<br />
interactions <strong>and</strong> isolate-isolate<br />
interactions.<br />
References<br />
Halperin, T., Schuster, S., Pnini-Cohen,<br />
S., Zilberstein, A., <strong>and</strong> Eyal, Z. 1996.<br />
The suppression <strong>of</strong> pycnidial<br />
production on wheat seedlings<br />
following sequential inoculation by<br />
isolates <strong>of</strong> <strong>Septoria</strong> tritici.<br />
Phytopathology 86:728-732.
Session 2: The Infection Process<br />
<strong>Stagonospora</strong> <strong>and</strong> <strong>Septoria</strong> Pathogens <strong>of</strong> <strong>Cereals</strong>: The<br />
Infection Process<br />
B.M. Cunfer<br />
Department <strong>of</strong> Plant Pathology, University <strong>of</strong> Georgia, Griffin, GA<br />
Abstract<br />
Definitive information on the infection process has been reported for <strong>Stagonospora</strong> nodorum, <strong>Septoria</strong> tritici, <strong>and</strong><br />
<strong>Septoria</strong> passerinii. Like other necrotrophic pathogens, they do not elicit the hypersensitive reaction. A significant difference<br />
in the infection process between <strong>Septoria</strong> <strong>and</strong> <strong>Stagonospora</strong> pathogens is that spore germination <strong>and</strong> penetration proceed<br />
much faster for S. nodorum than for S. tritici <strong>and</strong> S. passerinii. The <strong>Septoria</strong> pathogens penetrate the leaf primarily<br />
through stomata, whereas S. nodorum penetrates both directly <strong>and</strong> through stomata. <strong>Stagonospora</strong> nodorum kills the<br />
epidermal cells quickly, but S. tritici <strong>and</strong> S. passerinii do not kill epidermal cells until hyphae have ramified through the leaf<br />
mesophyll <strong>and</strong> rapid necrosis begins. Resistance slows host colonization but has no appreciable effect on the process <strong>of</strong> lesion<br />
development. The mechanisms controlling host response are still unclear. The infection process for ascospores is probably very<br />
similar to that for pycnidiospores. Ascospores <strong>of</strong> Phaeosphaeria nodorum germinate over a wide range <strong>of</strong> temperatures <strong>and</strong><br />
their germ tubes penetrate the leaf directly. However, unlike pycnidiospores, the ascospores do not germinate in free water.<br />
The infection process has been<br />
studied most intensely for<br />
<strong>Stagonospora</strong> nodorum <strong>and</strong> <strong>Septoria</strong><br />
tritici. One in-depth study on<br />
<strong>Septoria</strong> passerinii is available.<br />
Nearly all <strong>of</strong> the information<br />
reported is for infection by<br />
pycnidiospores. However, the<br />
infection process for other spore<br />
forms is quite similar. The<br />
information presented is mostly for<br />
infection <strong>of</strong> leaves under optimum<br />
conditions. Some studies were<br />
done with intact seedling plants,<br />
whereas others were conducted<br />
with detached leaves. Infection <strong>of</strong><br />
the wheat coleoptile <strong>and</strong> seedling<br />
by S. nodorum was described in<br />
detail by Baker (1971) <strong>and</strong><br />
reviewed by Cunfer (1983).<br />
Although no precise comparisons<br />
have been made, it appears that the<br />
infection process has many<br />
similarities in each host-parasite<br />
system <strong>and</strong> is typical <strong>of</strong> many<br />
necrotrophic pathogens.<br />
Information on factors influencing<br />
symptom development <strong>and</strong> disease<br />
expression are excluded but have<br />
been reviewed by other authors<br />
(Eyal et al., 1987; King et al., 1983;<br />
Shipton et al., 1971). A summary <strong>of</strong><br />
factors affecting spore longevity on<br />
the leaf surface is included.<br />
Role <strong>of</strong> the Cirrus <strong>and</strong><br />
Spore Survival on the<br />
Leaf Surface<br />
The most detailed information<br />
on the function <strong>of</strong> the cirrus<br />
encasing the pycnidiospores exuded<br />
from the pycnidium is for S.<br />
nodorum. The cirrus is a gel<br />
composed <strong>of</strong> proteinatous <strong>and</strong><br />
saccharide compounds. Its<br />
composition <strong>and</strong> function are<br />
similar to that <strong>of</strong> other fungi in the<br />
Sphaeropsidales (Fournet, 1969;<br />
Fournet et al., 1970; Griffiths <strong>and</strong><br />
Peverett, 1980).<br />
41<br />
The primary roles <strong>of</strong> cirrus<br />
components are protection <strong>of</strong><br />
pycnidiospores from dessication<br />
<strong>and</strong> prevention <strong>of</strong> premature<br />
germination. The cirrus protects the<br />
pycnidiospores so that some<br />
remain viable at least 28 days<br />
(Fournet, 1969). When the cirrus<br />
was diluted with water, if the<br />
concentration <strong>of</strong> cirrus solution was<br />
>20%, less that 10% <strong>of</strong><br />
pycnidiospores germinated. At a<br />
lower concentration, the<br />
components provide nutrients that<br />
stimulate spore germination <strong>and</strong><br />
elongation <strong>of</strong> germ tubes. Germ<br />
tube length increased up to 15%<br />
cirrus concentration, then declined<br />
moderately at higher<br />
concentrations (Harrower, 1976).<br />
Brennan et al. (1986) reported<br />
greater germination in dilute cirrus<br />
fluid. Cirrus components reduced<br />
germination at 10-60% relative<br />
humidity. Once spores are
42<br />
Session 2 — B.M. Cunfer<br />
dispersed, the stimulatory effects <strong>of</strong><br />
the cirrus fluid are probably<br />
negligible (Griffiths <strong>and</strong> Peverett,<br />
1980). At 35-45% relative humidity,<br />
spores <strong>of</strong> S. tritici in cirri remained<br />
viable at least 60 days (Gough <strong>and</strong><br />
Lee, 1985). The cirrus components<br />
may act as an inhibitor <strong>of</strong> spore<br />
germination, or the high osmotic<br />
potential <strong>of</strong> the cirrus may prevent<br />
germination.<br />
Pycnidiospores <strong>of</strong> S. nodorum<br />
did not survive for 24 hours at<br />
relative humidity above 80% at 20<br />
C. Spores survived two weeks or<br />
more at
<strong>Stagonospora</strong> nodorum releases a<br />
wide range <strong>of</strong> cell wall degrading<br />
enzymes including amylase, pectin<br />
methyl esterase,<br />
polygalacturonases, xylanases, <strong>and</strong><br />
cellulase in vitro <strong>and</strong> during<br />
infection <strong>of</strong> wheat leaves (Baker,<br />
1969; Lehtinen, 1993; Magro, 1984).<br />
The information related to cell wall<br />
degradation by enzymes agrees<br />
with histological observations.<br />
These enzymes may act in<br />
conjunction with toxins. Enzyme<br />
sensitivity may be related to<br />
resistance <strong>and</strong> rate <strong>of</strong> fungal<br />
colonization (Magro, 1984).<br />
Like many necrotrophs,<br />
<strong>Septoria</strong> <strong>and</strong> <strong>Stagonospora</strong><br />
pathogens produce phytotoxic<br />
compounds in vitro. Cell<br />
deterioration <strong>and</strong> death in advance<br />
<strong>of</strong> hyphal growth into mesophyll<br />
tissue (Bird <strong>and</strong> Ride, 1981) is<br />
consistent with toxin production.<br />
However, a definitive role for<br />
toxins in the infection process <strong>and</strong><br />
their relation to host resistance has<br />
not been established (Bethenod et<br />
al, 1982; Bousquet et al, 1980; Essad<br />
<strong>and</strong> Bousquet, 1981; King et al,<br />
1983). Differences in host range<br />
between wheat <strong>and</strong> barleyadapted<br />
strains <strong>of</strong> S. nodorum may<br />
be related to toxin production<br />
(Bousquet <strong>and</strong> Kollmann, 1998).<br />
Initiation <strong>of</strong> spore germination<br />
<strong>and</strong> percentage <strong>of</strong> spores<br />
germinated are not influenced by<br />
host susceptibility (Bird <strong>and</strong> Ride,<br />
1981; Morgan 1974; Straley, 1979;<br />
Straley <strong>and</strong> Scharen, 1979; Baker<br />
<strong>and</strong> Smith, 1978). Bird <strong>and</strong> Ride<br />
(1981) reported that extension <strong>of</strong><br />
germ tubes on the leaf surface was<br />
slower on resistant than on<br />
susceptible cultivars. This<br />
mechanism, expressed at least 48<br />
hours after spore deposition,<br />
indicates pre-penetration resistance<br />
to elongation <strong>of</strong> germ tubes. There<br />
were fewer successful penetrations<br />
in resistant cultivars, <strong>and</strong><br />
penetration proceeded more slowly<br />
on resistant cultivars (Baker <strong>and</strong><br />
Smith, 1978; Bird <strong>and</strong> Ride, 1981).<br />
Lignification was proposed to<br />
limit infection in both resistant <strong>and</strong><br />
susceptible cultivars, but other<br />
factors slowed fungal development<br />
in resistant lines. In susceptible<br />
lines, faster growing hyphae may<br />
escape lignification <strong>of</strong> host cells.<br />
Four days after inoculation <strong>of</strong><br />
barley with a wheat biotype isolate<br />
<strong>of</strong> S. nodorum, hyphae grew through<br />
the cuticle <strong>and</strong> sometimes in outer<br />
cellulose layers <strong>of</strong> epidermal cell<br />
walls. Thick papillae were<br />
deposited beneath the penetration<br />
hyphae <strong>and</strong> the cells were not<br />
penetrated (Keon <strong>and</strong> Hargreaves,<br />
1984).<br />
Infection by <strong>Septoria</strong><br />
tritici<br />
Pycnidiospores <strong>of</strong> S. tritici<br />
germinate in free water from both<br />
ends <strong>of</strong> the spore or from<br />
intercalary cells (Weber, 1922).<br />
Spore germination does not begin<br />
until about 12 hours after contact<br />
with the leaf. Germ tubes grow<br />
r<strong>and</strong>omly over the leaf surface.<br />
Weber (1922) observed only direct<br />
penetration between epidermal<br />
cells, but others concluded that<br />
penetration through both open <strong>and</strong><br />
closed stomata is the primary<br />
means <strong>of</strong> host penetration<br />
(Benedict, 1971; Cohen <strong>and</strong> Eyal,<br />
1993; Hilu <strong>and</strong> Bever, 1957). Kema<br />
<strong>Stagonospora</strong> <strong>and</strong> <strong>Septoria</strong> Pathogens <strong>of</strong> <strong>Cereals</strong>: The Infection Process 43<br />
et al. (1996) observed only stomatal<br />
penetration. Hyphae growing<br />
through stomata become<br />
constricted to about 1 µm diameter,<br />
then become wider after reaching<br />
the substomatal cavity.<br />
Hyphae grow parallel to the<br />
leaf surface under epidermal cells,<br />
then through the mesophyll to cells<br />
<strong>of</strong> lower the epidermis, but not into<br />
the epidermis. No haustoria are<br />
formed <strong>and</strong> hyphal growth is<br />
limited by sclerenchyma cells<br />
around the vascular bundles,<br />
except when hyphae are very<br />
dense. Vascular bundles are not<br />
invaded. Hyphae grow<br />
intercellularly along cell walls<br />
through the mesophyll, branching<br />
at a septum or middle <strong>of</strong> a cell. No<br />
macroscopic symptoms appear for<br />
about 9 days except for an<br />
occasional dead cell, but mesophyll<br />
cells die rapidly after 11 days.<br />
Pycnidia develop in substomatal<br />
chambers. Hyphae seldom grow<br />
into host cells (Hilu <strong>and</strong> Bever,<br />
1957; Kema et al, 1996;<br />
Weber, 1922).<br />
Successful infection only occurs<br />
after at least 20 hours <strong>of</strong> high<br />
humidity. Only a few brown flecks<br />
developed if leaves remained wet<br />
for 5-10 hours after spore<br />
deposition (Holmes <strong>and</strong> Colhoun,<br />
1974) or up to 24 hours (Kema et<br />
al., 1996). Host-parasite relations<br />
are the same on resistant or<br />
susceptible wheats. Spore<br />
germination on the leaf surface is<br />
the same regardless <strong>of</strong><br />
susceptibility. The number <strong>of</strong><br />
successful penetrations is about the<br />
same, but hyphal growth is faster<br />
in susceptible cultivars, resulting in<br />
more lesions. Hyphae extend
44<br />
Session 2 — B.M. Cunfer<br />
beyond the necrotic area in all<br />
cultivars. A toxin may play a role in<br />
pathogenesis (Cohen <strong>and</strong> Eyal,<br />
1993; Hilu <strong>and</strong> Bever, 1957). In<br />
contrast, colonization was greatly<br />
reduced on a resistant line (Kema et<br />
al., 1996).<br />
Infection by <strong>Septoria</strong><br />
passerinii<br />
Green <strong>and</strong> Dickson (1957)<br />
present a detailed description <strong>of</strong><br />
the infection process <strong>of</strong> S. passerinii<br />
on barley. The infection process is<br />
similar to S. tritici. Like S. tritici, the<br />
length <strong>of</strong> time required for leaf<br />
penetration is considerably longer<br />
than for S. nodorum.<br />
Germ tubes branch <strong>and</strong> grow<br />
over the leaf surface at r<strong>and</strong>om, but<br />
sometimes along depressions<br />
between epidermal cells. Leaf<br />
penetration is almost exclusively<br />
through stomata. Germination<br />
hyphae become swollen, <strong>and</strong> if<br />
penetration is unsuccessful, hyphae<br />
continue to elongate. No<br />
penetration occurs 48 hours after<br />
spore deposition. After 72 hours,<br />
germ tubes thicken over stomata,<br />
grow between guard cells <strong>and</strong> on<br />
surfaces <strong>of</strong> accessary cells <strong>and</strong> into<br />
the substomatal cavities. Direct<br />
penetration between epidermal<br />
cells is seen only rarely.<br />
Spore germination <strong>and</strong> host<br />
penetration are the same on<br />
resistant <strong>and</strong> susceptible cultivars.<br />
There is much less extension <strong>of</strong><br />
hyphae within leaves on resistant<br />
cultivars <strong>and</strong> papillae are observed<br />
on many but not all cell walls.<br />
Hyphae grow beneath the<br />
epidermis from one stoma to<br />
another, but do not penetrate<br />
between epidermal cells. The<br />
mesophyll is colonized, but no<br />
haustoria form. After the<br />
mesophyll cells become necrotic,<br />
epidermal cells collapse. Mycelial<br />
development in the leaf is sparse<br />
<strong>and</strong> usually blocked by vascular<br />
bundles. In younger leaves, if the<br />
vascular sheath is less developed,<br />
hyphae pass between the bundle<br />
<strong>and</strong> the epidermis. Pycnidia form<br />
in substomatal cavities, mostly on<br />
the upper leaf surface (Green <strong>and</strong><br />
Dickson, 1957).<br />
Factors Affecting Spore<br />
Longevity on the Leaf<br />
Surface<br />
Among the <strong>Stagonospora</strong> <strong>and</strong><br />
<strong>Septoria</strong> pathogens <strong>of</strong> cereals,<br />
definitive information on the<br />
infection process has been reported<br />
only for S. nodorum, S. tritici, <strong>and</strong> S.<br />
passerinii. Like many other<br />
necrotrophic pathogens, neither<br />
group <strong>of</strong> pathogens elicit the<br />
hypersensitive reaction. A<br />
significant difference in the<br />
infection process between <strong>Septoria</strong><br />
<strong>and</strong> <strong>Stagonospora</strong> pathogens is that<br />
spore germination <strong>and</strong> penetration<br />
proceeds much faster for S.<br />
nodorum than for S. tritici <strong>and</strong> S.<br />
passerinii. This has a significant<br />
influence on disease epidemiology.<br />
The <strong>Septoria</strong> pathogens penetrate<br />
the plant primarily through<br />
stomata, whereas S. nodorum<br />
penetrates both directly <strong>and</strong><br />
through stomata. S. nodorum<br />
penetrates <strong>and</strong> kills the epidermal<br />
cells quickly, but S. tritici <strong>and</strong> S.<br />
passerinii do not kill epidermal cells<br />
until hyphae have ramified<br />
through the leaf mesophyll <strong>and</strong><br />
rapid necrosis begins.<br />
Histological studies <strong>of</strong> fungal<br />
growth following host penetration<br />
match the data generated from<br />
epidemiological studies <strong>of</strong> host<br />
resistance. Resistance slows the rate<br />
<strong>of</strong> host colonization but has no<br />
appreciable effect on the process <strong>of</strong><br />
lesion development. The<br />
mechanisms controlling host<br />
response, whether related to<br />
enzymes <strong>and</strong> toxins or other<br />
metabolites released by the<br />
pathogens during infection, are still<br />
unclear.<br />
There is little information about<br />
infection by ascospores. The<br />
infection process is probably very<br />
similar to that for pycnidiospores.<br />
Ascospores <strong>of</strong> Phaeosphaeria nodorum<br />
germinate over a wide range <strong>of</strong><br />
temperatures, <strong>and</strong> their germ tubes<br />
penetrate the leaf directly. However,<br />
according to Rapilly et al. (1973),<br />
ascospores, unlike pycnidiospores,<br />
do not germinate in free water.<br />
References<br />
Baker, C.J. 1971. Morphology <strong>of</strong><br />
seedling infection by Leptosphaeria<br />
nodorum. Trans. Brit. Mycol. Soc.<br />
56:306-309.<br />
Baker, C.J. 1969. Studies on<br />
Leptosphaeria nodorum Müller <strong>and</strong><br />
<strong>Septoria</strong> tritici Desm. on wheat. Ph.<br />
D. thesis. University <strong>of</strong> Exeter.<br />
Baker E.A., <strong>and</strong> Smith, I.M. 1978.<br />
Development <strong>of</strong> the resistant <strong>and</strong><br />
susceptible reactions in wheat on<br />
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University <strong>of</strong> London.<br />
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Development <strong>of</strong> <strong>Septoria</strong> nodorum<br />
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585.
46<br />
Aggressiveness <strong>of</strong> Phaeosphaeria nodorum Isolates <strong>and</strong><br />
Their In Vitro Secretion <strong>of</strong> Cell-Wall-Degrading Enzymes<br />
P. Halama, a F. Lalaoui, a V. Dumortier, a <strong>and</strong> B. Paul b<br />
a Institut Supérieur d’Agriculture, Université Catholique de Lille, Lille, France<br />
b Laboratoire des Sciences de la Vigne, Institut Jules Guyot, Université de Bourgogne, Dijon, France<br />
Abstract<br />
The relationship between the in vitro production <strong>of</strong> cell-wall-degrading enzymes <strong>and</strong> the aggressiveness <strong>of</strong> three<br />
Phaeosphaeria nodorum isolates was studied. When grown in liquid medium containing 1% cell wall from wheat leaves as<br />
the only carbon source, the isolates secreted xylanase, α-arabinosidase, β-xylosidase, polygalacturonase, β-galactosidase,<br />
cellulase, β-1.3-glucanase, β-glucosidase, acetyl-esterase, <strong>and</strong> butyrate-esterase. Time course experiments showed different<br />
levels <strong>of</strong> enzyme production <strong>and</strong> different kinetics between isolates. Xylanase, cellulase, polygalacturonase, <strong>and</strong> butyrateesterase<br />
were positively correlated with isolate aggressiveness. The most aggressive isolate produced a higher proportion <strong>of</strong><br />
xylanase than the other two isolates, suggesting the role <strong>of</strong> this enzyme in the pathogenesis <strong>of</strong> P. nodorum.<br />
Studies have revealed that<br />
Phaeosphaeria nodorum populations<br />
have significant variability in<br />
pathogenicity that has been<br />
measured by the ability <strong>of</strong> isolates<br />
to cause symptoms on wheat<br />
(Griffith <strong>and</strong> Ao, 1980; Allinghan<br />
<strong>and</strong> Jackson, 1981;Yang <strong>and</strong><br />
Hughes, 1986; Krupinsky, 1997).<br />
As other plant-pathogenic<br />
fungi, P. nodorum produces a range<br />
<strong>of</strong> cell-wall-degrading enzymes<br />
(CWDE) that enables it to penetrate<br />
<strong>and</strong> infect host tissues (Lehtinen,<br />
1993; Magro, 1984). Very little is<br />
known about the variability <strong>of</strong><br />
enzyme production in P. nodorum<br />
populations; there is, however, a<br />
need to assess the diversity <strong>of</strong> these<br />
wall-degrading enzymes <strong>and</strong>, in<br />
particular, the extent to which this<br />
diversity could lead to differences<br />
in pathogenicity.<br />
The objective <strong>of</strong> this study was<br />
to compare enzymatic variation<br />
between two wild isolates <strong>and</strong> one<br />
mutant isolate <strong>of</strong> P. nodorum by<br />
examining CWDEs, <strong>and</strong> to<br />
establish the possible relationship<br />
between enzymes associated to<br />
pathogenesis <strong>and</strong> the<br />
aggressiveness <strong>of</strong> fungal strains.<br />
Materials <strong>and</strong> Methods<br />
Fungal strains<br />
Two wild strains (A/5 <strong>and</strong> 6/T)<br />
that differed significantly in their<br />
pathogenic behavior were isolated<br />
as described by Rapilly et al. (1992).<br />
The other isolate (300.2) is a<br />
carbendazim (MBC) resistant strain<br />
that was obtained using the<br />
protocol described in a previous<br />
study (Halama et al., 1999).<br />
Growth conditions <strong>and</strong><br />
preparation <strong>of</strong> fungal<br />
extracts<br />
The pycniospores used for<br />
inoculation were produced on<br />
synthetic (S) medium (Halama <strong>and</strong><br />
Lacoste, 1992). For enzyme<br />
production, isolates <strong>of</strong> S. nodorum<br />
were grown in liquid medium<br />
(Lehtinen, 1993) in which sucrose<br />
was replaced by 1% wheat cell<br />
walls. Wheat cell wall material was<br />
taken from 1-week-old seedlings <strong>of</strong><br />
cv. ‘Soissons’ <strong>and</strong> prepared<br />
according to Lehtinen’s modified<br />
procedure (Lehtinen, 1993). After<br />
incubating cultures at 24°C under<br />
constant shaking (72 rpm), liquid<br />
filtrates were centrifuged at 8000 g<br />
for 15 min at 4°C. Supernatants were<br />
used for enzyme activity assays, <strong>and</strong><br />
each culture experiment was<br />
replicated three times.<br />
Enzyme assays<br />
Cell-wall-degrading enzymes <strong>of</strong><br />
P. nodorum were assayed from the<br />
crude culture filtrates using<br />
different methods. The<br />
dinitrosalicylic acid (DNS) modified<br />
method, described by Miller (1959)<br />
for determining the reducing group,<br />
was used for the endo-enzyme<br />
assay. Xylanase, endopolygalacturonase,<br />
cellulase, <strong>and</strong> β-<br />
1.3-glucanase activities were<br />
measured respectively on oat spelts<br />
xylan, galacturonic acid,<br />
carboxymethyl cellulose, <strong>and</strong><br />
laminarin used as substrates.<br />
Absorbances were measured at 540<br />
nm. The reference sugar was<br />
glucose.<br />
Glycosidase activities were<br />
measured using p-nitrophenyl<br />
glycoside as a substrate (Poutanen,<br />
1988). β-glucosidase, β-
galactosidase, β-xylosidase, <strong>and</strong> αarabinosidase<br />
activities were<br />
assayed using p-nitrophenyl-β-Dglucopyranoside,<br />
p-nitrophenyl-β-<br />
D-galactopyranoside, pnitrophenyl-β-D-xylopyranoside,<br />
<strong>and</strong> p-nitrophenyl-α-Larabinopyranoside,<br />
respectively, as<br />
substrates.<br />
Esterase acetate activity was<br />
tested as described by Biely et al.<br />
(1988) using p-nitrophenylacetate.<br />
Degradation <strong>of</strong> pnitrophenylbutyrate<br />
was measured<br />
using p-nitrophenylbutyrate<br />
according to the procedure <strong>of</strong><br />
Kolattukudy et al. (1981). In each<br />
case, p-nitrophenol was used for<br />
the st<strong>and</strong>ard microtitre plate. For<br />
all enzyme assays, one unit <strong>of</strong><br />
enzyme was defined as the amount<br />
<strong>of</strong> enzyme that hydrolyzed 1 mmol<br />
<strong>of</strong> appropriate substrate, <strong>and</strong> each<br />
activity was expressed in mU.ml -1 .<br />
Pathogenicity test<br />
Isolates were tested for their<br />
relative pathogenicity on detached<br />
seedling leaves <strong>of</strong> the cultivar<br />
‘Soissons’ as previously described<br />
by Halama et al. (1999).<br />
Statistical analysis<br />
The analysis <strong>of</strong> variance was<br />
performed with the aid <strong>of</strong> the<br />
STAT-ITCF statistical s<strong>of</strong>tware.<br />
Differences between the isolates<br />
were assessed using the Newman-<br />
Keuls test. Correlations between<br />
data for aggressiveness rates <strong>and</strong><br />
for enzyme secretion were<br />
examined.<br />
Results<br />
The maximum secretions <strong>of</strong><br />
CWDEs are presented in Table 1,<br />
which shows that P. nodorum<br />
isolates were able to produce a<br />
wide range <strong>of</strong> enzymes on<br />
Aggressiveness <strong>of</strong> Phaeosphaeria nodorum Isolates <strong>and</strong> Their In Vitro Secretion <strong>of</strong> Cell-Wall-Degrading Enzymes 47<br />
synthetic medium supplemented<br />
with isolated cell wall. The secreted<br />
enzymes were determined every<br />
second day over the growth period<br />
(14 days).<br />
Time course for production<br />
<strong>of</strong> CWDEs<br />
The A/5 isolate produced, in<br />
order <strong>of</strong> decreasing activity,<br />
xylanase, butyrate-esterase, βglucosidase,<br />
β-1.3-glucanase,<br />
acetyl-esterase, cellulase,<br />
polygalacturonase, β-galactosidase,<br />
<strong>and</strong> β-xylosidase (Table 1). Culture<br />
filtrates <strong>of</strong> the A/5 isolate had<br />
significantly higher levels <strong>of</strong><br />
xylanase, butyrate-esterase,<br />
cellulase, <strong>and</strong> polygalacturonase<br />
activities than those <strong>of</strong> 6/T <strong>and</strong><br />
300.2 isolates. Levels <strong>of</strong> enzyme<br />
activity between 6/T <strong>and</strong> 300.2<br />
isolates were significantly different<br />
mainly for two peaks <strong>of</strong> β-1.3glucanase<br />
<strong>and</strong> acetyl-esterase<br />
activity on days 10 <strong>and</strong> 14<br />
produced respectively by the 6/T<br />
<strong>and</strong> the 300.2 isolates. Xylanase<br />
activity by the A/5 isolate on day<br />
10 was approximately seven times<br />
higher (1574 mU.m1 -1 ) than that <strong>of</strong><br />
the two other isolates.<br />
No α-arabinosidase activity<br />
could be detected in the<br />
filtrate <strong>of</strong> A/5 isolate,<br />
<strong>and</strong> very low levels were<br />
detected for 6/T <strong>and</strong><br />
300.2 isolates. Very low<br />
β-xylosidase activity was<br />
detected both by A/5<br />
<strong>and</strong> 6/T isolates.<br />
The polygalacturonase activities<br />
were highest on day 6 for A/5<br />
isolate (56 mU.ml –1 ), after which<br />
the activity decreased. For 6/T <strong>and</strong><br />
300.2 isolates, the highest activities<br />
were 27 <strong>and</strong> 12 mU.ml -1<br />
respectively, showing differences in<br />
their kinetics. As early as the<br />
second day, isolate 6/T produced a<br />
large amount <strong>of</strong> polygalacturonase,<br />
although isolate 300.2 secreted<br />
maximum polygalacturonase only<br />
after 12 days.<br />
β-1.3-glucanase assay was<br />
detected mainly for A/5 <strong>and</strong> 6/T<br />
isolates. β-glucosidase activity was<br />
produced by the three isolates with<br />
maximum activity on day 14 for the<br />
A/5 <strong>and</strong> 300.2 isolates <strong>and</strong> on day<br />
10 for the 6/T isolate. However, A/<br />
5 <strong>and</strong> 300.2 isolates may not have<br />
reached their peaks <strong>of</strong> βglucosidase<br />
activity at the end <strong>of</strong><br />
the time course.<br />
Where detected, the cellulase<br />
activity secreted by P. nodorum<br />
occurred transiently, <strong>and</strong> the<br />
highest activity took place during<br />
the early stages <strong>of</strong> fungal culture<br />
for A/5 isolate. The butyrateesterase<br />
activity was observed for<br />
the three isolates with a maximum<br />
Table 1. Maximum enzyme activity expressed by three<br />
isolates <strong>of</strong> Phaeosphaeria nodorum (A/5, 6/T, <strong>and</strong> 300.2) on<br />
medium supplemented with wheat cell walls.<br />
Isolate<br />
Enzyme A/5 6/T 300.2<br />
Xylanase 1574 1 (10) 2 213 (6) 200 (14)<br />
α-arabinosidase 0 5 (8) 7 (14)<br />
β-xylosidase 6 (10) 16 (14) 8 (14)<br />
Polygalacturonase 56 (6) 27(2) 12 (12)<br />
β-galactosidase 17 (12) 21 (10) 20 (14)<br />
Cellulase 106 (6) 0 3.5 (10)<br />
β-1,3 – glucanase 145 (12) 165 (10) 12 (12)<br />
β-glucosidase 153 (14) 158 (10) 118 (14)<br />
Acetyl esterase 126 (10) 44 (6) 124 (14)<br />
Butyrate esterase 527 (10) 409 (6) 379 (14)<br />
1 Data are expressed as mU.ml –1 culture medium filtrate.<br />
2 Enzyme activity was measured on the day given in<br />
parentheses.
48<br />
Session 2 — P. Halama, F. Lalaoui, V. Dumortier, <strong>and</strong> B. Paul<br />
at day 10, 6 <strong>and</strong> 14 for A/5, 6/T<br />
<strong>and</strong> 300.2 isolate respectively.<br />
Acetyl-esterase <strong>and</strong> β-galactosidase<br />
activities were also detectable for<br />
all three isolates.<br />
Aggressiveness <strong>of</strong> P.<br />
nodorum isolates<br />
The pathogenicity test allowed<br />
us to distinguish two groups <strong>of</strong><br />
isolates according to their<br />
aggressiveness (Table 2). Significant<br />
differences in the average <strong>of</strong><br />
necrosis symptoms were conclusive<br />
for differentiating the A/5 isolate<br />
from isolates 6/T <strong>and</strong> 300.2. The<br />
A/5 isolate <strong>of</strong> P. nodorum produced<br />
longer lesions on detached leaves<br />
than did the other two isolates 6/T<br />
<strong>and</strong> 300.2. Furthermore, there was<br />
partial correlation between the<br />
incubation period <strong>and</strong> length <strong>of</strong><br />
necrotic lesions; the longer lesion <strong>of</strong><br />
A/5 was related to a short latent<br />
period, but the similar lengths <strong>of</strong><br />
necrotic lesions caused by 6/T <strong>and</strong><br />
300.2 were unrelated to their<br />
incubation periods.<br />
Discussion<br />
Results reported here constitute<br />
the first report on the relationship<br />
between aggressiveness <strong>and</strong> in vitro<br />
production <strong>of</strong> CWDEs by P.<br />
nodorum. Our study indicated that<br />
Table 2. Lesion length <strong>and</strong> incubation period<br />
on detached leaves <strong>of</strong> wheat (cv. ‘Soissons’)<br />
caused by isolates <strong>of</strong> Phaeosphaeria nodorum<br />
10 days after inoculation.<br />
Lesion Incubation<br />
length period 1<br />
Isolate (mm) (hours)<br />
A/5 9.78 a 2 43<br />
6/T 3.60 b 63<br />
300.2 3.53 b 41<br />
1 The incubation period was determined by<br />
measuring the period between inoculation <strong>and</strong><br />
the moment when 50% <strong>of</strong> the detached leaves<br />
presented visible lesions.<br />
2 Within a column, means followed by different<br />
letters are significantly different according to t<br />
test (P
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Variation in pathogenicity,<br />
virulence, <strong>and</strong> aggressiveness <strong>of</strong><br />
<strong>Septoria</strong> nodorum in Florida.<br />
Phytopathology 71:1080-1085.<br />
Biely, P., Mackenzie, C.R., Schneider,<br />
H. 1988. Acetylxylan esterase <strong>of</strong><br />
Schizopyllum commune. Methods in<br />
Enzymology 160:700-707.<br />
Carder, J.H., Hignett, R.C., <strong>and</strong><br />
Swinburne, T.R. 1987. Relationship<br />
between the virulence <strong>of</strong> hop<br />
isolates <strong>of</strong> Verticillium albo-atrum<br />
<strong>and</strong> their in vitro secretion <strong>of</strong> cellwall<br />
degrading enzymes. Physiol.<br />
Mol. Plant Pathol. 31:441-452.<br />
Chan, Y.H., <strong>and</strong> Sackston, W.E. 1972.<br />
Production <strong>of</strong> pectolytic <strong>and</strong><br />
cellulotytic enzymes by virulent<br />
<strong>and</strong> avirulent isolates <strong>of</strong> Sclerotium<br />
bataticola during disease<br />
development in sunflowers. Can. J.<br />
Bot. 50:2449-2453.<br />
Cooper, R.M. 1977. Regulation <strong>of</strong><br />
synthesis <strong>of</strong> cell-degrading<br />
enzymes <strong>of</strong> plant pathogens. In:<br />
Cell Wall Biochemistry Related to<br />
Specificity in Host-Plant Pathogen<br />
Interactions. Solheim, B. <strong>and</strong> Raa,<br />
J. (eds.), pp. 163-211.<br />
Universitetsforlaget, Oslo.<br />
Cooper, R.M., Longman D.,<br />
Campdell, A., Henry, M., <strong>and</strong> Lees,<br />
P.E. 1988. Enzymic adaptation <strong>of</strong><br />
cereal pathogens to the<br />
monocotyledonous primary wall.<br />
Physiol. Mol. Plant Pathol. 32:37-<br />
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Aggressiveness <strong>of</strong> Phaeosphaeria nodorum Isolates <strong>and</strong> Their In Vitro Secretion <strong>of</strong> Cell-Wall-Degrading Enzymes 49<br />
Griffith, E., <strong>and</strong> Ao, H.C. 1980.<br />
Variation in <strong>Septoria</strong> nodorum. Ann.<br />
Appl. Biol. 94:294-296.<br />
Halama, P., <strong>and</strong> Lacoste, L. 1992. Etude<br />
des conditions optimales<br />
permettant la pycniogénèse de<br />
Phaeosphaeria (Leptosphaeria)<br />
nodorum, agent de la septoriose du<br />
blé. Agronomie 12:705-710.<br />
Halama, P., Skajennik<strong>of</strong>f, M., <strong>and</strong><br />
Dehorter, B. 1999. Tredad analysis<br />
<strong>of</strong> mating type, mutations <strong>and</strong><br />
aggressiveness in Phaeosphaeria<br />
nodorum. Mycol. Res. 1:43-49.<br />
Howel, H.E. 1975. Correlation <strong>of</strong><br />
virulence with secretion in vitro <strong>of</strong><br />
three wall-degrading enzymes in<br />
isolates <strong>of</strong> Sclerotium fructigena<br />
obtained after mutagen treatment. J.<br />
Gen. Microb. 90:32-40.<br />
Keon, J.P., Byrde, R.J.W., <strong>and</strong> Cooper,<br />
R.M. 1987. Some aspects <strong>of</strong> fungal<br />
enzymes that degrade plant cell<br />
walls. In: Fungal Infection <strong>of</strong> Plants.<br />
Pegg, G.F.<strong>and</strong> Ayres, P.G., (eds.),<br />
pp.133-157. Cambridge University<br />
Press.<br />
Kolattukudy, P.E., Purdy, R.E., <strong>and</strong><br />
Maiti, I.B. 1981. Cutinases from<br />
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Krupinsky, J.M. 1997. Aggressiveness<br />
<strong>of</strong> Stagnospora nodonum isolates<br />
obtained from wheat in the<br />
northern great plains. Plant Dis.<br />
81:1027-1031.<br />
Lehtinen, U. 1993. Plant cell wall<br />
degrading enzymes <strong>of</strong> <strong>Septoria</strong><br />
nodorum. Physiol. Mol. Plant Pathol.<br />
43:121-134.<br />
Magro, P. 1984. Production <strong>of</strong><br />
polysaccharide-degrading<br />
enzymes by <strong>Septoria</strong> nodorum in<br />
culture <strong>and</strong> during pathogenesis.<br />
Plant Sc. Let. 37:63-68.<br />
Martinez, M.J., Alconada, T., Guillen,<br />
F., Vasquez, C., <strong>and</strong> Reyes, F. 1991.<br />
Pectic activities from Fusarium<br />
oxysporum f. sp. melonis:<br />
purification <strong>and</strong> characterization<br />
<strong>of</strong> an exopolygalacturonase. FEMS<br />
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P., <strong>and</strong> Touraud, G. 1992. La<br />
reproduction sexuée et<br />
l’aggressivité de Phaeosphaeria<br />
nodorum Hedj (<strong>Septoria</strong> nodorum<br />
Berk). Agronomie 12:639-649.<br />
Senna, L.A., <strong>and</strong> Goodwin P.H. 1996.<br />
Comparison <strong>of</strong> cell wall-degrading<br />
enzymes produced by highly <strong>and</strong><br />
weakly virulent isolates <strong>of</strong><br />
Leptosphaeria maculans in culture.<br />
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Yang, R.C., <strong>and</strong> Hughes, G.R. 1986.<br />
Pathogenic variation among<br />
isolates <strong>of</strong> <strong>Septoria</strong> nodorum. Can. J.<br />
Plant Pathol. 8:356.
50<br />
Growth <strong>of</strong> <strong>Stagonospora</strong> nodorum Lesions<br />
A.M. Djurle (Poster)<br />
Department <strong>of</strong> Ecology <strong>and</strong> Crop Production Science/Plant Pathology, Swedish University <strong>of</strong> Agricultural<br />
Sciences, Uppsala, Sweden<br />
Disease severity is <strong>of</strong>ten<br />
expressed as a percentage or<br />
proportion <strong>of</strong> leaf area showing<br />
symptoms. Increase in disease<br />
severity <strong>of</strong> stagonospora nodorum<br />
glume blotch is caused by either an<br />
increase in the number <strong>of</strong> lesions as<br />
a result <strong>of</strong> new infection or by<br />
expansion <strong>of</strong> existing lesions. While<br />
new infections are still in the<br />
incubation period, existing lesions<br />
have time to exp<strong>and</strong> considerably<br />
(Lannou et al., 1994). Berger et al.<br />
(1997) suggested that lesion<br />
expansion should become the sixth<br />
component in describing polycyclic<br />
epidemics, <strong>and</strong> should be added to<br />
the already existing “epidemic<br />
quintet.” Lesion expansion has, for<br />
a long time, been considered as one<br />
<strong>of</strong> the most important factors for<br />
stagonospora nodorum disease<br />
increase (Rapilly et al., 1977;<br />
Rapilly et al., 1982).<br />
Materials <strong>and</strong> Methods<br />
In a field experiment with<br />
winter wheat inoculated with<br />
<strong>Stagonospora</strong> nodorum, length <strong>and</strong><br />
width <strong>of</strong> glume blotch lesions were<br />
measured repeatedly on the three<br />
uppermost leaves <strong>of</strong> tagged plants.<br />
The area <strong>of</strong> lesions was calculated<br />
assuming that they had an<br />
ellipsoidal shape. Lesion growth<br />
rates were analyzed in relation to<br />
current weather, lesion size, when a<br />
lesion was formed (first observed),<br />
<strong>and</strong> lesion age.<br />
Results <strong>and</strong> Discussion<br />
The results show that lesions<br />
followed a pattern <strong>of</strong> exponential<br />
growth <strong>and</strong> thus the rates were not<br />
constant over time. Among<br />
weather factors, mean temperature<br />
was the only factor that had a small<br />
effect on lesion growth rate, aside<br />
from the actual size <strong>of</strong> the lesions.<br />
Lesions that were formed (first<br />
observed) before or at heading<br />
(DC
Session 3A: Host-Parasite Interactions<br />
Genetic Control <strong>of</strong> Avirulence in Mycosphaerella<br />
graminicola (Anamorph <strong>Septoria</strong> tritici)<br />
G.H.J. Kema <strong>and</strong> E.C.P. Verstappen<br />
DLO-Research Institute for Plant Protection (IPO-DLO), Wageningen, The Netherl<strong>and</strong>s<br />
Mycosphaerella graminicola is a<br />
plant pathogenic bipolar<br />
heterothallic ascomycete (Kema et<br />
al., 1996c) that causes septoria<br />
tritici leaf blotch <strong>of</strong> wheat. The<br />
disease is currently considered the<br />
major threat to European wheat<br />
crops. Our main interest is to<br />
underst<strong>and</strong> the genetic control <strong>of</strong><br />
specificity in mating <strong>and</strong><br />
pathogenicity in this pathosystem.<br />
Therefore, we previously studied<br />
the histopathology <strong>of</strong> specific<br />
interactions <strong>and</strong> the genetic<br />
variation for virulence <strong>and</strong><br />
resistance in pathogen isolates <strong>and</strong><br />
host species <strong>and</strong> cultivars,<br />
respectively. We concluded that<br />
resistance to M. graminicola in the<br />
tested wheat cultivars is most<br />
probably not based on<br />
hypersensitivity (Kema et al.,<br />
1996d).<br />
Like others we observed<br />
numerous host-pathogen<br />
interactions, which were confirmed<br />
in repeated field experiments (Eyal<br />
<strong>and</strong> Levy, 1987; Saadaoui, 1987; van<br />
Ginkel <strong>and</strong> Scharen, 1988; Ahmed<br />
et al., 1995; Kema et al., 1996a, b;<br />
Kema <strong>and</strong> van Silfhout, 1997). The<br />
next step was to underst<strong>and</strong> the<br />
genetic control <strong>of</strong> these<br />
phenomena. Therefore, we had to<br />
determine the mating system <strong>of</strong> M.<br />
graminicola <strong>and</strong> find a way to cross<br />
particular isolates <strong>of</strong> the fungus,<br />
which eventually succeeded (Kema<br />
et al., 1996c).<br />
Since then, our group continues<br />
to study genome plasticity in M.<br />
graminicola <strong>and</strong> is exploring the<br />
possibility <strong>of</strong> dissecting the genetic<br />
factors that control the interaction<br />
between host <strong>and</strong> pathogen, as well<br />
as mating between pathogen<br />
strains (Kema et al., 1999). The<br />
current status <strong>and</strong> future prospects<br />
<strong>of</strong> this work will be discussed.<br />
Materials <strong>and</strong> Methods<br />
Wheat cultivars. Cultivars Shafir,<br />
Veranopolis, <strong>and</strong> Kavkaz were<br />
used as differentials <strong>and</strong> cvs.<br />
Taichung 29 <strong>and</strong> Kavkaz/K4500<br />
were used as susceptible <strong>and</strong><br />
resistant checks, respectively, in<br />
seedling experiments. In field<br />
experiments cvs. Vivant <strong>and</strong><br />
Hereward <strong>and</strong> the breeding lines<br />
NSL92-5719, RU51A, <strong>and</strong> CH76337<br />
were used.<br />
Table 1. Responses <strong>of</strong> wheat cultivars to the parental Mycosphaerella graminicola strains<br />
IPO323 <strong>and</strong> IPO94269 in seedling <strong>and</strong> adult plant experiments conducted in growth rooms <strong>and</strong> in<br />
the field, respectively. a<br />
Seedling Adult plant<br />
Cultivars IPO323 IPO94269 IPO323 IPO94269<br />
Taichung 29 + +<br />
Kavkaz/K4500 - -<br />
Kavkaz - +<br />
Veranopolis - +<br />
Shafir - + - +<br />
Hereward - +<br />
Vivant - +<br />
NSL92-5719 - +<br />
RU51A +/- -<br />
CH76337 +/- -<br />
a Pycnidial coverage: +=susceptible, -=resistant.<br />
51<br />
Fungal isolates. Isolate IPO323<br />
[MAT1-1] <strong>and</strong> IPO94269 [MAT1-2]<br />
were crossed to generate an F1<br />
population. Individual F1 progeny<br />
isolates were 1) backcrossed to both<br />
parents for mating type<br />
identification <strong>and</strong> to generate BC1<br />
populations, <strong>and</strong> 2) intercrossed to<br />
generate F2 populations. The<br />
avirulence/virulence <strong>of</strong> these<br />
parental isolates to the<br />
aforementioned cultivars is shown<br />
in Table 1.<br />
Experiments. F1, BC1, <strong>and</strong> F2<br />
populations were tested at the<br />
seedling stage in growth rooms <strong>and</strong><br />
repeated twice. Individual progeny<br />
isolates have been tested several<br />
times on the differential cultivars in<br />
the course <strong>of</strong> additional<br />
experiments. The F1 mapping<br />
population was also evaluated on<br />
differential wheat cultivars in the<br />
field. Each plot, with r<strong>and</strong>omized
52<br />
Session 3A — G.H.J. Kema <strong>and</strong> E.C.P. Verstappen<br />
cultivars, was replicated twice <strong>and</strong><br />
was inoculated with an individual<br />
F1 isolate.<br />
Results <strong>and</strong> Discussion<br />
In the seedling experiments all<br />
progeny isolates were virulent on<br />
cv. Taichung. None <strong>of</strong> these isolates<br />
carried virulence for cv. Kavkaz/<br />
K4500, indicating that the parental<br />
isolates carry the same avirulence<br />
factor(s) for this cultivar.<br />
Avirulence for each <strong>of</strong> the<br />
differentiating cultivars is inherited<br />
as a single gene. However, these<br />
avirulences co-segregated. Thus the<br />
entire F1, BC1, <strong>and</strong> F2 progenies<br />
showed the parental types. This<br />
suggests that the avirulences for<br />
these cultivars are tightly linked.<br />
There are two reasons for this<br />
hypothesis: 1) additional data<br />
indicate that cvs. Shafir,<br />
Veranopolis, <strong>and</strong> Kavkaz carry<br />
different resistance factors (Kema et<br />
al., 1996a, b) <strong>and</strong> 2) identification <strong>of</strong><br />
recombinant F1 progeny isolates in<br />
another cross<br />
(USDA50*IPO323.69.1) with<br />
avirulence for either Kavkaz or<br />
Veranopolis (unpublished data).<br />
Still, two alternative hypotheses<br />
cannot be excluded: 1) IPO323<br />
carries an avirulence factor for a<br />
common resistance factor in the<br />
tested differentials or 2) an<br />
avirulence gene product from<br />
IPO323 is recognized by different<br />
resistance factors in these cultivars.<br />
In order to investigate whether<br />
the avirulence in IPO323 for other<br />
wheat cultivars (Table 1) would<br />
segregate independently from the<br />
identified locus, a field experiment<br />
was conducted. Again, avirulence<br />
for the individual cultivars was<br />
controlled by a single locus. The<br />
avirulences for cvs. Vivant,<br />
Hereward, NSL92-5719, <strong>and</strong> Shafir<br />
co-segregated, indicating that they<br />
map to the same position as the<br />
locus identified in the seedling<br />
experiments. However, in addition<br />
to that, the avirulences for the<br />
breeding lines RU51A <strong>and</strong> CH76337<br />
inherited independently from that<br />
locus. Hence, recombinant progeny<br />
isolates were identified that were<br />
entirely virulent or avirulent on all<br />
cultivars.<br />
Hence, we hypothesize the<br />
presence <strong>of</strong> a complex major effect<br />
locus <strong>of</strong> tightly linked avirulence<br />
genes in M. graminicola IPO323 <strong>and</strong><br />
two additional independent loci<br />
carrying minor effect avirulence<br />
factors in IPO94269. The former<br />
locus was mapped on the M.<br />
graminicola genome <strong>and</strong> is currently<br />
being isolated following map-based<br />
cloning strategies.<br />
Acknowledgments<br />
We thank Jos Koeken, Suzanne<br />
Verhaegh, <strong>and</strong> the IPO-DLO<br />
technical <strong>and</strong> experimental farm<br />
staff for contributing to the<br />
presented data. Stimulating<br />
discussions with the “Wageningen”<br />
Mycosphaerella group, the “John<br />
Innes” Mycosphaerella group, <strong>and</strong><br />
the EU-SIS consortium are kindly<br />
acknowledged. Part <strong>of</strong> this project is<br />
funded by EU-BIOTECH PL096-352.<br />
References<br />
Ahmed, H.U., Mundt, C.C., <strong>and</strong><br />
Coakley, S.M. 1995. Host-pathogen<br />
relationship <strong>of</strong> geographically<br />
diverse isolates <strong>of</strong> <strong>Septoria</strong> tritici<br />
<strong>and</strong> wheat cultivars. Plant<br />
Pathology 44:838-847.<br />
Eyal, Z., <strong>and</strong> Levy, E. 1987. Variations<br />
in pathogenicity patterns <strong>of</strong><br />
Mycosphaerella graminicola within<br />
Triticum spp. in Israel. Euphytica<br />
36:237-250.<br />
Kema, G.H.J., Annone, J.G., Sayoud,<br />
R., van Silfhout, C.H., van Ginkel,<br />
M., <strong>and</strong> De Bree, J. 1996a. Genetic<br />
variation for virulence <strong>and</strong><br />
resistance in the wheat-<br />
Mycosphaerella graminicola<br />
pathosystem. I. Interactions<br />
between pathogen isolates <strong>and</strong><br />
host cultivars. Phytopathology<br />
86:200-212.<br />
Kema, G.H.J., Sayoud, R., Annone,<br />
J.G., <strong>and</strong> van Silfhout, C.H. 1996b.<br />
Genetic variation for virulence <strong>and</strong><br />
resistance in the wheat-<br />
Mycosphaerella graminicola<br />
pathosystem. II. Analysis <strong>of</strong><br />
interactions between pathogen<br />
isolates <strong>and</strong> host cultivars.<br />
Phytopathology 86:213-220.<br />
Kema, G.H.J., <strong>and</strong> van Silfhout, C.H.<br />
1997. Genetic variation for<br />
virulence <strong>and</strong> resistance in the<br />
wheat-Mycosphaerella graminicola<br />
pathosystem III. Comparative<br />
seedling <strong>and</strong> adult plant<br />
experiments. Phytopathology<br />
87:266-272.<br />
Kema, G.H.J., Verstappen, E.C.P.,<br />
Todorova, M., <strong>and</strong> Waalwijk, C.<br />
1996c. Successful crosses <strong>and</strong><br />
molecular tetrad <strong>and</strong> progeny<br />
analyses demonstrate<br />
heterothallism in Mycosphaerella<br />
graminicola. Current Genetics<br />
30:251-258.<br />
Kema, G.H.J., Verstappen, E.C.P.,<br />
Waalwijk, C., Bonants, P.J.M., De<br />
Koning, J.R.A., Hagenaar de<br />
Weerdt, M., Hamza, S., Koeken,<br />
J.G.P., <strong>and</strong> van der Lee, Th.A.J.<br />
1999. Genetics <strong>of</strong> biological <strong>and</strong><br />
molecular markers in<br />
Mycosphaerella graminicola, the<br />
cause <strong>of</strong> septoria tritici leaf blotch<br />
<strong>of</strong> wheat. In Lucas, J.A., Bowyer, P.,<br />
<strong>and</strong> Anderson, H.M. (eds.).<br />
<strong>Septoria</strong> on cereals: a study <strong>of</strong><br />
pathosystems. CABI Publishing,<br />
New York, NY, USA, pp. 161-180.<br />
Kema, G.H.J., Yu, D.Z., Rijkenberg,<br />
F.H.J., Shaw, M.W., <strong>and</strong> Baayen,<br />
R.P. 1996d. Histology <strong>of</strong> the<br />
pathogenesis <strong>of</strong> Mycosphaerella<br />
graminicola in wheat.<br />
Phytopathology 86:777-786.<br />
Saadaoui, E.M. 1987. Physiologic<br />
specialization <strong>of</strong> <strong>Septoria</strong> tritici in<br />
Morocco. Plant Disease 71:153-155.<br />
van Ginkel, M., <strong>and</strong> Scharen, A.L.<br />
1988. Host-pathogen relationships<br />
<strong>of</strong> wheat <strong>and</strong> <strong>Septoria</strong> tritici.<br />
Phytopathology 78:762-766.
Cytogenetics <strong>of</strong> Resistance <strong>of</strong> Wheat to <strong>Septoria</strong> Tritici<br />
Leaf Blotch<br />
L.S. Arraiano, A.J. Worl<strong>and</strong>, <strong>and</strong> J.K.M. Brown<br />
<strong>Cereals</strong> Research Department, John Innes Centre, Norwich, UK<br />
The John Innes Centre holds the<br />
world’s most comprehensive<br />
collection <strong>of</strong> precise genetic stocks<br />
<strong>of</strong> wheat. Elements <strong>of</strong> this<br />
collection, including intervarietal<br />
substitution <strong>and</strong> alien addition<br />
lines, are ideal for detecting<br />
chromosomes carrying<br />
agronomically important genes.<br />
These lines are being used in<br />
research on the genetics <strong>of</strong><br />
resistance to septoria tritici leaf<br />
blotch caused by Mycosphaerella<br />
graminicola (anamorph <strong>Septoria</strong><br />
tritici).<br />
A synthetic hexaploid wheat<br />
(Synthetic) was developed from a<br />
cross between Triticum dicoccoides<br />
(AABB) <strong>and</strong> Aegilops squarrosa (DD)<br />
(Sears, 1976). Synthetic carries<br />
genes for resistance to <strong>Stagonospora</strong><br />
nodorum (Nicholson et al., 1993),<br />
<strong>and</strong> a complete set <strong>of</strong> substitution<br />
lines <strong>of</strong> Synthetic chromosomes<br />
into a susceptible wheat variety,<br />
Chinese Spring (CS), has been<br />
developed (Worl<strong>and</strong> et al., 1996).<br />
To study resistance to M.<br />
graminicola, a detached seedling<br />
leaf technique (Arraiano et al.,<br />
1999) was used to test both Chinese<br />
Spring <strong>and</strong> Synthetic. Individual<br />
Dutch <strong>and</strong> Portuguese M.<br />
graminicola isolates supplied by<br />
IPO-DLO (Netherl<strong>and</strong>s) were<br />
tested. Synthetic was completely<br />
resistant to all isolates, except for<br />
IPO92006, whereas Chinese Spring<br />
was susceptible to all isolates<br />
except IPO323, to which it was<br />
moderately resistant. The results<br />
for Chinese Spring are consistent<br />
with field trial data (Brown et al.,<br />
1999). Baldus <strong>and</strong> Longbow, the<br />
susceptible controls, were very<br />
susceptible to all isolates.<br />
Based on these results, CS<br />
(Synthetic) substitution lines were<br />
tested using the detached leaf<br />
technique. Leaves were inoculated<br />
with the Dutch isolates IPO323 <strong>and</strong><br />
IPO94269, to identify the Synthetic<br />
chromosomes that carry genes for<br />
resistance to M. graminicola. The<br />
line carrying chromosome 7D (i.e.,<br />
CS background with the 7D<br />
chromosome substituted by that <strong>of</strong><br />
Synthetic) showed complete<br />
resistance to both isolates. Onehundred<br />
7D single-chromosome<br />
recombinant lines are now being<br />
tested to locate Synthetic’s<br />
resistance gene more precisely in<br />
relation to microsatellite <strong>and</strong> RFLP<br />
markers.<br />
Bezostaya 1 has been identified<br />
as a source <strong>of</strong> resistance to septoria<br />
tritici leaf blotch (Danon et al.,<br />
1982), <strong>and</strong> field trials showed it to<br />
be specifically resistant to IPO323<br />
(Brown et al., 1999). Substitution<br />
lines <strong>of</strong> Bezostaya 1 into Dwarf A, a<br />
susceptible line, were tested as<br />
adult plants, <strong>and</strong> chromosome 3A<br />
was identified as carrying genes for<br />
resistance to IPO323.<br />
Acknowledgment<br />
This research was supported by<br />
PRAXIS XXI – Fundação para a<br />
Ciência e a Tecnologia, Portugal,<br />
<strong>and</strong> the Ministry <strong>of</strong> Agriculture,<br />
Fisheries <strong>and</strong> Food, UK.<br />
References<br />
53<br />
Arraiano, L.S., P.A. Brading, <strong>and</strong><br />
J.K.M. Brown. 1999. A detached<br />
seedling leaf technique to screen<br />
for resistance to Mycosphaerella<br />
graminicola in field trials. In<br />
preparation.<br />
Brown, J.K.M., G.H.J. Kema, E.C.P.<br />
Verstappen, H.R. Forrer, L.S.<br />
Arraiano, P.A. Brading, E.M.<br />
Foster, A. Hecker, <strong>and</strong> E. Jenny.<br />
1999. Resistance to septoria tritici<br />
leaf blotch caused by isolates <strong>of</strong><br />
Mycosphaerella graminicola<br />
(anamorph <strong>Septoria</strong> tritici) in wheat<br />
varieties. In preparation.<br />
Danon, T., J.M. Sacks, <strong>and</strong> Z. Eyal.<br />
1982. The relationship among<br />
plant stature, maturity class <strong>and</strong><br />
susceptibility to <strong>Septoria</strong> leaf<br />
blotch <strong>of</strong> wheat. Phytopathology<br />
72:1037-1042.<br />
Nicholson, P., H.N. Rezanoor, <strong>and</strong> A.J.<br />
Worl<strong>and</strong>. 1993. Chromosomal<br />
location <strong>of</strong> resistance to <strong>Septoria</strong><br />
nodorum in a synthetic hexaploid<br />
wheat determined by the study <strong>of</strong><br />
chromosomal substitution lines in<br />
‘Chinese Spring’ wheat. Plant<br />
Breeding 110:177-184.<br />
Sears, E.R. 1976. A synthetic<br />
hexaploid wheat with fragile<br />
rachis. Wheat Information Service<br />
41:31-32.<br />
Worl<strong>and</strong>, A.J., S. Lewis, <strong>and</strong> C.<br />
Ellerbrook. 1996. <strong>Septoria</strong><br />
resistance in wheat. In John Innes<br />
Centre <strong>and</strong> Sainsbury Laboratory<br />
Annual Report 1995/96. Norwich,<br />
UK. p. 46.
54<br />
A Possible Gene-for-Gene Relationship for <strong>Septoria</strong> Tritici<br />
Leaf Blotch Resistance in Wheat<br />
P.A. Brading, 1 G.H.J. Kema, 2 <strong>and</strong> J.K.M. Brown 1<br />
1 <strong>Cereals</strong> Research Department, John Innes Centre, Norwich, UK<br />
2 DLO-Research Institute for Plant Protection (IPO-DLO), Wageningen, The Netherl<strong>and</strong>s<br />
Abstract<br />
In an F2 population <strong>of</strong> a cross between the resistant variety Flame <strong>and</strong> the susceptible variety Longbow it was shown that<br />
Flame carried one partially recessive gene for resistance to <strong>Septoria</strong> tritici isolate IPO323, as measured in a detached leaf<br />
assay. Work is underway to determine whether this resistance gene has a gene-for-gene relationship with the single avirulence<br />
locus identified in the IPO323 isolate.<br />
<strong>Septoria</strong> tritici leaf blotch,<br />
caused by the fungus<br />
Mycosphaerella graminicola<br />
(anamorph <strong>Septoria</strong> tritici), is one <strong>of</strong><br />
the most serious foliar pathogens <strong>of</strong><br />
wheat in Europe. In recent field<br />
trials using individual M.<br />
graminicola isolates, specific variety<br />
x isolate interactions have been<br />
observed (Kema <strong>and</strong> van Silfhout,<br />
1997). A number <strong>of</strong> wheat varieties<br />
show specific resistance to the<br />
Dutch isolate IPO323, including the<br />
British varieties Flame <strong>and</strong><br />
Hereward (Brown et al., 1999). In<br />
Holl<strong>and</strong>, crosses have been made<br />
between IPO323 <strong>and</strong> another<br />
isolate, IPO94269. Avirulence <strong>of</strong><br />
IPO323 on three specifically<br />
resistant cultivars, Shafir, Kavkaz,<br />
<strong>and</strong> Veranopolis, appears to be<br />
controlled at a single locus with<br />
simple inheritance (Kema et al.,<br />
1999). To investigate whether the<br />
specific resistances <strong>of</strong> Flame <strong>and</strong><br />
Hereward to IPO323 conform to a<br />
gene-for-gene relationship <strong>and</strong> also<br />
whether Flame <strong>and</strong> Hereward<br />
share the same resistance gene,<br />
crosses between Flame, Hereward,<br />
<strong>and</strong> a susceptible variety, Longbow,<br />
have been made.<br />
Eighty F 2 seedlings from a cross<br />
between Flame <strong>and</strong> Longbow were<br />
tested in a detached leaf assay<br />
(Arraiano et al., 1999) in which<br />
primary <strong>and</strong> secondary leaves were<br />
inoculated with IPO323. Leaf<br />
sections were scored according to<br />
the percentage leaf area covered<br />
with lesions bearing pycnidia 15-28<br />
days after inoculation. Each F 2<br />
plant was represented by four leaf<br />
sections tested in separate boxes.<br />
The parental varieties, Flame <strong>and</strong><br />
Longbow, were also included in<br />
each box. Comparison <strong>of</strong> infection<br />
levels on all four replicate leaf<br />
sections allowed individual plants<br />
to be rated as resistant, susceptible<br />
or intermediate. Flame was always<br />
resistant <strong>and</strong> Longbow always<br />
susceptible. Of the 80 progeny<br />
tested 23 were scored as resistant<br />
(less than 10% infection on all<br />
leaves). The other 57 progeny were<br />
scored as either susceptible (all leaf<br />
sections heavily infected) or<br />
intermediate (variable levels <strong>of</strong><br />
infection). The 1:3 ratio <strong>of</strong><br />
resistant:susceptible/intermediate<br />
is consistent with a single, partially<br />
recessive resistance gene in Flame.<br />
F 3 families generated from the<br />
80 F 2 individuals are being tested<br />
as seedlings (GS 25) in a polytunnel<br />
to test the prediction <strong>of</strong> a single<br />
gene for resistance to IPO323 in<br />
Flame. If this prediction is correct,<br />
F 3 families from resistant or<br />
susceptible F 2 individuals are<br />
expected to be uniformly resistant<br />
or susceptible, whereas<br />
intermediate F 2 individuals should<br />
produce F 3 families segregating 1:3<br />
for IPO 323<br />
resistance:susceptibility.<br />
To investigate whether<br />
Hereward carries the same IPO323<br />
resistance gene as Flame, F 2<br />
progeny <strong>of</strong> crosses between<br />
Hereward <strong>and</strong> Longbow <strong>and</strong><br />
between Hereward <strong>and</strong> Flame will<br />
be tested as detached leaves. The<br />
Hereward x Longbow cross will<br />
determine whether Hereward’s<br />
resistance is controlled by a single<br />
gene. The Hereward x Flame cross<br />
will test for allelism. The results<br />
from both tests will be presented.<br />
As IPO323 avirulence is<br />
controlled at a single locus (Kema<br />
et al., 1999) <strong>and</strong> Flame’s resistance<br />
to this isolate may be due to a<br />
single gene, we hypothesize that a<br />
gene-for-gene relationship exists<br />
between Flame <strong>and</strong> IPO323. To<br />
confirm this hypothesis, we are<br />
testing 61 progeny isolates from the<br />
IPO323 x IPO94269 cross on Flame<br />
<strong>and</strong> Longbow as detached leaves.
Acknowledgments<br />
This work was funded by the<br />
EU Framework 4 Biotechnology<br />
program <strong>and</strong> the Ministry <strong>of</strong><br />
Agriculture Fisheries <strong>and</strong> Foods<br />
(MAFF).<br />
A Possible Gene-for-Gene Relationship for <strong>Septoria</strong> Tritici Leaf Blotch Resistance in Wheat 55<br />
References<br />
Arraiano, L.S., P.A. Brading, <strong>and</strong><br />
J.K.M. Brown. 1999. A detached<br />
seedling leaf technique to screen<br />
for resistance to Mycosphaerella<br />
graminicola (anamorph <strong>Septoria</strong><br />
tritici) in wheat varieties. In<br />
preparation.<br />
Brown, J.K.M., G.H.J. Kema, H.R.<br />
Forrer, E.C.P. Verstappen, L.S.<br />
Arraiano, P.A. Brading <strong>and</strong> E.M.<br />
Foster. 1999. Resistance <strong>of</strong> wheat<br />
varieties to septoria tritici leaf<br />
blotch caused by isolates <strong>of</strong><br />
Mycosphaerella graminicola in field<br />
trials. In preparation.<br />
Kema, G.H.J., <strong>and</strong> C.H. Van Silfhout.<br />
1997. Genetic variation for<br />
virulence <strong>and</strong> resistance in the<br />
wheat-Mycosphaerella graminicola<br />
pathosystem III. Comparative<br />
seedling <strong>and</strong> adult plant<br />
experiments. Phytopathology<br />
87:266-272.<br />
Kema, G.H.J., E.C.P. Verstappen, C.<br />
Waalwijk, P.J.M. Bonants, J.R.A. de<br />
Koning, M. Hagenaar-de Weerdt,<br />
S. Hamza, J.G.P. Koeken, <strong>and</strong> T.A.J.<br />
van der Lee. 1999. Genetics <strong>of</strong><br />
biological <strong>and</strong> molecular markers<br />
in Mycosphaerella graminicola, the<br />
cause <strong>of</strong> septoria tritici leaf blotch<br />
<strong>of</strong> wheat. In: In <strong>Septoria</strong> on cereals:<br />
a Study <strong>of</strong> Pathosystems. Lucas,<br />
J.A., P. Bowyer, <strong>and</strong> H.M.<br />
Anderson (eds.). CABI Publishing,<br />
New York, NY, USA. pp.161-180.
56<br />
Diallel Analysis <strong>of</strong> <strong>Septoria</strong> Tritici Blotch Resistance in<br />
Winter Wheat<br />
X. Zhang, 1 S.D. Haley, 2 <strong>and</strong> Y. Jin 1 *<br />
1 Plant Science Department, South Dakota State University, Brookings, SD, USA<br />
2 Department <strong>of</strong> Soil <strong>and</strong> Crop Sciences, Colorado State University, Fort Collins, CO, USA<br />
Abstract<br />
In the winter wheat area <strong>of</strong> the northern Great Plains, leaf spot complex has been problematic in the past decade. In years<br />
with high precipitation from late April to July, septoria tritici blotch (STB), caused by <strong>Septoria</strong> tritici, is most prevalent. As<br />
part <strong>of</strong> our effort to improve STB resistance, inheritance <strong>of</strong> STB resistance was investigated by an eight-parent full diallel<br />
scheme. Parents, F 1 , <strong>and</strong> reciprocal F 1 were planted on three different dates. Within each planting date, three to five seeds <strong>of</strong><br />
each experimental unit were planted in the greenhouse. Materials were arranged in a r<strong>and</strong>omized complete block design<br />
(RCBD) with three replicates. Plants at the second-leaf stage were inoculated with a bulk <strong>of</strong> six S. tritici isolates. Significant<br />
general combining ability (GCA), specific combining ability (SCA), <strong>and</strong> reciprocal effects were observed in the analysis <strong>of</strong><br />
variance. The ratio <strong>of</strong> GCA sum <strong>of</strong> squares relative to SCA sum <strong>of</strong> squares suggested that GCA was more important than<br />
SCA. Additive effects played the major role in host response to STB, while non-additive effects were also detected. General<br />
combining ability effects <strong>of</strong> individual genotypes were in close agreement with parental performance. KS94U338, a genotype<br />
with resistance derived from Triticum tauschii, had the lowest STB score <strong>and</strong> the highest general combining ability. This<br />
result indicates that this genotype, possessing resistance distinct from other known sources, should prove useful in breeding<br />
efforts to improve STB resistance in wheat.<br />
Winter injury is the most<br />
adverse factor for winter wheat<br />
production in the northern Great<br />
Plains <strong>of</strong> the USA. Adoption <strong>of</strong><br />
conservation tillage practices, with<br />
winter wheat planting into spring<br />
wheat stubble, has increased<br />
steadily over the past decade.<br />
While this practice improves winter<br />
survival, accumulation <strong>of</strong> wheat<br />
residue on the soil surface has<br />
promoted the development <strong>of</strong> leaf<br />
spot diseases in this region. Severe<br />
epidemics <strong>of</strong> leaf spot complex on<br />
winter wheat have occurred during<br />
the last decade. <strong>Septoria</strong> tritici<br />
blotch has been predominant in<br />
years with above average<br />
precipitation from late April to July.<br />
Improvement <strong>of</strong> STB resistance was<br />
intensified in our project beginning<br />
in 1995. As part <strong>of</strong> this effort, we<br />
initiated studies to investigate<br />
combining ability <strong>of</strong> STB resistance<br />
* First author prevented from attending<br />
workshop by unforeseen travel<br />
problems.<br />
in known resistant genotypes. A<br />
better underst<strong>and</strong>ing <strong>of</strong> resistance<br />
could lead to more efficient<br />
deployment <strong>of</strong> germplasm<br />
resources.<br />
Materials <strong>and</strong> Methods<br />
Eight winter wheat genotypes<br />
(Table 1) were selected based on the<br />
level <strong>of</strong> resistance (field <strong>and</strong><br />
greenhouse) <strong>and</strong> their diverse<br />
origins <strong>of</strong> resistance. A total <strong>of</strong> 64<br />
genotypes (parents, F 1 , <strong>and</strong><br />
reciprocal F 1 ) were included in the<br />
test. The study consisted <strong>of</strong> three<br />
planting dates at three-day<br />
intervals. In each planting, three to<br />
five seeds <strong>of</strong> each experimental<br />
unit were planted in plastic<br />
conetainers filled with a peat-moss<br />
<strong>and</strong> perlite mixture. Each planting<br />
Table 1. Parental means, general combining ability (GCA), specific combining ability (SCA), <strong>and</strong><br />
reciprocal effects <strong>of</strong> septoria tritici blotch scores on the second leaf in an eight-parent diallel.<br />
Resistant<br />
Moderately<br />
resistant Susceptible<br />
Parent 1 2 3 4 5 6 7 8<br />
1 KS94U338 —† 0.5 0.1 0.3 -1.9** 0.9** 0.6* -0.6*<br />
2 Jagger 0.8* — -0.5 -1.0** -2.0** 1.0** 1.1** 2.0**<br />
3 KS91W005-1-4 -0.9* -0.9* — -2.0* 1.6** 0.8** 1.0** 0.7*<br />
4 KS91W0935-29-1 1.3** -0.6 0.0 — 1.3** 0.4 0.5 -0.6*<br />
5 KS87822-2-1 0.3 0.1 -0.6 -0.5 — 0.1 0.3 0.5<br />
6 SD93493 -0.1 -0.4 -0.2 0.1 0.2 — -0.8* -1.3**<br />
7 T<strong>and</strong>em -1.1* 0.3 0.2 -0.2 0.1 -0.2 — -0.9*<br />
8 SD93500 -1.2** -0.3 -0.1 -1.1** -0.2 -0.2 -0.1 —<br />
Parental means 1.4 1.4 1.9 5.8 5.8 8.0 8.3 8.7<br />
Parental GCA -2.2** -1.6** -1.1** -0.5** 0.0 1.8** 1.6** 1.8**<br />
*, ** Significantly different from zero at 0.05 <strong>and</strong> 0.01 probability levels, respectively.<br />
† SCA effects are above the diagonal line, <strong>and</strong> reciprocal effects are below.
consisted <strong>of</strong> three replicates<br />
arranged in a r<strong>and</strong>omized complete<br />
block design (RCBD).<br />
Six isolates were used<br />
throughout this study. Fresh<br />
inoculum was prepared each time<br />
before inoculation. Conidia <strong>of</strong> each<br />
isolate were produced on acidic<br />
potato dextrose agar petri plates.<br />
The plates were incubated on a<br />
laboratory bench for 12 h under<br />
cool white fluorescent lights at<br />
room temperature. When the edge<br />
<strong>of</strong> pink colonies tended to darken,<br />
conidia were harvested by flooding<br />
the plates with doubled-distilled<br />
water <strong>and</strong> gently scraping the<br />
colonies with a rubber spatula<br />
attached to a glass rod. The conidial<br />
suspensions from the six isolates<br />
were combined, filtered through<br />
two layers <strong>of</strong> cheesecloth, <strong>and</strong><br />
adjusted to approximately 5-10 x<br />
10 6 conidia ml -1 .<br />
Plants were inoculated when<br />
the second leaf was fully exp<strong>and</strong>ed.<br />
A volume <strong>of</strong> 100 ml <strong>of</strong> the conidial<br />
suspension was sprayed evenly<br />
onto 300 plants using an atomizer.<br />
Plants were maintained in a mist<br />
chamber for 96 h at 100% relative<br />
humidity <strong>and</strong> 21±1°C under a 12-h<br />
photoperiod. To avoid leaf<br />
senescence, the mist chamber was<br />
opened 30 min every 24 h while the<br />
plants were kept wet. Then the<br />
Table 2. <strong>Septoria</strong> tritici blotch reading scales used in this study.<br />
plants were incubated in a growth<br />
chamber with a 12-h photoperiod at<br />
21±1°C.<br />
Twenty-one days after<br />
inoculation, disease ratings <strong>of</strong> the<br />
second leaf were taken based on a 1<br />
to 9 scale (Table 2). The average<br />
disease scores <strong>of</strong> resistant<br />
genotypes (R) were 1.0 to 4.9; <strong>of</strong><br />
moderate resistant genotypes (MR)<br />
from 5.0 to 6.9; <strong>of</strong> moderate<br />
susceptible genotypes (MS) from<br />
7.0 to 7.9; <strong>and</strong> <strong>of</strong> susceptible<br />
genotypes from 8.0 to 9.0. The data<br />
were analyzed with the “Diallel<br />
analysis <strong>and</strong> simulation” program<br />
designed by Burow <strong>and</strong> Coors<br />
(1994) according to Griffing’s<br />
Model 1 (fixed model), Method 1<br />
(Griffing, 1956). Because these<br />
experiments were conducted in one<br />
controlled environment, the<br />
genotype by environment<br />
interaction was theoretically<br />
negligible. Thus, sets (planting<br />
dates) were treated as replicates,<br />
<strong>and</strong> the mean value <strong>of</strong> the three<br />
observations within each set<br />
represented the estimated STB score<br />
<strong>of</strong> the replicate.<br />
A t-test was used to test<br />
whether the GCA, SCA, <strong>and</strong><br />
reciprocal effects were significantly<br />
different from zero. The degrees <strong>of</strong><br />
freedom for estimated GCA effects<br />
(g i ) were (p-1), where p = number<br />
Rating Symptom description<br />
1. No visible symptoms are observed, <strong>and</strong> the leaf remains green <strong>and</strong> healthy.<br />
2. A few chlorotic lesions are present, <strong>and</strong> the infection site is a tan-colored spot.<br />
3. Extensive chlorotic lesions are present. Lesions occasionally have necrosis at the infection<br />
sites.<br />
6. Extensive chlorotic lesions merge with each other, <strong>and</strong> individual lesions are identified by<br />
initial infection sites. Necrosis <strong>and</strong> chlorosis both exist on the leaf.<br />
7. Lesions fully merge, <strong>and</strong> more than half <strong>of</strong> the leaf is desiccated by necrosis.<br />
8. The entire leaf is desiccated, <strong>and</strong> water-soaked lesions occupy the entire leaf.<br />
9. A few pycnidia are visible on the infected sites, <strong>and</strong> less than 30% <strong>of</strong> the leaf is occupied by<br />
pycnidia covered lesions that remain dry <strong>and</strong> green.<br />
10. Pycnidia occupy 50 to 70% <strong>of</strong> the leaf, <strong>and</strong> infected sites are dry yet still green.<br />
11. The entire leaf is covered by pycnidial lesions, <strong>and</strong> the leaf is dry yet still green.<br />
Diallel Analysis <strong>of</strong> <strong>Septoria</strong> Tritici Blotch Resistance in Winter Wheat 57<br />
<strong>of</strong> parents. The degrees <strong>of</strong> freedom<br />
for estimated SCA effects (s ij ) <strong>and</strong><br />
estimated reciprocal effects (r ij )<br />
were p(p-1) (Kang, 1994).<br />
Results <strong>and</strong> Discussion<br />
No significant differences<br />
between replicates (different<br />
planting dates) were detected (Table<br />
3). However, highly significant<br />
effects (P
58<br />
Session 3A — X. Zhang, S.D. Haley, <strong>and</strong> Y. Jin<br />
with resistance derived from T.<br />
tauschii, should prove useful in<br />
breeding efforts to improve STB<br />
resistance in wheat. The other two<br />
resistant genotypes, ‘Jagger’ <strong>and</strong><br />
‘KS91W005-1-4’, had large,<br />
negative, highly significant GCA<br />
effects. Of the moderate resistant<br />
parents, ‘KS91W0935-29-1’ <strong>and</strong><br />
‘KS87822-2-1’, only KS91W0935-29-<br />
1 had significant negative GCA<br />
effects. Each <strong>of</strong> the susceptible<br />
parents (‘SD93493’, ‘SD93500’, <strong>and</strong><br />
‘T<strong>and</strong>em’) had high GCA values.<br />
Specific combining ability<br />
effects were observed in 20 <strong>of</strong> the 28<br />
possible combinations, indicating<br />
that non-additive effects may exist<br />
for STB resistance. Reciprocal<br />
effects were present in five <strong>of</strong> the<br />
eight combinations involving<br />
KS94U338, crosses <strong>of</strong> KS91W005-1-<br />
4 with Jagger, <strong>and</strong> SD93500 with<br />
KS91W0935-29-1. When crossed<br />
with KS94U338 as female, the<br />
hybrids <strong>of</strong> KS91W005-1-4 <strong>and</strong> the<br />
two susceptible parents (SD93500<br />
<strong>and</strong> T<strong>and</strong>em) had lower disease<br />
score than those <strong>of</strong> the reciprocal<br />
crosses (with reciprocal effects <strong>of</strong> -<br />
0.9, -1.1, <strong>and</strong> -1.2). Apparently<br />
KS91W005-1-4, T<strong>and</strong>em, <strong>and</strong><br />
SD93500 could contribute<br />
additional resistance when used as<br />
female. The maternal effects <strong>of</strong><br />
SD93500 were also observed when<br />
it was crossed with KS91W0935-29-<br />
1. The maternal effects in<br />
combinations <strong>of</strong> KS94U338 with<br />
Jagger <strong>and</strong> KS94U338 with<br />
KS91W0935-29-1 are ascribed to<br />
KS94U338, due to the positive<br />
reciprocal effects between crosses<br />
<strong>of</strong> KS94U338 used as male <strong>and</strong><br />
female. The existence <strong>of</strong> reciprocal<br />
effects in resistance to STB was also<br />
observed by Jlibene et al. (1994).<br />
With the observation <strong>of</strong><br />
predominant GCA effects <strong>and</strong><br />
reciprocal effects for enhanced<br />
resistance, improvement <strong>of</strong> STB<br />
resistance can be achieved by<br />
crossing two parents having good<br />
resistance, while selecting resistant<br />
progeny from particular crosses<br />
based on the direction <strong>of</strong> the<br />
crosses is also predictable.<br />
Acknowledgments<br />
The authors gratefully<br />
acknowledge Drs. Rollin Sears, Joe<br />
Martin, <strong>and</strong> Stan Cox (Kansas State<br />
University, USDA-ARS) for<br />
providing some <strong>of</strong> the germplasm<br />
used in our studies.<br />
References<br />
Burow, M.D., <strong>and</strong> J.G. Coors. 1994.<br />
DIALLEL: A microcomputer<br />
program for the simulation <strong>and</strong><br />
analysis <strong>of</strong> diallel crosses. Agron. J.<br />
86:154-159.<br />
Griffing, B. 1956. Concept <strong>of</strong> general<br />
<strong>and</strong> specific combining ability in<br />
relation to diallel crossing systems.<br />
Aust. J. Biol. Sci. 9:463-493.<br />
Jlibene, M., J.P. Gustafson, <strong>and</strong> S.<br />
Rajaram. 1994. Inheritance <strong>of</strong><br />
resistance to Mycosphaerella<br />
graminicola in hexaploid wheat.<br />
Plant Breed. 112:301-310.<br />
Kang, M.S. 1994. Applied quantitative<br />
genetics. Kang M.S. Publisher,<br />
Baton Rouge, LA.
Analysis <strong>of</strong> the <strong>Septoria</strong> Monitoring Nursery<br />
L. Gilchrist, C. Velazquez, <strong>and</strong> J. Crossa<br />
<strong>CIMMYT</strong> Wheat Program, El Batan, Mexico<br />
Abstract<br />
The <strong>Septoria</strong> Monitoring Nursery (SMN) was divided into two sections: 1) the best sources <strong>of</strong> resistance identified by<br />
<strong>CIMMYT</strong> <strong>and</strong> national agricultural research breeding programs (renewed every three years) <strong>and</strong> 2) a tentative group <strong>of</strong><br />
differentials. The nursery included two susceptible durum varieties <strong>and</strong> a resistant one, as well as two susceptible bread wheat<br />
varieties <strong>and</strong> a resistant one. Readings <strong>of</strong> septoria leaf blotch were taken using the double digit modified scale, <strong>and</strong> analyses<br />
were done separately by digits. The variance component estimate <strong>and</strong> the F test for the first <strong>and</strong> the second digits were highly<br />
significant for site, as well as for the site x genotype interaction component for the 1 st <strong>and</strong> 2 nd SMN. An analysis <strong>of</strong> the group<br />
<strong>of</strong> tentative differentials across the seven years <strong>and</strong> all sites also gave significant differences between sites <strong>and</strong> between sites<br />
<strong>and</strong> genotypes. The contrast between durum <strong>and</strong> bread wheat for the 1 st SMN was not significant, which indicates that at<br />
least at those locations there were no host-specific pathogen populations. The contrast between durum <strong>and</strong> bread wheat was<br />
significant in the 2 nd SMN, indicating the existence <strong>of</strong> specific pathogen populations for each <strong>of</strong> the two crops in some sites.<br />
The most resistant lines across sites for the 1 st SMN were Trap#1/Bow <strong>and</strong> #1959, <strong>of</strong> Chinese origin, <strong>and</strong> for the 2 nd<br />
SMN, Eg-AH567.71// 4*Eg-A/3/2*CMH79.243, Cal/NH/H567.71/3/2*Ning 7840/4/CMH83.2277/5/Bow/2*Ning 7840//<br />
CMH83.2277, <strong>and</strong> Sha5/Bow. A more detailed analysis <strong>of</strong> genotype x site interaction will be carried out to identify the<br />
differential response <strong>of</strong> some lines to the pathogen populations in different sites.<br />
The <strong>Septoria</strong> Monitoring<br />
Nursery was initiated in 1992<br />
(Gilchrist, 1994). Based on virulence<br />
studies on seedlings involving<br />
<strong>Septoria</strong> tritici isolates obtained from<br />
hot spot locations in many countries<br />
around the world, Kema et al.<br />
(1996; 1997) found significant<br />
interaction between pathogen<br />
isolates <strong>and</strong> host cultivars. The<br />
same authors also confirmed the<br />
specificity <strong>of</strong> the relationship<br />
between bread wheat genotypes<br />
<strong>and</strong> isolates taken from bread<br />
wheat, <strong>and</strong> between durum wheat<br />
genotypes <strong>and</strong> isolates collected<br />
from durum wheat, as<br />
demonstrated by Eyal et al. (1973),<br />
Saadaoui (1987), <strong>and</strong> van Ginkel<br />
<strong>and</strong> Scharen (1988).<br />
A better underst<strong>and</strong>ing <strong>of</strong><br />
specificity in the <strong>Septoria</strong>-wheat<br />
pathosystem may facilitate<br />
quantifying its magnitude <strong>and</strong><br />
underst<strong>and</strong>ing its role in providing<br />
protection at the crop level rather<br />
than under controlled conditions<br />
(Eyal, 1999). According to Eyal<br />
(1999), “the International<br />
Monitoring Nursery philosophy<br />
executed by <strong>CIMMYT</strong> can provide<br />
information on pathogen x cultivar<br />
interaction in the regional, national<br />
<strong>and</strong> international domain.<br />
Futhermore, it may serve as a<br />
common basis for broad-base<br />
evaluation <strong>of</strong> germplasm to diverse<br />
pathogen populations under<br />
variable environmental<br />
conditions.”<br />
The information generated by<br />
this nursery could be extremely<br />
useful to <strong>CIMMYT</strong>’s wheat<br />
breeding program <strong>and</strong> those <strong>of</strong><br />
national agricultural research<br />
systems (NARS) in their attempts to<br />
increase stable resistance over time.<br />
Material <strong>and</strong> Methods<br />
The <strong>Septoria</strong> Monitoring<br />
Nursery was divided into two<br />
sections: 1) one (renewed every<br />
three years) with the best sources <strong>of</strong><br />
59<br />
resistance identified by <strong>CIMMYT</strong><br />
<strong>and</strong> NARS breeding programs <strong>and</strong><br />
2) a tentative group <strong>of</strong> differentials<br />
proposed by Dr. Z. Eyal, Tel Aviv<br />
University, as a fixed group. They<br />
were Ald/Pvn, Enkoy (K=4500),<br />
Colotana, IAS 20, Bow CM33203-K-<br />
9M-2Y-1M-1Y-2M-0Y-1Ptz, Don<br />
Ernesto (Bow) CM33203-K-9M-33Y-<br />
1M-500Y-0M-OJ-1J, Seri 82, Kvz-<br />
K500.L.6.A.4, Beth Lehem, Lakhish,<br />
Kauz, Penjamo 62, Etit 38 (D), Inbar<br />
(D), Glennson 81, <strong>and</strong> Barrigon<br />
Yaqui (D).<br />
The same nursery with two<br />
replications was sent to each site<br />
during three years to avoid<br />
environmental <strong>and</strong> year effects,<br />
<strong>and</strong> to obtain more reliable data.<br />
The first <strong>Septoria</strong> Monitoring<br />
Nursery was sent to 23 locations.<br />
The following group <strong>of</strong> resistant<br />
lines selected under Patzcuaro<br />
(Michoacan state) <strong>and</strong> Toluca<br />
(Mexico state) conditions were<br />
included:
60<br />
Session 3A — L. Gilchrist, C. Velazquez, <strong>and</strong> J. Crossa<br />
1. Trap #1/Bow<br />
2. Thb//Ias 20/H567.71<br />
3. PF70354/Bow<br />
4. Lfn/1158.57//Prl/3/Hahn<br />
5. Ias 58/4/Kal/Bb//Cj/3/Ald/5/Bow<br />
6. Br 14*2/Sumai 3<br />
7. Sushoe #6//Ald/Pav<br />
8. Br 14*2//Nobre*2/Tp<br />
9. M2A/Cml//Nyubay/3/CMH 72A .576/Mrg<br />
10. M2A/Cml//2* Nyubay<br />
11. CMH80A.253/Sx<br />
12. CMH80.278/3/Ssfm/H567.71//2*Ssmf<br />
13. #1959<br />
14. Sumai#3<br />
15. CS/Th. curv.//Glenn81/3/Ald/Pvn<br />
The second <strong>Septoria</strong> Monitoring<br />
Nursery was sent to 22 locations<br />
<strong>and</strong> included the following resistant<br />
lines:<br />
1. Bobwhite (CM 33203-K-10M-7Y-3M-2Y-1M-<br />
0M)<br />
2. Cno79/4/CS//Th. curv.//Glenn81/3/Ald/Pvn<br />
3. TIA.2/4/CS/Th. curv.//Glenn81/3/Ald/Pvn<br />
4. CS/Th. curv.//Glenn81/3/ Ald/Pvn/4/ CS/Le.<br />
Rac//*…<br />
5. Chirya 3<br />
6. Chirya 1<br />
7. Chirya 4<br />
8. CS/Th. curv.//Glenn81/3/Ald/Pvn/4/ Suz8<br />
9. CS/Th.curv.//Glenn81/3/Ald/Pvn/4/Nanjing<br />
8401<br />
10. Eg-A/H567.71//4*Eg-A/3/2*CMH79.243<br />
11. Cal/NH//H567.71/3/2*Ning 7840/4/…<br />
12. CMH72A.576/Mrng//CMH78.443/3/<br />
CMH79.243/…<br />
13. CMH77.308/CMH82.205<br />
14. Ald/Pvn//Ymi #6<br />
15. Sha 5/Bow<br />
The nursery included two<br />
susceptible durum varieties <strong>and</strong> a<br />
resistant one, as well as two<br />
susceptible bread wheat varieties<br />
<strong>and</strong> a resistant one. Readings <strong>of</strong><br />
septoria tritici leaf blotch were<br />
taken using the double digit<br />
modified scale (Eyal et al., 1987),<br />
<strong>and</strong> the analysis was done<br />
separately by digits. Height <strong>and</strong><br />
spike emergence measurements<br />
were used as covariants in the<br />
analysis.<br />
Results <strong>and</strong> Discussion<br />
Seven <strong>of</strong> the 23 locations were<br />
included in the analysis <strong>of</strong> first<br />
<strong>Septoria</strong> Monitoring Nursery.<br />
Practically no infection was<br />
detected at the other locations due<br />
to low moisture conditions. The<br />
sites where weather conditions<br />
produced medium to good<br />
epidemics were Argentina (Balcarce<br />
<strong>and</strong> Pergamino), Chile (Hidango<br />
<strong>and</strong> Chillan), Guatemala<br />
(Quetzaltenango), Mexico (Toluca,<br />
four years), <strong>and</strong> Uruguay (La<br />
Estanzuela). Some sites were<br />
eliminated because there was<br />
strong interference from other<br />
diseases. Toluca was the only site<br />
that had no such interference, <strong>and</strong> it<br />
allowed four diseases evaluations.<br />
The variance component<br />
estimate <strong>and</strong> the F test for the first<br />
<strong>and</strong> the second digits were highly<br />
significant for the factor site, as was<br />
the site x genotype interaction<br />
component, although the latter was<br />
much smaller than the former<br />
(Table 1).<br />
Infection averages per country<br />
<strong>and</strong> site are presented in Table 2.<br />
Results indicate that La Estanzuela<br />
(Uruguay) <strong>and</strong> Toluca (Mexico) had<br />
the heaviest epidemic compared<br />
with the other sites.<br />
Table 1. Variance <strong>and</strong> F test for digits 1<br />
<strong>and</strong> 2 <strong>of</strong> the response to infection by<br />
<strong>Septoria</strong> tritici for site <strong>and</strong> genotype x site<br />
interaction (1 st SMN).<br />
Variance<br />
component F test<br />
Digit 1<br />
Site 2.68 82.98***<br />
Site x genotype 1.12 1.95***<br />
Digit 2<br />
Site 1.34 40.53***<br />
Site x genotype 1.64 2.44***<br />
The responses <strong>of</strong> durum <strong>and</strong><br />
bread wheat lines in the first<br />
<strong>Septoria</strong> Monitoring Nursery were<br />
not significantly different, which<br />
indicates that at least at those<br />
locations there were no host-specific<br />
pathogen populations. There were<br />
no significant differences between<br />
the susceptible bread wheat checks<br />
(Seri 82 <strong>and</strong> Lakish) <strong>and</strong> the<br />
resistant check (Beth Lehem). This<br />
suggests that the resistant entries<br />
were not always resistant, <strong>and</strong> that<br />
our universal resistant check was<br />
not always resistant. In the case <strong>of</strong><br />
durum wheat, this difference was<br />
significant. The resistant variety Etit<br />
38 was always resistant, while<br />
susceptible varieties Inbar <strong>and</strong><br />
Barrigon Yaqui were always<br />
susceptible.<br />
The mean infection (first <strong>and</strong><br />
second digit) <strong>of</strong> the varieties across<br />
sites <strong>and</strong> years are shown in Table 3.<br />
The most resistant varieties across<br />
sites were Trap#1/Bow <strong>and</strong> #1959,<br />
<strong>of</strong> Chinese origin.<br />
Fourteen <strong>of</strong> a total <strong>of</strong> twentytwo<br />
sites sampled were included in<br />
the second <strong>Septoria</strong> Monitoring<br />
Nursery data analysis. The sites not<br />
included had problems with<br />
interference by rust infection or<br />
experienced low moisture<br />
conditions. The countries <strong>and</strong><br />
Table 2. <strong>Septoria</strong> tritici infection average per<br />
country <strong>and</strong> site, <strong>and</strong> disease evaluation for<br />
digits 1 <strong>and</strong> 2 (1 st SMN).<br />
Infection average<br />
Country <strong>and</strong> site Digit 1 Digit 2<br />
Toluca (Mexico) 7.25 6.63<br />
La Estanzuela (Uruguay) 6.06 6.96<br />
Chillan (Chile) 6.91 5.32<br />
SNA-Hidango (Chile) 3.08 5.67<br />
Balcarce (Argentina) 5.64 4.32<br />
Pergamino (Argentina) 6.07 4.92<br />
Quetzaltenango (Guatemala) 2.45 4.07
Table. 3. Infection averages (digits 1 <strong>and</strong> 2)<br />
across sites <strong>and</strong> years (1 st SMN).<br />
Variety Digit 1 Digit 2<br />
Trap #1/Bow 3.56 1.49<br />
Thb//Ias 20/H567.71 4.78 2.70<br />
PF70354/Bow 4.62 2.73<br />
Lfn/1158.57//Prl/3/Hahn 4.97 2.61<br />
Ias 58/4/Kal/Bb//Cj/3/Ald/5/Bow 5.81 3.25<br />
Br 14*2/Sumai 3 4.25 2.06<br />
Sushoe #6//Ald/Pvn 4.42 2.92<br />
Br 14*2//Nobre*2/Tp<br />
M2A/Cml//Nyubay/3/<br />
4.72 2.70<br />
CMH 72A.576/Mrg 5.36 3.75<br />
M2A/Cml//2* Nyubay 5.39 3.34<br />
CMH80A.253/Sx<br />
CMH80.278/3/Ssfm/<br />
6.24 4.79<br />
H567.71//2*Ssmf 5.51 3.93<br />
#1959 4.03 1.94<br />
Sumai#3<br />
CS/Th. curv. //Glenn81/3/<br />
5.25 4.20<br />
Ald/Pvn<br />
Tentative differentials<br />
5.37 3.55<br />
Ald/Pvn 5.03 3.72<br />
Enkoy (K=4500) 5.18 3.09<br />
Colotana 3.06 1.56<br />
IAS 20<br />
Bow CM33203-K-9M-2Y-1M-<br />
3.67 2.31<br />
1Y-2M-0Y-1Ptz<br />
Don Ernesto(Bow)CM33203-<br />
6.29 4.52<br />
K-9M-33Y-1M-500Y-0M-OJ-1J 6.62 5.50<br />
Seri 82 7.07 6.33<br />
Kvz-K500.L6.A.4 6.16 4.48<br />
Beth Lehem 7.17 6.77<br />
Lakhish 7.03 6.78<br />
Kauz 6.92 6.50<br />
Penjamo 62 7.28 6.75<br />
Etit 38 (D) 4.04 3.06<br />
Inbar (D) 4.37 3.15<br />
Glennson 81 6.44 5.53<br />
Barrigon Yaqui (D) 7.00 5.74<br />
locations selected were Argentina<br />
(Tres Arroyos, La Plata), Chile<br />
(Temuco), Ethiopia (Holetta),<br />
Mexico (Toluca, Patzcuaro),<br />
Portugal (Elvas), Russia<br />
(Krasnoda), Switzerl<strong>and</strong> (Zurich),<br />
<strong>and</strong> Uruguay (La Estanzuela,<br />
Tarariras). Some <strong>of</strong> them were<br />
included for more than one year.<br />
The variance component<br />
estimate <strong>and</strong> the F test for digits 1<br />
<strong>and</strong> 2 S. tritici evaluation were<br />
highly significant for the factor site,<br />
as was the genotype x site<br />
interaction component (Table 4).<br />
Again, the interaction component<br />
was much smaller than the main<br />
factor component.<br />
The S. tritici infection averages<br />
per country <strong>and</strong> site for digits 1<br />
<strong>and</strong> 2 are presented in Table 5.<br />
Mexico (Toluca <strong>and</strong> Patzcuaro) <strong>and</strong><br />
Uruguay (La Estanzuela) had the<br />
lowest infection averages. This was<br />
to be expected, given that data for<br />
these three locations were<br />
considered in selecting resistant<br />
lines.<br />
The contrast between durum<br />
<strong>and</strong> bread wheats was significant,<br />
which indicated that most sites<br />
included in the second nursery had<br />
specific pathogen populations for<br />
every crop (Table 6). As in the first<br />
nursery, there were no differences<br />
between the susceptible bread<br />
wheat checks (Seri 82 <strong>and</strong> Lakish)<br />
Table 4. Variance <strong>and</strong> F test for digits 1<br />
<strong>and</strong> 2 <strong>of</strong> the response to infection by<br />
<strong>Septoria</strong> tritici for site <strong>and</strong> genotype x site<br />
interaction (2 nd SMN).<br />
Variance<br />
component F test<br />
Digit 1<br />
Site 1.12 45.07***<br />
Site x genotype<br />
Digit 2<br />
1.20 2.64***<br />
Site 0.78 22.55***<br />
Site x genotype 1.18 2.11***<br />
Table 5. <strong>Septoria</strong> tritici infection averages per<br />
country <strong>and</strong> site for digits 1 <strong>and</strong> 2 (2 nd SMN).<br />
Infection average<br />
Country <strong>and</strong> site Digit 1 Digit 2<br />
Argentina Tres Arroyos 7.81 4.05<br />
La Plata 6.45 3.46<br />
Chile Temuco 6.43 5.11<br />
Ethiopia Holetta 7.64 5.04<br />
Mexico Toluca 4.87 3.52<br />
Patzcuaro 6.75 4.90<br />
Portugal Elvas 7.40 3.94<br />
Russia Krasnovar 7.36 5.10<br />
Switzerl<strong>and</strong> Zurich 7.45 3.23<br />
Uruguay La Estanzuela 6.45 4.47<br />
Tarariras 7.37 4.98<br />
Analysis <strong>of</strong> the <strong>Septoria</strong> Monitoring Nursery 61<br />
<strong>and</strong> the resistant check (Beth<br />
Lehem). This confirms that the<br />
resistant check was not always<br />
resistant, <strong>and</strong> that our resistant<br />
check is not universally resistant<br />
<strong>and</strong> should be changed. Results<br />
confirmed that the resistant durum<br />
variety Etit 38 was always resistant,<br />
<strong>and</strong> varieties Inbar <strong>and</strong> Barrigon<br />
Yaqui were always<br />
susceptible(Table 6).<br />
Infection averages (digits 1 <strong>and</strong><br />
2) <strong>of</strong> the varieties in the second<br />
<strong>Septoria</strong> Monitoring Nursery<br />
across sites <strong>and</strong> years are shown in<br />
Table 7. The most resistant lines<br />
across sites were: 1) Eg-AH567.71//<br />
4*Eg-A/3/2*CMH79.243, 2) Cal/<br />
NH/H567.71/3/2*Ning 7840/4/<br />
CMH83.2277/5/Bow/2*Ning<br />
7840//CMH83.2277, <strong>and</strong> 3) Sha 5/<br />
Bow.<br />
Table 6. Differences between average durum<br />
<strong>and</strong> bread wheat <strong>and</strong> resistant (R) <strong>and</strong><br />
susceptible (S) checks for each crop for<br />
the 1 st SMN, 2 nd SMN, <strong>and</strong> the combination <strong>of</strong><br />
all sites for the 1 st <strong>and</strong> the 2 nd SMN.<br />
Significance<br />
levels<br />
Source<br />
1<br />
Digit 1 Digit 2<br />
st SMN<br />
Bread <strong>and</strong> durum wheat<br />
Bread wheat (S) <strong>and</strong><br />
1.93 NS 2.64 NS<br />
bread wheat (R)<br />
Durum wheat (S) <strong>and</strong><br />
0.04 NS -0.22 NS<br />
durum wheat (R)<br />
2<br />
1.64 ** 1.38 *<br />
nd SMN<br />
Bread <strong>and</strong> durum wheat<br />
Bread wheat (S) <strong>and</strong><br />
1.77 *** 1.89 NS<br />
bread wheat (R)<br />
Durum wheat (S) <strong>and</strong><br />
-0.07 NS 0.25 NS<br />
durum wheat (R)<br />
1<br />
2.02 *** 0.99 **<br />
st <strong>and</strong> 2nd SMN combined<br />
Bread <strong>and</strong> durum wheat<br />
Bread wheat (S) <strong>and</strong><br />
1.83 *** 2.16 **<br />
bread wheat (R)<br />
Durum wheat (S) <strong>and</strong><br />
-0.08 NS 0.23 NS<br />
durum wheat (R)<br />
NS: Not significant.<br />
* Significant at the 5% level.<br />
1.91 *** 1.11 ***<br />
** Significant at the 1% level.<br />
*** Significant at the 0.1% level.
62<br />
Session 3A — L. Gilchrist, C. Velazquez, <strong>and</strong> J. Crossa<br />
An analysis <strong>of</strong> the differential<br />
group across the seven years <strong>and</strong><br />
all sites also gave significant<br />
differences between sites <strong>and</strong> a<br />
significant sites x varieties value.<br />
The values <strong>of</strong> the S. tritici infection<br />
averages for digits 1 <strong>and</strong> 2 for each<br />
variety are in Table 8.<br />
Table 7. Infection averages (digits 1 <strong>and</strong> 2)<br />
across sites <strong>and</strong> years (2 nd SMN).<br />
Variety<br />
Bow CM 33203-K-10M-7Y-<br />
Digit 1 Digit 2<br />
3M-2Y-1M-0M<br />
Cno79/4/CS/Th. curv.//<br />
6.10 3.26<br />
Glenn81/3/Ald/Pvn<br />
TIA.2/4/CS/Th. curv.//Glenn81/<br />
6.61 3.62<br />
3/Ald/Pvn<br />
CS/Th. curv//Glenn81/3/Ald/<br />
6.81 3.92<br />
Pvn/4/CS/Le.Rac//*… 6.64 3.56<br />
Chirya 3 6.87 3.75<br />
Chirya 1 6.82 4.27<br />
Chirya 4<br />
CS/Th. curv. //Glenn81/3/Ald/<br />
6.94 3.86<br />
Pvn/4/ Suz8<br />
CS/Th.curv. //Glenn81/3/Ald/<br />
6.26 3.37<br />
Pvn/4/Nanjing 8401<br />
Eg-A/H567.71//4*EG-A/3/<br />
6.74 4.08<br />
2*CMH79.243<br />
Cal/NH//H567.71/3/<br />
5.25 2.60<br />
2*Ning 7840/4/…<br />
CMH72A.576/Mrng//CMH78.443/<br />
5.47 3.38<br />
3/CMH79.243/… 7.04 5.53<br />
CMH77.308/CMH82.205 6.67 3.78<br />
Ald/Pvn//Ymi #6 6.27 3.12<br />
Sha 5/Bow<br />
Tentative differentials<br />
5.96 3.17<br />
Ald/Pvn 6.10 3.34<br />
Enkoy (K=4500) 5.37 2.52<br />
Colotana 3.74 2.16<br />
IAS 20<br />
Bow CM33203-K-9M-2Y-1M-<br />
4.51 2.34<br />
1Y-2M-0Y-1Ptz<br />
Don Ernesto (Bow) CM33203-K<br />
6.35 3.87<br />
-9M-33Y-1M-500Y-0M-OJ-1J 6.85 4.45<br />
Seri 82 7.27 5.39<br />
Kvz-K500.L.6.A.4 6.60 3.67<br />
Beth Lehem 7.61 5.62<br />
Lakhish 7.82 5.35<br />
Kauz 7.27 4.74<br />
Penjamo 62 7.78 5.60<br />
Etit 38 (D) 4.44 2.90<br />
Inbar (D) 5.89 3.35<br />
Glennson 81 6.74 4.75<br />
Barrigon Yaqui (D) 7.42 4.44<br />
A more detailed analysis <strong>of</strong><br />
variety x site interactions will be<br />
carried out in the near future to<br />
identify the differential response <strong>of</strong><br />
some lines to the pathogen<br />
populations in different sites.<br />
Acknowledgments<br />
The authors fondly remember<br />
Dr. Zahir Eyal <strong>and</strong> recognize his<br />
positive influence, his wise advice<br />
to follow this line <strong>of</strong> research, <strong>and</strong><br />
his keen visualization <strong>of</strong> its<br />
potential impact. They also thank<br />
the NARS cooperators who sent in<br />
the data that made it possible to<br />
obtain the reported results. Last,<br />
but not least, the authors wish to<br />
thank Dr. Jesse Dubin for<br />
contributing his well-considered<br />
ideas on the data analyses.<br />
Table 8. <strong>Septoria</strong> tritici infection averages for<br />
differential varieties across the seven years<br />
<strong>of</strong> evaluation covering 33 locations.<br />
Variety number Digit 1 Digit 2<br />
Ald/Pvn 5.71 3.43<br />
Enkoy (K=4500) 5.29 2.69<br />
Colotana 3.51 1.96<br />
IAS 20<br />
Bow CM33203-K-9M-2Y-1M-<br />
4.23 2.33<br />
1Y-2M-0Y-1Ptz<br />
Don Ernesto (Bow) CM33203-K-<br />
6.30 4.08<br />
9M-33Y-1M-500Y-0M-OJ-1J 6.76 4.78<br />
Seri 82 7.19 5.70<br />
Kvz-K500.L.6.A.4 6.44 3.90<br />
Beth Lehem 7.45 5.98<br />
Lakhish 7.55 5.81<br />
Kauz 7.15 5.30<br />
Penjamo 62 7.61 5.96<br />
Etit 38 (D) 4.28 2.93<br />
Inbar (D) 5.37 3.26<br />
Glennson 81 6.59 4.96<br />
Barrigon Yaqui (D) 7.01 4.83<br />
References<br />
Eyal, Z., Amiri, Z., <strong>and</strong> Wahl, I. 1973.<br />
Physiologic specialization <strong>of</strong><br />
<strong>Septoria</strong> tritici. Phytopathology<br />
63:1087-1091.<br />
Eyal, Z., Sharen, A.L., Prescott, J.M.,<br />
<strong>and</strong> van Ginkel, M. 1987. The<br />
<strong>Septoria</strong> <strong>Diseases</strong> <strong>of</strong> Wheat:<br />
Concepts <strong>and</strong> Methods <strong>of</strong> Disease<br />
Management. Mexico, D.F.:<br />
<strong>CIMMYT</strong>. 46 pp.<br />
Eyal, Z. 1999. Breeding for resistance<br />
to <strong>Septoria</strong> <strong>and</strong> <strong>Stagonospora</strong>. In:<br />
<strong>Septoria</strong> on <strong>Cereals</strong>: A Study <strong>of</strong><br />
Pathosystems. Lucas, J.A., Bowyer,<br />
P. <strong>and</strong> Anderson, H.M. (eds.).<br />
CABI Publishing. Wallingford, UK.<br />
pp. 1-25.<br />
Gilchrist, L. 1994. New <strong>Septoria</strong> tritici<br />
resistance sources in <strong>CIMMYT</strong><br />
germplasm <strong>and</strong> its incorporation<br />
in the <strong>Septoria</strong> Monitoring<br />
Nursery. In: Proceedings <strong>of</strong> the 4 th<br />
International Workshop on:<br />
<strong>Septoria</strong> <strong>of</strong> <strong>Cereals</strong>. July 4-7, 1994.<br />
Arseniuk, E., Goral, T., <strong>and</strong><br />
Czembor, P. (eds.). Ihar Radzikow,<br />
Pol<strong>and</strong>. pp. 187-190.<br />
Kema, G.H.J., Annone, G.J., Sayoud,<br />
R., van Silfhout, C.H., van Ginkel,<br />
M.,<strong>and</strong> de Bree, J. 1996. Genetic<br />
variation for virulence <strong>and</strong><br />
resistance in the wheat-<br />
Mycosphaerella graminicola<br />
pathosystem I. Interaction between<br />
pathogen isolates <strong>and</strong> host<br />
cultivars Phytopathology 86:200-<br />
212.<br />
Kema, G.H.J., <strong>and</strong> van Silfhout, C.H.<br />
1997. Genetic variation for<br />
virulence <strong>and</strong> resistance in the<br />
wheat-Mycosphaerella graminicola<br />
pathosystem III. Comparative<br />
seeedling <strong>and</strong> adult plant<br />
experiments. Phytopathology<br />
87:266-272.<br />
Saadaoui, E.M. 1987. Physiologic<br />
specialization <strong>of</strong> <strong>Septoria</strong> tritici in<br />
Morocco. Plant Disease 71:153-155.<br />
Van Ginkel, M., <strong>and</strong> A.L. Scharen.<br />
1988. Host-pathogen relationships<br />
<strong>of</strong> wheat <strong>and</strong> <strong>Septoria</strong> tritici.<br />
Phytopathology 78(6):762-766.
Session 3B: Host-Parasite Interactions<br />
Host – Parasite Interactions: <strong>Stagonospora</strong> nodorum<br />
E. Arseniuk <strong>and</strong> P.C. Czembor<br />
Plant Breeding <strong>and</strong> Acclimatization Institute, Radzików, Pol<strong>and</strong><br />
Abstract<br />
Relative virulence <strong>of</strong> 15 <strong>Stagonospora</strong> (<strong>Septoria</strong>) nodorum (SNB) monopycnidiospore isolates <strong>and</strong> their mixture was<br />
studied under field conditions on a differential set <strong>of</strong> triticale <strong>and</strong> bread wheat cultivars. The isolates originated from diseased<br />
triticale <strong>and</strong> wheat plants sampled in diverse geographical regions <strong>of</strong> Pol<strong>and</strong>. Significant effects <strong>of</strong> isolates, cultivars, <strong>and</strong><br />
cultivar by isolate interactions were detected. The interaction between S. nodorum isolates <strong>and</strong> wheat <strong>and</strong> triticale<br />
genotypes appeared to be to a great extent a statistical interaction, rather than a well defined physiological specialization.<br />
The detection <strong>of</strong> the interaction was influenced by the amount <strong>of</strong> disease inflicted by the pathogen isolates on selected host<br />
genotypes <strong>and</strong> the effect <strong>of</strong> environmental conditions on disease development, as well as by the tools <strong>and</strong> precision with<br />
which the host was examined. Nonetheless, since the isolate by cultivar interaction was significant in most cases, the term<br />
virulence is used. The mixture <strong>of</strong> isolates was classified among isolates showing intermediate virulence on the cultivars<br />
tested. In comparison to single isolates, the differential capacity <strong>of</strong> the isolate mixture was also reduced. Relationships<br />
between mean isolate virulences <strong>and</strong> cultivar variances <strong>of</strong> SNB reactions were not significant for leaves (moderate SNB<br />
level) <strong>and</strong> significant for heads (low SNB level). A single pathogenic isolate used for screening breeding materials may<br />
provide reliable information on their SNB resistance.<br />
<strong>Stagonospora</strong> nodorum (Berk.)<br />
Castellani et E.G. Germano<br />
(=<strong>Septoria</strong> nodorum (Berk. Berk. in<br />
Berk. & Broome) [teleomorph<br />
Phaeosphaeria nodorum (E. Müller)<br />
Hedjaroude (=Leptosphaeria nodorum<br />
E. Müller) is a necrotrophic<br />
pathogen <strong>of</strong> graminaceous plant<br />
species. The pathogen, together<br />
with S. avenae <strong>and</strong> <strong>Septoria</strong> tritici,<br />
belongs to a group <strong>of</strong> cereal<br />
pathogens sometimes called<br />
<strong>Septoria</strong> complex. Among these<br />
pathogens special forms have been<br />
recognized only in S. avenae: f.sp.<br />
avenae <strong>and</strong> f.sp. triticea. In the other<br />
two pathogens, i.e. S. nodorum <strong>and</strong><br />
S. tritici, host specificity is generally<br />
low, but more pronounced in S.<br />
tritici (Eyal et al., 1987; Skajennik<strong>of</strong>f<br />
<strong>and</strong> Rapilly, 1983).<br />
The presence <strong>of</strong> specificity in<br />
these pathogen populations is<br />
based on the analyses <strong>of</strong> the<br />
statistical interaction terms between<br />
cultivars <strong>and</strong> isolates. The isolate<br />
by cultivar interaction terms in S.<br />
nodorum are usually <strong>of</strong> a smaller<br />
magnitude than for S. tritici<br />
(Yechilevich-Auster et al., 1983;<br />
Scharen <strong>and</strong> Eyal, 1983; Scharen et<br />
al., 1985). Therefore, distinguishing<br />
specificity (virulence) from<br />
aggressiveness is more difficult for<br />
S. nodorum. Since specificity in the<br />
S. nodorum/cereal systems at the<br />
cultivar level is not so clear, it<br />
might be called genome specificity.<br />
Isolate by genotype interactions<br />
between S. nodorum isolates <strong>and</strong><br />
cereal species have been reported<br />
by a number <strong>of</strong> authors under<br />
controlled environment as well as<br />
63<br />
field conditions (Arseniuk et. al.,<br />
1994; Krupinsky, 1986, 1994,<br />
1997a,b,c). Information on the<br />
magnitude <strong>of</strong> such interaction is<br />
important for inoculum<br />
composition <strong>and</strong> resistance<br />
management in breeding programs.<br />
Although genetic protection is<br />
considered the main pillar <strong>of</strong> host<br />
defense, the majority <strong>of</strong> commercial<br />
wheat cultivars do not have<br />
suitable protection.<br />
The lack <strong>of</strong> resistant germplasm,<br />
high pathogen variability, <strong>and</strong> low<br />
specificity have all contributed to<br />
the slow progress in breeding for<br />
resistance <strong>and</strong> the deficient genetic<br />
protection in wheat <strong>and</strong> triticale.<br />
Therefore, the objective <strong>of</strong> this<br />
study was to compare the screening<br />
efficiency <strong>of</strong> triticale <strong>and</strong> wheat<br />
cultivars for stagonospora
64<br />
Session 3B — E. Arseniuk <strong>and</strong> P.C. Czembor<br />
nodorum blotch (SNB) resistance<br />
with single isolates <strong>and</strong> their<br />
mixture under field conditions.<br />
Concurrently, the relative virulence<br />
<strong>of</strong> S. nodorum isolates, the<br />
magnitude <strong>of</strong> cultivar by isolate<br />
interaction, <strong>and</strong> the relative<br />
resistance <strong>of</strong> the cereal genotypes<br />
used in the study were examined.<br />
Materials <strong>and</strong> Methods<br />
The relative virulence <strong>of</strong> 15<br />
single pycnidiospore isolates <strong>of</strong><br />
<strong>Stagonospora</strong> (= <strong>Septoria</strong>) nodorum<br />
<strong>and</strong> their mixture was studied in<br />
1993-1995 under field conditions on<br />
both winter <strong>and</strong> spring triticale <strong>and</strong><br />
bread wheat. For the purpose 11<br />
winter triticale <strong>and</strong> 4 winter wheat<br />
cultivars <strong>and</strong> germplasm lines<br />
(Tables 1 <strong>and</strong> 2) <strong>and</strong> 5 spring<br />
triticale <strong>and</strong> 3 spring wheat<br />
cultivars (Tables 3 <strong>and</strong> 4) were<br />
used. The genotypes had been<br />
preselected earlier based on their<br />
capacity to differentiate S. nodorum<br />
isolates in regard to their<br />
virulence/aggressiveness<br />
(pathogenicity). (The term<br />
virulence will be used from this<br />
point on in this paper, since the<br />
interaction <strong>of</strong> isolates by cultivars<br />
was significant.)<br />
Fourteen isolates used for the<br />
study originated from different<br />
geographical regions <strong>of</strong> Pol<strong>and</strong> <strong>and</strong><br />
one from Switzerl<strong>and</strong> (87TS-1-1).<br />
As for host association, <strong>of</strong> those 15<br />
isolates, 14 were derived from<br />
diseased hexaploid triticale plants<br />
<strong>and</strong> one (Wen-1-1) from a hexaploid<br />
bread wheat plant cv. ‘Weneda’<br />
(Table 5). No record was made <strong>of</strong><br />
the specific triticale genotypes from<br />
which the isolates were derived. All<br />
isolates used in the study were<br />
derived from single pycnidiospores<br />
Table 1. Analysis <strong>of</strong> variance for the amount <strong>of</strong> SNB on leaves <strong>and</strong> heads <strong>of</strong> 11 winter triticale<br />
<strong>and</strong> 4 winter wheat cultivars scored on a 0-9 digit scale (0 = resistant, 9 = susceptible) under<br />
field conditions.<br />
Leaves Heads<br />
Source <strong>of</strong> Mean F Proba- Mean F Probavariation<br />
D. f. Square value bility D. f. Square value bility<br />
Years (Y) 2 427.5 21.8 0.01 2 1957.8 2696.1 0.00<br />
Blocks (B)<br />
Error 1 (Y × B) 3 19.6 3 0.7<br />
Scoring dates (S.d.) 3 4217.7 866.7 0.00 2 650.3 4.1 0.07<br />
Y × S.d. 6 77.7 16.0 0.02 4 55.0 0.3 0.83<br />
Error 2 (Y×B×S.d.) 9 4.9 6 159.7<br />
Cultivars (C) 14 113.1 321.3 0.00 14 139.3 346.4 0.00<br />
Isolates (I) 15 157.3 446.7 0.00 15 131.4 326.8 0.00<br />
C × I 210 1.4 3.9 0.00 210 1.1 2.8 0.00<br />
Y × C 28 17.1 48.6 0.00 28 20.2 50.2 0.00<br />
Y × I 30 26.5 75.4 0.00 28 3.4 8.5 0.00<br />
S.d. × C 42 4.5 12.7 0.00 30 46.3 115.1 0.00<br />
S.d. × I 45 2.2 6.3 0.00 30 3.7 9.1 0.00<br />
Y × C × I 420 0.7 1.9 0.00 56 1.2 3.1 0.00<br />
Y × S.d. × I 90 1.4 3.9 0.00 60 1.8 4.6 0.00<br />
Y × S.d. × C 84 1.8 5.2 0.00 420 0.3 0.7 1.00<br />
S.d. × C × I 630 0.3 0.8 1.00 420 0.7 1.8 0.00<br />
Y × S.d.× C × I) 1260 0.3 0.7 1.00 840 0.3 0.7 1.00<br />
Error(Y×B×S.d.×C×I) 2868 0.4 2151 0.4<br />
Table 2. The expression <strong>of</strong> virulence by 15 <strong>Stagonospora</strong> nodorum isolates <strong>and</strong> their mixture (a composite isolate) on leaves <strong>and</strong> heads <strong>of</strong> 11<br />
winter triticale (t) <strong>and</strong> 4 winter wheat (w) cultivars under field conditions.<br />
Almo Ugo Malno Lasko Grado Dagro Bolero Presto Largo LAD- DAD- Alba Jawa Begra Liwilla<br />
No. Isolate (t) (t) (t) (t) (t) (t) (t) (t) (t) 285 (t) 187 (t) (w) (w) (w) (w) Mean<br />
1 Lb-2-1 3.9* 4.1 3.7 2.9 4.3 4.5 3.0 3.1 3.5 4.4 3.8 3.5 4.2 4.9 3.4 3.8<br />
2 Lb-3-1 4.8 4.7 4.1 3.6 4.8 5.0 4.0 3.8 4.2 5.1 4.7 3.9 4.7 5.3 3.5 4.4<br />
3 Lb-4-1 4.8 4.6 4.3 3.5 4.9 4.9 3.8 3.8 4.3 5.0 4.5 3.9 4.4 5.6 3.6 4.4<br />
4 Z-20-1 3.4 3.5 3.4 2.6 3.8 3.9 2.9 2.8 3.3 3.6 3.5 3.3 4.1 4.9 3.1 3.5<br />
5 Z-22-1 4.1 4.2 4.2 3.6 4.9 5.0 3.3 4.1 4.6 5.4 4.8 3.8 4.7 5.6 3.5 4.4<br />
6 Gw-3-1 5.9 5.8 5.5 4.9 6.2 5.9 4.6 5.3 5.3 5.9 5.6 4.5 5.4 6.1 4.3 5.4<br />
7 TS87-1-1 5.4 5.4 5.3 4.8 5.8 5.9 4.3 5.2 5.0 5.7 5.1 4.7 5.6 6.1 4.7 5.3<br />
8 Wen-1-1 5.1 5.3 5.3 4.3 6.0 5.9 4.5 5.3 5.1 5.2 5.4 4.3 5.2 5.6 4.1 5.1<br />
9 Ml-2-1 3.5 3.3 3.4 2.9 3.9 4.0 2.8 2.8 3.7 3.7 3.5 3.2 4.3 5.0 3.1 3.5<br />
10 Ch-9-1 3.2 3.5 3.3 2.8 3.7 3.7 2.6 2.8 3.2 3.3 3.3 2.8 4.0 4.8 3.3 3.3<br />
11 Wr-6-1 4.4 4.2 4.0 3.5 4.5 4.5 3.5 3.6 4.2 4.5 4.4 3.8 4.6 4.9 3.8 4.2<br />
12 Ch90-3-1 5.1 4.9 4.8 4.1 5.8 5.9 4.1 4.8 4.8 5.6 8.6 3.8 5.1 5.4 3.8 5.1<br />
13 Rd93-1-1 4.0 3.8 3.7 3.0 4.3 4.3 3.1 3.2 3.5 4.0 4.0 3.4 4.3 5.4 3.7 3.8<br />
14 T91-1-4 3.4 3.9 3.3 2.8 3.9 4.1 2.8 2.7 3.5 4.1 4.0 3.3 4.3 4.9 3.0 3.6<br />
15 Lu90-1-1 4.3 4.5 4.3 3.4 5.3 5.3 3.4 4.4 4.7 4.8 4.3 3.8 4.8 5.2 3.6 4.4<br />
16 Mixture 5.0 4.6 4.5 3.9 5.4 5.3 3.7 4.3 5.0 5.4 4.8 4.0 4.9 5.4 3.5 4.6<br />
Mean 4.4 4.4 4.2 3.5 4.8 4.9 3.5 3.9 4.2 4.7 4.6 3.7 4.7 5.3 3.6 4.3<br />
LSD 0.05 = 0.114 for virulence <strong>of</strong> isolates on leaves; LSD 0.01 = 0.110 for reaction <strong>of</strong> cultivars on leaves.<br />
* Mean score on a 0-9 scale <strong>of</strong> 4 SNB scorings per season × 6 blocks over 3 growing seasons.
<strong>and</strong>, as determined after the<br />
research was completed, all were <strong>of</strong><br />
the wheat biotype.<br />
The isolates used for inoculation<br />
were increased on bread wheat<br />
grain. The composite isolate (a<br />
volumetric mixture <strong>of</strong> isolates) was<br />
prepared by mixing equal volumes<br />
<strong>of</strong> spore suspensions <strong>of</strong> all isolates<br />
adjusted to equal concentrations <strong>of</strong><br />
spores/ml. Plants were inoculated<br />
at the late boot stage (5 × 106 spores/ml) <strong>and</strong> after heading (2 ×<br />
106 spores/ml). SNB was rated<br />
visually on a 0 (resistant) - 9<br />
(susceptible) scale at about weekly<br />
intervals. Separate notes were taken<br />
for leaves <strong>and</strong> heads four <strong>and</strong> three<br />
times, respectively, on winter types,<br />
<strong>and</strong> four <strong>and</strong> two times,<br />
respectively, on spring types. All<br />
Table 3.The expression <strong>of</strong> virulence by 15 <strong>Stagonospora</strong> nodorum isolates <strong>and</strong> their mixture (a<br />
composite isolate) on leaves <strong>and</strong> heads <strong>of</strong> 5 spring triticale <strong>and</strong> 3 spring wheat cultivars un-der<br />
field conditions.<br />
Jago Maja Gabo Grego Migo Henika Eta Sigma<br />
No. Isolate (t) (t) (t) (t) (t) (w) (w) (w) Mean<br />
1 Lb-2-1 2.8* 2.6 2.2 2.9 2.8 3.6 2.4 2.8 2.8<br />
2 Lb-3-1 2.7 2.8 2.4 3.2 2.7 3.5 3.0 2.8 2.9<br />
3 Lb-4-1 2.8 2.1 2.3 2.8 2.8 3.4 2.6 2.6 2.7<br />
4 Z-20-1 1.9 2.1 2.2 2.5 2.7 3.2 2.2 2.3 2.4<br />
5 Z-22-1 2.2 2.2 2.3 2.3 2.5 2.8 2.2 2.1 2.3<br />
6 Gw-3-1 3.3 3.3 3.6 3.4 3.7 4.2 3.2 3.0 3.4<br />
7 TS87-1-1 4.4 4.3 4.4 4.3 4.9 4.9 4.1 4.1 4.4<br />
8 Wen-1-1 3.8 3.8 3.9 4.2 4.0 4.8 3.9 3.8 4.0<br />
9 Ml-2-1 2.7 2.8 2.5 2.5 3.2 2.9 2.7 2.9 2.8<br />
10 Ch-9-1 2.8 2.9 2.7 2.5 3.2 2.7 3.1 2.9 2.8<br />
11 Wr-6-1 2.7 3.1 2.6 2.3 2.9 2.9 2.6 2.7 2.7<br />
12 Ch-90-3 2.2 2.4 2.2 2.0 2.4 2.6 2.3 2.1 2.3<br />
13 Rd91-1-3 2.1 2.5 2.1 2.0 2.3 2.2 2.0 2.2 2.2<br />
14 T91-1-4 2.9 3.6 3.4 2.9 3.9 3.8 3.3 3.6 3.4<br />
15 Lu90-1-1 3.8 4.5 4.1 4.3 4.9 4.8 4.2 4.4 4.4<br />
16 Mixture 3.6 4.1 3.7 3.6 4.3 4.6 3.7 3.9 3.9<br />
Mean 2.9 3.1 2.9 3.0 3.3 3.6 3.0 3.0 3.1<br />
LSD0.01 = 0.243 - for virulence <strong>of</strong> isolates on leaves; LSD0.01 = 0.172 - for reactions <strong>of</strong> cultivars on leaves.<br />
* Mean score on a 0-9 scale <strong>of</strong> 4 SNB scorings per season x 5 blocks over 3 growing seasons.<br />
Table 4. The expression <strong>of</strong> virulence by 15 <strong>Stagonospora</strong> nodorum isolates <strong>and</strong> their mixture (a<br />
composite isolate) on heads <strong>of</strong> 5 spring triticale <strong>and</strong> 3 spring wheat cultivars under field<br />
conditions.<br />
Jago Maja Gabo Grego Migo Henika Eta Sigma<br />
No. Isolate (t) (t) (t) (t) (t) (w) (w) (w) Mean<br />
1 Lb-2-1 0.2 0.0 0.1 0.3 0.1 1.3 2.0 2.0 0.7<br />
2 Lb-3-1 0.1 0.1 0.3 0.2 0.0 2.2 2.0 2.0 0.9<br />
3 Lb-4-1 0.1 0.0 0.3 0.0 0.0 2.6 3.1 2.0 1.0<br />
4 Z-20-1 0.0 0.5 0.1 0.4 0.0 2.6 2.8 2.0 1.0<br />
5 Z-22-1 0.3 0.2 0.1 0.3 0.1 1.7 2.9 2.0 0.9<br />
6 Gw-3-1 0.7 0.2 0.0 1.0 0.8 2.9 3.5 2.0 1.4<br />
7 TS87-1-1 0.3 0.2 0.3 0.3 0.1 2.2 3.3 3.5 1.3<br />
8 Wen-1-1 0.2 0.2 0.6 0.4 0.3 2.9 3.2 3.5 1.4<br />
9 Ml-2-1 0.3 0.0 0.1 0.1 0.1 1.8 2.2 2.5 0.9<br />
10 Ch-9-1 0.0 0.1 0.1 0.0 0.0 1.6 1.6 2.0 0.7<br />
11 Wr-6-1 0.1 0.2 0.1 0.1 0.0 1.6 1.8 1.5 0.7<br />
12 Ch-90-3 0.4 0.4 0.2 0.3 0.3 1.9 1.9 2.5 1.0<br />
13 Rd91-1-3 0.0 0.0 0.0 0.6 0.0 1.8 2.0 2.5 0.9<br />
14 T91-1-4 0.1 0.2 0.2 0.0 0.0 2.2 1.9 2.0 0.8<br />
15 Lu-90-1 0.1 0.1 0.1 0.0 0.2 2.1 1.8 2.0 0.8<br />
16 Mixture 0.0 0.3 0.0 0.3 0.2 2.0 2.4 3.0 1.0<br />
Mean 0.2 0.2 0.2 0.3 0.1 2.1 2.4 2.3 1.0<br />
LSD 0.01 = 0.268 - for virulence <strong>of</strong> isolates on heads; LSD 0.01 = 0.189 - for reactions <strong>of</strong> cultivars on heads.<br />
* Mean score on 0 - 9 scale <strong>of</strong> 2 SNB scorings per season ¥ 5 blocks over 3 growing seasons.<br />
Host – Parasite Interactions: <strong>Stagonospora</strong> nodorum 65<br />
the tabulated data (Tables 2, 6, 3, 4)<br />
are averages <strong>of</strong> the numbers <strong>of</strong><br />
replications <strong>and</strong> disease scoring<br />
dates.<br />
A split-plot experimental design<br />
was used. One experimental block<br />
comprised a number <strong>of</strong> main plots<br />
equal to the number <strong>of</strong> individual S.<br />
nodorum isolates studied. The main<br />
plots (rows <strong>of</strong> parcels) contained 1m2<br />
parcels (subplots) planted to<br />
assigned cereal cultivars <strong>and</strong><br />
germplasm lines. Individual isolates<br />
<strong>of</strong> the pathogen, considered as the<br />
main plots, were r<strong>and</strong>omly<br />
assigned to the sets <strong>of</strong> subplots. The<br />
number <strong>of</strong> subplots within the main<br />
plot was equal to the number <strong>of</strong><br />
cereal genotypes studied (15: 11<br />
triticales + 4 wheat genotypes) <strong>and</strong><br />
8 (5 triticales + 3 wheat genotypes)<br />
for both winter <strong>and</strong> spring types,<br />
respectively. Each isolate (main<br />
plot) had its own control, treated<br />
with a fungicide (Tilt 250 EC, 0.1%<br />
a.i., 500 l/ha). In fact, the disease<br />
scores were actually differences<br />
between disease levels on cereal<br />
subpolts inoculated with individual<br />
isolates <strong>and</strong> its mirror images<br />
protected chemically. Isolate main<br />
plots containing the cereal subplots<br />
(cultivars/lines) were replicated<br />
twice for both winter <strong>and</strong> spring<br />
types (in 1994 only once).<br />
Data were subjected to an<br />
analysis <strong>of</strong> variance using an<br />
MSUSTAT computer program,<br />
Version 5.25 (developed by R. Lund,<br />
Montana State University, MT<br />
59715-002, USA) for a split-plot<br />
experiment. The variance among<br />
cereal cultivars/germplasm lines<br />
for disease reaction to individual S.<br />
nodorum isolates was regressed
66<br />
Session 3B — E. Arseniuk <strong>and</strong> P.C. Czembor<br />
Table 5. Origin <strong>of</strong> <strong>Stagonospora</strong> nodorum isolates.<br />
against mean virulence <strong>of</strong> the<br />
No. Isolate name Host Geographic origin (location)<br />
isolates. Similarly, the variance<br />
1 Lb-2-1 triticale Lubinicko, woj. zielonogórskie<br />
among isolates for virulence to<br />
2<br />
3<br />
4<br />
Lb-3-1<br />
Lb-4-1<br />
Z-20-1<br />
triticale<br />
triticale<br />
triticale<br />
Lubinicko, woj. zielonogórskie<br />
Lubinicko, woj. zielonogórskie<br />
Zadabrowie. woj. przemyskie<br />
individual cultivars/lines was<br />
regressed against mean resistance<br />
5<br />
6<br />
7<br />
Z-22-1<br />
Gw-3-1<br />
TS87-1-1<br />
triticale<br />
triticale<br />
triticale<br />
Zadabrowie. woj. przemyskie<br />
Grodkowice, woj. krakowskie<br />
Z¸rich, Switzerl<strong>and</strong><br />
<strong>of</strong> the cultivars/lines expressed on<br />
the 0-9 scale.<br />
8 Wen-1-1 winter wheat Weneda Elblag, woj. torunskie<br />
9<br />
10<br />
Ml-2-1<br />
Ch-9-1<br />
triticale<br />
triticale<br />
Malyszyn, woj. gorzowskie<br />
Choryn, woj. poznanskie Results<br />
11 Wr-6-1 triticale Wrocikowo, woj. olsztynskie<br />
12 Ch90-3-1 triticale Chelm, woj. chelmskie<br />
13 Rd93-1-1 triticale Radzików, woj. warszawskie<br />
All isolates <strong>of</strong> S. nodorum used<br />
14<br />
15<br />
T91-1-4<br />
Lu90-1-1<br />
triticale<br />
triticale<br />
Torun, woj. torunskie<br />
Lucmierz, woj. lódzkie<br />
in the study were pathogenic to<br />
leaves <strong>and</strong> to heads <strong>of</strong> all triticale<br />
<strong>and</strong> bread wheat cultivars (Tables<br />
2, 6, 3, 4). On average, the disease<br />
Table 6. The expression <strong>of</strong> virulence by 15 <strong>Stagonospora</strong> nodorum isolates <strong>and</strong> their mixture (a composite<br />
isolate) on heads <strong>of</strong> 11 winter triticale (t) <strong>and</strong> 4 winter wheat (w) cultivars under field conditions.<br />
on leaves was 2-3 times<br />
more severe than on<br />
No. Isolate<br />
Almo Ugo<br />
(t) (t)<br />
Malno Lasko Grado Dagro Bolero Presto Largo<br />
(t) (t) (t) (t) (t) (t) (t)<br />
LAD-<br />
285 (t)<br />
DAD-<br />
187 (t)<br />
Alba Jawa Begra Liwilla<br />
(w) (w) (w) (w) Mean<br />
heads. Some <strong>of</strong> the<br />
isolates did not cause<br />
1 Lb-2-1 0.7* 0.8 1.5 1.4 1.3 1.4 0.7 0.8 0.9 1.6 1.1 1.8 2.9 2.7 1.8 1.4<br />
disease on heads <strong>of</strong><br />
2 Lb-3-1 1.2 1.0 1.7 1.7 1.7 2.2 1.2 1.5 1.5 2.5 1.6 2.7 3.6 3.6 1.9 2.0 spring triticale genotypes<br />
3<br />
4<br />
5<br />
Lb-4-1<br />
Z-20-1<br />
Z-22-1<br />
1.3<br />
0.5<br />
0.9<br />
1.4<br />
0.6<br />
0.8<br />
2.2<br />
0.7<br />
1.1<br />
2.1<br />
0.6<br />
1.2<br />
2.5<br />
1.9<br />
1.2<br />
2.8<br />
1.1<br />
1.8<br />
1.8<br />
0.7<br />
0.8<br />
1.8<br />
0.8<br />
1.3<br />
1.8<br />
0.9<br />
1.0<br />
3.1<br />
1.2<br />
2.1<br />
1.8<br />
0.9<br />
1.6<br />
2.3<br />
1.6<br />
2.3<br />
3.6<br />
2.3<br />
3.2<br />
3.2<br />
2.0<br />
2.9<br />
2.3<br />
1.6<br />
1.9<br />
2.3<br />
1.2<br />
1.6<br />
(Table 4). The lack <strong>of</strong><br />
disease was due to low<br />
6 Gw-3-1 2.1 2.1 3.2 2.7 2.5 3.6 2.1 2.8 2.0 3.5 2.6 4.2 5.2 4.4 3.8 3.1 rainfall <strong>and</strong> drought<br />
7<br />
8<br />
TS87-1-1<br />
Wen-1-1<br />
1.8<br />
1.6<br />
2.1<br />
2.2<br />
3.2<br />
2.7<br />
2.8<br />
2.6<br />
3.3<br />
3.3<br />
3.7<br />
3.4<br />
2.2<br />
1.8<br />
2.8<br />
2.3<br />
2.6<br />
2.2<br />
4.0<br />
3.6<br />
2.5<br />
2.6<br />
3.3<br />
3.4<br />
4.9<br />
4.5<br />
4.1<br />
3.9<br />
3.7<br />
2.7<br />
3.1<br />
2.9<br />
during the summers. In<br />
9 Ml-2-1 0.7 0.6 0.9 0.9 2.3 1.3 0.6 0.6 0.8 1.8 1.0 1.7 2.7 2.2 1.7 1.3 spite <strong>of</strong> the adverse<br />
10<br />
11<br />
12<br />
Ch-9-1<br />
Wr-6-1<br />
Ch90-3-1<br />
0.7<br />
1.4<br />
1.4<br />
0.5<br />
1.3<br />
1.3<br />
0.6<br />
1.8<br />
2.2<br />
0.7<br />
1.6<br />
2.3<br />
0.9<br />
1.5<br />
2.8<br />
0.9<br />
2.4<br />
3.2<br />
0.7<br />
1.3<br />
1.5<br />
0.7<br />
1.7<br />
1.8<br />
0.6<br />
1.4<br />
1.6<br />
1.3<br />
2.4<br />
3.1<br />
1.0<br />
1.7<br />
2.1<br />
1.2<br />
2.4<br />
2.7<br />
2.1<br />
3.5<br />
4.3<br />
1.9<br />
3.2<br />
3.3<br />
2.9<br />
2.8<br />
2.6<br />
1.1<br />
2.0<br />
2.4<br />
weather, the same<br />
isolates were able to<br />
13 Rd93-1-1 0.8 0.9 0.8 1.1 2.3 1.8 0.8 1.2 1.1 1.9 1.4 2.1 2.6 2.4 3.6 1.7 cause the disease on<br />
14<br />
15<br />
T91-1-4 0.6<br />
Lu90-1-1 1.0<br />
0.5<br />
0.9<br />
1.0<br />
1.6<br />
1.4<br />
1.5<br />
1.6<br />
1.7<br />
1.7<br />
2.1<br />
0.8<br />
1.0<br />
1.0<br />
1.4<br />
1.1<br />
1.6<br />
1.8<br />
2.6<br />
1.2<br />
1.4<br />
2.1<br />
2.4<br />
2.8<br />
3.5<br />
2.6<br />
2.7<br />
1.8<br />
2.4<br />
1.5<br />
1.9<br />
heads <strong>of</strong> more SNB<br />
16 Mixture 1.1 1.3 2.4 2.3 2.4 3.0 1.6 1.7 2.1 2.6 1.7 2.7 4.0 3.3 2.8 2.3 susceptible wheat<br />
Mean 1.1 1.1 1.7 1.7 2.1 2.3 1.2 1.5 1.4 2.4 1.6 2.4 3.5 3.0 2.5 2.0 cultivars.<br />
LSD0.01 = 0.141 - for virulence <strong>of</strong> isolates on heads; LSD0.01 = 0.136 for reaction <strong>of</strong> cultivars on heads.<br />
* Mean score on a 0-9 scale <strong>of</strong> 3 SNB scorings per season ¥ 6 blocks over 3 growing seasons.<br />
Due to a large<br />
number degrees <strong>of</strong> freedom <strong>and</strong><br />
Table 7. Analysis <strong>of</strong> variance for the amount <strong>of</strong> SNB on leaves <strong>and</strong> heads <strong>of</strong> 5 spring triticale low error values, most <strong>of</strong> the<br />
<strong>and</strong> 3 spring wheat cultivars scored on a 0-9 digit scale (0 = resistant, 9 = susceptible) under<br />
field conditions.<br />
Leaves Heads<br />
Source <strong>of</strong> Mean F Proba- Mean F Probavariation<br />
D. f. Square value bility D. f. Square value bility<br />
sources <strong>of</strong> variation included in the<br />
analysis <strong>of</strong> variance (Tables 1 <strong>and</strong> 7)<br />
proved to be significant. The main<br />
effects <strong>of</strong> cultivars <strong>and</strong> isolates<br />
Blocks (B) 4 844.03 18.13 0.01 4 13.17 1.84 0.28<br />
were highly significant. It is<br />
Scoring dates (R.d.) 3 1553.60 33.37 0.00 1 39.55 5.52 0.08 noticeable that the effect <strong>of</strong> isolates<br />
Error (B × S.d.)<br />
Cultivars (C)<br />
Isolates (I)<br />
12<br />
7<br />
15<br />
46.56<br />
193.32<br />
11.3<br />
272.50<br />
15.65<br />
0.00<br />
0.00<br />
4<br />
7<br />
15<br />
7.17<br />
238.49<br />
4.58<br />
552.86<br />
10.62<br />
0.00<br />
0.00<br />
was smaller than that <strong>of</strong> cultivars.<br />
This would indicate that the<br />
C × I<br />
S.d. × C<br />
S.d. × I<br />
105<br />
21<br />
45<br />
0.636<br />
4.95<br />
0.693<br />
0.90<br />
6.97<br />
0.98<br />
0.76<br />
0.00<br />
0.51<br />
105<br />
7<br />
15<br />
0.71<br />
2.55<br />
0.167<br />
1.65<br />
5.91<br />
0.39<br />
0.01<br />
0.00<br />
0.98<br />
reaction <strong>of</strong> cultivars to inoculation<br />
with different isolates <strong>of</strong> S. nodorum<br />
S.d. × I × C<br />
Error (B×S.d.×C×I)<br />
315<br />
2032<br />
0.239<br />
0.709<br />
0.34 0.00 105<br />
1016<br />
0.129<br />
0.431<br />
0.30 1.00 was determined mainly by the
cultivar genotype. Nonetheless, the<br />
differences in virulence among<br />
isolates were large enough to make<br />
cultivar response range from<br />
resistant to susceptible, depending<br />
on the isolate used (Tables 2, 6, 3, 4).<br />
The effect <strong>of</strong> cultivar by isolate<br />
interaction was not significant only<br />
for SNB assessment on leaves <strong>of</strong><br />
spring genotypes (Table 7). In all<br />
other cases, the latter effect, though<br />
highly significant, contributed only<br />
slightly to the total variance (Tables<br />
1 <strong>and</strong> 7).<br />
The significance <strong>of</strong> cultivar by<br />
isolate interaction indicated that<br />
there is some degree <strong>of</strong> specificity<br />
in the host-pathogen relationship.<br />
The low specificity in the<br />
pathosystem was confirmed<br />
through Spearman rank<br />
correlations. They revealed that<br />
rankings <strong>of</strong> the genotypes on their<br />
disease reaction to inoculation with<br />
individual S. nodorum isolates, <strong>and</strong><br />
conversely, the rankings <strong>of</strong> isolates<br />
on their virulence expressed on the<br />
genotypes were correlated.<br />
The analyses <strong>of</strong> variance <strong>of</strong> the<br />
disease scores revealed significant<br />
differences between disease scoring<br />
dates, primarily on leaves. This<br />
means that the disease progressed<br />
with the passing <strong>of</strong> time. For SNB<br />
on heads, scoring dates were<br />
bordering on significance for both<br />
winter <strong>and</strong> spring genotypes. This<br />
indicates that, due to adverse<br />
environmental conditions<br />
(primarily summer droughts),<br />
disease progress over time was<br />
slower on heads than on leaves<br />
(Tables 1 <strong>and</strong> 7). The highly<br />
significant interactions between<br />
disease scoring dates <strong>and</strong> other<br />
components <strong>of</strong> the analysis <strong>of</strong><br />
variance indicate that the disease<br />
response progressed at different<br />
rates (Tables 1 <strong>and</strong> 7) on individual<br />
genotypes (S.d. by C) between<br />
consecutive scoring dates, <strong>and</strong> that<br />
the isolates (S.d. by I) caused more<br />
disease during the intervals<br />
between consecutive scoring dates.<br />
This may have something to do<br />
with the susceptibility <strong>of</strong> plant<br />
tissue at different growth stages.<br />
Older tissue may be more<br />
susceptible. The significance <strong>of</strong><br />
second <strong>and</strong> third degree<br />
interactions among years, scoring<br />
dates, isolates, <strong>and</strong> cultivars<br />
indicated that disease progress was<br />
influenced by all factors involved,<br />
i.e. host <strong>and</strong> pathogen genotypes,<br />
as well as the environment.<br />
It should be pointed out that the<br />
interactions <strong>of</strong> years with isolates<br />
<strong>and</strong> cultivars (Table 1 <strong>and</strong> 7),<br />
although significant, were low <strong>and</strong><br />
contributed only slightly to total<br />
variation. Thus rankings <strong>of</strong><br />
cultivars based on their SNB<br />
reaction <strong>and</strong> isolates on their<br />
Cultivar variances<br />
4.6<br />
4.2<br />
3.8<br />
3.4<br />
3.0<br />
2.6<br />
Cv. variance = 1.306 + 0.488 (mean isolate virulence)<br />
r = 0.682, p
68<br />
Cultivar variances<br />
Cultivar variances<br />
Isolate variances<br />
5.6<br />
5.4<br />
5.2<br />
7<br />
6<br />
5<br />
4<br />
3.2<br />
2.8<br />
Session 3B — E. Arseniuk <strong>and</strong> P.C. Czembor<br />
Mixture<br />
5.0<br />
Gw-3-1 TS87-1-1<br />
4.8<br />
4.6<br />
Lb-4-1<br />
Ch90-3-1<br />
Z-22-1<br />
4.4 T91-1-4<br />
Lu90-1-1<br />
Lb-3-1<br />
4.2<br />
4.0<br />
3.8<br />
2.6<br />
Wr-6-1<br />
Ch-9-1<br />
2.8<br />
MI-2-1<br />
Lb-2-1<br />
Z-20-1<br />
3.0<br />
Rd93-1-1<br />
3.2<br />
Cv. variance = 1.203 + 1.094 (mean isolate virulence)<br />
r = 0.607, p
Isolate variances<br />
Isolate variances<br />
4.6<br />
4.0<br />
3.4<br />
2.8<br />
2.2<br />
1.6<br />
3.2 3.6 4.0 4.4 4.8 5.2 5.6<br />
Mean cultivars reactions on leaves (0-9 scale)<br />
Figure 5. Relationship between the mean reactions <strong>of</strong> 11 winter triticale (t) <strong>and</strong> 4 winter wheat<br />
(w) cultivars <strong>and</strong> variances <strong>of</strong> SNB induction on leaves <strong>of</strong> the cultivars by 15 <strong>Stagonospora</strong><br />
nodorum isolates <strong>and</strong> their mixture (3 years’ data).<br />
5.5<br />
5.0<br />
4.5<br />
4.0<br />
3.5<br />
3.0<br />
2.5<br />
2.0<br />
1.5<br />
r = 0.044, n.s.<br />
Lasko (t)<br />
Bolero (t)<br />
Presto (t)<br />
Largo (t)<br />
Bolero (t)<br />
Ugo (t)<br />
Almo (t)<br />
Presto (t)<br />
Liwilla (w)<br />
Alba (w)<br />
Lasko (t)<br />
Malno (t)<br />
DAD-187 (t)<br />
Malno (t)<br />
Almo (t)<br />
Largo (t)<br />
Grado (t)<br />
Ugo (t)<br />
DAD-187 (t)<br />
Dagro (t)<br />
LAD-285 (t)<br />
Alba (w)<br />
Liwilla (t)<br />
LAD-285 (t)<br />
Dagro (t)<br />
Jawa (w)<br />
Grado (t)<br />
Begra (w)<br />
Begra (w)<br />
Jawa (w)<br />
Isolate variance = 0.285 + 11.022<br />
(mean cultivar reaction)<br />
r = 0.624, p < 0.03<br />
1.0<br />
0.8 1.4 2.0 2.6 3.2 3.8<br />
Mean cultivars reaction on heads (0-9 scale)<br />
Figure 6. Relationship between the mean reactions <strong>of</strong> 11 winter triticale (t) <strong>and</strong> 4 winter wheat<br />
(w) cultivars <strong>and</strong> variances <strong>of</strong> SNB induction on heads <strong>of</strong> the cultivars by 15 <strong>Stagonospora</strong><br />
nodorum isolates <strong>and</strong> their mixture (3 years’ data).<br />
Cultivar variances<br />
7.0<br />
6.5<br />
6.0<br />
5.5<br />
r = 0.577, n.s.<br />
Sigma (w)<br />
5.0<br />
Henika (w)<br />
4.5<br />
4.0<br />
Gabo (t)<br />
Eta (w)<br />
3.5<br />
Jago (t) Maja (t)<br />
3.0 Grego (t)<br />
Migo (t)<br />
2.5<br />
2.0 2.4 2.8 3.2 3.6 4.0 4.4 4.8<br />
Mean cultivar reaction on leaves (0-9 scale)<br />
Figure 7. Relationship between mean reactions <strong>of</strong> 5 spring triticale (t) <strong>and</strong> 3 spring wheat (w)<br />
cultivars <strong>and</strong> variances <strong>of</strong> SNB induction on leaves <strong>of</strong> the cultivars by 15 <strong>Stagonospora</strong> nodorum<br />
isolates <strong>and</strong> their mixture (3 years’ data).<br />
Host – Parasite Interactions: <strong>Stagonospora</strong> nodorum 69<br />
showed two distinct resistance<br />
groups (Figures 7 <strong>and</strong> 8).<br />
Correlations between mean host<br />
reactions on leaves <strong>and</strong> heads <strong>and</strong><br />
the variances in SNB induction<br />
among isolates were significant<br />
only for heads (Figures 5 <strong>and</strong> 7) <strong>and</strong><br />
not for leaves (Figures 6 <strong>and</strong> 8). It<br />
was found that a highly susceptible<br />
genotype is not necessarily efficient<br />
in differentiating virulence among<br />
isolates (Figures 5, 4, 7, 8).<br />
Conclusions<br />
The results produced in the<br />
study do not seem to support that<br />
using a mixture <strong>of</strong> S. nodorum<br />
isolates is the most efficient way to<br />
differentiate cereal breeding<br />
materials on their SNB resistance.<br />
The finding for S. nodorum is in<br />
accordance with results obtained by<br />
Zelikovitch <strong>and</strong> Eyal (1991) that<br />
inoculation <strong>of</strong> wheat seedlings <strong>and</strong><br />
adult plants with a mixture <strong>of</strong> S.<br />
tritici isolates resulted in significant<br />
suppression <strong>of</strong> symptoms (necrosis<br />
<strong>and</strong> pycnidial coverage) compared<br />
to the most virulent single isolate in<br />
the mixture.<br />
1. Screening for SNB resistance<br />
with a few virulent isolates that<br />
also show good differential<br />
capacity seems most appropriate.<br />
Screening breeding materials<br />
with a single highly virulent<br />
isolate may provide reliable<br />
information on their SNB<br />
resistance.<br />
2. Sets <strong>of</strong> selected isolates <strong>and</strong><br />
cultivars differentiated each<br />
other sufficiently well, but<br />
according to cluster analyses<br />
similar results could have been<br />
obtained with a lower number <strong>of</strong><br />
both isolates <strong>and</strong> germplasm<br />
lines.
70<br />
Session 3B — E. Arseniuk <strong>and</strong> P.C. Czembor<br />
3. The reported data indicate that<br />
the interaction between S.<br />
nodorum isolates <strong>of</strong> the wheat<br />
biotype <strong>and</strong> wheat <strong>and</strong> triticale<br />
genotypes to a great extent is<br />
more a matter <strong>of</strong> statistics than a<br />
well defined physiological<br />
specialization. Whether the<br />
interaction is detected or not<br />
depends upon the amount <strong>of</strong><br />
disease inflicted by the pathogen<br />
isolates on selected host<br />
genotypes, the effect <strong>of</strong><br />
environmental conditions on<br />
disease development, <strong>and</strong> the<br />
tools <strong>and</strong> precision with which<br />
the host has been examined.<br />
Acknowledgments<br />
This study was financially<br />
supported by the ICD-RSE-FAS-<br />
USDA (Project no. PL-AES-241,<br />
Grant no. MR-USDA-93-139) <strong>and</strong><br />
by the Plant Breeding <strong>and</strong><br />
Acclimatization Institute. The<br />
technical assistance <strong>of</strong> T. Bajor, E.<br />
Matyska, <strong>and</strong> E. Pawióska is<br />
gratefully acknowledged.<br />
Isolate variances<br />
2.4<br />
2.0<br />
1.6<br />
1.2<br />
0.8<br />
Isolate variance = 0.048 + 0.503 (mean cultivar reaction)<br />
r = 0.928, p = 0.01<br />
References<br />
Arseniuk, E., A L. Scharen, P.M. Fried,<br />
<strong>and</strong> H.J. Czembor. 1994.<br />
Pathogenic interactions in X<br />
Triticosecale - <strong>Septoria</strong> spp. <strong>and</strong><br />
Triticum aestivum - <strong>Septoria</strong> spp.<br />
systems. Hod. Rol. Aklim., Nasien.<br />
(Special Edition) 38(3/4):101-108.<br />
Eyal, Z., A.L. Scharen, J.M. Prescott,<br />
<strong>and</strong> M. van Ginkel. 1987. The<br />
<strong>Septoria</strong> <strong>Diseases</strong> <strong>of</strong> Wheat:<br />
Concepts <strong>and</strong> Methods <strong>of</strong> Disease<br />
Management. Mexico D.F.:<br />
<strong>CIMMYT</strong>.<br />
Eyal, Z. 1992. The response <strong>of</strong> fieldinoculated<br />
wheat cultivars to<br />
mixtures <strong>of</strong> <strong>Septoria</strong> tritici isolates.<br />
Euphytica 61:25-35.<br />
Krupinsky, J.M. 1986. Virulence on<br />
wheat <strong>of</strong> Leptosphaeria nodorum<br />
isolates from Bromus inermis. Can.<br />
J. Plant Path. 8:201-207.<br />
Krupinsky, J.M. 1994. Aggressiveness<br />
<strong>of</strong> <strong>Stagonospora</strong> nodorum from<br />
alternative hosts after passage<br />
through wheat. Hod Rol. Aklim.,<br />
Nasien. (Special Edition) 38(3/<br />
4):123-126.<br />
Krupinsky, J.M. 1997a.<br />
Aggressiveness <strong>of</strong> <strong>Stagonospora</strong><br />
nodorum isolates obtained from<br />
wheat in the Northern Great<br />
Plains. Plant Dis. 81:1027-1031.<br />
Henika (t)<br />
Eta (w)<br />
Sigma (w)<br />
0.4<br />
Migo (t)<br />
Jago (t) Maja (t)<br />
Gabo (t) Grego (t)<br />
0.0<br />
-0.2 0.4 1.0 1.6 2.2 2.8 3.4<br />
Mean cultivar reaction on heads (0-9 scale)<br />
Figure 8. Relationship between mean reactions <strong>of</strong> 5 spring triticale (t) <strong>and</strong> 3 spring wheat (w)<br />
cultivars <strong>and</strong> variances <strong>of</strong> SNB induction on heads <strong>of</strong> the cultivars by 15 <strong>Stagonospora</strong> nodorum<br />
isolates <strong>and</strong> their mixture (3 years’ data).<br />
Krupinsky, J.M. 1997b. Stability <strong>of</strong><br />
<strong>Stagonospora</strong> nodorum isolates from<br />
perennial grass hosts after passage<br />
through wheat. Plant Dis. 81:1037-<br />
1041.<br />
Krupinsky, J.M. 1997c. Aggressiveness<br />
<strong>of</strong> <strong>Stagonospora</strong> nodorum isolates<br />
from perennial grasses on wheat.<br />
Plant Dis. 81:1032-1036.<br />
Scharen, L.A., <strong>and</strong> Z. Eyal. 1983.<br />
Analysis <strong>of</strong> symptoms on spring<br />
<strong>and</strong> winter wheat cultivars<br />
inoculated with different isolates<br />
<strong>of</strong> <strong>Septoria</strong> nodorum.<br />
Phytopathology 73:143-147.<br />
Scharen, L.A., Z. Eyal, M.D. Huffman,<br />
<strong>and</strong> J.M. Prescott. 1985. The<br />
distribution <strong>and</strong> frequency <strong>of</strong><br />
virulence genes in geographically<br />
separated populations <strong>of</strong><br />
Leptosphaeria nodorum.<br />
Phytopathology 75:1463-1468.<br />
Skajennik<strong>of</strong>f, M., <strong>and</strong> F. Rapilly. 1983.<br />
Aggressivness <strong>of</strong> <strong>Septoria</strong> nodorum<br />
on wheat <strong>and</strong> triticale. Effects <strong>of</strong><br />
the host <strong>and</strong> infected organs.<br />
Agronomie 3:131-140.<br />
Yechilevich-Auster, M., E. Levi, <strong>and</strong><br />
Z. Eyal. 1983. Assessment <strong>of</strong><br />
interactions between cultivated<br />
<strong>and</strong> wild wheats <strong>and</strong> <strong>Septoria</strong><br />
tritici. Phytopathology 73:1077-<br />
1083.<br />
Zelikovitch, N., <strong>and</strong> Z. Eyal. 1991.<br />
Reduction in pycnidial coverage<br />
after inoculation <strong>of</strong> wheat with<br />
mixtures <strong>of</strong> isolates <strong>of</strong> <strong>Septoria</strong><br />
tritici. Plant Dis. 75:907-910.
Identification <strong>of</strong> a Molecular Marker Linked to <strong>Septoria</strong><br />
Nodorum Blotch Resistance in Triticum tauschii Using<br />
F2 Bulked Segregant<br />
N.E.A. Murphy, 1 R. Loughman, 2 R. Wilson, 2 E.S. Lagudah, 3<br />
R. Appels, 3 <strong>and</strong> M.G.K. Jones1 1 WA State Agriculture Biotechnology Centre, Division <strong>of</strong> Science <strong>and</strong> Engineering, Murdoch University,<br />
Murdoch, Australia<br />
2 Agriculture Western Australia, Bentley Delivery Centre, Western Australia<br />
3 Plant Industries, CSIRO, Canberra, Australia<br />
Abstract<br />
A search was conducted for a molecular marker linked to a gene for resistance to septoria nodorum blotch in the<br />
Triticum tauschii accession RL5271. DNA was extracted from leaves <strong>of</strong> F2 plants which were progeny tested to identify<br />
homozygous resistant <strong>and</strong> homozygous susceptible F2 plants. The DNA from the homozygous resistant plants was pooled<br />
together <strong>and</strong> the low copy sequences were enriched using renaturation kinetics <strong>and</strong> hydroxyapatite to remove the repetitive<br />
DNA sequences. The pooled DNA from the homozygous susceptible plants was treated in the same manner. The pooled<br />
DNA <strong>and</strong> the parental DNA were screened using RAPD primers. Two markers present in the pooled resistant DNA <strong>and</strong><br />
in the parental DNA were identified <strong>and</strong> cloned. These markers were verified using RFLPs with the cloned marker as a<br />
probe. One <strong>of</strong> the probes did not produce any polymorphism as a RFLP, even when a number <strong>of</strong> different restriction<br />
enzymes were used. The second marker was polymorphic between the two parents when the DNA was restricted with<br />
HindIII <strong>and</strong> then MseI. In the 14 homozygous F2 plants tested, the marker was completely linked to the resistance gene.<br />
This marker may be valuable in introgressing the resistance gene from T. tauschii into a commercial bread wheat cultivar.<br />
<strong>Septoria</strong> nodorum blotch is the<br />
major disease <strong>of</strong> wheat (Triticum<br />
aestivum L.) in the Western<br />
Australian wheat belt. It is caused<br />
by <strong>Stagonospora</strong> nodorum (Berk.)<br />
Castellani & E.G. Germano<br />
(teleomorph Phaeosphaeria nodorum<br />
(E. Müller) Hedjaroude). The<br />
preferred method <strong>of</strong> control is the<br />
use <strong>of</strong> resistant cultivars, but this<br />
has been limited in the past by the<br />
lack <strong>of</strong> major genes for resistance.<br />
In bread wheat inheritance <strong>of</strong><br />
resistance to septoria nodorum<br />
blotch is frequently complex<br />
(Mullaney et al., 1982; Scharen <strong>and</strong><br />
Krupinsky, 1978).<br />
The complex <strong>and</strong> additive<br />
nature <strong>of</strong> septoria nodorum<br />
resistance in bread wheats has<br />
made the selection for resistance<br />
difficult. Relatively small<br />
improvements in the overall<br />
resistance can be difficult to<br />
identify, as they can be masked by<br />
strong environmental effects.<br />
Selection <strong>of</strong> resistant plants is<br />
complicated by the association <strong>of</strong><br />
resistance <strong>and</strong> both plant height<br />
<strong>and</strong> maturity (Rosielle <strong>and</strong> Brown,<br />
1980; Scott et al., 1982). The use <strong>of</strong> a<br />
molecular marker would help<br />
overcome these problems by<br />
avoiding interpretation <strong>of</strong><br />
phenotype under environmental<br />
influences (Allen, 1994).<br />
Furthermore, if the molecular<br />
marker is co-dominant, it will make<br />
it possible to identify an individual<br />
plant as homozygous for resistance<br />
without further progeny testing.<br />
This ability to identify homozygous<br />
71<br />
plants would be particularly useful<br />
in any program where extensive<br />
backcrossing would be required,<br />
such as the introgression <strong>of</strong> a<br />
resistance gene from wild wheats.<br />
The aim <strong>of</strong> this work was to<br />
identify a molecular marker that is<br />
linked to a single gene conferring<br />
resistance to septoria nodorum<br />
blotch identified in the accession<br />
RL5271 <strong>of</strong> the wild wheat Triticum<br />
tauschii.<br />
Materials <strong>and</strong> Methods<br />
A cross between the resistant<br />
accession RL5271 <strong>and</strong> the<br />
susceptible accession CPI110889<br />
was progeny tested. The F2 plants<br />
were progeny tested using F3
72<br />
Session 3B — N.E.A. Murphy, R. Loughman, R. Wilson, E.S. Lagudah, R. Appels, <strong>and</strong> M.G.K. Jones<br />
families to identify homozygous F2<br />
plants. The DNA <strong>of</strong> eight<br />
homozygous resistant F2 plants<br />
<strong>and</strong> 14 homozygous susceptible F2<br />
plants was selected for the analysis.<br />
Equal amounts <strong>of</strong> DNA from<br />
the homozygous resistant F2 plants<br />
were bulked together as was the<br />
DNA from the homozygous<br />
susceptible F2 plants. Each bulked<br />
sample <strong>and</strong> the DNA from each<br />
parent were divided into two<br />
aliquots. One aliquot was left<br />
untreated. The second aliquot was<br />
treated to enrich the proportion <strong>of</strong><br />
low copy sequence DNA by<br />
removing the repetitive DNA<br />
sequences using hydroxyapatite<br />
after selective renaturation. The<br />
technique followed Eastwood et al.<br />
(1994). The double-str<strong>and</strong>ed DNA<br />
was removed from the samples<br />
after a Cot value <strong>of</strong> 120 had been<br />
achieved (Smith <strong>and</strong> Flavell, 1975).<br />
The DNA was re-suspended in<br />
0.1xTE buffer <strong>and</strong> diluted to a final<br />
concentration <strong>of</strong> 5ng/ml <strong>of</strong> DNA.<br />
RAPD reactions were carried<br />
out on a Perkin-Elmer PE9600. The<br />
r<strong>and</strong>om primers were produced by<br />
Operon Technologies (Alameda,<br />
Ca.). The RAPD products were<br />
electrophoresed on polyacrylamide<br />
gels, <strong>and</strong> the gels were stained<br />
using ethidium bromide with gel<br />
images recorded digitally using a<br />
Bio-Rad Gel Doc 1000 system. For<br />
each primer nine reactions were<br />
carried out: a negative control,<br />
sterile distilled water, was used<br />
instead <strong>of</strong> template DNA, the next<br />
four reactions used the total<br />
genomic DNA as template, <strong>and</strong> the<br />
final four reactions used the nonrepetitive<br />
DNA as template.<br />
If a polymorphism was<br />
identified, the polymorphic b<strong>and</strong><br />
was stabbed with a sterile syringe<br />
needle. The needle was washed in<br />
sterile distilled water which was<br />
used as template for a further<br />
RAPD reaction using the same<br />
primer <strong>and</strong> reaction conditions that<br />
had generated the polymorphism.<br />
The amplified fragment was ligated<br />
into the plasmid pGEM-T. The<br />
plasmids were transformed into E.<br />
coli cells by heat shock. The<br />
following day colonies were<br />
screened using PCR primers, <strong>and</strong><br />
permanent cultures were<br />
established from insert-containing<br />
clones.<br />
The cloned polymorphic b<strong>and</strong><br />
was used as an RFLP probe to<br />
verify <strong>and</strong> determine the linkage <strong>of</strong><br />
the potential marker to the<br />
resistance gene. Total genomic<br />
DNA <strong>of</strong> the parental accessions was<br />
digested with a number <strong>of</strong><br />
restriction enzymes, singly <strong>and</strong> in<br />
combination, to find a restriction<br />
enzyme that would produce a<br />
polymorphism with the marker<br />
between the resistant <strong>and</strong> the<br />
susceptible parent. The marker<br />
b<strong>and</strong> was prepared using an alkali-<br />
SDS minprep, double digested with<br />
NcoI <strong>and</strong> SacI, separated on an<br />
agarose gel <strong>and</strong> purified using<br />
agarase. The purified b<strong>and</strong> was<br />
labeled using r<strong>and</strong>om nonamer<br />
primers with 32P as dCTP. The<br />
membranes were hybridized<br />
overnight, washed the following<br />
morning, <strong>and</strong> exposed to<br />
autoradiograph film for up to one<br />
week.<br />
When a suitable restriction<br />
enzyme had been selected, the<br />
DNA from the selected seven<br />
homozygous resistant F2 plants<br />
<strong>and</strong> seven homozygous susceptible<br />
F2 plants was digested,<br />
electrophoresed <strong>and</strong> blotted using<br />
an alkali capillary blot. The<br />
membranes were autoradiographed<br />
for up to two weeks.<br />
Results<br />
Sixty r<strong>and</strong>om primers were<br />
used to screen the DNA samples.<br />
Two polymorphic b<strong>and</strong>s were<br />
identified by comparing the<br />
b<strong>and</strong>ing pattern <strong>of</strong> the resistant<br />
parent <strong>and</strong> the resistant bulk with<br />
the susceptible parent <strong>and</strong> the<br />
susceptible bulk. The first was<br />
amplified using primer OPJ6 <strong>and</strong><br />
was approximately 770bp long<br />
(OPJ6770 ). The second polymorphic<br />
b<strong>and</strong> was amplified using primer<br />
OPJ18 <strong>and</strong> the fragment was<br />
approximately 350bp (OPJ18350 ).<br />
Both b<strong>and</strong>s were ligated into<br />
pGEM-T.<br />
For the first marker, OPJ6770 , no<br />
restriction enzyme nor combination<br />
<strong>of</strong> enzymes tested was able to<br />
produce a polymorphism between<br />
the resistant <strong>and</strong> the susceptible<br />
parent. Consequently the marker<br />
could not be tested for its linkage to<br />
resistance. The second marker,<br />
OPJ18350 , produced a<br />
polymorphism between the<br />
resistant <strong>and</strong> susceptible parent<br />
when the DNA was double<br />
restricted with HindIII <strong>and</strong><br />
followed by MseI. The<br />
polymorphism was present as an<br />
extra b<strong>and</strong> in the resistant parent.<br />
All <strong>of</strong> the F2 plants tested were<br />
correctly classified as resistant or<br />
susceptible when the marker was<br />
used.
Discussion<br />
A marker linked to a resistance<br />
gene to septoria nodorum blotch in<br />
the Triticum tauschii accession<br />
RL5271 has been found. The marker<br />
<strong>and</strong> the resistance gene appear to<br />
be completely linked; however, a<br />
greater number <strong>of</strong> plants need to be<br />
tested to develop a more accurate<br />
estimate <strong>of</strong> the linkage. The<br />
multiple b<strong>and</strong>s produced when the<br />
marker was used as an RFLP probe<br />
indicate that regions <strong>of</strong> repetitive<br />
DNA exist within the marker,<br />
which is not uncommon (Eastwood<br />
et al., 1994).<br />
The RFLP probe is currently<br />
being sequenced <strong>and</strong> primers<br />
designed that will allow the<br />
development <strong>of</strong> a sequence<br />
characterized amplified region<br />
(SCAR). This will allow a simpler<br />
<strong>and</strong> faster test to be developed. The<br />
simpler assay will be required if it<br />
is to be used extensively in a<br />
marker-assisted selection program.<br />
If the designed primers do amplify<br />
a polymorphism between the<br />
Identification <strong>of</strong> a Molecular Marker Linked to <strong>Septoria</strong> Nodorum Blotch Resistance in Triticum tauschii 73<br />
resistant <strong>and</strong> the susceptible plants,<br />
it may be one <strong>of</strong> several types. The<br />
primers may amplify a region that<br />
is present in the resistant plants but<br />
not in the susceptible plants. This<br />
marker will be dominant <strong>and</strong> will<br />
not allow for the identification <strong>of</strong><br />
heterozygotes; however, this will<br />
enable simple alternatives to<br />
running the PCR product on a gel<br />
to determine if the b<strong>and</strong> is present<br />
or not to be used (Dedryver et al.,<br />
1996). This will remove one <strong>of</strong> the<br />
major time constraints <strong>and</strong> limiting<br />
factors in the screening <strong>of</strong> large<br />
numbers <strong>of</strong> plants. Alternatively,<br />
the SCAR primers could amplify<br />
different size fragments between<br />
the resistant <strong>and</strong> the susceptible<br />
plants. This type <strong>of</strong> polymorphism<br />
will allow heterozygotes to be<br />
identified (co-dominant), as both<br />
alleles will be amplified. This in<br />
turn will allow the selection <strong>of</strong><br />
homozygotes for use in a<br />
backcrossing population, where the<br />
direct <strong>and</strong> accurate selection <strong>of</strong><br />
homozygotes will be <strong>of</strong> greatest<br />
benefit.<br />
References<br />
Allen, F.L. 1994. Usefulness <strong>of</strong> plant<br />
genome mapping to plant<br />
breeding. In Plant Genome<br />
Analysis (G.M. Gressh<strong>of</strong>f, ed.).<br />
CRC Press, London.<br />
Dedryver, F., M.F. Jubier, J.<br />
Thouvenin, <strong>and</strong> H. Goyeau. 1996.<br />
Molecular markers linked to the<br />
leaf rust resistance gene Lr24 in<br />
different wheats. Genome 39:830-<br />
835.<br />
Eastwood, R.F., E.S. Lagudah, <strong>and</strong> R.<br />
Appels. 1994. A directed search for<br />
DNA sequences tightly linked to<br />
cereal cyst nematode (CCN)<br />
resistance in Triticum tauschii.<br />
Genome 37:311-319.<br />
Mullaney, J., M. Martin, <strong>and</strong> A.L.<br />
Scharen. 1982. Generation mean<br />
analysis to identify <strong>and</strong> partition<br />
the components <strong>of</strong> genetic<br />
resistance to <strong>Septoria</strong> nodorum in<br />
wheat. Euphytica 31:539-545.<br />
Rosielle, A.A., <strong>and</strong> A.G.P. Brown.<br />
1980. Selection for resistance to<br />
<strong>Septoria</strong> nodorum in wheat.<br />
Euphytica 29:337-346.<br />
Scharen, A.L., <strong>and</strong> J.M. Krupinsky.<br />
1978. Detection <strong>and</strong> manipulation<br />
<strong>of</strong> resistance to <strong>Septoria</strong> nodorum in<br />
wheat. Phytopathology 68:245-248.<br />
Scott, P.R., P.W. Benedikz, <strong>and</strong> C.J.<br />
Cox. 1982. A genetic study <strong>of</strong> the<br />
relationship between height, time<br />
<strong>of</strong> ear emergence <strong>and</strong> resistance to<br />
<strong>Septoria</strong> nodorum in wheat. Plant<br />
Pathology 31:45-60.<br />
Smith, D.B., <strong>and</strong> R.B. Flavell. 1975.<br />
Characterisation <strong>of</strong> the wheat<br />
genome by renaturation kinetics.<br />
Chromosoma 50:223-242.
74<br />
Inheritance <strong>of</strong> <strong>Septoria</strong> Nodorum Blotch Resistance in a<br />
Triticum tauschii Accession Controlled by a Single Gene<br />
N.E.A. Murphy, 1 R. Loughman, 2 R. Wilson, 2 E.S. Lagudah, 3<br />
R. Appels, 3 <strong>and</strong> M.G.K. Jones1 1 WA State Agriculture Biotechnology Centre, Division <strong>of</strong> Science <strong>and</strong> Engineering, Murdoch University,<br />
Murdoch, Australia<br />
2 Agriculture Western Australia, Bentley Delivery Centre, Western Australia<br />
3 Plant Industries, CSIRO, Canberra, Australia<br />
Abstract<br />
A potentially useful source <strong>of</strong> resistance has been identified in an accession <strong>of</strong> Triticum tauschii. A cross was made<br />
between the resistant T. tauschii accession, RL5271, <strong>and</strong> a susceptible accession, CPI110889, to study the genetics <strong>of</strong><br />
resistance from this source. The parental accessions <strong>and</strong> the F1 <strong>and</strong> F3 progeny were screened in the glasshouse as seedlings.<br />
The resistant parent took significantly longer to develop symptoms, developed significantly fewer lesions, <strong>and</strong> expressed<br />
significantly lower levels <strong>of</strong> disease than the susceptible parent. The F1 mean response for disease severity indicated there was<br />
no complete dominance. The genotypic ratios generated by screening the F3 families were not significantly different from the<br />
genotypic ratio expected for a single gene. The effectiveness <strong>and</strong> simple genetic control <strong>of</strong> the resistance in the T. tauschii<br />
accession RL5271 may be useful as a resistance source in a bread wheat breeding program.<br />
<strong>Septoria</strong> nodorum blotch,<br />
caused by Phaeosphaeria nodorum (E.<br />
Müller) Hedjaroude (anamorph<br />
<strong>Stagonospora</strong> nodorum (Berk.)<br />
Castellani, <strong>and</strong> E.G. Germano), is<br />
the principal foliar disease <strong>of</strong> wheat<br />
(Triticum aestivum L.) in the cereal<br />
belt <strong>of</strong> Western Australia (Murray<br />
<strong>and</strong> Brown, 1987). The preferred<br />
method <strong>of</strong> control is the use <strong>of</strong><br />
resistant cultivars. In bread wheat<br />
inheritance <strong>of</strong> resistance to septoria<br />
nodorum blotch is reported to be<br />
complex, with an additive action <strong>of</strong><br />
the genes (Mullaney et al., 1982;<br />
Scharen <strong>and</strong> Krupinsky, 1978).<br />
Resistance is <strong>of</strong>ten linked to a<br />
number <strong>of</strong> traits such as height <strong>and</strong><br />
maturity (Rosielle <strong>and</strong> Brown, 1980;<br />
Scott et al., 1982), which makes<br />
breeding for resistance difficult.<br />
Promising levels <strong>of</strong> resistance to<br />
septoria nodorum blotch were<br />
found when a collection <strong>of</strong> T.<br />
tauschii accessions was investigated<br />
for resistance to septoria nodorum<br />
blotch. The ability to use these<br />
potential resistance sources in a<br />
wheat breeding program will be<br />
influenced by the number <strong>of</strong> genes<br />
controlling the resistance <strong>and</strong> its<br />
expression in a bread wheat<br />
background. The aim <strong>of</strong> this work<br />
was to investigate the differences<br />
between the parents in the<br />
components <strong>of</strong> resistance <strong>and</strong> to<br />
determine the number <strong>of</strong> genes<br />
controlling resistance to septoria<br />
nodorum blotch in the Triticum<br />
tauschii accession RL5271.<br />
Materials <strong>and</strong> Methods<br />
The resistant T. tauschii<br />
accession RL5271 was crossed to a<br />
susceptible accession CPI110889.<br />
The 17 F1 progenies were selfed,<br />
producing 300 F2s which were<br />
selfed to produce the F3 families.<br />
The F1 plants were screened for<br />
resistance to assess dominance, <strong>and</strong><br />
the F3 plants were screened to<br />
determine the genotypic ratio,<br />
which was used to estimate the<br />
number <strong>of</strong> genes controlling<br />
resistance.<br />
The T. tauschii seed was<br />
germinated on sterile filter papers<br />
in petri dishes <strong>and</strong> vernalized<br />
before being sown into pots in a<br />
glasshouse. In the F3 generation, a<br />
r<strong>and</strong>om selection <strong>of</strong> approximately<br />
200 F3 families was screened<br />
sequentially in two subpopulations.<br />
Each sub-population<br />
was grown on a single bench in the<br />
glasshouse, <strong>and</strong> each F3 family<br />
contained 10 plants.<br />
The plants were inoculated<br />
when the third leaf on the main<br />
stem had emerged. A single isolate<br />
<strong>of</strong> <strong>Stagonospora</strong> nodorum was used.<br />
The inoculum was produced using<br />
V8 Czapek-Dox agar (V8CDA) as a
modified method <strong>of</strong> Cooke <strong>and</strong><br />
Jones (1970). The plants were<br />
inoculated with 106 spores/mL,<br />
with a surfactant, until the point <strong>of</strong><br />
run-<strong>of</strong>f using an airless spraypainting<br />
gun while the pots were<br />
revolving on a turntable at 33 rpm.<br />
After inoculation the plants were<br />
placed in a humidity chamber for<br />
48 hours <strong>and</strong> then returned to the<br />
bench. The plants were rated using<br />
the youngest fully emerged leaf at<br />
the time <strong>of</strong> inoculation on the main<br />
tiller eight days after inoculation.<br />
The leaves were rated using disease<br />
score (DS), a 0 to 5 severity scale<br />
where 0 was an immune response<br />
<strong>and</strong> a score <strong>of</strong> 5 was given if the<br />
leaves were fully necrosed. The DS<br />
is based upon the number, size, <strong>and</strong><br />
type <strong>of</strong> lesions, as well as the area<br />
<strong>of</strong> the leaf covered by the lesions.<br />
The two parental accessions<br />
were assessed twice for two<br />
components <strong>of</strong> resistance, <strong>and</strong> the<br />
F1 plants were assessed once. The<br />
components were the incubation<br />
period (IP, the number <strong>of</strong> days from<br />
the time <strong>of</strong> inoculation till<br />
symptoms first appear) <strong>and</strong> the<br />
infection frequency (IF, the number<br />
<strong>of</strong> lesions per square centimeter <strong>of</strong><br />
leaf tissue at IP). In the first<br />
screening the IF had to be<br />
transformed using a natural<br />
logarithmic function, <strong>and</strong> during<br />
the second screening it was<br />
transformed using a square root<br />
function to remove the trends in the<br />
residuals.<br />
The F3 families were classified<br />
using the response <strong>of</strong> individual<br />
plants within an F3 family. During<br />
the rating it was evident from the<br />
parental accession’s response that a<br />
Inheritance <strong>of</strong> <strong>Septoria</strong> Nodorum Blotch Resistance in a Triticum tauschii Accession Controlled by a Single Gene 75<br />
score <strong>of</strong> less than 2.5 was resistant<br />
<strong>and</strong> a score <strong>of</strong> equal to or greater<br />
than 2.5 was susceptible. A F3<br />
family was classified as resistant if<br />
all its plants had a DS <strong>of</strong> less than<br />
2.5 <strong>and</strong> as susceptible if all the<br />
plants had a disease score greater<br />
than or equal to 2.5. Any family<br />
with plants with a DS <strong>of</strong> less than<br />
2.5 <strong>and</strong> greater than 2.5 were<br />
classified as segregating.<br />
Results<br />
In both tests the components <strong>of</strong><br />
resistance showed significant<br />
differences between the resistant<br />
<strong>and</strong> susceptible parents in their<br />
response to infection. The IP for the<br />
parents was not significantly<br />
different in the first screening but<br />
the resistant parent (RL5271) had a<br />
Table 1. The incubation period (IP), infection<br />
frequency (IF), <strong>and</strong> disease score (DS) <strong>of</strong> the<br />
F1 mean response during screening 1, <strong>and</strong> the<br />
parental accessions, RL5271 <strong>and</strong> CPI110889,<br />
during screenings 1 <strong>and</strong> 2.<br />
IF<br />
IP (lesions/<br />
(days) cm2 ) i DS<br />
Screening 1<br />
F1 8.5bii RL5271<br />
0.8a 1.3b<br />
(Resistant parent)<br />
CPI110889<br />
6.8a 0.8a 0.4a<br />
(Susceptible parent)<br />
Screening 2<br />
RL5271<br />
5.9a 2.0b 2.2c<br />
(Resistant parent)<br />
CPI110889<br />
5.5a 2.4a 0.9a<br />
(Susceptible parent) 3.5b 5.8b 3.3b<br />
i Back transformed values are presented for the<br />
IF in both screenings.<br />
ii Numbers from the same screening <strong>and</strong> for the<br />
same trait followed by different letters are<br />
significantly different from each other at p
76<br />
Session 3B — N.E.A. Murphy, R. Loughman, R. Wilson, E.S. Lagudah, R. Appels, <strong>and</strong> M.G.K. Jones<br />
different from the expected<br />
genotypic ratio for a single gene<br />
(X2 =0.81, p=0.67) (Table 2). When<br />
the genotypic ratios <strong>of</strong> the two subpopulations<br />
were compared using<br />
a Pearson’s chi-squared test for<br />
homogeneity, they were not<br />
significantly different (X2 =0.84,<br />
p=0.66). The combined ratio (45<br />
resistant F3 families, 97<br />
segregating, <strong>and</strong> 52 susceptible<br />
families) was not significantly<br />
different from the genotypic ratio<br />
expected for a single gene (X2 =0.50,<br />
p=0.78) (Table 2).<br />
Discussion<br />
Resistance to septoria nodorum<br />
blotch in the Triticum tauschii<br />
accession RL5271 is controlled by a<br />
single gene. From the F1 mean<br />
response there was no evidence <strong>of</strong><br />
complete dominance or<br />
recessiveness for resistance. The<br />
resistant parent took significantly<br />
longer to develop symptoms,<br />
developed significantly fewer<br />
infections <strong>and</strong> expressed<br />
significantly lower levels <strong>of</strong> disease<br />
than the susceptible parent. The<br />
variation in the resistance<br />
components between the two<br />
parents was consistent with<br />
previous studies reporting a<br />
resistance response. The IP has<br />
been shown to vary significantly<br />
among wheats with longer IP being<br />
associated with resistance<br />
(Wilkinson et al., 1990; Bruno <strong>and</strong><br />
Nelson, 1990). The IF was<br />
significantly lower for the resistant<br />
parent, which is consistent with<br />
previous work where a low IF has<br />
been associated with resistance<br />
(Wilkinson et al., 1990; Ma <strong>and</strong><br />
Hughes, 1993; Loughman et al.,<br />
1996). The inheritance <strong>of</strong> the IF has<br />
been found to be clearly dominant<br />
in the F1 (Ma <strong>and</strong> Hughes, 1993)<br />
which is consistent with this study.<br />
Resistance in Triticum tauschii<br />
accession RL5271 is the first singlegene<br />
resistance to septoria<br />
nodorum blotch identified in the D<br />
genome. Resistance to septoria<br />
nodorum blotch has been<br />
previously identified in another<br />
Triticum tauschii accession<br />
(=Aegilops squarrosa Tausch). It was<br />
found to be controlled by three<br />
genes, located on chromosomes 3D,<br />
5D, <strong>and</strong> 7D, with 3D being the most<br />
important <strong>of</strong> the three (Nicholson<br />
et al., 1993). The simplicity <strong>of</strong> the<br />
inheritance <strong>and</strong> strong expression<br />
<strong>of</strong> the resistance gene in T. tauschii<br />
warrants making an attempt at<br />
introgressing the resistance into a<br />
bread wheat background.<br />
Acknowledgment<br />
This work was funded by the<br />
Grains Research <strong>and</strong> Development<br />
Corporation <strong>of</strong> Australia.<br />
References<br />
Bruno, H.H., <strong>and</strong> L.R. Nelson. 1990.<br />
Partial resistance to septoria glume<br />
blotch analyzed in winter wheat<br />
seedlings. Crop Science 30:54-59.<br />
Cooke, B.M., <strong>and</strong> D.G. Jones. 1970.<br />
The effect <strong>of</strong> near-ultraviolet<br />
irradiation <strong>and</strong> agar medium on<br />
the sporulation <strong>of</strong> <strong>Septoria</strong> nodorum<br />
<strong>and</strong> <strong>Septoria</strong> tritici. Transactions <strong>of</strong><br />
the British Mycological Society<br />
54:221-226.<br />
Loughman, R., R.E. Wilson, <strong>and</strong> G.J.<br />
Thomas. 1996. Components <strong>of</strong><br />
resistance to Mycosphaerella<br />
graminicola <strong>and</strong> Phaeosphaeria<br />
nodorum in spring wheats.<br />
Euphytica 89:377-385.<br />
Ma, H., <strong>and</strong> G.R. Hughes. 1993.<br />
Resistance to septoria nodorum<br />
blotch in several Triticum species.<br />
Euphytica 70:151-157.<br />
Mullaney, J., M. Martin, <strong>and</strong> A.L.<br />
Scharen. 1982. Generation mean<br />
analysis to identify <strong>and</strong> partition<br />
the components <strong>of</strong> genetic<br />
resistance to <strong>Septoria</strong> nodorum in<br />
wheat. Euphytica 31:539-545.<br />
Murray, G.M., <strong>and</strong> J.F. Brown. 1987.<br />
The incidence <strong>and</strong> relative<br />
importance <strong>of</strong> wheat diseases in<br />
Australia. Australasian Plant<br />
Pathology 16:34-37.<br />
Nicholson, H., N. Rezanoor, <strong>and</strong> A.J.<br />
Worl<strong>and</strong>. 1993. Chromosomal<br />
location <strong>of</strong> resistance to <strong>Septoria</strong><br />
nodorum in a synthetic hexaploid<br />
wheat determined by the study <strong>of</strong><br />
chromosomal substitution lines in<br />
Chinese Spring wheat. Plant<br />
Breeding 110:177-184.<br />
Rosielle, A.A., <strong>and</strong> A.G.P. Brown.<br />
1980. Selection for resistance to<br />
<strong>Septoria</strong> nodorum in wheat.<br />
Euphytica 29:337-346.<br />
Scharen, A.L., <strong>and</strong> J.M. Krupinsky.<br />
1978. Detection <strong>and</strong> manipulation<br />
<strong>of</strong> resistance to <strong>Septoria</strong> nodorum in<br />
wheat. Phytopathology 68:245-248.<br />
Scott, P.R., P.W. Benedikz, <strong>and</strong> C.J.<br />
Cox. 1982. A genetic study <strong>of</strong> the<br />
relationship between height, time<br />
<strong>of</strong> ear emergence <strong>and</strong> resistance to<br />
<strong>Septoria</strong> nodorum in wheat. Plant<br />
Pathology 31:45-60.<br />
Wilkinson, C.A., J.P. Murphy, <strong>and</strong><br />
R.C. Rufty. 1990. Diallel analysis <strong>of</strong><br />
components <strong>of</strong> partial resistance to<br />
<strong>Septoria</strong> nodorum. Plant Disease<br />
74:47-50.
Session 4: Population Dynamics<br />
Population Genetics <strong>of</strong> Mycosphaerella graminicola <strong>and</strong><br />
Phaeosphaeria nodorum<br />
B.A. McDonald, 1 C.C. Mundt, 2 <strong>and</strong> J. Zhan2 1 Institute <strong>of</strong> Plant Sciences, Phytopathology Group, Zürich, Switzerl<strong>and</strong><br />
2 Department <strong>of</strong> Botany <strong>and</strong> Plant Pathology, Oregon State University, Corvallis, OR, USA<br />
Abstract<br />
Restriction fragment length polymorphisms (RFLPs) in the nuclear (nu) <strong>and</strong> mitochondrial (mt) genomes were used to<br />
determine the genetic structure <strong>of</strong> populations <strong>of</strong> Mycosphaerella graminicola <strong>and</strong> Phaeosphaeria nodorum from<br />
around the world. Both fungi have genetic structures consistent with a regular sexual cycle <strong>and</strong> a high degree <strong>of</strong> gene flow<br />
occurring on a global scale. Gene as well as genotype diversity in the nuDNA are high for both fungi. There was no<br />
evidence for widespread clones within field populations <strong>of</strong> either fungus. While both fungi had less diversity in the mtDNA,<br />
M. graminicola exhibited significantly less diversity for the mtDNA compared to P. nodorum. Mycosphaerella<br />
graminicola populations from Patzcuaro, Mexico, <strong>and</strong> Australia exhibited significantly lower gene diversity, suggesting<br />
that these populations originated from a limited number <strong>of</strong> founders. Collections <strong>of</strong> M. graminicola taken from the same<br />
field between 1990 <strong>and</strong> 1995 showed that genetic drift is negligible, suggesting that effective population sizes are very large.<br />
A replicated field experiment showed that selection can cause significant changes in genotype frequencies during the course<br />
<strong>of</strong> a growing season, <strong>and</strong> that the contributions <strong>of</strong> immigration <strong>and</strong> recombination to genetic diversity in field populations<br />
can change over the growing season.<br />
Ten years ago, we began<br />
developing DNA-based markers as<br />
tools to learn about the population<br />
genetics <strong>of</strong> the wheat leaf blotch<br />
pathogen Mycosphaerella<br />
graminicola. One year later, we<br />
began parallel studies using the<br />
same genetic tools for the wheat<br />
glume blotch pathogen<br />
Phaeosphaeria nodorum. We began<br />
with two elementary questions<br />
regarding the population genetics<br />
<strong>of</strong> both fungi. How much genetic<br />
diversity is present within<br />
populations? How is genetic<br />
diversity distributed within <strong>and</strong><br />
among populations? As our<br />
knowledge <strong>of</strong> the genetic structure<br />
<strong>of</strong> both pathogens deepened, we<br />
addressed more complex questions.<br />
What are the relative contributions<br />
<strong>of</strong> sexual <strong>and</strong> asexual reproduction<br />
to the genetic structure <strong>of</strong><br />
populations? How stable are<br />
populations over time? Does<br />
selection for specific pathogen<br />
genotypes occur on particular host<br />
genotypes? Is there evidence for<br />
host specialization in these<br />
pathosystems?<br />
To address the latter questions,<br />
we utilized increasingly<br />
sophisticated field experiments to<br />
differentiate among the various<br />
evolutionary forces acting on<br />
populations <strong>of</strong> these fungi. In this<br />
manuscript, I will briefly review<br />
our underst<strong>and</strong>ing <strong>of</strong> the<br />
population genetics <strong>of</strong> both fungi at<br />
this point in time. The majority <strong>of</strong><br />
this manuscript was distilled from a<br />
review chapter written for the Long<br />
Ashton Symposium on <strong>Septoria</strong> in<br />
<strong>Cereals</strong> held in 1997. Detailed data<br />
to support our interpretations are<br />
presented in that chapter<br />
(McDonald et al., 1999).<br />
Materials <strong>and</strong> Methods<br />
77<br />
DNA markers<br />
The RFLP markers utilized for<br />
these studies were developed in the<br />
same way for both fungi utilizing<br />
the methods described in<br />
McDonald <strong>and</strong> Martinez (1990b).<br />
Single-locus probes were used to<br />
measure gene diversity for<br />
individual RFLP loci <strong>and</strong> to<br />
measure population subdivision<br />
<strong>and</strong> genetic similarity among<br />
populations (McDonald <strong>and</strong><br />
Martinez, 1990a; Boeger et al., 1993;<br />
McDonald et al., 1994; Keller et al.,<br />
1997a,b). Probes that hybridized to<br />
repetitive elements were used for
78<br />
Session 4 — B.A. McDonald, C.C. Mundt, <strong>and</strong> J. Zhan<br />
DNA fingerprinting <strong>and</strong> to make<br />
measures <strong>of</strong> genotype diversity<br />
(McDonald <strong>and</strong> Martinez, 1991;<br />
Keller et al., 1997a,b). To measure<br />
variation in the mitochondrial (mt)<br />
genome, we purified mtDNA <strong>and</strong><br />
used it as a probe to hybridize to<br />
the entire digested mitochondrial<br />
genome for each individual.<br />
Sampling methods<br />
Hierarchical sampling methods<br />
now are commonly used in plant<br />
pathology (Kohli et al., 1995;<br />
McDonald et al., 1995; McDonald,<br />
1997). For many <strong>of</strong> the collections<br />
described in this manuscript, a<br />
st<strong>and</strong>ardized six-site hierarchy was<br />
used to sample field populations<br />
(McDonald et al., 1999). For other<br />
field collections, long transects were<br />
arranged in a field <strong>and</strong> leaves were<br />
sampled at 1-2-meter intervals<br />
along each transect. For some<br />
collections, leaves were collected at<br />
r<strong>and</strong>om in a field. All isolates in a<br />
collection were taken from a single<br />
field at a single time point for the<br />
majority <strong>of</strong> collections. I refer to<br />
these collections as field<br />
populations. Four collections <strong>of</strong><br />
isolates came from the same<br />
country or geographical region, but<br />
did not originate from a single field.<br />
I refer to these as regional<br />
populations to distinguish them<br />
from field populations.<br />
For P. nodorum, the Arkansas<br />
population was made up <strong>of</strong> isolates<br />
collected from infected seed from<br />
two different seed lots distributed<br />
in Arkansas. The crested<br />
wheatgrass population consisted <strong>of</strong><br />
isolations made from the weed<br />
crested wheatgrass over a two-year<br />
period in the state <strong>of</strong> North Dakota.<br />
The goal for each collection was to<br />
have a large enough field sample to<br />
be confident that allele frequencies<br />
for individual RFLP loci were<br />
representative <strong>of</strong> the entire field<br />
population. Only populations<br />
having at least 25 isolates were<br />
included in the analysis. A<br />
summary <strong>of</strong> the M. graminicola <strong>and</strong><br />
P. nodorum isolates in our collection<br />
was presented previously<br />
(McDonald et al., 1999).<br />
Data analysis<br />
Methods used to measure gene<br />
<strong>and</strong> genotype diversity, genetic<br />
similarity, population subdivision,<br />
<strong>and</strong> gametic disequilibrium have<br />
been published elsewhere<br />
(McDonald <strong>and</strong> Martinez, 1990a;<br />
Boeger et al., 1993; Chen et al., 1994;<br />
Chen <strong>and</strong> McDonald, 1996). The<br />
DNA fingerprint probes were<br />
validated for both fungi in previous<br />
experiments (McDonald <strong>and</strong><br />
Martinez, 1991; Keller et al., 1997b).<br />
Here we will pay special attention<br />
to the collection <strong>of</strong> M. graminicola<br />
isolates from Mexico.<br />
Results <strong>and</strong> Discussion<br />
Genetic diversity <strong>and</strong><br />
mating/reproduction systems<br />
Both gene <strong>and</strong> genotype<br />
diversity are high for both fungi.<br />
On average, 18 alleles were present<br />
for 11 RFLP loci across ~2000<br />
isolates <strong>of</strong> M. graminicola, <strong>and</strong> five<br />
alleles were present for seven loci<br />
across ~950 isolates <strong>of</strong> P. nodorum.<br />
In each population, between 3-4<br />
alleles were present in ~90% <strong>of</strong> the<br />
isolates (McDonald et al., 1999).<br />
Nei’s measure <strong>of</strong> gene diversity<br />
across all populations averaged 0.44<br />
across eight RFLP loci for M.<br />
graminicola <strong>and</strong> 0.51 across seven<br />
RFLP loci for P. nodorum. Though<br />
both fungi exhibited high gene<br />
diversities, M. graminicola had a<br />
significantly greater number <strong>of</strong><br />
alleles per locus then P. nodorum<br />
<strong>and</strong> P. nodorum had a more even<br />
distribution <strong>of</strong> allele frequencies.<br />
Comparisons <strong>of</strong> gene diversities<br />
across populations revealed some<br />
interesting patterns. The Israeli<br />
population had significantly higher<br />
gene diversity than other<br />
populations around the world<br />
(McDonald et al., 1999). We<br />
interpret this as evidence that the<br />
Middle East is a center <strong>of</strong> diversity<br />
for M. graminicola, <strong>and</strong> the likely<br />
center <strong>of</strong> origin for this fungus. The<br />
Mexican population exhibited<br />
lower gene diversity than other<br />
populations. It was made from<br />
leaves collected by Dr. Lucy<br />
Gilchrist at a <strong>CIMMYT</strong> disease<br />
nursery in Patzcuaro, a location<br />
remote from other wheat fields (L.<br />
Gilchrist, personal communication).<br />
The Mexican population also<br />
exhibited a lower genotype<br />
diversity than expected (McDonald<br />
et al., 1999), having a higher<br />
incidence <strong>of</strong> a few widespread<br />
clones than any other population<br />
surveyed. These findings are<br />
consistent with a small founding<br />
population that has not undergone<br />
regular sexual reproduction. We<br />
hypothesized that these isolates<br />
represent an historic inoculation <strong>of</strong><br />
the Patzcuaro site with a few<br />
isolates that were not sexually<br />
compatible. We hope to collect<br />
another M. graminicola field<br />
population from wheat-growing<br />
areas <strong>of</strong> Mexico to obtain a more<br />
representative sample <strong>of</strong> this<br />
fungus in that region.
The gene diversity in the P.<br />
nodorum population was lowest in<br />
the collections from crested<br />
wheatgrass <strong>and</strong> from Mexico<br />
(McDonald et al., 1999). These were<br />
the only two collections that were<br />
fixed for one allele at an RFLP<br />
locus. These populations also<br />
exhibited substantial differences in<br />
allele frequencies for some RFLP<br />
loci when compared to other<br />
populations (McDonald et al.,<br />
1999). It is possible that the lower<br />
gene diversity <strong>and</strong> different allele<br />
frequencies in the crested<br />
wheatgrass population resulted<br />
from selection due to host<br />
specialization. However, the<br />
finding that common alleles are<br />
shared between isolates taken from<br />
crested wheatgrass <strong>and</strong> wheat<br />
suggests that crested wheatgrass<br />
may serve as a reservoir <strong>of</strong><br />
inoculum for P. nodorum that infects<br />
wheat. The differences in the<br />
Patzcuaro population may reflect<br />
founder events <strong>and</strong> the geographic<br />
isolation <strong>of</strong> this location as<br />
described previously for M.<br />
graminicola.<br />
Genotype diversity. Genotype<br />
diversity in the nuclear genome<br />
was high for both fungi (McDonald<br />
et al., 1999). In the majority <strong>of</strong><br />
populations surveyed, each leaf<br />
was colonized by a unique fungal<br />
genotype. When isolates shared the<br />
same DNA fingerprint, they usually<br />
were sampled from the same leaf.<br />
The only evidence for a widespread<br />
clone in M. graminicola was in the<br />
Patzcuaro population. The only<br />
evidence for a widespread clone in<br />
P. nodorum was from our first<br />
collection in a Texas field<br />
(McDonald et al., 1994).<br />
Population Genetics <strong>of</strong> Mycosphaerella graminicola <strong>and</strong> Phaeosphaeria nodorum 79<br />
It appears that the typical<br />
population <strong>of</strong> both fungi exhibits a<br />
low degree <strong>of</strong> clonality at the field<br />
level. Clones were usually<br />
distributed across an area <strong>of</strong><br />
approximately 1 square meter <strong>and</strong><br />
did not become widespread. This<br />
finding is consistent with limited<br />
spread <strong>of</strong> the splash-dispersed<br />
conidia for both fungi.<br />
Our interpretation <strong>of</strong> the high<br />
degree <strong>of</strong> genotype diversity is that<br />
populations <strong>of</strong> both fungi undergo<br />
regular sexual cycles <strong>and</strong> that the<br />
primary inoculum for both fungi is<br />
likely to be ascospores (Chen <strong>and</strong><br />
McDonald, 1996; Keller et al.,<br />
1997b). We have used tests for the<br />
r<strong>and</strong>omness <strong>of</strong> associations among<br />
RFLP loci to indicate the frequency<br />
<strong>of</strong> sexual recombination for both<br />
fungi (Chen <strong>and</strong> McDonald, 1996).<br />
Associations were r<strong>and</strong>om for both<br />
fungi in the cases where sample<br />
sizes were adequate to make robust<br />
tests for disequilibrium, supporting<br />
the hypothesis <strong>of</strong> r<strong>and</strong>om mating.<br />
Though the sample size was<br />
smaller (N=160), the Israel<br />
population also showed the<br />
gametic equilibrium <strong>and</strong> high<br />
genotypic diversity typical <strong>of</strong> a<br />
r<strong>and</strong>om mating fungus (McDonald<br />
et al., 1999), suggesting that the<br />
teleomorph is present in the Middle<br />
East.<br />
After a field experiment<br />
provided evidence that the<br />
teleomorph was contributing<br />
significantly to infection during the<br />
growing season (Zhan et al., 1998),<br />
we sampled lower leaves in the<br />
crop canopy in Oregon <strong>and</strong> found<br />
the teleomorph existing as a<br />
significant fraction <strong>of</strong> all fruiting<br />
bodies on wheat leaves (C. Cowger,<br />
C.C. Mundt, <strong>and</strong> B.A. McDonald,<br />
unpublished).<br />
mtDNA diversity. For both fungi,<br />
the mtDNA exhibited less diversity<br />
then the nuDNA. The mtDNA<br />
genome ranges from 48-62 kb in size<br />
for both fungi. Among 385 isolates<br />
<strong>of</strong> P. nodorum from Switzerl<strong>and</strong>,<br />
Texas, <strong>and</strong> Oregon, we found only<br />
46 different mtDNA haplotypes<br />
compared to over 300 nuDNA<br />
haplotypes. Fifteen <strong>of</strong> the mtDNA<br />
haplotypes were shared among<br />
these field populations (S. Keller<br />
<strong>and</strong> B. A. McDonald, unpublished<br />
data).<br />
The mtDNA <strong>of</strong> M. graminicola<br />
exhibited even lower diversity. We<br />
routinely found only 2-3 mtDNA<br />
haplotypes in field populations <strong>of</strong><br />
M. graminicola (McDonald et al.,<br />
1999). We sometimes found only<br />
one M. graminicola mtDNA<br />
haplotype in a field (McDonald et<br />
al., 1999). We picked the three most<br />
common mtDNA haplotypes from<br />
around the world <strong>and</strong> digested the<br />
mtDNA with 10 different restriction<br />
enzymes that sampled ~5% <strong>of</strong> the<br />
entire 48 kb <strong>of</strong> mtDNA sequence in<br />
an attempt to detect cryptic<br />
variation. Types 1, 2, <strong>and</strong> 3 mtDNA<br />
haplotypes produced identical<br />
digestion patterns with all enzymes<br />
in all populations tested, suggesting<br />
that these mtDNA haplotypes are<br />
identical globally (B.A. McDonald<br />
<strong>and</strong> K. Hogan, unpublished). We<br />
have proposed that the limited<br />
mtDNA diversity is due to a<br />
selective sweep that occurred as M.<br />
graminicola populations became<br />
specialized to infect bread wheat<br />
(McDonald et al., 1999).
80<br />
Session 4 — B.A. McDonald, C.C. Mundt, <strong>and</strong> J. Zhan<br />
Genetic drift<br />
The high nuclear gene diversity<br />
found in most populations for both<br />
fungi suggests that population<br />
sizes are very large <strong>and</strong> the effects<br />
<strong>of</strong> genetic drift are small. In a<br />
previous experiment, we showed<br />
that the population genetic<br />
structure <strong>of</strong> an M. graminicola<br />
population did not change over a<br />
three-year period <strong>of</strong> 1990-1992<br />
(Chen et al., 1994). We have<br />
extended data from this population<br />
to 1995 <strong>and</strong> find the same result.<br />
The frequencies <strong>of</strong> neutral RFLP<br />
alleles did not change significantly<br />
over time (Table 1), <strong>and</strong> the degree<br />
<strong>of</strong> population subdivision across<br />
years is less than 1% (Table 2),<br />
demonstrating that population<br />
sizes are large enough to make<br />
genetic drift insignificant as an<br />
evolutionary force in populations<br />
<strong>of</strong> this fungus.<br />
Population subdivision<br />
<strong>and</strong> gene flow<br />
The distribution <strong>of</strong> genetic<br />
diversity across populations was<br />
consistent with a significant level<br />
<strong>of</strong> gene flow for both fungi.<br />
Common alleles at individual<br />
RFLP loci were shared among<br />
nearly all populations (McDonald<br />
et al., 1999), <strong>and</strong> allele frequencies<br />
<strong>of</strong>ten were quite similar though<br />
populations were separated by<br />
thous<strong>and</strong>s <strong>of</strong> kilometers<br />
(McDonald et al., 1999). We have<br />
interpreted this similarity as<br />
indicating a significant degree <strong>of</strong><br />
gene flow among populations <strong>of</strong><br />
both fungi on an international scale<br />
(McDonald et al., 1995; Keller et al.,<br />
1997). Global estimates <strong>of</strong> GST were<br />
0.06 <strong>and</strong> 0.07 for M. graminicola <strong>and</strong><br />
P. nodorum, respectively. These<br />
values <strong>of</strong> GST are consistent with<br />
movement <strong>of</strong> 11 <strong>and</strong> 7 individuals<br />
among populations globally every<br />
generation.<br />
Table 1. Allele frequencies for seven RFLP loci in Mycosphaerella graminicola collections from<br />
Corvallis, Oregon, in 1990, 1991, 1992, <strong>and</strong> 1995. Alleles present at a frequency <strong>of</strong> less than 0.05 in<br />
all four populations were pooled into one category (allele “p”). Sample sizes (N) used to<br />
calculate allele frequencies are shown for each locus. The chi-square values <strong>and</strong> their degrees<br />
<strong>of</strong> freedom (in parentheses) for measurement <strong>of</strong> population differentiation are also included.<br />
Populations<br />
RFLP Locus Allele 1990 1991 1992 1995 c2 pSTS192-PstIA 1 0.943 0.877 0.911 0.953 6.36(6)<br />
2 0.030 0.088 0.054 0.047<br />
p 0.027 0.035 0.036 0.000<br />
N 401 57 56 43<br />
pSTS192-PstIB 1 0.980 1.000 0.981 1.000 1.99(3)<br />
P 0.020 0.000 0.019 0.000<br />
N 406 56 56 44<br />
pSTS14-PstI 1 0.808 0.825 0.821 0.818 0.85(6)<br />
2 0.187 0.175 0.179 0.182<br />
p 0.005 0.000 0.000 0.000<br />
N 407 57 56 44<br />
pSTS2-PstI 1 0.648 0.667 0.571 0.818 8.87(9)<br />
2 0.085 0.056 0.125 0.023<br />
3 0.203 0.204 0.214 0.136<br />
p 0.065 0.074 0.089 0.023<br />
N 400 54 56 44<br />
pSTL10-PstI 1 0.680 0.649 0.518 0.643 14.51(9)<br />
2 0.051 0.053 0.107 0.000<br />
3 0.220 0.281 0.357 0.286<br />
p 0.049 0.018 0.018 0.071<br />
N 409 57 56 43<br />
pSTL53-PstI 1 0.475 0.561 0.536 0.465 26.86(18)<br />
2 0.119 0.070 0.107 0.023<br />
3 0.161 0.158 0.232 0.070<br />
5 0.042 0.053 0.000 0.047<br />
6 0.119 0.140 0.107 0.209<br />
7 0.027 0.000 0.000 0.047<br />
p 0.057 0.018 0.018 0.140<br />
N 404 57 56 43<br />
pSTS197-PstI 1 0.632 0.649 0.589 0.591<br />
2 0.227 0.175 0.286 0.364 9.65(9)<br />
3 0.108 0.158 0.107 0.045<br />
p 0.033 0.018 0.018 0.000<br />
N 259 57 56 44<br />
Table 2. Nei’s measures <strong>of</strong> gene diversity <strong>and</strong> population subdivision for 7 RFLP loci in<br />
Mycosphaerella graminicola populations collected from Corvallis, Oregon, in 1990, 1991, 1992,<br />
<strong>and</strong> 1995.<br />
Hi a<br />
The present lack <strong>of</strong> subdivision<br />
in the nuclear genome probably<br />
reflects historic movement <strong>of</strong> both<br />
fungi around the world. It is<br />
possible that gene flow is not a<br />
RFLP Loci 1990 1991 1992 1995 HTb HSc Dstd Gste pSTS192A-PstI 0.110 0.222 0.166 0.091 0.126 0.125 0.001 0.008<br />
pSTS192B-PstI 0.039 0.000 0.104 0.000 0.039 0.039 0.000 0.007<br />
pSTS14-PstI 0.312 0.289 0.294 0.298 0.307 0.307 0.000 0.000<br />
pSTS2-PstI 0.531 0.508 0.610 0.323 0.524 0.520 0.004 0.007<br />
pSTL10-PstI 0.486 0.497 0.592 0.526 0.506 0.501 0.005 0.010<br />
pSTL53-PstI 0.716 0.633 0.636 0.722 0.704 0.700 0.004 0.006<br />
pSTS197-PstI 0.537 0.523 0.560 0.517 0.540 0.536 0.004 0.007<br />
Pooled 0.390 0.382 0.423 0.354 0.392 0.390 0.003 0.006<br />
Notes:a= gene diversity for each population; b= combined gene diversity across all collections; c= gene<br />
diversity within populations; d= gene diversity among populations; e= population differentiation
significant evolutionary force at<br />
present. However, we consider it<br />
more likely that some gene flow<br />
continues as a result <strong>of</strong> the global<br />
commerce in grain. The obvious<br />
mechanism for gene flow on a<br />
regional basis is air-dispersed<br />
ascospores. In the case <strong>of</strong> P.<br />
nodorum, the most likely<br />
mechanism for intercontinental<br />
dispersal is infected seed (King et<br />
al., 1983). Since it has been shown<br />
that M. graminicola can infect seed<br />
(Brokenshire, 1975) we consider it<br />
likely that this also is the<br />
mechanism for long distance gene<br />
flow in M. graminicola. Whatever<br />
the mechanism, the high degree <strong>of</strong><br />
similarity in populations around<br />
the world for both fungi suggests<br />
that they have been transported<br />
around the world by humans.<br />
Evidence for selection<br />
We recently completed an<br />
experiment to measure competition<br />
among 10 genotypes <strong>of</strong> M.<br />
graminicola in a field setting. The 10<br />
isolates were inoculated onto three<br />
host treatments consisting <strong>of</strong> a<br />
moderately resistant wheat variety<br />
(Madsen), a susceptible wheat<br />
variety (Stephens) <strong>and</strong> a 1:1<br />
mixture <strong>of</strong> these cultivars. Our<br />
most important finding in this<br />
experiment was that intense<br />
competition appeared to occur<br />
among the different genotypes.<br />
Significant changes in the<br />
frequencies <strong>of</strong> specific pathogen<br />
genotypes occurred over the season<br />
(McDonald et al., 1999). Some<br />
isolates showed evidence for<br />
adaptation to particular hosts. The<br />
results from this experiment<br />
provided our first direct evidence<br />
Population Genetics <strong>of</strong> Mycosphaerella graminicola <strong>and</strong> Phaeosphaeria nodorum 81<br />
that selection operates on specific<br />
M. graminicola pathogen genotypes<br />
in a field setting.<br />
We have not yet conducted<br />
similar replicated field experiments<br />
to measure selection in P. nodorum.<br />
But we have indirect evidence that<br />
selection does not result in<br />
widespread clones that are adapted<br />
to specific host genotypes. In an<br />
experiment conducted in<br />
Switzerl<strong>and</strong> in collaboration with<br />
Martin Wolfe’s group, we sampled<br />
50 isolates <strong>of</strong> P. nodorum from each<br />
<strong>of</strong> nine wheat fields near Zurich.<br />
Three different wheat varieties were<br />
represented three times each among<br />
the nine wheat fields. Though fields<br />
planted to the same variety used the<br />
same source <strong>of</strong> seed, no genotypes<br />
were shared among field<br />
populations. Only six pairs <strong>of</strong> clones<br />
were found among the 432 isolates<br />
that were assayed. Isolates with the<br />
same DNA fingerprints always<br />
came from the same site within a<br />
field (Keller et al., 1997b).<br />
Taken together, all <strong>of</strong> our<br />
experiments suggest that nuclear<br />
genotypes do not persist through<br />
time for either fungus. Instead, the<br />
genes are the units <strong>of</strong> selection that<br />
are carried forward across<br />
generations. Selection operates on<br />
the population instead <strong>of</strong> the<br />
individual. In order to gain a<br />
representative spectrum <strong>of</strong> the<br />
diversity for virulence in natural<br />
populations, plant breeders should<br />
include the widest possible<br />
diversity <strong>of</strong> strains when screening<br />
germplasm for resistance to these<br />
fungi. Similarly, chemical<br />
companies should include at least<br />
several hundred strains in their<br />
screens for resistance to fungicides.<br />
Conclusions<br />
Given our present data, we have<br />
drawn the following conclusions<br />
regarding the evolutionary forces<br />
that affect the population genetics<br />
<strong>of</strong> M. graminicola <strong>and</strong> P. nodorum:<br />
• For both fungi, the mating system<br />
includes both sexual <strong>and</strong> asexual<br />
reproduction. Asexual<br />
reproduction may have an<br />
important impact over an area <strong>of</strong><br />
a few square meters, but the<br />
sexual reproduction has much<br />
greater consequences for the<br />
evolutionary biology <strong>of</strong> both<br />
fungi. Genotypes are ephemeral<br />
but genes persist in populations<br />
through time.<br />
• Population sizes are large enough<br />
to make genetic drift negligible<br />
for both fungi. Large population<br />
sizes also ensure that ample<br />
mutations are present in every<br />
population to allow for a rapid<br />
response to selection, e.g.<br />
mutations from avirulence to<br />
virulence for major resistance<br />
genes. The population from<br />
Patzcuaro, Mexico, exhibits a<br />
genetic structure consistent with<br />
a founder effect.<br />
• Gene flow is sufficient to unite<br />
large geographical areas into a<br />
single genetic population. If gene<br />
flow is ongoing, then breeders<br />
should continue to test their<br />
resistant lines over the widest<br />
possible geographical area. If<br />
gene flow is episodic, continued<br />
vigilance is needed to limit the<br />
spread <strong>of</strong> new virulence genes<br />
<strong>and</strong> fungicide resistance genes.<br />
Quarantines in areas with low<br />
gene diversity, such as Australia,<br />
should be enforced to limit the<br />
evolutionary potential <strong>of</strong> these<br />
populations. If <strong>CIMMYT</strong><br />
continues to use Patzcuaro as a<br />
field site to screen for resistance
82<br />
Session 4 — B.A. McDonald, C.C. Mundt, <strong>and</strong> J. Zhan<br />
to M. graminicola <strong>and</strong> P. nodorum,<br />
it may want to consider<br />
introducing more diverse fungal<br />
populations from other parts <strong>of</strong><br />
Mexico into this disease nursery.<br />
• Selection appears to operate on<br />
both nuclear <strong>and</strong> mitochondrial<br />
genomes in M. graminicola.<br />
Though selection may increase<br />
the frequency <strong>of</strong> particular<br />
genotypes over the course <strong>of</strong> a<br />
growing season, it appears that<br />
particular genotypes are unlikely<br />
to reach high frequencies within<br />
field populations because <strong>of</strong> the<br />
limited dispersal potential for<br />
conidia. But the genes in the most<br />
fit individuals will persist <strong>and</strong> be<br />
recombined to create novel<br />
genotypes in the next growing<br />
season. Over the course <strong>of</strong> many<br />
growing seasons, selection will<br />
change the frequency <strong>of</strong> genes<br />
that affect adaptation to the<br />
wheat host, but new genotypes<br />
will appear each season. It is too<br />
early to say if selection operates<br />
the same way in P. nodorum.<br />
In summary, the population<br />
genetics <strong>of</strong> P. nodorum <strong>and</strong> M.<br />
graminicola are very similar. This<br />
probably reflects the similarity in<br />
their life histories. Both fungi<br />
produce airborne sexual ascospores<br />
<strong>and</strong> splash-dispersed asexual<br />
spores. They both infect aboveground<br />
plant parts on the same<br />
host <strong>and</strong> they both infect seeds that<br />
can be transported globally as part<br />
<strong>of</strong> the world commerce in wheat.<br />
Use <strong>of</strong> multi-allelic, neutral genetic<br />
markers combined with<br />
hierarchical sampling has allowed<br />
us to achieve a much greater<br />
underst<strong>and</strong>ing <strong>of</strong> the population<br />
biology <strong>of</strong> both fungi.<br />
Acknowledgments<br />
BAM gratefully acknowledges<br />
the many collectors <strong>and</strong><br />
collaborators around the world who<br />
responded to his request for<br />
infected leaf material. Funding for<br />
this project came from the USDA<br />
National Research Initiative<br />
Competitive Grants Program (Grant<br />
# 93-37303-9039), the National<br />
Science Foundation (Grant # DEB-<br />
9306377), the Texas Agricultural<br />
Experiment Station (Hatch project<br />
#6928), <strong>and</strong> the Swiss National<br />
Fund (Grant # 5002-38966).<br />
References<br />
Boeger, J.M., Chen, R.S., <strong>and</strong><br />
McDonald, B.A. 1993. Gene flow<br />
between geographic populations <strong>of</strong><br />
Mycosphaerella graminicola<br />
(anamorph <strong>Septoria</strong> tritici) detected<br />
with RFLP markers.<br />
Phytopathology 83:1148-1154.<br />
Brokenshire, T. 1975. Wheat seed<br />
infection by <strong>Septoria</strong> tritici.<br />
Transactions <strong>of</strong> the British<br />
Mycological Society 64:331-335.<br />
Chen, R.S., <strong>and</strong> McDonald, B.A. 1996.<br />
Sexual reproduction plays a major<br />
role in the genetic structure <strong>of</strong><br />
populations <strong>of</strong> the fungus<br />
Mycosphaerella graminicola. Genetics<br />
142:1119-1127.<br />
Chen, R.S., Boeger, J.M., <strong>and</strong><br />
McDonald, B.A. 1994. Genetic<br />
stability in a population <strong>of</strong> a plant<br />
pathogenic fungus over time.<br />
Molecular Ecology 3:209-218.<br />
Keller, S.M., McDermott, J.M.,<br />
Pettway, R.E., Wolfe, M.S., <strong>and</strong><br />
McDonald, B.A. 1997a. Gene flow<br />
<strong>and</strong> sexual reproduction in the<br />
wheat glume blotch pathogen<br />
Phaeosphaeria nodorum (anamorph<br />
<strong>Stagonospora</strong> nodorum).<br />
Phytopathology 87:353-358.<br />
Keller, S.M., Wolfe, M.S., McDermott,<br />
J.M., <strong>and</strong> McDonald, B.A. 1997b.<br />
High genetic similarity among<br />
populations <strong>of</strong> Phaeosphaeria<br />
nodorum across wheat cultivars <strong>and</strong><br />
regions in Switzerl<strong>and</strong>.<br />
Phytopathology 87:1134-1139.<br />
King, J.E., Cook, R.J., <strong>and</strong> Melville, S.C.<br />
1983. A review <strong>of</strong> <strong>Septoria</strong> diseases<br />
<strong>of</strong> wheat <strong>and</strong> barley. Annals<br />
Applied Biology 103:345-373.<br />
Kohli, Y., Brunner, L.J., Yoell, H.,<br />
Milgroom, M.G., Anderson, J.B.,<br />
Morrall, R.A.A., <strong>and</strong> Kohn, L.M.<br />
1995. Clonal dispersal <strong>and</strong> spatial<br />
mixing in populations <strong>of</strong> the plant<br />
pathogenic fungus, Sclerotinia<br />
sclerotiorum. Molecular Ecology<br />
4:69-77.<br />
McDonald, B.A. 1997. The population<br />
genetics <strong>of</strong> fungi: tools <strong>and</strong><br />
techniques. Phytopathology 87:448-<br />
453.<br />
McDonald, B.A., <strong>and</strong> Martinez, J.P.<br />
1990a. DNA restriction fragment<br />
length polymorphisms among<br />
Mycosphaerella graminicola<br />
(anamorph <strong>Septoria</strong> tritici) isolates<br />
collected from a single wheat field.<br />
Phytopathology 80:1368-1373.<br />
McDonald, B.A., <strong>and</strong> Martinez, J.P.<br />
1990b. Restriction fragment length<br />
polymorphisms in <strong>Septoria</strong> tritici<br />
occur at a high frequency. Current<br />
Genetics 17:133-138.<br />
McDonald, B.A., <strong>and</strong> Martinez, J.P.<br />
1991. DNA fingerprinting <strong>of</strong> the<br />
plant pathogenic fungus<br />
Mycosphaerella graminicola<br />
(anamorph <strong>Septoria</strong> tritici).<br />
Experimental Mycology 15:146-158.<br />
McDonald, B.A., Miles, J., Nelson, L.R.,<br />
<strong>and</strong> Pettway, R.E. 1994. Genetic<br />
variability in nuclear DNA in field<br />
populations <strong>of</strong> <strong>Stagonospora</strong><br />
nodorum. Phytopathology 84:250-<br />
255.<br />
McDonald, B.A., Pettway, R.E., Chen,<br />
R.S., Boeger, J.M., <strong>and</strong> Martinez, J.P.<br />
1995. The population genetics <strong>of</strong><br />
<strong>Septoria</strong> tritici (teleomorph<br />
Mycosphaerella graminicola).<br />
Canadian Journal <strong>of</strong> Botany 73<br />
(supplement), S292-S301.<br />
McDonald, B.A., Zhan J., Yarden O.,<br />
Hogan K., Garton J., <strong>and</strong> Pettway<br />
R.E. 1999. The population genetics<br />
<strong>of</strong> Mycosphaerella graminicola <strong>and</strong><br />
Phaeosphaeria nodorum. pp. 44-69 In:<br />
<strong>Septoria</strong> in <strong>Cereals</strong>: a Study <strong>of</strong><br />
Pathosystems. Lucas, J.A., Bowyer,<br />
P., Anderson, H.M., eds. CABI<br />
Publishing, Wallingford, UK.<br />
Zhan, J., Mundt, C.C., <strong>and</strong> McDonald,<br />
B.A. 1998. Measuring immigration<br />
<strong>and</strong> sexual reproduction in field<br />
populations <strong>of</strong> Mycosphaerella<br />
graminicola. Phytopathology<br />
88:1330-1337.
Characterization <strong>of</strong> Less Aggressive <strong>Stagonospora</strong><br />
nodorum Isolates from Wheat<br />
E. Arseniuk, 1 H.S. Tsang, 2 J.M. Krupinsky, 3 <strong>and</strong> P.P. Ueng 4<br />
1 Plant Breeding <strong>and</strong> Acclimatization Institute, Radzików, Pol<strong>and</strong><br />
2 Department <strong>of</strong> Biochemistry, University <strong>of</strong> Maryl<strong>and</strong>, MD, USA<br />
3 USDA-ARS, Northern Great Plains Research Lab, M<strong>and</strong>an, ND, USA<br />
4 USDA-ARS, Molecular Plant Pathology Lab, Beltsville, MD, USA<br />
Abstract<br />
Two less aggressive <strong>Stagonospora</strong> nodorum isolates, 9074 <strong>and</strong> 9076, were characterized by inoculation tests, mating<br />
ability, <strong>and</strong> molecular tools. As expected, they caused mild symptom severity on wheat, triticale, <strong>and</strong> rye. They crossed<br />
with other S. nodorum isolates <strong>and</strong> had “+” mating type determinant(s). These two isolates also had the same restriction<br />
patterns <strong>and</strong> sequences in the internal transcribed spacer (ITS) region <strong>of</strong> rDNA similar to other S. nodorum isolates.<br />
With AFLP analysis, it was concluded that these less aggressive S. nodorum isolates were closely related <strong>and</strong> genetically<br />
different from highly aggressive ones.<br />
<strong>Stagonospora</strong> nodorum (Berk.)<br />
Castellani & E.G. Germano<br />
(teleomorph Phaeosphaeria nodorum<br />
(E. Müller) Hedjaroude) causes<br />
septoria nodorum blotch on wheat,<br />
barley, <strong>and</strong> other small grains<br />
(Sprague, 1950). Genetic variation<br />
<strong>of</strong> S. nodorum isolates has been<br />
reported (Allingham <strong>and</strong> Jackson,<br />
1981; Krupinsky, 1997a,b; Scharen et<br />
al., 1985). Underst<strong>and</strong>ing this<br />
genetic variation would help in the<br />
selection <strong>and</strong>/or development <strong>of</strong><br />
resistant germplasm. This study<br />
was undertaken to determine<br />
whether less aggressive isolates<br />
could be characterized <strong>and</strong> how<br />
they relate to highly aggressive<br />
isolates.<br />
Materials <strong>and</strong> Methods<br />
Inoculation tests<br />
<strong>Stagonospora</strong> nodorum isolates<br />
were obtained from lesions on<br />
infected wheat (Sn26-1, 9074, <strong>and</strong><br />
9076) <strong>and</strong> rye (Sn48-1) in Pol<strong>and</strong><br />
<strong>and</strong> USA. Isolates 9074 <strong>and</strong> 9076<br />
were collected from Richl<strong>and</strong> <strong>and</strong><br />
Gallatin counties in Montana,<br />
respectively. These two isolates<br />
were associated with mild symptom<br />
severity on detached wheat leaves<br />
<strong>and</strong> seedlings, <strong>and</strong> considered to be<br />
less aggressive isolates (Krupinsky,<br />
1997a,b). Plants were inoculated by<br />
spraying a 3-ml pycnidiospore<br />
suspension (3 x 10 6 spores/ml) per<br />
pot onto 10-day-old seedlings.<br />
Three wheat cultivars (Alba, Begra,<br />
<strong>and</strong> Liwilla), two triticales (Bogo<br />
<strong>and</strong> Pinokio) <strong>and</strong> one rye (Zduno)<br />
were used as host plants. The<br />
plants were maintained in a high<br />
humidity environment for 72 h<br />
after spraying. Fourteen days after<br />
inoculation, 10 primary leaves were<br />
assessed for percentage necrosis<br />
<strong>and</strong> scored using a 1 (resistant) to 9<br />
(susceptible) scale. The experiments<br />
were repeated three times.<br />
Determination <strong>of</strong> mating type<br />
Isolates 9074 <strong>and</strong> 9076 were<br />
mated in vitro with reference “+”<br />
<strong>and</strong> “–“ strains <strong>of</strong> the pathogen<br />
with a procedure developed in the<br />
lab (Arseniuk et al., 1997).<br />
DNA isolation <strong>and</strong><br />
characterization <strong>of</strong> ITS <strong>of</strong><br />
nuclear rDNA<br />
Growth <strong>of</strong> fungal culture in a<br />
liquid medium, isolation <strong>and</strong><br />
purification <strong>of</strong> genomic DNA<br />
mainly followed the procedures<br />
83<br />
described earlier (Ueng et al., 1992).<br />
The internal transcribed spacer<br />
(ITS) region <strong>of</strong> the nuclear rDNA<br />
repeat units was amplified by PCR<br />
following the protocol reported<br />
earlier (Ueng et al., 1998; White et<br />
al., 1990). Two highly aggressive S.<br />
nodorum isolates, 8408 <strong>and</strong> 9506-2<br />
(Krupinsky, 1997a; Krupinsky,<br />
unpublished data) were compared<br />
with the two less aggressive ones,<br />
9074 <strong>and</strong> 9076. The PCR products <strong>of</strong><br />
the ITS region were restricted with<br />
endonuclease enzymes, DraI,<br />
HaeIII, HpaII, <strong>and</strong> PvuII. The<br />
sequences <strong>of</strong> PCR-amplified ITS<br />
regions were determined using a<br />
DNA sequencer (Applied<br />
Biosystem 373A, Perkin Elmer,<br />
Foster City, CA).<br />
AFLP analysis<br />
The amplified restriction<br />
fragment polymorphism (AFLP)<br />
technique was used to explore<br />
DNA polymorphisms in four S.<br />
nodorum isolates with different<br />
degrees <strong>of</strong> aggressiveness on<br />
wheat. The AFLP Analysis<br />
System I (Life Technologies,<br />
Gaithersburg, MD) kit was used<br />
following the protocol provided by
84<br />
Session 4 — E. Arseniuk, H.S. Tsang, J.M. Krupinsky, <strong>and</strong> P.P. Ueng<br />
the manufacturer. Cluster analysis<br />
using the unweighted pair group<br />
method with an arithmetic average<br />
(UPGMA) algorithm was<br />
performed to produce a phenogram<br />
based on the b<strong>and</strong>ing patterns<br />
produced by AFLP (Rohlf, 1990).<br />
Results <strong>and</strong> Discussion<br />
In the greenhouse seedling<br />
inoculations, isolates 9074 <strong>and</strong> 9076<br />
generally showed lower symptom<br />
severity on wheats, triticales, <strong>and</strong><br />
rye (Table 1). The results agreed<br />
with the previous work on<br />
detached wheat leaves <strong>and</strong><br />
seedlings (Krupinsky, 1997a,b). It<br />
was also shown that Polish isolate<br />
Sn48-1, originally isolated from rye,<br />
caused severe symptom severity on<br />
rye (Table 1) <strong>and</strong> less symptom<br />
severity on triticale (a cross<br />
between wheat <strong>and</strong> rye).<br />
After the isolates mated,<br />
pseudothecia with mature<br />
ascospores were formed after 50-80<br />
days <strong>of</strong> incubation. Isolates 9074<br />
<strong>and</strong> 9076 were both shown to be<br />
“+” mating types. The ITS regions<br />
<strong>of</strong> rDNA in these two isolates had<br />
the same endonuclease restriction<br />
patterns <strong>and</strong> DNA sequences as the<br />
two highly aggressive isolates 8408<br />
<strong>and</strong> 9506-2, <strong>and</strong> other wheatbiotype<br />
S. nodorum isolates (Beck<br />
<strong>and</strong> Ligon, 1995; Ueng et al., 1998).<br />
This would confirm that these two<br />
less aggressive isolates are wheatbiotype<br />
S. nodorum isolates. Of a<br />
total 390 b<strong>and</strong>s generated by 48<br />
AFLP reaction combinations, only<br />
78 b<strong>and</strong>s were shared by all four<br />
isolates in this study. The genetic<br />
diversity was high in highly<br />
aggressive isolates, <strong>and</strong> between<br />
highly <strong>and</strong> less aggressive ones<br />
(Figure 1). Cluster analysis showed<br />
great similarity between the two<br />
less aggressive isolates. The<br />
presence <strong>of</strong> differential AFLP<br />
fragments in the highly aggressive<br />
isolates may be related to<br />
aggressiveness <strong>and</strong> may be useful<br />
to identify virulent elements in the<br />
future.<br />
Acknowledgments<br />
The authors thank Yan Zhao <strong>of</strong><br />
USDA-ARS, MPPL, Beltsville, MD,<br />
for his technical support <strong>and</strong><br />
Weidong Chen <strong>of</strong> the University <strong>of</strong><br />
Illinois for his comments <strong>and</strong><br />
suggestions.<br />
References<br />
Allingham, E.A., <strong>and</strong> L.F. Jackson.<br />
1981. Variation in pathogenicity,<br />
virulence, <strong>and</strong> aggressiveness <strong>of</strong><br />
<strong>Septoria</strong> nodorum in Florida.<br />
Phytopathology 71:1080-1085.<br />
Arseniuk, E., P.C. Czembor, <strong>and</strong> B.M.<br />
Cunfer. 1997. Segregation <strong>and</strong><br />
recombination <strong>of</strong> PCR-based<br />
markers in progenies <strong>of</strong> in vitro<br />
mated isolates <strong>of</strong> Phaeosphaeria<br />
nodorum (Müller) Hedjaroude.<br />
Phytopathology 87 (Supplement)<br />
S5.<br />
Beck, J.J., <strong>and</strong> J.M. Ligon. 1995.<br />
Polymerase chain reaction assays<br />
for the detection <strong>of</strong> <strong>Stagonospora</strong><br />
nodorum <strong>and</strong> <strong>Septoria</strong> tritici in<br />
wheat. Phytopathology 85:319-324.<br />
Table 1. Comparison <strong>of</strong> <strong>Stagonospora</strong> nodorum isolates on cereal seedlings.<br />
Wheat Triticale Rye<br />
Isolates Alba Begra Liwilla Bogo Pinokio Zduno<br />
Sn26-1 4.1+1.63 6.2+1.70 1.8+0.70b 4.3+1.24 3.1+1.55 3.0+1.97f<br />
Sn48-1 2.2+1.00 4.3+1.46 2.1+0.96b 6.6+0.94 6.4+1.72 8.3+1.03<br />
9074 1.5+0.57 3.1+1.92a 1.0+0.56c 1.7+0.99d 1.8+0.73e 2.2+2.02f, g<br />
9076 2.1+0.52 2.9+0.92a 1.0+0.53c 1.4+0.57d 1.8+0.71e 2.0+1.80g<br />
The rating scale for fungal infection is from 1 (resistant) to 9 (susceptible).With T-test at the confidence<br />
level <strong>of</strong> 0.05, the pairs with the same letters are not significantly different.<br />
Krupinsky, J.M. 1997. Aggressiveness<br />
<strong>of</strong> <strong>Stagonospora</strong> nodorum isolates<br />
obtained from wheat in the<br />
northern great plains. Plant Dis.<br />
81:1027-1031.<br />
Krupinsky, J.M. 1997. Aggressiveness<br />
<strong>of</strong> <strong>Stagonospora</strong> nodorum isolates<br />
from perennial grasses on wheat.<br />
Plant Dis. 81:1032-1036.<br />
Rohlf, F.J. 1990. Numerical Taxonomy<br />
<strong>and</strong> Multivariate Analysis System.<br />
Version 1.6. Exeter, Setauket, NY.<br />
Scharen, A.L., Z. Eyal, M.D. Huffman,<br />
<strong>and</strong> J.M. Prescott. 1985. The<br />
distribution <strong>and</strong> frequency <strong>of</strong><br />
virulence genes in geographically<br />
separated populations <strong>of</strong><br />
Leptosphaeria nodorum.<br />
Phytopathology 75:1463-168.<br />
Sprague, R. 1950. <strong>Diseases</strong> <strong>of</strong> cereals<br />
<strong>and</strong> grasses in North America.<br />
Ronald Press Co., New York.<br />
Ueng, P.P., G.C. Bergstrom, R.M. Slay,<br />
E.A. Geiger, G. Shaner, <strong>and</strong> A.L.<br />
Scharen. 1992. Restriction fragment<br />
length polymorphisms in the<br />
wheat glume blotch fungus,<br />
Phaeosphaeria nodorum.<br />
Phytopathology 82:1302-1305.<br />
Ueng, P.P., K. Subramaniam, W. Chen,<br />
E. Arseniuk, L. Wang, A.M.<br />
Cheung, G.M. H<strong>of</strong>fmann, <strong>and</strong> G.C.<br />
Bergstrom. 1998. Intraspecific<br />
genetic variation <strong>of</strong> <strong>Stagonospora</strong><br />
avenae <strong>and</strong> its differentiation from<br />
S. nodorum. Mycol. Res. 102:607-<br />
614.<br />
White, T.J., T. Bruns, S. Lee, <strong>and</strong> J.<br />
Taylor. 1990. Amplification <strong>and</strong><br />
direct sequencing <strong>of</strong> fungal<br />
ribosomal RNA genes for<br />
phylogenetics. PCR Protocols.<br />
Innis, M.A., D.H. Gelf<strong>and</strong>, J.J.<br />
Sninsky, <strong>and</strong> T.J. White (eds.). pp.<br />
315-322.<br />
Dice Similarity<br />
0.5 0.6 0.7 0.8 0.9 1.0<br />
8408<br />
9506-2<br />
9074<br />
9076<br />
Figure 1. UPGMA phenogram <strong>of</strong> four<br />
<strong>Stagonospora</strong> nodorum isolates based on the<br />
Dice Similarity Coefficients (Sd ) <strong>of</strong> 390<br />
individual AFLP DNA fragments.
A Vertically Resistant Wheat Selects for Specifically<br />
Adapted Mycosphaerella graminicola Strains<br />
C. Cowger, C.C. Mundt, <strong>and</strong> M.E. H<strong>of</strong>fer<br />
Department <strong>of</strong> Botany <strong>and</strong> Plant Pathology, Oregon State University, Corvallis, OR, USA<br />
Abstract<br />
At its commercial release in Oregon, USA, in 1992, the winter bread wheat cultivar Gene was highly resistant to<br />
Mycosphaerella graminicola. Within just four years, that resistance had substantially disintegrated. In 1997, we collected<br />
M. graminicola isolates from experimental field plots <strong>of</strong> Gene <strong>and</strong> two other cultivars in Corvallis, Oregon, <strong>and</strong> in a<br />
greenhouse experiment inoculated seedlings <strong>of</strong> the same three cultivars with those isolates. Gene seedlings were resistant to all<br />
isolates derived from the other two cultivars, but were susceptible to six <strong>of</strong> seven isolates derived from Gene. A similar<br />
experiment using a small number <strong>of</strong> 1992 isolates from the same three cultivars had previously shown no isolates virulent on<br />
Gene, in keeping with other published data on Gene’s early resistance. Commercial cultivation <strong>of</strong> Gene, which increased to<br />
about 15% <strong>of</strong> the local wheat area during the period 1992-1995, appears to have selected for strains <strong>of</strong> M. graminicola<br />
adapted to specific virulence on Gene. Cultivar specificity is a feature <strong>of</strong> the M. graminicola-wheat pathosystem.<br />
The issue <strong>of</strong> whether M.<br />
graminicola is specific in its<br />
interactions with cultivars <strong>of</strong> a<br />
single wheat species (wheat or<br />
durum) has been widely debated.<br />
Recent studies (Ahmed et al., 1995;<br />
Ahmed, et al., 1996; Kema, et al.,<br />
1996; Kema, et al., 1997)<br />
consistently indicate the existence<br />
<strong>of</strong> interactions between cultivars <strong>of</strong><br />
a single species <strong>and</strong> isolates<br />
derived from that species.<br />
However, most experiments where<br />
interaction has been measured<br />
have utilized tester cultivars <strong>and</strong><br />
isolates not originating on them.<br />
Such experiments can reveal only<br />
the potential for cultivar-specific<br />
adaptation. Here, we report the<br />
appearance <strong>of</strong> cultivar specificity<br />
when tester cultivars were<br />
challenged with isolates selected on<br />
those same cultivars in the field.<br />
Materials <strong>and</strong> Methods<br />
We conducted two experiments<br />
involving the s<strong>of</strong>t white winter<br />
wheat cultivars Gene (P.I. 560129),<br />
Madsen (P.I. 511673), <strong>and</strong> Stephens<br />
(C.I. 017596). Monopycnidial<br />
isolates <strong>of</strong> M. graminicola were<br />
obtained from Corvallis, Oregon,<br />
field plots <strong>of</strong> these cultivars in 1992<br />
<strong>and</strong> again in 1997. Greenhouse<br />
experiments were performed with<br />
these isolates in 1994 <strong>and</strong> 1998,<br />
respectively. We used two isolates<br />
derived from each cultivar in 1992,<br />
<strong>and</strong> seven or eight isolates from<br />
each cultivar in 1997. Seedlings <strong>of</strong><br />
each cultivar were grown in pots in<br />
the greenhouse, <strong>and</strong> each tester pot<br />
was inoculated at 21 days with an<br />
individual isolate in a factorial<br />
design. Both experiments were<br />
replicated four times. The percent<br />
<strong>of</strong> leaf area covered by lesions was<br />
assessed at 21 days after<br />
inoculation. Data were log e -<br />
transformed <strong>and</strong> subjected to<br />
analysis <strong>of</strong> variance.<br />
Results<br />
Of the six 1992 isolates tested,<br />
including two from Gene, none<br />
was virulent to Gene (data not<br />
shown). By contrast, the 1997<br />
isolates derived from Gene were<br />
generally virulent to Gene, while<br />
85<br />
the 1997 isolates derived from the<br />
other two cultivars were avirulent<br />
to Gene (Table 1). Analysis <strong>of</strong><br />
variance showed no significant<br />
cultivar-<strong>of</strong>-origin x tester<br />
interaction for the 1992 isolates, <strong>and</strong><br />
a significant cultivar-<strong>of</strong>-origin x<br />
tester interaction for the 1997<br />
isolates (not shown). Linear<br />
contrasts (not shown) were used to<br />
dissect the cultivar-<strong>of</strong>-origin x tester<br />
interaction, <strong>and</strong> they revealed a<br />
highly significant interactive effect<br />
<strong>of</strong> the tester Gene vs. testers<br />
Madsen <strong>and</strong> Stephens (P = 0.0068).<br />
Discussion<br />
From its release in 1992, Gene<br />
exp<strong>and</strong>ed to occupy 15% <strong>of</strong> the<br />
Willamette Valley wheat area by<br />
1995, but then declined (Figure 1)<br />
as its resistance to M. graminicola<br />
broke down (<strong>and</strong> incidence <strong>of</strong><br />
septoria nodorum blotch, to which<br />
Gene was always susceptible,<br />
increased).
86<br />
Session 4 — C. Cowger, C.C. Mundt, <strong>and</strong> M.E. H<strong>of</strong>fer<br />
Our first greenhouse<br />
experiment used only six isolates,<br />
but it supports published field data<br />
on Gene’s resistance during the<br />
years leading up to its release in<br />
1992 (Ahmed et al., 1995; Mundt et<br />
al., 1999). Our data suggest that<br />
cultivar specificity can develop in<br />
the M. graminicola-wheat<br />
pathosystem, <strong>and</strong> that this<br />
pathogen is capable <strong>of</strong> rapidly<br />
adapting to qualitative host<br />
resistance. Past observations<br />
suggested that resistance to M.<br />
graminicola does not break down<br />
rapidly or completely (Johnson,<br />
1992; Kema et al., 1996). However,<br />
the resistance <strong>of</strong> Gene succumbed<br />
within less than five years. We<br />
believe that Gene’s resistance is<br />
probably under monogenic or<br />
oligogenic control, <strong>and</strong> is perhaps<br />
due to the Stb4 gene (Kronstad et<br />
al., 1994). Further, M. graminicola<br />
strains able to defeat Gene’s<br />
vertical resistance appear to persist<br />
in the local population despite the<br />
limited continuing commercial<br />
production <strong>of</strong> Gene.<br />
References<br />
Ahmed, H.U., Mundt, C.C., <strong>and</strong><br />
Coakley, S.M. 1995. Host-pathogen<br />
relationship <strong>of</strong> geographically<br />
diverse isolates <strong>of</strong> <strong>Septoria</strong> tritici<br />
<strong>and</strong> wheat cultivars. Plant Pathol.<br />
44:838-847.<br />
Table 1. Percent diseased leaf area <strong>of</strong> greenhouse-grown wheat seedlings inoculated with<br />
Mycosphaerella graminicola isolates collected in 1997. a<br />
Cultivar <strong>of</strong> origin Tester cultivar<br />
& isolate nbr. Gene Madsen Stephens Mean<br />
Gene<br />
1 19.9 26.4 9.4 18.6<br />
2 3.4 18.5 16.7 12.8<br />
3 13.0 29.5 23.4 21.9<br />
4 11.2 17.9 29.6 19.5<br />
5 43.6 63.4 63.4 56.8<br />
6 21.1 32.9 33.0 29.0<br />
7 37.2 61.3 56.6 51.7<br />
Meanb 21.3ac Madsen<br />
35.7a 33.1a 30.1a<br />
8 0.9 77.1 56.0 44.7<br />
9 3.0 7.4 11.5 7.3<br />
10 4.2 30.6 28.4 21.0<br />
11 10.7 38.0 31.5 26.8<br />
12 1.0 30.1 28.7 19.9<br />
13 1.3 30.1 22.6 18.0<br />
14 0.2 19.6 7.8 9.2<br />
15 0.7 15.1 13.4 9.8<br />
Mean<br />
Stephens<br />
2.8b 31.0a 25.0a 19.6b<br />
16 3.3 22.7 24.7 16.9<br />
17 1.0 13.2 7.9 7.4<br />
18 0.4 31.0 21.7 17.7<br />
19 2.5 31.4 24.9 19.6<br />
20 2.7 22.0 16.1 13.6<br />
21 0.7 15.5 14.3 10.2<br />
22 2.2 36.1 28.3 22.2<br />
Mean 1.8b 24.6a 19.7a 15.4b<br />
Gr<strong>and</strong> mean 8.4 30.5 25.9 21.6<br />
a Experiment conducted in 1998.<br />
b Means are the untransformed percent diseased leaf area over four replications. Statistical analyses<br />
c<br />
were conducted on loge-transformed data.<br />
Within a column, means followed by the same letter are not significantly different at the 95%<br />
confidence level based on Fisher’s protected least significant difference.<br />
Ahmed, H.U., Mundt, C.C., H<strong>of</strong>fer,<br />
M.E., <strong>and</strong> Coakley, S.M. 1996.<br />
Selective influence <strong>of</strong> wheat<br />
cultivars on pathogenicity <strong>of</strong><br />
Mycosphaerella graminicola<br />
(anamorph <strong>Septoria</strong> tritici).<br />
Phytopathology 86:454-458.<br />
Johnson, R. 1992. Past, present <strong>and</strong><br />
future opportunities in breeding for<br />
disease resistance, with examples<br />
from wheat. Euphytica 63:3-22.<br />
Kema, G.H.J., Sayoud, R., Annone,<br />
J.G., <strong>and</strong> van Silfhout, C.H. 1996.<br />
Genetic variation for virulence <strong>and</strong><br />
resistance in the wheat-<br />
Mycosphaerella graminicola<br />
pathosystem. II. Analysis <strong>of</strong><br />
interactions between pathogen<br />
isolates <strong>and</strong> host cultivars.<br />
Phytopathology 86:213-220.<br />
Kema, G.H.J., <strong>and</strong> van Silfhout, C.H.<br />
1997. Genetic variation for<br />
virulence <strong>and</strong> resistance in the<br />
wheat-Mycosphaerella graminicola<br />
pathosystem. III. Comparative<br />
seedling <strong>and</strong> adult plant<br />
experiments. Phytopathology<br />
87:266-272.<br />
Kronstad, W.E., Kolding, M.F., Zwer,<br />
P.K., <strong>and</strong> Karow, R.S. 1994.<br />
Registration <strong>of</strong> ‘Gene’ wheat. Crop<br />
Sci. 34:538.<br />
Mundt, C.C., H<strong>of</strong>fer, M.E., Ahmed,<br />
H.U., Coakley, S.M., DiLeone, J.A.,<br />
<strong>and</strong> Cowger, C.C. 1999. Population<br />
genetics <strong>and</strong> host resistance. Pages<br />
115-130 in: <strong>Septoria</strong> on <strong>Cereals</strong>: A<br />
Study <strong>of</strong> Pathosystems. J.A. Lucas,<br />
P. Bowyer, <strong>and</strong> A.M. Anderson, eds.<br />
CAB International, Wallingford,<br />
United Kingdom.<br />
Percent <strong>of</strong> valley wheat area<br />
70<br />
60<br />
50<br />
40<br />
30<br />
20<br />
10<br />
Gene<br />
Madsen<br />
Stephens<br />
0<br />
1990 91 92 93 94 95 96 97<br />
Figure 1. Percent <strong>of</strong> s<strong>of</strong>t white winter wheat<br />
area occupied by three cultivars in the<br />
Willamette Valley <strong>of</strong> Oregon during the period<br />
1990-97.
Genetic Variability in a Collection <strong>of</strong> <strong>Stagonospora</strong><br />
nodorum Isolates from Western Australia<br />
N.E.A. Murphy, 1 R. Loughman, 2 E.S. Lagudah, 3 R. Appels, 3 <strong>and</strong> M.G.K. Jones1 1 WA State Agriculture Biotechnology Centre, Division <strong>of</strong> Science <strong>and</strong> Engineering, Murdoch University,<br />
Murdoch, Australia<br />
2 Agriculture Western Australia, Western Australia<br />
3 Plant Industries, CSIRO, Canberra, Australia<br />
Abstract<br />
<strong>Stagonospora</strong> nodorum isolates were collected from the Western Australian cereal-belt during 1993. These isolates<br />
<strong>and</strong> a subset <strong>of</strong> isolates taken from a single location were used to assay the level <strong>of</strong> variation within the pathogen<br />
population. The isolates were compared using anonymous nuclear DNA markers. Three low copy number <strong>and</strong> a single<br />
high copy number probe were used to generate restriction fragment length polymorphisms (RFLPs). The collection<br />
exhibited a high genotypic diversity for the high copy number probe. This is consistent with the high level <strong>of</strong> sexual<br />
reproduction previously found in the fungal population. Only minor differences between the total collection <strong>of</strong> isolates<br />
<strong>and</strong> the subset <strong>of</strong> isolates taken from the single location were found.<br />
<strong>Septoria</strong> nodorum blotch is one<br />
<strong>of</strong> the most important leaf diseases<br />
<strong>of</strong> wheat (Triticum aestivum L.) in the<br />
Western Australian cereal-belt<br />
(Murray <strong>and</strong> Brown, 1987). It is<br />
caused by <strong>Stagonospora</strong> nodorum<br />
(Berk.) Castellani & E. Germano<br />
(teleomorph Phaeosphaeria nodorum<br />
(E.Muller) Hedjaroude). Previous<br />
work on the variability <strong>of</strong> S. nodorum<br />
has been conducted principally on<br />
phenotypic traits such as<br />
aggressiveness (Allingham <strong>and</strong><br />
Jackson, 1981) <strong>and</strong> culture color<br />
(Scharen <strong>and</strong> Krupinsky, 1970).<br />
These studies have shown that there<br />
is considerable variation between<br />
isolates collected within relatively<br />
small distances (Allingham et al.,<br />
1981). More recently studies have<br />
assayed the genotypic variability<br />
using molecular techniques.<br />
RFLPs were used to look at two<br />
populations <strong>of</strong> S. nodorum in the USA<br />
using a hierarchical system <strong>of</strong><br />
sampling (McDonald et al., 1994).<br />
Considerable genetic variation was<br />
found to occur on a relatively small<br />
scale. The same set <strong>of</strong> eight probes<br />
were used to study a population <strong>of</strong><br />
S. nodorum isolates gathered in<br />
Switzerl<strong>and</strong> <strong>and</strong> two populations<br />
from the USA using a similar<br />
hierarchical sampling system. The<br />
Swiss population was genetically<br />
similar to the populations from the<br />
USA, providing evidence for gene<br />
flow between the populations (Keller<br />
et al., 1997a) <strong>and</strong> the gene diversity<br />
for four <strong>of</strong> the seven RFLP loci tested<br />
was not significantly different<br />
between the populations (Keller et<br />
al., 1997a).<br />
The aim <strong>of</strong> this investigation was<br />
to examine the level <strong>of</strong> genetic<br />
diversity among a collection <strong>of</strong><br />
Western Australian S. nodorum<br />
isolates using RFLPs <strong>and</strong> to compare<br />
the results obtained with similar<br />
work done in other regions <strong>of</strong> the<br />
world.<br />
Materials <strong>and</strong> Methods<br />
A total <strong>of</strong> 118 isolates from 38<br />
locations were used in the study.<br />
Thirty-nine <strong>of</strong> the isolates were<br />
obtained from an existing collection.<br />
Of these 39 historical isolates, 26<br />
87<br />
were from a single site at<br />
Badgingarra Research Station,<br />
recognized as an area where high<br />
levels <strong>of</strong> disease severity could be<br />
expected. These isolates were<br />
captured as single ascospores in<br />
spore traps during 1991. The<br />
remaining 79 isolates were collected<br />
from 26 locations throughout the<br />
cereal belt in 1993. They were<br />
isolated as ascospores from wheat<br />
stubble remaining from the 1992<br />
crop.<br />
The cultures <strong>of</strong> S. nodorum were<br />
revived on wheat meal agar plates to<br />
confirm their identity through<br />
pycnidiospore production. Mycelia<br />
was cultured in liquid broth <strong>and</strong> the<br />
DNA extracted (Lagudah et al., 1991).<br />
The genomic DNA was digested<br />
with HindIII <strong>and</strong> run on an agarose<br />
gel. A capillary alkali method was<br />
used to transfer the DNA onto the<br />
membrane. Three low copy number<br />
probes (pASN15, pASN125 <strong>and</strong><br />
pJSN73) were used to produce a<br />
multilocus haplotype, <strong>and</strong> a single<br />
high copy number probe (pSNS4)<br />
was used to generate a fingerprint for
88<br />
each isolate. Two <strong>of</strong> the probes used<br />
were from the genomic library <strong>of</strong> a<br />
Western Australian isolate <strong>of</strong> S.<br />
nodorum (pASN15 <strong>and</strong> pASN125)<br />
<strong>and</strong> the remaining two probes were a<br />
gift from Bruce McDonald <strong>of</strong> Texas<br />
A&M University (pJSN73, pSNS4).<br />
Both genomic libraries were set up<br />
by the restriction <strong>of</strong> genomic DNA<br />
from a S. nodorum isolate using PstI.<br />
Each probe was labelled with alpha<br />
32 P. Hybridization was carried out<br />
for a minimum <strong>of</strong> 12 hours. The<br />
membranes were washed <strong>and</strong> placed<br />
on x-ray film for up to 14 days.<br />
Each probe was assumed to<br />
represent a single RFLP locus <strong>and</strong><br />
each restriction fragment length<br />
variant were treated as an allele<br />
(McDonald et al., 1994). Each allele<br />
for each RFLP locus was designated<br />
an arbitrary number. The high copy<br />
number probe (pSNS4) was used to<br />
identify clones within the same<br />
multilocus haplotype (McDonald et<br />
al., 1994). The subset <strong>of</strong> the isolates<br />
from Badgingarra was treated as a<br />
separate collection to determine if<br />
the same level <strong>of</strong> genetic diversity<br />
present in the total collection was<br />
also present at a single location. The<br />
allelic frequencies, Nei’s measure <strong>of</strong><br />
gene diversity (Nei, 1973), the<br />
genotypic diversity, <strong>and</strong> the gametic<br />
disequilibrium were all calculated as<br />
described in McDonald et al. (1994).<br />
Results<br />
There were two alleles for probes<br />
pASN125 <strong>and</strong> pJSN73 <strong>and</strong> four for<br />
probe pASN15. Only four S. nodorum<br />
isolates had the same DNA<br />
fingerprint using the high copy<br />
number probe pSNS4. Nei’s measure<br />
<strong>of</strong> gene diversity was 0.23 for<br />
pJSN73-HindIII, 0.50 for pASN15-<br />
HindIII <strong>and</strong> 0.56 for pASN15-HindIII.<br />
The average over all three loci was<br />
0.43 in the clone-corrected data. For<br />
the Badgingarra subset no clones <strong>of</strong><br />
S. nodorum were identified. The<br />
allelic frequencies were very similar<br />
for the three loci compared with the<br />
total population (being 0.27, 0.49,<br />
<strong>and</strong> 0.60, respectively) <strong>and</strong> Nei’s<br />
measure <strong>of</strong> gene diversity averaged<br />
0.45 over the three loci.<br />
For the clone-corrected data <strong>of</strong><br />
the total population, 25% <strong>of</strong> the<br />
allele pairs were in significant<br />
gametic disequilibrium (Table 1).<br />
When all <strong>of</strong> the alleles were added<br />
together for each locus pair in the<br />
clone-corrected data, one <strong>of</strong> the<br />
locus pairs was in significant<br />
gametic disequilibrium (Table 1). In<br />
the Badgingarra subset, 20% <strong>of</strong> all<br />
the allelic combinations had a<br />
significant disequilibrium<br />
coefficient, <strong>and</strong> for each locus pair,<br />
one pair were in significant gametic<br />
disequilibrium (Table 1).<br />
There were 100 different<br />
fingerprint haplotypes produced<br />
from 103 isolates, giving a genotypic<br />
diversity <strong>of</strong> 92.2, 90% <strong>of</strong> its<br />
maximum value. In the subset<br />
collection from Badgingarra, the<br />
genotypic diversity was 15, 100% <strong>of</strong><br />
its maximum value.<br />
Table 1. Gametic disequilibrium for the clonecorrected<br />
data <strong>of</strong> the total population <strong>and</strong> the<br />
Badgingarra subset for each <strong>of</strong> pair <strong>of</strong> alleles<br />
between each pair <strong>of</strong> loci <strong>and</strong> for each pair <strong>of</strong> loci.<br />
Probe pASN15 pJSN73 pASN125<br />
pASN15 4/8 0/8<br />
15.7** (3) 7.15 (3)<br />
pJSN73 0/8 1,2 1/4<br />
2.05 (3) 3.70 (1)<br />
pASN125 3/8 0/4<br />
9.62 (3) 1.71 (1)<br />
1 Clone-corrected data only; values above the<br />
diagonal are for the total population <strong>and</strong> values<br />
below the diagonal are for the Badgingarra subset.<br />
2 For each set <strong>of</strong> comparisons, the first line shows the<br />
number <strong>of</strong> allele pairs that are in significant gametic<br />
disequilibrium (p
ecombination plays in the<br />
population dynamics. This level <strong>of</strong><br />
variation may have implications for<br />
the cereal plant breeding <strong>and</strong><br />
selection program if it is a reflection<br />
<strong>of</strong> the potential variability for<br />
aggressiveness in the pathogen. If<br />
the level <strong>of</strong> genotypic variation does<br />
reflect the level <strong>of</strong> variation in<br />
aggressiveness, selecting resistance<br />
using the widest possible range <strong>of</strong> S.<br />
nodorum isolates would be desirable.<br />
The most practical method to<br />
screen breeding lines for a wide<br />
selection <strong>of</strong> S. nodorum isolates is to<br />
use straw from previously infected<br />
wheat as a source <strong>of</strong> inoculum<br />
(Holmes <strong>and</strong> Colhoun, 1975).<br />
Previous work investigating the<br />
effect <strong>of</strong> the host genotype upon the<br />
selection <strong>of</strong> S. nodorum provided no<br />
evidence <strong>of</strong> specialization <strong>of</strong> the<br />
pathogen with the host cultivar<br />
(Keller et al., 1997b) according to<br />
neutral RFLP markers. It would also<br />
be unnecessary to use straw from<br />
numerous sites, as the genotypic<br />
variation encountered at a single site<br />
can account for as much as 95% <strong>of</strong><br />
the total genotypic variation within a<br />
population (Keller et al., 1997b).<br />
Using straw from a collection <strong>of</strong><br />
cultivars <strong>and</strong> from a number <strong>of</strong> sites<br />
may be unnecessary, as it would<br />
provide only a modest increase in<br />
the overall genotypic diversity.<br />
References<br />
Allingham, E.A., <strong>and</strong> L.F. Jackson. 1981.<br />
Variation in pathogenicity, virulence<br />
<strong>and</strong> aggressiveness <strong>of</strong> <strong>Septoria</strong><br />
nodorum in Florida. Phytopathology<br />
71:1080-1085.<br />
Bathgate, J.A., <strong>and</strong> R. Loughman. 1995.<br />
Ascospores as primary inoculum <strong>of</strong><br />
Phaeosphaeria spp. in Western<br />
Australia. 10th Biennial Australasian<br />
Plant Pathology Society Conference.<br />
New Zeal<strong>and</strong>, Lincoln University,<br />
28-30th August. p. 100.<br />
Holmes, S.J.I., <strong>and</strong> J. Colhoun. 1975.<br />
Straw-borne inoculum <strong>of</strong> <strong>Septoria</strong><br />
nodorum <strong>and</strong> <strong>Septoria</strong> tritici in<br />
relation to incidence <strong>of</strong> disease on<br />
wheat plants. Plant Pathology 24:63-<br />
66.<br />
Keller, S.M., J.M. McDermott, R.E.<br />
Pettway, M.S. Wolfe, <strong>and</strong> B.A.<br />
McDonald. 1997a. Gene flow <strong>and</strong><br />
sexual reproduction in the wheat<br />
glume blotch pathogen Phaesophaeria<br />
nodorum (anamorph Stagonosopora<br />
nodorum). Phytopathology 87:353-<br />
358.<br />
89<br />
Keller, S.M., M.S. Wolfe, J.M.<br />
McDermott, <strong>and</strong> B.A. McDonald.<br />
1997b. High genetic similarity<br />
among populations <strong>of</strong> Phaeosphaeria<br />
nodorum across wheat cultivars <strong>and</strong><br />
regions in Switzerl<strong>and</strong>.<br />
Phytopathology 87:1134-1139.<br />
Lagudah, E.S., R. Appels, <strong>and</strong> D.<br />
McNeil. 1991. The Nor-D3 locus <strong>of</strong><br />
Triticum tauschii: natural variation<br />
<strong>and</strong> genetic linkage to markers in<br />
chromosome 5. Genome 34:387-395.<br />
McDonald, B.A., J. Miles, L.R. Nelson,<br />
<strong>and</strong> R.E. Pettway. 1994. Genetic<br />
variability in nuclear DNA in field<br />
populations <strong>of</strong> <strong>Stagonospora</strong><br />
nodorum. Phytopathology 84:250-<br />
255.<br />
Murray, G.M., <strong>and</strong> J.F. Brown. 1987.<br />
The incidence <strong>and</strong> relative<br />
importance <strong>of</strong> wheat diseases in<br />
Australia. Australasian Plant<br />
Pathology 16:34-37.<br />
Nei, M. 1973. Analysis <strong>of</strong> gene diversity<br />
in subdivided populations.<br />
Proceedings <strong>of</strong> the National<br />
Academy <strong>of</strong> Science, USA 70:3321-<br />
3323.<br />
Scharen, A.L., <strong>and</strong> J.M. Krupinsky.<br />
1970. Cultural <strong>and</strong> Inoculation<br />
studies <strong>of</strong> <strong>Septoria</strong> nodorum cause <strong>of</strong><br />
glume blotch <strong>of</strong> wheat.<br />
Phytopathology 60:1480-1485.
90<br />
Mating Type-Specific PCR Primers for <strong>Stagonospora</strong><br />
nodorum Field Studies<br />
Bennett, R.S., 1 S.-H. Yun, 1 T.Y. Lee, 1 B.G. Turgeon, 1 B. Cunfer, 2<br />
E. Arseniuk, 3 <strong>and</strong> G.C. Bergstrom 1 * (Poster)<br />
1 Department <strong>of</strong> Plant Pathology, Cornell University, Ithaca, NY, USA<br />
2 Department <strong>of</strong> Plant Pathology, University <strong>of</strong> Georgia, Griffin, GA, USA<br />
3 Plant Breeding <strong>and</strong> Acclimatization Institute, Radzików, Pol<strong>and</strong><br />
* corresponding author<br />
Abstract<br />
Conserved regions <strong>of</strong> the mating type genes (MAT) in Cochliobolus heterostrophus, Mycosphaerella zeaemaydis,<br />
<strong>and</strong> Alternaria alternata were used to design primers to identify the mating type genes <strong>and</strong> thus assign<br />
mating types to <strong>Stagonospora</strong> nodorum. These primers successfully distinguished between mating types <strong>of</strong> S.<br />
nodorum isolates that had been identified through traditional crosses. The primers were used to screen a small sample <strong>of</strong><br />
S. nodorum isolates from New York, thus revealing the presence <strong>of</strong> both mating types. This efficient method <strong>of</strong><br />
identifying mating types may help determine the role <strong>of</strong> sexual recombination in the epidemiology <strong>of</strong> S. nodorum, as<br />
well as elucidate phylogenetic relationships among related species.<br />
<strong>Stagonospora</strong> nodorum (Berk.)<br />
Castellani & E.G. Germano<br />
(teleomorph = Phaeosphaeria<br />
nodorum (E. Müller) Hedjaroude) is<br />
the causal agent <strong>of</strong> stagonospora<br />
nodorum blotch, a major disease <strong>of</strong><br />
wheat around the world.<br />
Phaeosphaeria nodorum is<br />
heterothallic <strong>and</strong> thus requires<br />
strains <strong>of</strong> the two different mating<br />
types to produce ascospores<br />
(Müller, 1989). The potential<br />
significance <strong>of</strong> sexual reproduction<br />
in the epidemiology <strong>of</strong><br />
stagonospora nodorum blotch has<br />
been heightened by the high RFLP<br />
variability found among field<br />
isolates (McDonald et al., 1994).<br />
Fungi that regularly reproduce<br />
sexually have more opportunities<br />
to develop fungicide resistance <strong>and</strong><br />
to overcome cultivar resistance.<br />
Furthermore, airborne ascospores<br />
would permit dissemination over<br />
longer distances than splashdispersed<br />
conidia.<br />
MAT genes have been identified<br />
for several species <strong>of</strong><br />
Loculoascomycetes including<br />
Setosphaeria turcica, Mycosphaerella<br />
zeae-maydis, Alternaria alternata, <strong>and</strong><br />
several Cochliobolus spp. (Arie et al.,<br />
1997). Both genes, MAT-1 <strong>and</strong> MAT-<br />
2, contain conserved regions that<br />
allow the relatively rapid<br />
identification <strong>of</strong> these genes from an<br />
increasing group <strong>of</strong> ascomycetes. A<br />
high mobility group (HMG) <strong>and</strong> abox<br />
motif are the conserved regions<br />
in MAT-2 <strong>and</strong> MAT-1, respectively<br />
(Turgeon, 1998). These sequences,<br />
besides elucidating sexual<br />
mechanisms <strong>and</strong> phylogeny, may<br />
also help address epidemiological<br />
questions.<br />
We have developed primers to<br />
identify the mating types <strong>of</strong> P.<br />
nodorum from the conserved MAT<br />
regions <strong>of</strong> related fungi. Preliminary<br />
data on the mating type prevalence<br />
in a small sample <strong>of</strong> New York field<br />
isolates is also presented.<br />
Materials <strong>and</strong> Methods<br />
Fungal isolates <strong>and</strong> media<br />
Three S. nodorum isolates <strong>of</strong><br />
different mating types (two (-)<br />
(SN435PL-98, SN437GA-98) <strong>and</strong><br />
one (+) (SN436GA-98)) (Arseniuk<br />
et al., 1997a,b) were grown on V-8<br />
juice agar (200 ml V8 juice, 3 g<br />
CaCO3 , 15 g agar per 800 ml <strong>of</strong><br />
distilled water). After<br />
approximately seven days, mycelial<br />
plugs were taken <strong>and</strong> placed into<br />
yeast-malt-sucrose broth (5 g yeast<br />
extract, 5 g malt extract, 20 g<br />
sucrose per 1 liter <strong>of</strong> distilled<br />
water), <strong>and</strong> placed on a shaker (150<br />
rpm) at room temperature. Mycelia<br />
were harvested after 5-7 days by<br />
filtering through 3-ply sterile<br />
cheesecloth lining a Buchner funnel<br />
<strong>and</strong> were rinsed with distilled<br />
water. The samples were then<br />
frozen <strong>and</strong> lyophilized overnight.<br />
MAT-1 <strong>and</strong> MAT-2 C. heterostrophus<br />
laboratory strains (C5 <strong>and</strong> C4,<br />
respectively) were used as controls.
Thirty-eight arbitrarily chosen<br />
field isolates <strong>of</strong> S. nodorum collected<br />
from New York in different places<br />
<strong>and</strong> years were grown on<br />
cellophane discs overlaid on V-8<br />
juice agar. After approximately<br />
seven days, mycelia were scraped<br />
<strong>of</strong>f the cellophane <strong>and</strong> placed in<br />
microcentrifuge tubes to be<br />
lyophilized.<br />
DNA extraction<br />
Lyophilized mycelia were<br />
ground in liquid nitrogen. The field<br />
isolates were ground directly in<br />
microcentrifuge tubes with a small<br />
amount <strong>of</strong> white quartz s<strong>and</strong> (-50 +<br />
70 mesh, Sigma Chemical<br />
Company, St. Louis, MO). DNA<br />
was extracted by a miniprep<br />
procedure (Wirsel et al., 1996).<br />
Amplification <strong>of</strong> the HMG<br />
<strong>and</strong> a-boxes by PCR<br />
PCR primers (Sn-HMG1 <strong>and</strong><br />
Sn-HMG2) for the HMG box (Table<br />
1) were designed from conserved<br />
regions in the HMG box <strong>of</strong> the MAT<br />
gene <strong>of</strong> Cochliobolus heterostrophus,<br />
M. zeae-maydis, <strong>and</strong> A. alternata,<br />
whereas the primers (Sn-ab1 <strong>and</strong><br />
Sn-ab2) for the a-box were designed<br />
from the a-boxes <strong>of</strong> M. zeae-maydis<br />
<strong>and</strong> A. alternata. The primers were<br />
synthesized by the Cornell<br />
University DNA Services Facility<br />
<strong>and</strong> were dissolved (100 µM) in<br />
Table 1. PCR primers used to amplify<br />
fragments <strong>of</strong> MAT genes in <strong>Stagonospora</strong><br />
nodorum.<br />
MAT-1<br />
Sn-ab1 5’ AA(A/G)GCN(C/T)TNAA(C/<br />
T)GCNTT (C/T)GTNGG 3’<br />
Sn-ab2 5’ TC(C/T)TTNCC(A/G/T)AT(C/T)<br />
TG(A/G)TCNCG(A/G/T)AT 3’<br />
MAT-2<br />
Sn-HMG1 5’ AA(A/G)GCNCCN(AC)<br />
GNCCNATGAA 3’<br />
Sn-HMG2 5’ TT(C/T)TT(C/T)TT(C/T)T(CG)<br />
NCCNGG(C/T)TT 3’<br />
sterile distilled water <strong>and</strong> stored at<br />
–20C. Each PCR reaction mixture<br />
had approximately 20 ng <strong>of</strong><br />
genomic DNA in 50 µl reaction<br />
buffer [1X PCR Buffer (Perkin<br />
Elmer, Norwalk, CT), 0.2 mM<br />
dNTPs, 2.5 mM MgCl2, , 2 µM each<br />
primer, <strong>and</strong> 0.025 U Taq<br />
polymerase]. The samples were<br />
denatured at 95C for 2 min, <strong>and</strong><br />
then subjected to 30 cycles <strong>of</strong> 95C<br />
for 1 min, 50C for 30 sec, <strong>and</strong> 72C<br />
for 1.5 min. After extension at 72C<br />
for 10 min, the samples were kept<br />
at 4C. 5 µl aliquots <strong>of</strong> the PCR<br />
products were analyzed on a 2%<br />
agarose gel in TAE buffer.<br />
Cloning, sequencing, <strong>and</strong><br />
analysis <strong>of</strong> PCR products<br />
The MAT sequences from<br />
related fungi predicted that a PCR<br />
product <strong>of</strong> ~270 bp for the HMG<br />
box <strong>and</strong> a product <strong>of</strong> ~230 bp for<br />
the a-boxes would result if the<br />
amplifications were successful.<br />
Products <strong>of</strong> these sizes were cloned<br />
from the PCR reaction into the<br />
vector pCR2.1, using the guidelines<br />
Mating Type-Specific PCR Primers for <strong>Stagonospora</strong> nodorum Field Studies 91<br />
<strong>of</strong> the manufacturer (Invitrogen<br />
Co., San Diego, CA). DNA<br />
sequences were determined at the<br />
Cornell University DNA Services<br />
Facility. Sequences were aligned<br />
with the LaserGene program<br />
MegAlign (DNASTAR Inc.,<br />
Madison, WI), using the clustal<br />
method. A BLAST search was done<br />
using the NCBI/Genbank internet<br />
databases.<br />
Gel blot hybridization<br />
A st<strong>and</strong>ard DNA gel blot<br />
hybridization procedure was<br />
followed (Sambrook et al., 1989).<br />
Results<br />
HMG alpha<br />
The primers for the HMG box<br />
were used with genomic DNA <strong>of</strong><br />
(+) <strong>and</strong> (-) S. nodorum strains as<br />
templates. A PCR product<br />
approximately 0.3 kb, matching the<br />
~270 bp predicted, was present in<br />
the (+) (MAT-2) strain, but was not<br />
present in the (-) (MAT-1) strains<br />
(Figure 1). The primers for the abox,<br />
however, amplified the<br />
1 2 3 4 5 6 7 8 9 101112131415161718<br />
Figure 1. PCR products amplified with the HMG <strong>and</strong> a-box primers. Lanes 2-9 were amplified using<br />
HMG (MAT-2) primers, <strong>and</strong> lanes 10-17 were amplified with a-box (MAT-1) primers. Lanes 2,10 <strong>and</strong><br />
3,11: C. heterostrophus C5 (MAT-1) <strong>and</strong> C. heterostrophus C4 (MAT-2), respectively; lanes 4,12 <strong>and</strong><br />
5,13: plasmid with cloned a-box from SN435PL-98 <strong>and</strong> plasmid with cloned HMG box from SN436GA-<br />
98, respectively; lanes 6 <strong>and</strong> 14: SN436GA-98; lanes 7 <strong>and</strong> 15: SN437GA-98; lanes 8 <strong>and</strong> 16: SN005NY-<br />
85; lanes 9 <strong>and</strong> 17: SN197NY-88. A ~0.3-kb b<strong>and</strong> (arrowhead) was amplified in the MAT-2 isolates<br />
only, <strong>and</strong> a b<strong>and</strong> above 0.2-kb (asterisk) was amplified only in the MAT-1 isolates.
92<br />
Session 4 — Bennett, R.S., S.-H. Yun, T.Y. Lee, B.G. Turgeon, B. Cunfer, E. Arseniuk, <strong>and</strong> G.C. Bergstrom<br />
expected product (~230 bp) from<br />
only the (-) (MAT-1) strains. The<br />
primers were also able to<br />
distinguish between the MAT-1 <strong>and</strong><br />
MAT-2 C. heterostrophus controls.<br />
The BLAST search <strong>of</strong> the S.<br />
nodorum cloned products showed<br />
highest homology to the MAT<br />
genes <strong>of</strong> Cochliobolus spp.<br />
In gel blot hybridization, a<br />
single b<strong>and</strong> was obtained from<br />
DNA <strong>of</strong> the (+) SN436GA-98 isolate<br />
only when probed with the HMG<br />
box; no hybridization occurred on<br />
the (-) SN435PL-98 isolate.<br />
Likewise, a single b<strong>and</strong> was<br />
obtained when SN435PL-98 was<br />
probed with the a-box, <strong>and</strong> no<br />
hybridization occurred to the DNA<br />
<strong>of</strong> SN436GA-98.<br />
When these primers were used<br />
to test the 38 field isolates, 20 were<br />
shown to be <strong>of</strong> the MAT-1 mating<br />
type <strong>and</strong> 18 were <strong>of</strong> the MAT-2<br />
mating type.<br />
Discussion<br />
A PCR technique that readily<br />
identifies the mating type can be a<br />
useful tool for studying S. nodorum.<br />
Surveying for the distribution <strong>of</strong><br />
mating types in the field may result<br />
in a better underst<strong>and</strong>ing <strong>of</strong> the<br />
role <strong>of</strong> sexual reproduction in the<br />
disease cycle. The preliminary data<br />
given here tested S. nodorum<br />
isolates that were collected in New<br />
York wheat fields between 1985<br />
<strong>and</strong> 1995. These isolates are not<br />
only temporally r<strong>and</strong>om, but<br />
spatially unrelated. More<br />
informative surveys <strong>of</strong> spatially<br />
distributed isolates from single<br />
fields are being conducted.<br />
However, the preliminary data<br />
presented here do indicate that<br />
both mating types exist in New<br />
York <strong>and</strong> that sexual recombination<br />
is possible.<br />
Mating types may also serve as<br />
an additional marker for<br />
experiments requiring genetically<br />
characterized isolates. As<br />
previously established (Arie et<br />
al,.1997), we refer to (-) isolates<br />
containing the a-box as MAT-1, <strong>and</strong><br />
(+) isolates containing the HMG<br />
box as MAT-2. Finally, as outlined<br />
in a review by Turgeon (1998), MAT<br />
gene sequences may provide<br />
valuable information for<br />
phylogenetic analyses, particularly<br />
<strong>of</strong> related species, the study <strong>of</strong><br />
pathogenesis, <strong>and</strong> the underlying<br />
mechanisms <strong>of</strong> reproductive life<br />
styles in fungi.<br />
References<br />
Arie, T., S.K. Christiansen, O.C. Yoder,<br />
<strong>and</strong> B.G. Turgeon. 1997. Efficient<br />
cloning <strong>of</strong> ascomycete mating type<br />
genes by PCR amplification <strong>of</strong> the<br />
conserved MAT HMG box. Fungal<br />
Genetics <strong>and</strong> Biology 21:118-130.<br />
Arseniuk, E., B.M. Cunfer, S. Mitchell,<br />
<strong>and</strong> S. Kresovich. 1997a.<br />
Characterization <strong>of</strong> genetic<br />
similarities among isolates <strong>of</strong><br />
<strong>Stagonospora</strong> spp. <strong>and</strong> <strong>Septoria</strong> tritici<br />
by amplified fragment length<br />
polymorphism analysis.<br />
Phytopathology 87 (Supplement) S5.<br />
Arseniuk, E., P.C. Czembor, <strong>and</strong> B.M.<br />
Cunfer. 1997b. Segregation <strong>and</strong><br />
recombination <strong>of</strong> PCR-based<br />
markers inprogenies <strong>of</strong> in vitro<br />
mated isolates <strong>of</strong> Phaeosphaeria<br />
nodorum (Müller) Hedjaroude.<br />
Phytopathology 87(Supplement), S5<br />
.<br />
McDonald, B.A., J. Miles, L.R. Nelson,<br />
<strong>and</strong> R.E. Pettway. 1994. Genetic<br />
variability in nuclear DNA in field<br />
populations <strong>of</strong> <strong>Stagonospora</strong><br />
nodorum. Phytopathology 84:250-<br />
255.<br />
Müller, E. 1989. On the taxonomic<br />
position <strong>of</strong> <strong>Septoria</strong> nodorum <strong>and</strong><br />
<strong>Septoria</strong> tritici, p. 11-12. In:<br />
Proceedings from the Third<br />
International Workshop on <strong>Septoria</strong><br />
<strong>Diseases</strong> <strong>of</strong> <strong>Cereals</strong>. Zurich,<br />
Switzerl<strong>and</strong>.<br />
Sambrook, J., E.F. Fritsch, <strong>and</strong> T.<br />
Maniatis. 1989. Molecular Cloning:<br />
A Laboratory Manual, 2 nd ed. Cold<br />
Spring Harbor Laboratory Press:<br />
Cold Spring Harbor, NY.<br />
Turgeon, B.G. 1998. Application <strong>of</strong><br />
mating type gene technology to<br />
problems in fungal biology. Annual<br />
Review <strong>of</strong> Phytopathology 36:115-<br />
137.<br />
Wirsel, S., B.G. Turgeon, <strong>and</strong> O.C.<br />
Yoder. 1996. Deletion <strong>of</strong> the<br />
Cochliobolus heterostrophus mating<br />
type (MAT) locus promotes function<br />
<strong>of</strong> MAT transgenes. Current<br />
Genetics 29:241-249.
Session 5: Epidemiology<br />
Epidemiology <strong>of</strong> Mycosphaerella graminicola <strong>and</strong><br />
Phaeosphaeria nodorum: An Overview<br />
M.W. Shaw<br />
Department <strong>of</strong> Agricultural Botany, School <strong>of</strong> Plant Sciences, The University <strong>of</strong> Reading, Reading, Engl<strong>and</strong><br />
Abstract<br />
The within-season <strong>and</strong> between-crop methods <strong>of</strong> multiplication <strong>and</strong> survival, <strong>and</strong> their environmental relations are<br />
reviewed. Mycosphaerella graminicola multiplies within a season by conidia which are primarily but not exclusively<br />
dispersed by rain. Arguments are given that the influence <strong>of</strong> ascospores within a crop will be minor, but they are the<br />
major source <strong>of</strong> movement <strong>of</strong> the pathogen into new crops. Phaeosphaeria nodorum also multiplies within a season by<br />
conidia, but has clearer associations with wet weather. The role <strong>of</strong> ascospores in movement between crops may vary<br />
geographically; seed transmission seems to be very important in some areas.<br />
In this contribution, I have tried<br />
to summarize what is understood<br />
<strong>of</strong> the epidemiology <strong>of</strong> these two<br />
diseases. My aim has been to<br />
produce a concise summary to<br />
introduce the detailed <strong>and</strong> novel<br />
contributions that follow. I have<br />
tried to give slightly more extended<br />
discussion <strong>of</strong> those areas where<br />
new ideas have arisen or our<br />
underst<strong>and</strong>ing has changed<br />
substantially in the last few years.<br />
The relevant questions for both<br />
diseases fall into two classes. First,<br />
qualitative: what conditions allow<br />
inoculum transfer, permit infection,<br />
<strong>and</strong> encourage sporulation?<br />
Second: quantitative: in a given<br />
agro-ecosystem, what factors in<br />
practice regulate pathogen<br />
population size? The two questions<br />
are related, but both need to be<br />
answered if the diseases are to be<br />
managed most effectively. The<br />
answers also depend greatly on<br />
scale: within a region <strong>and</strong> over<br />
several years, very different<br />
processes <strong>and</strong> factors may need to<br />
be considered from those operating<br />
within a crop <strong>and</strong> within a season.<br />
The paper is restricted to wheat.<br />
Mycosphaerella<br />
graminicola<br />
Within a crop<br />
As discussed later, infection <strong>of</strong> a<br />
crop is usually initiated by airborne<br />
ascospores (Shaw <strong>and</strong> Royle, 1989).<br />
The density <strong>of</strong> initial infections is<br />
such that once a few sporulating<br />
lesions per square meter exist, a<br />
polycyclic epidemic on successive<br />
leaf layers follows (Shaw <strong>and</strong><br />
Royle, 1993). Pycnidia are produced<br />
within roughly 14 to 40 days,<br />
depending on both temperature<br />
<strong>and</strong> host cultivar. Conidia may be<br />
dispersed by single rain splashes<br />
within a circle <strong>of</strong> about 1 m radius,<br />
the number dispersed decreasing<br />
exponentially with distance, with<br />
half distances <strong>of</strong> the order <strong>of</strong> 10 cm<br />
(Bannon <strong>and</strong> Cooke, 1998; Brennan<br />
et al., 1985a; Brennan et al., 1985b).<br />
Initial dispersal is to the ground or<br />
to surface water on a leaf, whence<br />
further dispersal is possible.<br />
However, spores contacting leaf<br />
surfaces are bound to the surface<br />
within a short time. The average<br />
number <strong>of</strong> splashes moving a spore<br />
during rain <strong>of</strong> given intensity <strong>and</strong><br />
duration is hard to estimate, but is<br />
93<br />
unlikely to be large, so half<br />
distances for effective horizontal<br />
dispersal will be <strong>of</strong> the order <strong>of</strong> 20-<br />
50 cm at most. Each initial infection<br />
may plausibly produce 50,000 to<br />
500,000 conidia (10-100 pycnidia <strong>of</strong><br />
ca. 5000 spores) (Eyal, 1971).<br />
Although most <strong>of</strong> these are not<br />
dispersed far, considering them as<br />
evenly dispersed over a circle <strong>of</strong> 0.5<br />
m radius gives 5-50 spores per<br />
square centimeter from initial<br />
infections spaced at about 1/m 2 . If<br />
they had 2-20% infection efficiency,<br />
the crop would be saturated with<br />
latent lesions. Fortunately, infection<br />
efficiency is usually lower than this,<br />
but if few spores are present, 1% <strong>of</strong><br />
those applied may cause infection<br />
under good infection conditions.<br />
The actual environmental<br />
conditions permitting infection are<br />
lax because the pathogen tolerates<br />
extended breaks in humidity<br />
during the infection process (Shaw,<br />
1991a; Shaw <strong>and</strong> Royle, 1993).<br />
Certainly, within two infection<br />
cycles the pathogen population in<br />
crops with moderate initial<br />
amounts <strong>of</strong> disease will be limited<br />
by the rate <strong>of</strong> growth <strong>of</strong> leaf area
94<br />
Session 5 — M.W. Shaw<br />
<strong>and</strong> the favorability <strong>of</strong> the<br />
environment rather than by<br />
shortage <strong>of</strong> inoculum. However,<br />
spread by splash is very inefficient<br />
over other than short distances, <strong>and</strong><br />
if initial infections are widely<br />
spaced, then the disease should<br />
remain patchy <strong>and</strong> therefore cause<br />
limited damage.<br />
The preceding paragraph<br />
ignored vertical spread, which has<br />
been argued to be the key factor<br />
determining severe disease on the<br />
uppermost leaf layers <strong>of</strong> a wheat<br />
crop. Vertical movement <strong>of</strong> splash<br />
droplets is limited, declining<br />
exponentially with typical halfdistances<br />
<strong>of</strong> 5 cm, but varies<br />
between storms (Bannon <strong>and</strong><br />
Cooke, 1998; Shaw, 1987; Shaw,<br />
1991b). Probably more critically, the<br />
distance between leaf layers with<br />
<strong>and</strong> without infection varies greatly<br />
according to both the architecture<br />
<strong>of</strong> the wheat cultivar <strong>and</strong> the latent<br />
period <strong>of</strong> the pathogen on the<br />
cultivar. Lovell et al. (1997) have<br />
shown how the interaction <strong>of</strong> these<br />
causes great variation in the<br />
potential for spread <strong>of</strong> pathogen to<br />
the upper part <strong>of</strong> the crop, where it<br />
becomes damaging.<br />
Once a few sporulating lesions<br />
are present on a reasonable<br />
proportion <strong>of</strong> the uppermost leaves<br />
<strong>of</strong> a wheat crop, there is potential<br />
for multiplication leading to<br />
widespread infection <strong>and</strong><br />
premature leaf death, except in very<br />
dry weather.<br />
The major advance since the last<br />
Workshop is the recognition that<br />
pseudothecia are produced<br />
regularly throughout the year<br />
under at least some conditions on<br />
at least some cultivars (Hunter et<br />
al., 1999; Kema et al., 1996). This<br />
means that sparse initial infections<br />
could spread much more effectively<br />
by windblown ascospores, <strong>and</strong> that<br />
multiplication <strong>of</strong> the disease could<br />
take place efficiently in the absence<br />
<strong>of</strong> rain, dew alone providing<br />
wetness for infection by dry<br />
dispersed spores. This is a<br />
potentially dramatic change to our<br />
view <strong>of</strong> the ecology <strong>of</strong> the organism.<br />
However, the effect on epidemics<br />
within a season is probably limited,<br />
because the pseudothecia are<br />
always produced long after<br />
pycnidia. Two extreme situations<br />
can be imagined: one in which<br />
pycnidia can infect <strong>of</strong>ten during the<br />
season, one in which they can never<br />
infect.<br />
In the first situation, even in the<br />
fastest quoted latent periods for<br />
perithecia, the effect on the<br />
epidemic rate <strong>of</strong> ascospore<br />
production is equivalent to perhaps<br />
1000-10,000 extra spores from a<br />
single infection, produced at<br />
roughly the time when the next<br />
generation <strong>of</strong> lesions from the<br />
pycnidia produced earlier are<br />
themselves producing pycnidia. If<br />
the multiplication ratio <strong>of</strong> lesions<br />
from one generation to the next is R,<br />
then the pycnidial route is<br />
producing <strong>of</strong> the order <strong>of</strong> R * 10,000<br />
conidia in the time during which<br />
the sexual route is producing 10,000<br />
spores: a ratio <strong>of</strong> 1:R. Order <strong>of</strong><br />
magnitude estimates <strong>of</strong> R can be<br />
obtained in at least two ways. First,<br />
the ratio between the number <strong>of</strong><br />
lesions a latent period after disease<br />
first entered a crop <strong>and</strong> the initial<br />
number <strong>of</strong> lesions. For the crops<br />
observed by Shaw <strong>and</strong> Royle (1989)<br />
this ratio was about 100. Second, the<br />
ratio between the initial number <strong>of</strong><br />
lesions on the uppermost leaves <strong>of</strong> a<br />
crop, <strong>and</strong> the number one latent<br />
period later (Shaw <strong>and</strong> Royle 1993):<br />
this too suggests a large number.<br />
The effect on the epidemic rate<br />
must be very small. This is borne<br />
out by detailed modelling (Eriksen<br />
et al., in prep.).<br />
In the second situation,<br />
pseudothecia are the only route by<br />
which infection can progress. In<br />
this case, the latent period is at least<br />
double the latent period for an<br />
epidemic otherwise similar but<br />
driven by pycnidia. Since the latent<br />
period is at least doubled, <strong>and</strong> any<br />
gains in infection or dispersal<br />
efficiency are <strong>of</strong>fset by the reduced<br />
number <strong>of</strong> ascospores produced,<br />
the rate <strong>of</strong> the epidemic is at least<br />
halved. A rather more qualitative<br />
prediction would be that where<br />
conditions prevent conidial<br />
dispersal, epidemics will develop<br />
very slowly, <strong>and</strong> be damaging only<br />
where initial disease is rather<br />
widespread. It will clearly be very<br />
interesting to study epidemic<br />
progress in areas where pycnidial<br />
progression is not possible, <strong>and</strong> see<br />
whether this prediction is true.<br />
In fact, even this may overstate<br />
the case, since the fungus is<br />
heterothallic (Kema et al., 1996) <strong>and</strong><br />
presumably therefore requires two<br />
infections <strong>of</strong> opposite mating type<br />
to be adjacent in order to produce<br />
ascospores. It is not known how<br />
close together infections must be to<br />
mate effectively. Metcalfe (1999)<br />
found internal hyphae spanning an<br />
average maximum <strong>of</strong> 11 mm in a<br />
susceptible wheat cultivar just<br />
before pycnidia were produced, so<br />
infections more than 20 mm apart<br />
may be isolated. In a sparse, slow<br />
epidemic this may be rare. Mating<br />
will be commonest in situations<br />
where inoculum is not limiting the<br />
pathogen population in any way,<br />
but in these cases, the extra<br />
inoculum provided by ascospores<br />
will not affect epidemic rate.
When inoculum is common low<br />
in the canopy, but transport is<br />
potentially limiting infection <strong>of</strong><br />
upper leaves, ascospores could<br />
pr<strong>of</strong>oundly alter the risk <strong>of</strong> severe<br />
infection on the uppermost leaves,<br />
because rainfall is not required for<br />
their movement to these leaves.<br />
However, recent work confirms<br />
(under UK conditions at least) the<br />
apparent association <strong>of</strong> severe M.<br />
graminicola with rain (Lovell et al.,<br />
1997); <strong>and</strong> windblown transport <strong>of</strong><br />
spores from close to the ground to<br />
the upper leaves <strong>of</strong> a dense canopy<br />
is not efficient because windspeeds<br />
are not high within the canopy.<br />
Nonetheless, the simple picture <strong>of</strong><br />
rain moving spores from the base <strong>of</strong><br />
the canopy onto the upper leaves as<br />
the only risk factor requires<br />
modification <strong>and</strong> may well be<br />
misleading under some<br />
circumstances.<br />
Interactions between genetics<br />
<strong>and</strong> ecology are <strong>of</strong> interest, <strong>and</strong><br />
could complicate the population<br />
dynamics <strong>of</strong> the pathogen<br />
considerably, at worst rendering<br />
forecasts almost impossible. The<br />
fittest isolate on a given cultivar can<br />
vary with temperature (B. al-<br />
Hamar, University <strong>of</strong> Reading,<br />
personal communication). This<br />
means that equivalent sized<br />
populations could have<br />
intrinsically different growth-rates<br />
according to the combination <strong>of</strong><br />
weather conditions which gave rise<br />
to them, so that identical<br />
environmental conditions <strong>and</strong><br />
initial population sizes would give<br />
rise to different population<br />
responses.<br />
Between crops<br />
Windblown ascospores appear<br />
to be the main mechanism by<br />
which M. graminicola moves<br />
between crops, both from one<br />
Epidemiology <strong>of</strong> Mycosphaerella graminicola <strong>and</strong> Phaeosphaeria nodorum: An Overview 95<br />
season to the next <strong>and</strong> within a<br />
season. However, there is little<br />
documentation <strong>of</strong> the relative<br />
importance <strong>of</strong> local debris <strong>and</strong><br />
widely dispersed, generalized<br />
sources <strong>of</strong> inoculum for new crops.<br />
In northern Europe, with most<br />
wheat grown in rotation <strong>and</strong> with<br />
full tillage, the generalized air<br />
spora appear to dominate sources.<br />
However, this situation must differ<br />
in different farming systems. Where<br />
wheat is a scarcer crop, the<br />
generalized concentration <strong>of</strong><br />
ascospores in the air must be lower,<br />
<strong>and</strong> where no-till <strong>and</strong> continuous<br />
cropping is common but the wheat<br />
fields are scattered, one would<br />
expect local sources to dominate.<br />
There is relatively little evidence<br />
published about this outside<br />
Europe <strong>and</strong> northern America. A<br />
curious feature <strong>of</strong> the disease in, for<br />
example, the UK seems to be that<br />
the extremely efficient control <strong>of</strong><br />
the pathogen achieved on the<br />
upper part <strong>of</strong> the plant by fungicide<br />
does not appear to have reduced<br />
the ability <strong>of</strong> the fungus to produce<br />
ascospores <strong>and</strong> infect subsequent<br />
crops. This could have two<br />
explanations: 1) in stubbles,<br />
ascospores are produced as much<br />
on the lower parts <strong>of</strong> plants, never<br />
managed by fungicide, as on the<br />
upper parts; or 2) the dominant<br />
triazole fungicides act only as<br />
fungistats <strong>and</strong> after the death <strong>of</strong><br />
leaves, fungal development <strong>and</strong> the<br />
production <strong>of</strong> pseudothecia can<br />
continue as if the treatment had<br />
never taken place. Genetic <strong>and</strong><br />
physiological evidence suggests<br />
that alternate hosts are unimportant<br />
in perpetuating the disease on<br />
wheat.<br />
As noted in a previous review<br />
(Shaw, 1999), the proportion <strong>of</strong> l<strong>and</strong><br />
devoted to wheat in a region could<br />
affect the importance <strong>of</strong> M.<br />
graminicola as a pathogen. As the<br />
proportion increases, so the<br />
distance between last year’s fields<br />
<strong>and</strong> this year’s will decline. Even<br />
by wind, the number <strong>of</strong> spores<br />
moving a given distance declines<br />
rapidly with distance, so moderate<br />
changes in average distance<br />
between fields could cause<br />
substantial changes in the density<br />
<strong>of</strong> initial infections. In turn, at low<br />
densities, this could affect the<br />
importance <strong>of</strong> the disease at the end<br />
<strong>of</strong> the cropping season. The detail<br />
depends on how cropl<strong>and</strong> is<br />
organized <strong>and</strong> how new<br />
wheatl<strong>and</strong>s tend to be disposed<br />
relative to old, as well as on the<br />
strength <strong>of</strong> linkages between initial<br />
<strong>and</strong> end-<strong>of</strong>-season disease.<br />
At the Long Ashton conference<br />
in 1997, a crucial question in<br />
underst<strong>and</strong>ing the uniformity in<br />
genetic composition <strong>of</strong> M.<br />
graminicola worldwide was whether<br />
seed-borne transport could ever<br />
occur. This apparently simple<br />
question remains open, <strong>and</strong> is far<br />
from simple, since very, very rare<br />
events could provide enough<br />
migration to leave populations<br />
genetically linked. Suppose a<br />
million seeds each from a diseased<br />
crop <strong>and</strong> a fungicide treated crop<br />
are grown, <strong>and</strong> a lesion appears on<br />
one plant from the diseased crop.<br />
Can we confidently say that lesion<br />
arose from seed transmission or<br />
could it have been attributable to a<br />
failure <strong>of</strong> isolation <strong>and</strong> a stray<br />
ascospore? Some form <strong>of</strong> gene<br />
exchange between widely<br />
dispersed populations apparently<br />
occurs, so the geneticists tell us, but<br />
it may be almost impossible to<br />
distinguish the various categories<br />
<strong>of</strong> very rare events giving rise to<br />
this.
96<br />
Session 5 — M.W. Shaw<br />
The pathogen appears to have<br />
been becoming more widespread<br />
worldwide. A most interesting<br />
series <strong>of</strong> data is provided by Gilbert<br />
(1998) showing a more or less<br />
linear increase in M. graminicola<br />
incidence between 1990 <strong>and</strong> 1995 in<br />
southern Manitoba, from a very<br />
scarce disease to the most<br />
prevalent. Coupled with the<br />
genetic evidence that populations<br />
worldwide are genetically very<br />
similar (McDonald et al., 1995) it<br />
does seem possible that a novel<br />
form <strong>of</strong> the pathogen has been<br />
spreading worldwide. If true, this<br />
would raise the interesting<br />
question as to what<br />
epidemiological characteristic<br />
confers the new form’s<br />
invasiveness.<br />
Phaeosphaeria<br />
nodorum<br />
Within crops<br />
Multiplication is more rain<br />
dependent than M. graminicola,<br />
since pycnidia are produced in<br />
response to rain as well as<br />
dispersed by it. Conidia require<br />
wet periods to infect <strong>and</strong> do not<br />
tolerate interruptions well. The<br />
pathogen also appears to multiply<br />
ineffectively in cold conditions.<br />
Correlations with weather<br />
conditions are much better (Djurle<br />
et al., 1996; Gilbert et al., 1998) <strong>and</strong><br />
more closely linked in time to<br />
serious outbreaks than for M.<br />
graminicola. In wet, warm weather,<br />
typical <strong>of</strong> summer in some regions,<br />
latent periods can be shorter than<br />
in M. graminicola. Progress up the<br />
growing plant is correspondingly<br />
faster <strong>and</strong> more regular<br />
(Mittermeier <strong>and</strong> H<strong>of</strong>fmann, 1984).<br />
Like M. graminicola spores,<br />
spores <strong>of</strong> some isolates <strong>of</strong> P.<br />
nodorum infect with a startlingly<br />
high probability when rare on a leaf<br />
surface, the infection efficiency per<br />
spore falling rapidly with total<br />
numbers attacking in a droplet<br />
(Jeger et al., 1985)(MWS,<br />
unpublished). The ecological<br />
advantage is presumably that<br />
reinfection <strong>of</strong> a new crop is very<br />
effective, after which survival <strong>of</strong> the<br />
isolate is more or less certain. The<br />
epidemiological consequence is<br />
surprisingly rapid early spread <strong>of</strong><br />
the pathogen with subsequent rapid<br />
decline in apparent epidemic rate.<br />
Between crops<br />
Phaeosphaeria nodorum can be<br />
seedborne, <strong>and</strong> before routine<br />
fungicide treatment <strong>of</strong> seed <strong>and</strong> the<br />
upper parts <strong>of</strong> the crop, substantial<br />
numbers <strong>of</strong> seeds were infected. In<br />
some regions, such as northern<br />
Europe, the disease appears to have<br />
become much less important during<br />
the 1980s <strong>and</strong> 1990s. It is tempting<br />
to ascribe this decline to the<br />
increased use <strong>of</strong> fungicides<br />
reducing seed infection both in the<br />
ear <strong>and</strong> at planting. Recent<br />
experiments in other parts <strong>of</strong> the<br />
world suggest that seedborne<br />
inoculum is crucial in many places<br />
(Milus <strong>and</strong> Chalkley, 1997).<br />
Alternate hosts seem to harbor<br />
isolates <strong>of</strong> the pathogen capable <strong>of</strong><br />
attacking wheat, but there is<br />
sufficient partial specialization that<br />
these do not act as the source <strong>of</strong><br />
most infections on wheat as well as<br />
isolates clearly partially specialized<br />
to other hosts (Krupinsky, 1997a;<br />
1997b). Crop residues have been<br />
shown to be important (Cunfer,<br />
1998) but international survey<br />
evidence does not suggest they are<br />
the dominant source <strong>of</strong> inoculum<br />
(Leath et al., 1994).<br />
Ascospores have been found,<br />
but the evidence for their ubiquity<br />
<strong>and</strong> epidemiological importance<br />
seems to vary geographically.<br />
Trapping experiments like those<br />
used to show how airborne<br />
inoculum <strong>of</strong> P. nodorum varies<br />
through the year have been<br />
published by Cunfer (1998). These<br />
showed that in Georgia, USA,<br />
inoculum persisted for long periods<br />
in stubble, detectable sporadically<br />
for at least 18 months after the crop<br />
was harvested. As little as 17 m<br />
from a field edge, inoculum was<br />
trapped only once, in March, even<br />
though both mating types <strong>of</strong> the<br />
fungus were present <strong>and</strong> could<br />
potentially form perithecia. This<br />
suggests that sexual reproduction is<br />
unusual, though possibly not<br />
absent.<br />
These data are consistent with<br />
the relatively old published data on<br />
ascospore release in France (Rapilly<br />
et al., 1973). Anecdotally, near<br />
Reading in 1994 we observed a<br />
sudden patch <strong>of</strong> the disease in May<br />
in a first wheat. Although all<br />
isolations were from a single plot,<br />
<strong>and</strong> the disease had not been<br />
previously observed in that crop, 13<br />
genotypes tested using RFLPs were<br />
<strong>of</strong> different genotypes (C.N.F.M.R.J<br />
Pijls <strong>and</strong> M.W. Shaw, unpublished).<br />
However, the most extensive<br />
study made, in Pol<strong>and</strong>,<br />
demonstrated a quite different<br />
picture, with ascospores <strong>of</strong> P.<br />
nodorum present throughout the<br />
year <strong>and</strong> at densities that appeared<br />
to be independent <strong>of</strong> distance from<br />
the nearest infected plot (Arseniuk<br />
et al., 1998). Ascospores were also<br />
found throughout the year in<br />
northern Germany, but the trap was<br />
operated over an unharvested field,<br />
so it is not possible to say whether
the spores were from local or<br />
distant sources (Mittelstadt <strong>and</strong><br />
Fehrmann, 1987).<br />
References<br />
Arseniuk, E., T. Goral, <strong>and</strong> A.L.<br />
Scharen. 1998. Seasonal patterns <strong>of</strong><br />
spore dispersal <strong>of</strong> Phaeosphaeria<br />
spp. <strong>and</strong> <strong>Stagonospora</strong> spp. Plant<br />
Disease 82:187-194.<br />
Bannon, F.J., <strong>and</strong> B.M. Cooke. 1998.<br />
Studies on dispersal <strong>of</strong> <strong>Septoria</strong><br />
tritici pycnidiospores in wheatclover<br />
intercrops. Plant Pathology<br />
47:49-56.<br />
Brennan, R.M., B.D.L. Fitt, G.S. Taylor,<br />
<strong>and</strong> J. Colhoun. 1985a. Dispersal <strong>of</strong><br />
<strong>Septoria</strong> nodorum pycnidiospores by<br />
simulated rain <strong>and</strong> wild. Journal <strong>of</strong><br />
Phytopathology 112:291-297.<br />
Brennan, R.M., B.D.L. Fitt, G.S. Taylor,<br />
<strong>and</strong> J. Colhoun. 1985b. Dispersal <strong>of</strong><br />
<strong>Septoria</strong> nodorum pycnidiospores by<br />
simulated raindrops in still air.<br />
Journal <strong>of</strong> Phytopathology 112:281-<br />
290.<br />
Cunfer, B.M. 1998. Seasonal<br />
availability <strong>of</strong> inoculum <strong>of</strong><br />
<strong>Stagonospora</strong> nodorum in the field in<br />
the southeastern USA. Cereal<br />
Research Communications 26:259-<br />
263.<br />
Djurle, A., B. Ekbom, <strong>and</strong> J.E. Yuen.<br />
1996. The relationship <strong>of</strong> leaf<br />
wetness duration <strong>and</strong> disease<br />
progress <strong>of</strong> glume blotch, caused<br />
by <strong>Stagonospora</strong> nodorum, in winter<br />
wheat to st<strong>and</strong>ard weather data.<br />
European Journal <strong>of</strong> Plant<br />
Pathology 102:9-20.<br />
Eriksen, L., M.W. Shaw, <strong>and</strong> H.<br />
Østergård. 1999. A model <strong>of</strong> the<br />
effect <strong>of</strong> pseudothecia on epidemic<br />
progress <strong>and</strong> genetic composition<br />
in Mycosphaerella graminicola. (In<br />
preparation.)<br />
Eyal, Z. 1971. The kinetics <strong>of</strong><br />
pycnospore liberation in <strong>Septoria</strong><br />
tritici. Canadian Journal <strong>of</strong> Botany<br />
49:1095-1099.<br />
Gilbert, J., S.M. Woods, <strong>and</strong> A. Tekauz.<br />
1998. Relationship between<br />
environmental variables <strong>and</strong> the<br />
prevalence <strong>and</strong> isolation frequency<br />
<strong>of</strong> leaf-spotting pathogen in spring<br />
wheat. Canadian Journal <strong>of</strong> Plant<br />
Pathology 20:158-164.<br />
Epidemiology <strong>of</strong> Mycosphaerella graminicola <strong>and</strong> Phaeosphaeria nodorum: An Overview 97<br />
Hunter, T., R.R. Coker, <strong>and</strong> D.J. Royle.<br />
1999. The teleomorph stage,<br />
Mycosphaerella graminicola, in<br />
epidemics <strong>of</strong> septoria tritici blotch<br />
on winter wheat in the UK. Plant<br />
Pathology 48:51-57.<br />
Jeger, M.J., E. Griffiths, <strong>and</strong> D.G.<br />
Jones. 1985. The effects <strong>of</strong> postinoculation<br />
wet <strong>and</strong> dry periods,<br />
<strong>and</strong> inoculum concentration, on<br />
lesion numbers <strong>of</strong> <strong>Septoria</strong> nodorum<br />
in spring wheat seedlings. Annals<br />
<strong>of</strong> Applied Biology 106:55-63.<br />
Kema, G.H.J., E.C.P. Verstappen, M.<br />
Todorova, <strong>and</strong> C. Waalwijk. 1996.<br />
Successful crosses <strong>and</strong> molecular<br />
tetrad <strong>and</strong> progeny analyses<br />
demonstrate heterothallism in<br />
Mycosphaerella graminicola. Current<br />
Genetics 30:251-258.<br />
Krupinsky, J.M. 1997a. Aggressiveness<br />
<strong>of</strong> <strong>Stagonospora</strong> nodorum isolates<br />
from perennial grasses on wheat.<br />
Plant Disease 81:1032-1036.<br />
Krupinsky, J.M. 1997b. Stability <strong>of</strong><br />
<strong>Stagonospora</strong> nodorum isolates from<br />
perennial grass hosts after passage<br />
through wheat. Plant Disease<br />
81:1037-1041.<br />
Leath, S., A.L. Scharen, M.E.<br />
Dietzholmes, <strong>and</strong> R.E. Lund. 1994.<br />
Factors associated with global<br />
occurrences <strong>of</strong> septoria nodorum<br />
blotch <strong>and</strong> septoria tritici blotch <strong>of</strong><br />
wheat. Plant Disease 77:1266-1270.<br />
Lovell, D.J., S.R. Parker, T. Hunter, D.J.<br />
Royle, <strong>and</strong> R.R. Coker. 1997.<br />
Influence <strong>of</strong> crop growth <strong>and</strong><br />
structure on the risk <strong>of</strong> epidemics<br />
by Mycosphaerella graminicola<br />
(<strong>Septoria</strong> tritici) in winter wheat.<br />
Plant Pathology 46:126-138.<br />
McDonald, B.A., R.E. Pettway, R.S.<br />
Chen, J.M. Boerger, <strong>and</strong> J.P.<br />
Martinez. 1995. The population<br />
genetics <strong>of</strong> <strong>Septoria</strong> tritici<br />
(teleomorph Mycosphaerella<br />
graminicola). Canadian Journal <strong>of</strong><br />
Plant Pathology 73:292-301.<br />
Metcalfe, R.J. 1999. Selection for<br />
resistance to demethylation<br />
inhibitor fungicides in<br />
Mycosphaerella graminicola on<br />
wheat. PhD, The University <strong>of</strong><br />
Reading.<br />
Milus, E.A., <strong>and</strong> D.B. Chalkley. 1997.<br />
Effect <strong>of</strong> previous crop, seedborne<br />
inoculum, <strong>and</strong> fungicides on<br />
development <strong>of</strong> <strong>Stagonospora</strong><br />
blotch. Plant Disease 81:1279-1283.<br />
Mittelstadt, A., <strong>and</strong> H. Fehrmann.<br />
1987. Zum Auftreten der<br />
Hauptfruchtform von <strong>Septoria</strong><br />
nodorum in der Bundesrepublik<br />
Deutschl<strong>and</strong>. Zeitschrift für<br />
Pflanzenkrankheiten und<br />
Pflanzenschütz 94:380-385.<br />
Mittermeier, L., <strong>and</strong> G.M. H<strong>of</strong>fmann.<br />
1984. Untersuchungen zur<br />
Populationsentwicklung von<br />
<strong>Septoria</strong> nodorum in Feldbestund<br />
von Weizen. Zeitschrift fur<br />
Pflanzenkrankheiten und<br />
Pflanzenschutz 91:629-640.<br />
Rapilly, F., B. Foucault, <strong>and</strong> J.<br />
Lacazedieux. 1973. Études sur<br />
l’inoculum de <strong>Septoria</strong> nodorum<br />
Berk. (Leptosphaeria nodorum<br />
Müller) agent de la septoriose du<br />
blé. I. Les ascospores. Annales de<br />
Phytopathologie 5:131-141.<br />
Shaw, M.W. 1987. Assessment <strong>of</strong><br />
upward movement <strong>of</strong> rain splash<br />
using a fluorescent tracer method<br />
<strong>and</strong> its application to the<br />
epidemiology <strong>of</strong> cereal pathogens.<br />
Plant Pathology 36:201-213.<br />
Shaw, M.W. 1991a. Interacting effects<br />
<strong>of</strong> interrupted humid periods <strong>and</strong><br />
light on infection <strong>of</strong> wheat leaves<br />
by <strong>Septoria</strong> tritici (Mycosphaerella<br />
graminicola). Plant Pathology<br />
40:595-607.<br />
Shaw, M.W. 1991b. Variation in the<br />
height to which tracer is moved by<br />
splash during natural summer rain<br />
in the UK. Agricultural <strong>and</strong> Forest<br />
Meteorology 55:1-14.<br />
Shaw, M.W. 1999. Population<br />
dynamics <strong>of</strong> septoria in the crop<br />
ecosystem. In <strong>Septoria</strong> on <strong>Cereals</strong>:<br />
A Study <strong>of</strong> Pathosystems (J.A.<br />
Lucas, P. Bowyer <strong>and</strong> H.A.<br />
Anderson, eds.). CABI Publishing,<br />
Wallingford, UK. pp. 82-95.<br />
Shaw, M.W., <strong>and</strong> D.J. Royle. 1989.<br />
Airborne inoculum as a major<br />
source <strong>of</strong> <strong>Septoria</strong> tritici<br />
(Mycosphaerella graminicola)<br />
infections in winter wheat crops in<br />
the UK. Plant Pathology 38:35-43.<br />
Shaw, M.W., <strong>and</strong> D.J. Royle. 1993.<br />
Factors determining the severity <strong>of</strong><br />
Mycosphaerella graminicola (<strong>Septoria</strong><br />
tritici) on winter wheat in the UK.<br />
Plant Pathology 42:882-899.
98<br />
Spore Dispersal <strong>of</strong> Leaf Blotch Pathogens <strong>of</strong> Wheat<br />
(Mycosphaerella graminicola <strong>and</strong> <strong>Septoria</strong> tritici)<br />
C.A. Cordo, 1, 3 M.R. Simón, 2 A.E. Perelló, 1, 4 <strong>and</strong> H.E. Alippi1 1 Centro de Investigaciones de Fitopatología (CIDEFI), Facultad de Ciencias Agrarias y Forestales de La Plata,<br />
La Plata, Argentina<br />
2 Laboratorio de Cerealicultura, Facultad de Ciencias Agrarias y Forestales de La Plata, La Plata, Argentina<br />
3 Comisión de Investigaciones Científicas (CIC), Provincia de Buenos Aires, Argentina<br />
4 Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET), Provincia de Buenos Aires,<br />
Argentina<br />
Abstract<br />
The spatial <strong>and</strong> temporal patterns <strong>of</strong> discharge <strong>and</strong> dissemination <strong>of</strong> air-borne Mycosphaerella graminicola spores<br />
<strong>and</strong> <strong>of</strong> <strong>Septoria</strong> tritici spores through rain splash were studied. Spore traps were used to monitor both ascospores <strong>and</strong><br />
pycnidiospores when the wheat crop was in the vegetative <strong>and</strong> debris states. Relationships between distance from a point<br />
source <strong>and</strong> weather variables such as rainfall, relative humidity, <strong>and</strong> air temperature were analyzed. The release <strong>of</strong><br />
pycnidiospores was favored by rainfall, as was explained through the multiple regression model (18% <strong>of</strong> the variation). Air<br />
dispersal <strong>of</strong> ascospores <strong>and</strong> splash dispersal <strong>of</strong> pycnidiospores were significantly influenced by each <strong>of</strong> the weather<br />
variables. The number <strong>of</strong> air-borne ascospores increased in association with rainfall. The multiple regression model<br />
explained 59% <strong>of</strong> the variation. The correlation analysis showed significant association with temperature, humidity, <strong>and</strong><br />
rainfall; the regression coefficients <strong>of</strong> the climatic variables were significant. The effect <strong>of</strong> different distances from the<br />
inoculum source on the density <strong>of</strong> rain-splashed pycnidiospores <strong>and</strong> wind-borne ascospores was not significant.<br />
Pycnidiospores were the omnipresent inoculum in the cereal-producing area during the observed period. Thus this<br />
inoculum poses a risk to crop production <strong>and</strong> may be important to the epidemiology <strong>of</strong> septoria diseases under the climatic<br />
conditions in the wheat-producing areas <strong>of</strong> Argentina.<br />
Leaf blotch, caused by <strong>Septoria</strong><br />
tritici Rob.ex Desm. (teleomorph<br />
Mycosphaerella graminicola (Fuckel)<br />
Schroeter, in Cohn) is an important<br />
wheat (Triticum aestivum) disease<br />
that causes yield losses in different<br />
regions <strong>of</strong> the world every year<br />
(Shipton et al., 1971; Eyal, 1981;<br />
Eyal et al., 1987). Its incidence<br />
depends on cultivar susceptibility,<br />
inoculum availability, crop<br />
management practices, <strong>and</strong><br />
favorable environmental conditions<br />
(cool temperature, high humidity,<br />
<strong>and</strong> frequent rain). Climatic factors,<br />
especially precipitation, affect<br />
fungal growth <strong>and</strong> the amount <strong>and</strong><br />
timing <strong>of</strong> spore production, as well<br />
as the release, dispersal, <strong>and</strong><br />
deposition <strong>of</strong> spores. Unusually<br />
intense rain may cause the onset <strong>of</strong><br />
a S. tritici epidemic in a wheat crop.<br />
The greatest risk to a crop is related<br />
to the occurrence <strong>of</strong> conditions that<br />
favor spore dispersal during <strong>and</strong><br />
shortly after flag leaf emergence.<br />
Spore dispersal <strong>and</strong> infection at this<br />
time favors a second generation <strong>of</strong><br />
pathogens. Spore dissemination<br />
patterns <strong>of</strong> Phaeosphaeria spp. <strong>and</strong><br />
<strong>Stagonospora</strong> spp. have been<br />
described (Arseniuk <strong>and</strong> Góral,<br />
1998; Góral <strong>and</strong> Arseniuk, 1998;<br />
1991). The release <strong>of</strong> M. graminicola<br />
ascospores occurred at two peak<br />
times <strong>of</strong> the year (at crop<br />
emergence <strong>and</strong> emergence <strong>of</strong> the<br />
upper two leaves) (T. Hunter<br />
unpublished).<br />
This work aimed at producing a<br />
mathematical model <strong>of</strong><br />
pycnidiospore <strong>and</strong> ascospore<br />
dispersal by:<br />
1. determining the pattern <strong>of</strong> spore<br />
dispersal during a 6-month<br />
period;<br />
2. investigating the effect <strong>of</strong><br />
climatic variables (rainfall,<br />
temperature, humidity) on<br />
density <strong>of</strong> air-borne spores; <strong>and</strong><br />
3. doing a preliminary analysis <strong>of</strong><br />
the dispersal distance for both<br />
types <strong>of</strong> spores.<br />
Materials <strong>and</strong> Methods<br />
In the experiment station<br />
situated in Los Hornos, near La<br />
Plata, Buenos Aires Province, airborne<br />
spores were collected on 13<br />
spore traps made <strong>of</strong> PVC capsules<br />
containing slides covered with<br />
petroleum jelly. The capsules were<br />
fixed to wooden stakes 0.7 m above<br />
the soil surface. Glass tubes 0.03 m<br />
in diameter <strong>and</strong> 0.16 m long were
placed near the capsules to catch<br />
rain-splashed spores. The capsules<br />
moved with the wind to ensure that<br />
the slides caught spores in different<br />
weather conditions. The spore traps<br />
were located at different distances<br />
(11 <strong>of</strong> them at 1.5 m <strong>and</strong> 2 at 12 m)<br />
from a wheat plot 30 m long <strong>and</strong> 10<br />
m wide. Spore samplers were<br />
arranged parallel to the length <strong>of</strong><br />
the test plot.<br />
The plot was sown on 24 June<br />
1998 <strong>and</strong> inoculated twice (17 July<br />
<strong>and</strong> 12 August) to obtain high<br />
disease incidence. Plants were<br />
inoculated when the second leaf<br />
unfolded (GS12). The main shoot<br />
<strong>and</strong> second side shoot (GS 22)<br />
(Zadoks et al., 1974) were<br />
inoculated by spraying a<br />
pycnidiospore suspension (5 x 10 6<br />
spores/ml) <strong>of</strong> one S. tritici isolate.<br />
Slides were collected <strong>and</strong> examined<br />
weekly from October 1998 to March<br />
1999. The rain-splashed spores were<br />
identified by observing one drop <strong>of</strong><br />
rain water stained with aniline blue<br />
(1/100) solution under the<br />
microscope. Four spore counts (1<br />
cm 2 each) were done on every<br />
sample. Furthermore, ascospores<br />
<strong>and</strong> pycnidiospores on the entire<br />
slide area covered with petroleum<br />
jelly were counted.<br />
Different populations <strong>of</strong> spores<br />
were observed under a light<br />
microscope at 100x magnification.<br />
Identification was based on the<br />
morphology <strong>of</strong> Mycosphaerella spp.<br />
<strong>and</strong> Phaeosphaeria spp. ascospores<br />
<strong>and</strong> <strong>Septoria</strong> spp. pycnidiospores.<br />
Viability <strong>of</strong> air-borne spores was<br />
determined by recording their<br />
germination directly on the<br />
petroleum jelly on the slides.<br />
Climatic variables (rainfall,<br />
temperature, <strong>and</strong> relative humidity)<br />
were recorded at the meteorological<br />
observatory in La Plata.<br />
Spore Dispersal <strong>of</strong> Leaf Blotch Pathogens <strong>of</strong> Wheat (Mycosphaerella graminicola <strong>and</strong> <strong>Septoria</strong> tritici) 99<br />
Multiple regression analyses<br />
were used to explore the<br />
relationship between the number <strong>of</strong><br />
pycnidiospores in rain water <strong>and</strong><br />
petroleum jelly, <strong>and</strong> ascospores in<br />
petroleum jelly, <strong>and</strong> the three<br />
weather variables. An analysis <strong>of</strong><br />
variance was performed to<br />
determine significant differences<br />
between spore traps placed at<br />
different distances. Pycnidiospore<br />
<strong>and</strong> ascospore dispersion was<br />
plotted in relation to climatic<br />
variables.<br />
Results <strong>and</strong> Discussion<br />
During the observed period, the<br />
weekly mean temperature ranged<br />
between 14ºC <strong>and</strong> 25ºC, relative<br />
humidity between 64 <strong>and</strong> 84%, <strong>and</strong><br />
rainfall between 0 <strong>and</strong> 135 mm.<br />
Figure 1a <strong>and</strong> b, <strong>and</strong> Figure 2a,<br />
b, <strong>and</strong> c show the distribution <strong>of</strong><br />
pycnidospores in rain water <strong>and</strong><br />
petroleum jelly <strong>and</strong> <strong>of</strong> ascospores in<br />
no. spores/slide/week<br />
no. spores/slide/week<br />
14<br />
12<br />
10<br />
8<br />
6<br />
4<br />
2<br />
0<br />
40<br />
35<br />
30<br />
25<br />
20<br />
15<br />
10<br />
5<br />
0<br />
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8/11/98<br />
Pycnidiospores<br />
Ascospores<br />
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25/10/98<br />
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8/11/98<br />
15/11/98<br />
22/11/98<br />
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29/11/98<br />
5/12/98<br />
29/11/98<br />
5/12/98<br />
12/12/98<br />
12/12/98<br />
rain water with weather variables<br />
throughout the 24 weeks <strong>of</strong><br />
sampling.<br />
During the 6-month period,<br />
pycnidiospores were always<br />
present in both types <strong>of</strong> sampling.<br />
Figure 1a shows an increase in rainsplashed<br />
pycnidiospores around<br />
the week <strong>of</strong> 21 December. This<br />
increase was associated with a high<br />
rainfall regimen in which intense<br />
rains lasting as long as five hours<br />
without interruption occurred.<br />
However, correlation coefficients<br />
<strong>and</strong> partial correlation coefficients<br />
with weather variables were not<br />
significant (Table 1). Application <strong>of</strong><br />
a multiple regression model did not<br />
yield significant numbers either<br />
(Table 2). The increased amount <strong>of</strong><br />
pycnidiospores was associated with<br />
the increase in rainfall between 22<br />
January <strong>and</strong> 9 February 1999. The<br />
pycnidiospore pattern showed a<br />
marked reduction from 30<br />
December to 9 February. This was<br />
Mean <strong>of</strong> pycnidiospores<br />
21/12/98<br />
30/12/98<br />
Sampling date<br />
21/12/98<br />
30/12/98<br />
6/1/99<br />
15/1/99<br />
6/1/99<br />
15/1/99<br />
22/1/99<br />
29/1/99<br />
Mean <strong>of</strong> pycnidiospores <strong>and</strong> ascospores<br />
Sampling date<br />
22/1/99<br />
29/1/99<br />
9/2/99<br />
17/2/99<br />
9/2/99<br />
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26/2/99<br />
5/3/99<br />
12/3/99<br />
19/3/99<br />
12/3/99<br />
19/3/99<br />
Figure 1. Mean counts <strong>of</strong> pycnidiospores in rainwater (a), <strong>and</strong> pycnidiospores <strong>and</strong> ascospores in<br />
petroleum jelly (b) settled on microscope slides.<br />
A<br />
27/3/99<br />
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Session 5 — C.A. Cordo, M.R. Simón, A.E. Perelló, <strong>and</strong> H.E. Alippi<br />
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180<br />
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40<br />
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25<br />
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Rainfall (mm)<br />
12/12/98<br />
21/12/98<br />
30/12/98<br />
6/1/99<br />
Sampling date<br />
Temperature (°C)<br />
12/12/98<br />
21/12/98<br />
30/12/98<br />
6/1/99<br />
Sampling date<br />
Relative humidity (%)<br />
12/12/98<br />
21/12/98<br />
30/12/98<br />
6/1/99<br />
Sampling date<br />
15/1/99<br />
22/1/99<br />
15/1/99<br />
22/1/99<br />
15/1/99<br />
22/1/99<br />
29/1/99<br />
9/2/99<br />
29/1/99<br />
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29/1/99<br />
9/2/99<br />
17/2/99<br />
26/2/99<br />
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26/2/99<br />
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26/2/99<br />
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12/3/99<br />
19/3/99<br />
12/3/99<br />
19/3/99<br />
12/3/99<br />
19/3/99<br />
Figure 2. Weekly mean rainfall (a), weekly mean temperature (b), <strong>and</strong> weekly mean relative<br />
humidity (c).<br />
related to the end <strong>of</strong> the crop<br />
vegetative period <strong>and</strong> the advent <strong>of</strong><br />
saprophytic pathogens on the<br />
wheat debris. A new increase in<br />
pycnidiospores on 5 March was<br />
related to the first natural infection<br />
<strong>of</strong> seedlings by septoria leaf blotch.<br />
The pycnidiospore pattern in<br />
petroleum jelly was constant from<br />
the first week <strong>of</strong> observation<br />
(Figure 1b). The increased density<br />
was associated with the amount <strong>of</strong><br />
rain water. The correlation<br />
A<br />
27/3/99<br />
B<br />
27/3/99<br />
C<br />
27/3/99<br />
coefficient <strong>and</strong> the partial<br />
regression coefficient between<br />
pycnidiospores <strong>and</strong> rainfall were<br />
significant (Table 1), as was the<br />
regression coefficient for rainfall in<br />
the multiple regression model. This<br />
model explained 18% <strong>of</strong> the<br />
variance (Table 2). Temperature <strong>and</strong><br />
humidity during this period were<br />
probably not so high as to affect the<br />
number <strong>of</strong> pycnidiospores released.<br />
Similar to observations by Arseniuk<br />
<strong>and</strong> Góral (1998), we recorded high<br />
Table 1. Correlation coefficients <strong>and</strong> partial<br />
correlation coefficients between number <strong>of</strong><br />
pycnidiospores in rain water <strong>and</strong> petroleum<br />
jelly <strong>and</strong> number <strong>of</strong> ascospores <strong>of</strong> <strong>Septoria</strong><br />
tritici <strong>and</strong> Mycosphaerella graminicola with<br />
weather variables.<br />
Temp- Relative<br />
erature humidity Rainfall<br />
Pycnidiospores/<br />
rain water -0.20 ns a 0.06 ns 0.26 ns<br />
-0.19 ns b 0.04 ns 0.04 ns<br />
Pycnidiospores/<br />
p. jelly 0.12 ns 0.11 ns 0.53**<br />
0.27 ns 0.01 ns 0.46*<br />
Ascospores 0.53** 0.46* 0.57**<br />
0.62** 0.42* 0.60**<br />
a= correlation coefficient.<br />
b= partial correlation coefficient.<br />
Table 2. Multiple regression model <strong>of</strong><br />
weather variables <strong>and</strong> number <strong>of</strong><br />
pycnidiospores in rain water, petroleum jelly,<br />
<strong>and</strong> ascospores <strong>of</strong> <strong>Septoria</strong> tritici <strong>and</strong><br />
Mycosphaerella graminicola.<br />
Significance<br />
Coefficient level<br />
No. <strong>of</strong> pycnidiospores<br />
in water<br />
Constant 9.33<br />
Temperature -0.26 ns<br />
Relative humidity -0.01 ns<br />
Rainfall<br />
No. <strong>of</strong> pycnidiospores<br />
in p. jelly<br />
0.02 ns<br />
Constant 11.31 ns<br />
Temperature 0.20 ns<br />
Relative humidity 0.07 ns<br />
Rainfall<br />
No. <strong>of</strong> ascospores<br />
0.08 P
ainfall increased. Periods <strong>of</strong> low<br />
rainfall were associated with low<br />
ascospore density (30 December to<br />
15 January, 17 February, <strong>and</strong> 5<br />
March). Temperatures after week 12<br />
were in general above 20ºC,<br />
whereas in the previous period they<br />
were generally lower. It is thus<br />
likely that both the disease cycle<br />
<strong>and</strong> the increase in temperature<br />
affected ascospore release. Analysis<br />
<strong>of</strong> the partial correlation, which<br />
considers variables as being<br />
independent, showed significant<br />
association with temperature,<br />
relative humidity, <strong>and</strong> rainfall<br />
(Table 1). The multiple regression<br />
model (Table 2) explained 59% <strong>of</strong><br />
the variance, <strong>and</strong> the regression<br />
coefficients for climatic variables<br />
were significant. As Arseniuk <strong>and</strong><br />
Góral established in 1998, the<br />
abundance <strong>of</strong> ascospores reached<br />
its peak at harvest time. After<br />
harvest, plant debris decomposed<br />
<strong>and</strong> the amount <strong>of</strong> air-borne spores<br />
was reduced. Despite this<br />
reduction, ascospores were also<br />
released when there was low<br />
rainfall <strong>and</strong> adequate relative<br />
humidity. Arseniuk <strong>and</strong> Góral<br />
(1998) observed that less than 1 mm<br />
<strong>of</strong> rainfall <strong>and</strong> high relative<br />
humidity were enough to increase<br />
the number <strong>of</strong> air-borne ascospores.<br />
Spore Dispersal <strong>of</strong> Leaf Blotch Pathogens <strong>of</strong> Wheat (Mycosphaerella graminicola <strong>and</strong> <strong>Septoria</strong> tritici) 101<br />
Finally, the effect <strong>of</strong> different<br />
distance from the inoculum source<br />
(1 m <strong>and</strong> 12 m) on the density <strong>of</strong><br />
ascospores <strong>and</strong> pycnidiospores was<br />
not significant, in agreement with<br />
Góral <strong>and</strong> Arseniuk (in press 1998;<br />
Arseniuk <strong>and</strong> Góral, 1998) (Table<br />
3). The number <strong>of</strong> spores near the<br />
inoculum source <strong>and</strong> at 12 m was<br />
similar.<br />
It is concluded that air dispersal<br />
<strong>of</strong> ascospores <strong>and</strong> pycnidiospores<br />
plays a role in the epidemiology <strong>of</strong><br />
Mycosphaerella (<strong>Septoria</strong>) blotch<br />
under the climatic conditions east<br />
<strong>of</strong> Buenos Aires, Argentina.<br />
Pycnidiospores were produced in<br />
greater abundance than ascospores<br />
<strong>and</strong> they were the predominant<br />
source <strong>of</strong> inoculum. The abundance<br />
<strong>of</strong> pycnidiospores probably reflects<br />
their greater importance in the<br />
epidemiology <strong>of</strong> leaf blotch <strong>of</strong><br />
wheat.<br />
Table 3. Analysis <strong>of</strong> variance for mean values<br />
<strong>of</strong> pycnidiospores <strong>and</strong> ascospores sampled at<br />
different distances from an inoculum source.<br />
Source <strong>of</strong> variation Mean square<br />
Distance 53.7 ns<br />
Error 35.0<br />
ns= not significant according to the F test. P=0.05.<br />
Acknowledgments<br />
We are grateful to the Comisión<br />
de Investigaciones Científicas,<br />
Buenos Aires Province, <strong>and</strong> the<br />
Consejo Nacional de<br />
Investigaciones Científicas y<br />
Tecnológicas for their financial<br />
support. We also thank Ing. Agr. D.<br />
Bayo, Mrs. N. Kripelz, Mr. B. Cordo<br />
López, <strong>and</strong> J. Balonga for their<br />
technical assistance.<br />
References<br />
Arseniuk, E., <strong>and</strong> T. Góral. 1998.<br />
Seasonal patterns <strong>of</strong> spore<br />
dispersal <strong>of</strong> Phaeosphaeria spp. <strong>and</strong><br />
<strong>Stagonospora</strong> spp. Plant Dis. 82:187-<br />
194.<br />
Eyal, Z. 1981 Integrated control <strong>of</strong><br />
<strong>Septoria</strong> diseases <strong>of</strong> wheat. Plant<br />
Dis. 65:763-768.<br />
Eyal, Z., A.L. Scharen, M.J. Prescott,<br />
<strong>and</strong> M. van Ginkel. 1987. The<br />
<strong>Septoria</strong> <strong>Diseases</strong> <strong>of</strong> Wheat:<br />
Concepts <strong>and</strong> Methods <strong>of</strong> Disease<br />
Management. Mexico, D.F.:<br />
<strong>CIMMYT</strong>.<br />
Góral, T., <strong>and</strong> E. Arseniuk. 1991.<br />
Effect <strong>of</strong> climatic conditions on<br />
liberation <strong>and</strong> dispersal <strong>of</strong> spores<br />
<strong>of</strong> Leptosphaeria spp. in the air.<br />
Phytopath. Polonica 2 (XIV):28-34.<br />
Góral, T., <strong>and</strong> E. Arseniuk. 1998. The<br />
study <strong>of</strong> relationship between<br />
distance from a point inoculum<br />
source, weather variables, <strong>and</strong> the<br />
air presence <strong>of</strong> Phaeosphaeria spp.<br />
ascospores in warmer <strong>and</strong> cooler<br />
sub-periods <strong>of</strong> a growing season.<br />
Plant Breeding <strong>and</strong> Seed Science<br />
(in press).<br />
Shipton, W.A., W.J.R. Boyd, A.A.<br />
Rossielle, <strong>and</strong> B.I. Shearer. 1971.<br />
The common <strong>Septoria</strong> diseases <strong>of</strong><br />
wheat. Bot. Rev. 27:231-262.<br />
Zadoks, J.C., Chang, T.T., <strong>and</strong><br />
Konzak, C.F. 1974. A decimal code<br />
for the growth stages <strong>of</strong> cereals.<br />
Weed Research 14: 415-421.
102<br />
Epidemiology <strong>of</strong> Seedborne <strong>Stagonospora</strong> nodorum:<br />
A Case Study on New York Winter Wheat<br />
D.A. Shah <strong>and</strong> G.C. Bergstrom<br />
Department <strong>of</strong> Plant Pathology, Cornell University, Ithaca, New York, USA<br />
Abstract<br />
<strong>Stagonospora</strong> nodorum blotch is the most important component <strong>of</strong> the foliar disease complex that attacks New York winter<br />
wheat. Ascospores <strong>of</strong> the teleomorph Phaeosphaeria nodorum are not commonly observed in New York, where wheat is<br />
generally preceded in sequence by several years <strong>of</strong> nonhost, rotational crops. Wheat seed infection by <strong>Stagonospora</strong><br />
nodorum is common, the extent <strong>and</strong> range depending mainly on rainfall during the production season. Transmission <strong>of</strong> the<br />
pathogen from infected seeds to coleoptiles can approach 100% over a wide soil temperature range; transmission to the first<br />
leaves is less than 50% <strong>and</strong> is most efficient at soil temperatures below 17 ºC. Nevertheless, under the high densities at which<br />
wheat is sown, a significant number <strong>of</strong> infected seedlings per unit area can originate from relatively low initial seed infection<br />
levels <strong>and</strong> transmission efficiencies. <strong>Stagonospora</strong> nodorum blotch epidemics arising from infected seed potentially can be<br />
managed by reducing initial seedborne inoculum <strong>and</strong> its transmission. Wheat cultivars exhibit differential responses to<br />
infection <strong>of</strong> seed by S. nodorum, <strong>and</strong> this seed resistance appears to be under genetic control separate from foliar resistance.<br />
Breeding for reduced frequencies <strong>of</strong> seed infection <strong>and</strong> transmission, along with improved disease management in seed<br />
production fields <strong>and</strong> the application <strong>of</strong> seed fungicides, may comprise an effective integrated strategy for managing<br />
stagonospora nodorum blotch in wheat production systems similar to that in New York.<br />
The Pathosystem<br />
in New York<br />
Approximately 53,000 hectares<br />
<strong>of</strong> s<strong>of</strong>t winter wheat (mainly white<br />
cultivars for pastry flour) are<br />
cultivated annually as a rotational<br />
crop on vegetable, dairy, <strong>and</strong> cash<br />
grain farms in western <strong>and</strong> central<br />
New York. <strong>Stagonospora</strong> nodorum<br />
blotch is the most prevalent <strong>and</strong><br />
severe foliar disease affecting the<br />
crop (Schilder <strong>and</strong> Bergstrom,<br />
1989). The disease is currently<br />
undermanaged. Adapted wheat<br />
cultivars are susceptible, <strong>and</strong><br />
because <strong>of</strong> low grain prices <strong>and</strong><br />
inconsistent returns on fungicide<br />
expenditure, few producers apply<br />
foliar fungicide.<br />
Several observations led to the<br />
hypothesis that infected seed is the<br />
primary inoculum source for<br />
stagonospora nodorum blotch<br />
epidemics in New York. Firstly,<br />
infected wheat debris, regarded as<br />
the primary inoculum source in<br />
continuously or frequently cropped<br />
wheat systems, is eliminated from<br />
wheat fields by three- to six-year<br />
rotations. New York wheat fields<br />
are also relatively small <strong>and</strong><br />
scattered, reducing the chances <strong>of</strong><br />
infected debris blowing from one<br />
wheat field to another. <strong>Stagonospora</strong><br />
nodorum infects several grasses, but<br />
their role in epidemic initiation on<br />
wheat is likely minimal (Krupinsky,<br />
1997), <strong>and</strong> there is some evidence<br />
<strong>of</strong> host specificity (Ueng et al., 1994;<br />
Krupinsky, 1997). The teleomorphic<br />
stage, P. nodorum, formed typically<br />
on wheat straw, has not been<br />
recovered in New York, despite<br />
concerted searches <strong>of</strong> debris <strong>and</strong><br />
aerial spore trapping from fields<br />
affected heavily by stagonospora<br />
nodorum blotch (Bergstrom,<br />
unpublished). Moreover, most<br />
straw is baled <strong>and</strong> removed from<br />
fields shortly after harvest, <strong>and</strong> the<br />
remaining stubble is sometimes<br />
plowed under in late summer<br />
before planting alfalfa or grass, or<br />
is plowed the following spring<br />
prior to planting <strong>of</strong> corn,<br />
vegetables, or soybean.<br />
About 40% <strong>of</strong> New York winter<br />
wheat is underseeded with red<br />
clover, which serves as a winter<br />
cover crop. Clover attains a height<br />
<strong>of</strong> 30 to 40 cm, enough to<br />
completely cover unplowed<br />
stubble 20 to 25 cm high, thus<br />
impeding spore dispersal.<br />
Assuming that the P. nodorum stage<br />
does occur undetected, current<br />
cultural practices would diminish<br />
substantially its role in disease<br />
epidemiology in New York wheat.<br />
Shah et al. (1995), using DNA<br />
fingerprinted isolates <strong>of</strong> the<br />
pathogen, demonstrated that<br />
seedborne S. nodorum could initiate<br />
foliar epidemics under New York<br />
field conditions <strong>and</strong> foliar disease<br />
severity during grain development<br />
may be correlated with seed<br />
infection incidence (Shah et al.,<br />
1995).
Seed Surveys<br />
Most New York wheat<br />
producers sow certified seed that<br />
has passed minimal st<strong>and</strong>ards for<br />
germination, cultivar purity,<br />
contamination by weed seed <strong>and</strong><br />
foreign materials, <strong>and</strong> infection by<br />
smuts <strong>and</strong> bunts. Infection by S.<br />
nodorum is not part <strong>of</strong> the<br />
certification st<strong>and</strong>ard. Winter<br />
wheat seedlots were surveyed from<br />
1990 to 1996 for S. nodorum by<br />
assaying seed samples on SNAW<br />
medium (Man<strong>and</strong>har <strong>and</strong> Cunfer,<br />
1991). Parts <strong>of</strong> the survey have<br />
been published (Shah <strong>and</strong><br />
Bergstrom, 1993). In some years, all<br />
sampled lots contained seed<br />
infected by S. nodorum, <strong>and</strong> no<br />
cultivar was completely resistant to<br />
seed infection. Seasons with higher<br />
rainfall during wheat elongation<br />
through grain formation stages<br />
resulted in elevated seed infection<br />
incidences compared to low rainfall<br />
seasons (Figure 1).<br />
Transmission from<br />
Infected Seed<br />
Four lots (representing the cvs.<br />
Cayuga, Geneva, <strong>and</strong> Harus),<br />
ranging in infection incidence from<br />
Seed infection incidence (%)<br />
50<br />
140<br />
40<br />
120<br />
100<br />
30<br />
20<br />
1-71<br />
2-77<br />
80<br />
60<br />
40<br />
10<br />
0-19<br />
0-42<br />
20<br />
0<br />
1990 1991 1995<br />
Year<br />
1996<br />
0<br />
Figure 1. Mean seed infection incidence by<br />
<strong>Stagonospora</strong> nodorum in New York winter<br />
wheat lots in four production seasons.<br />
Incidence <strong>of</strong> infection range is shown over<br />
each bar. Rainfall averaged over the months<br />
<strong>of</strong> April-June <strong>and</strong> the 15 major wheat<br />
growing counties in western <strong>and</strong> central<br />
New York are shown by the line graph.<br />
Rain (mm)<br />
Epidemiology <strong>of</strong> Seedborne <strong>Stagonospora</strong> nodorum: A Case Study on New York Winter Wheat 103<br />
53 to 96%, were sown in soil at five<br />
temperatures (9, 13, 17, 21, 25 ºC),<br />
at 90 % relative humidity <strong>and</strong> a 16h<br />
photoperiod. All seedlings were<br />
harvested at the second leaf<br />
emerging stage, <strong>and</strong> coleoptiles<br />
were inspected for lesions induced<br />
by S. nodorum. Segments <strong>of</strong> the first<br />
leaf were plated onto Bannon’s<br />
medium (Bannon, 1978). Plates<br />
were incubated for 14 d under near<br />
ultraviolet light at room<br />
temperature. Leaf pieces were then<br />
inspected for S. nodorum pycnidia,<br />
which confirmed infection <strong>of</strong> the<br />
leaf. Transmission frequencies to<br />
the coleoptiles <strong>and</strong> first leaves were<br />
analyzed by SAS Proc Mixed<br />
(version 6.12; SAS Institute Inc.,<br />
Cary, NC).<br />
Germination was slightly, but<br />
not significantly, lower at 9 ºC.<br />
Transmission to the coleoptiles was<br />
high at each temperature, but the<br />
effect <strong>of</strong> temperature was<br />
significant (P = 0.0001), reflecting<br />
differences in transmission at the<br />
extremes <strong>of</strong> 9 <strong>and</strong> 25 ºC. Pycnidia<br />
were found sometimes on the<br />
coleoptiles. Transmission to the<br />
first leaf was much less frequent<br />
<strong>and</strong> was temperature dependent.<br />
The average transmission<br />
frequency at 9 ºC was 0.39, but<br />
dropped to 0.03 at 25 ºC. The<br />
pathogen was not distributed<br />
evenly in leaf tissue. It was<br />
recovered at higher frequency from<br />
sections <strong>of</strong> the first leaf proximal to<br />
the stem than from sections taken<br />
from the leaf apex region.<br />
Approximately 50% <strong>of</strong> infected first<br />
leaves were asymptomatic.<br />
At a transmission frequency <strong>of</strong><br />
0.1, based conservatively on our<br />
results, a seedlot with 5% infected<br />
seed would give rise to an average<br />
<strong>of</strong> one infected seedling per m 2 in a<br />
st<strong>and</strong> <strong>of</strong> 200 seedlings per m 2 .<br />
Simulations<br />
We simulated stagonospora<br />
nodorum blotch epidemics from<br />
infected seedlings at main shoot <strong>and</strong><br />
3 tillers to early dough, a period <strong>of</strong><br />
about 60 to 70 d for New York wheat,<br />
using apparent infection rates (sensu<br />
van der Plank, 1963) derived from or<br />
reported in the literature (Jeger et al.,<br />
1983; Rambow, 1989) <strong>and</strong> the logistic<br />
model (van der Plank, 1963). Near<br />
100% incidence <strong>of</strong> infection by the<br />
early dough stage is possible from as<br />
low as one infected plant per m 2<br />
(Figure 2). Relatively low seed<br />
infection incidences <strong>and</strong> transmission<br />
rates, without any increase in disease<br />
incidence over the winter, could<br />
conceiveably result in 50% <strong>of</strong> all<br />
plants infected by flowering (Figure<br />
3). Decreasing the initial seedling<br />
disease incidence can delay the<br />
epidemic significantly (Figure 3).<br />
Differential Seed<br />
Infection Among<br />
Cultivars<br />
To examine the possibility <strong>of</strong><br />
cultivar differential response to seed<br />
infection, we assayed seed samples<br />
Disease incidence<br />
1<br />
0.8<br />
0.6<br />
0.4<br />
0.2<br />
0<br />
0 10 20 30 40 50 60 70<br />
Days<br />
Figure 2. Increase in stagonospora nodorum<br />
blotch incidence with apparent infection rates<br />
<strong>of</strong> 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, or 0.19, starting<br />
from an initial seedling disease incidence <strong>of</strong><br />
0.005, which corresponds to 1 infected plant<br />
per m2 at a density <strong>of</strong> 200 per m2 . A period <strong>of</strong> 70<br />
days corresponds to the interval from main<br />
shoot <strong>and</strong> 3-tiller stage to early dough stage<br />
under New York winter wheat production<br />
conditions.
Session 5 — D.A. Shah <strong>and</strong> G.C. Bergstrom<br />
104<br />
from each <strong>of</strong> four New York winter<br />
wheat trial locations in 1995 <strong>and</strong><br />
1996. Data for two 1995 sites are<br />
shown in Figure 4. Results for 1996<br />
were similar. The effect <strong>of</strong> local<br />
environment is significant; seed<br />
infection at site B is greater for all<br />
cultivars than at site A. The initial<br />
inoculum sources were the same at<br />
both sites since seed used for<br />
sowing came from the same lot for<br />
any given cultivar. Some cultivars<br />
(Delaware <strong>and</strong> Houser, for<br />
instance) had consistently less seed<br />
infection than others, even across<br />
different production environments<br />
(Figure 4).<br />
Screening <strong>of</strong> the cultivars for<br />
differential responses to seed<br />
infection by S. nodorum was<br />
continued under glasshouse<br />
conditions. Flag leaves <strong>and</strong> ears<br />
were inoculated with spore<br />
suspensions <strong>of</strong> a single isolate at<br />
10 6 spores/ml. Flag leaves were<br />
assessed for percent leaf area<br />
diseased 15 d post-inoculation, <strong>and</strong><br />
seeds were assayed on SNAW<br />
medium upon maturity. Seed<br />
infection was high, but again, some<br />
cultivars had less seed infection<br />
than others. There was a strong<br />
correlation between the field based<br />
Disease incidence<br />
1<br />
0.8<br />
0.6<br />
0.4<br />
0.2<br />
0<br />
0 10 20 30 40 50 60 70<br />
Days<br />
Figure 3. Increase in stagonospora nodorum<br />
blotch incidence from initial seedling<br />
disease incidences <strong>of</strong> 0.001, 0.003, 0.005, 0.01,<br />
0.02, or 0.04, given an apparent infection rate<br />
<strong>of</strong> 0.14. The 70 day period corresponds to the<br />
interval from main shoot <strong>and</strong> 3-tiller stage to<br />
early dough stage under New York winter<br />
wheat production conditions.<br />
observations <strong>and</strong> the results <strong>of</strong> the<br />
quantitative glasshouse<br />
inoculations when cultivars were<br />
ranked according to seed infection<br />
level. There appears to be genetic<br />
mechanisms <strong>of</strong> resistance to seed<br />
infection by S. nodorum. The poor<br />
correlation between flag leaf<br />
disease severity <strong>and</strong> seed infection<br />
incidence suggests that seed <strong>and</strong><br />
foliar resistance are under separate<br />
genetic control.<br />
Potential for Integrated<br />
Management<br />
No single control tactic is likely<br />
to reduce seedborne inoculum<br />
below levels that could initiate<br />
epidemics under favorable<br />
environments. The most effective<br />
triazole seed fungicides can reduce<br />
pathogen transmission in grossly<br />
infected seedlots so that only 1 to<br />
2% <strong>of</strong> seedlings are infected, still<br />
enough for epidemics to develop<br />
(Bergstrom, unpublished). A<br />
strategy integrating breeding for<br />
reduced seed infection <strong>and</strong><br />
transmission frequencies, improved<br />
disease management in seed<br />
production fields, <strong>and</strong> the<br />
application <strong>of</strong> seed fungicides may<br />
Seed infection incidence (%)<br />
60<br />
50<br />
40<br />
30<br />
20<br />
10<br />
0<br />
Site A<br />
Site B<br />
Delaware<br />
Houser<br />
Annett<br />
Chelsea<br />
ACRon<br />
Pioneer 2737W<br />
NYBatavia<br />
Karena<br />
Harus<br />
Caledonia<br />
Geneva<br />
Arkona<br />
Diana<br />
Year<br />
Figure 4. Differential seed infection <strong>of</strong> winter<br />
wheat cultivars by <strong>Stagonospora</strong> nodorum at<br />
two New York sites (A <strong>and</strong> B), located 5 km<br />
apart but differing in microclimate, in 1995.<br />
be employed to manage<br />
stagonospora nodorum blotch<br />
effectively in wheat production<br />
systems similar to that in New York.<br />
References<br />
Bannon, E. 1978. A method <strong>of</strong> detecting<br />
<strong>Septoria</strong> nodorum on symptomless<br />
leaves <strong>of</strong> wheat. Ir. J. Agric. Res.<br />
17:323-325.<br />
Jeger, M.J., Gareth-Jones, D., <strong>and</strong><br />
Griffiths, E. 1983. Disease spread <strong>of</strong><br />
non-specialised fungal pathogens<br />
from inoculated point sources in<br />
intraspecific mixed st<strong>and</strong>s <strong>of</strong> cereal<br />
cultivars. Ann. Appl. Biol. 102:237-<br />
244.<br />
Krupinsky, J.M. 1997. Stability <strong>of</strong><br />
<strong>Stagonospora</strong> nodorum isolates from<br />
perennial grass hosts after passage<br />
through wheat. Plant Dis. 81:1037-<br />
1041.<br />
Man<strong>and</strong>har, J.B., <strong>and</strong> Cunfer, B.M. 1991.<br />
An improved selective medium for<br />
the assay <strong>of</strong> <strong>Septoria</strong> nodorum from<br />
wheat seed. Phytopathology 81:771-<br />
773.<br />
Rambow, M. 1989. Befallsentwicklung<br />
von <strong>Septoria</strong> nodorum Berk. in<br />
winterweizenbeständen.<br />
Nachrichtenbl. Pflanzenschutz DDR<br />
43:209-212.<br />
Schilder, A.M.C., <strong>and</strong> Bergstrom, G.C.<br />
1989. Distribution, prevalence, <strong>and</strong><br />
severity <strong>of</strong> fungal leaf <strong>and</strong> spike<br />
diseases <strong>of</strong> winter wheat in New<br />
York in 1986 <strong>and</strong> 1987. Plant Dis.<br />
73:177-182.<br />
Shah, D., <strong>and</strong> Bergstrom, G.C. 1993.<br />
Assessment <strong>of</strong> seedborne<br />
<strong>Stagonospora</strong> nodorum in New York<br />
s<strong>of</strong>t white winter wheat. Plant Dis.<br />
77:468-471.<br />
Shah, D., Bergstrom, G.C., <strong>and</strong> Ueng,<br />
P.P. 1995. Initiation <strong>of</strong> <strong>Septoria</strong><br />
nodorum blotch epidemics in winter<br />
wheat by seedborne <strong>Stagonospora</strong><br />
nodorum. Phytopathology 85:452-<br />
457.<br />
Ueng, P.P., Cunfer, B.M., <strong>and</strong> Chen, W.<br />
1994. Identification <strong>of</strong> the wheat <strong>and</strong><br />
barley biotypes <strong>of</strong> <strong>Stagonospora</strong><br />
nodorum using restriction fragment<br />
length polymorphisms <strong>and</strong><br />
biological characteristics.<br />
Phytopathology 84:1146.<br />
Van der Plank, J.E. 1963. Plant <strong>Diseases</strong>:<br />
Epidemics <strong>and</strong> Control. New York<br />
<strong>and</strong> London. Academic Press. 349<br />
pp.
Sessions 6A <strong>and</strong> 6B: Cultural Practices <strong>and</strong> Disease Management<br />
Influence <strong>of</strong> Cultural Practices on <strong>Septoria</strong>/<strong>Stagonospora</strong><br />
<strong>Diseases</strong><br />
J.M. Krupinsky<br />
USDA-Agricultural Research Service, Northern Great Plains Research Lab, M<strong>and</strong>an, ND, USA<br />
Abstract<br />
The use <strong>of</strong> cultural management practices is probably one <strong>of</strong> the oldest approaches to controlling plant diseases. How<br />
cultural management practices such as crop rotation, tillage (residue level), fertilizer application, seeding operations, <strong>and</strong><br />
disease-free seed can influence the incidence <strong>and</strong> severity <strong>of</strong> <strong>Septoria</strong>/<strong>Stagonospora</strong> diseases are reviewed. Considering that<br />
early reports on cultural practices have been reviewed previously, recent research will be emphasized. Management practices<br />
such as the use <strong>of</strong> resistant cultivars, cultivar blends, <strong>and</strong> the use <strong>of</strong> fungicides will be covered by others at the workshop.<br />
From ancient times various<br />
cultural practices have been used to<br />
reduce plant diseases <strong>and</strong> have<br />
paralleled the development <strong>of</strong><br />
agriculture (Howard, 1996).<br />
Disease severity can be modified<br />
by various cultural practices, but<br />
the magnitude <strong>and</strong> direction <strong>of</strong><br />
these effects are not always<br />
consistent (King et al., 1983;<br />
Shipton et al., 1971; Tompkins et al.,<br />
1993). In some studies, disease<br />
severity can be affected more by<br />
trial location than by the agronomic<br />
practices being tested (Tompkins et<br />
al., 1993).<br />
Considering that early reports<br />
on cultural practices have been<br />
reviewed (Eyal, 1981; King et al.,<br />
1983; Shipton et al., 1971), more<br />
recent research will be emphasized<br />
in this paper. A number <strong>of</strong> cultural<br />
practices such as crop rotation,<br />
tillage, fertilization, seeding, <strong>and</strong><br />
disease-free seed will be reviewed.<br />
Other practices, including the use<br />
<strong>of</strong> resistant cultivars, cultivar<br />
blends, <strong>and</strong> fungicides are<br />
management practices that are<br />
covered by other authors in these<br />
proceedings.<br />
Crop Rotation: A Key<br />
Factor<br />
The survival <strong>of</strong> <strong>Septoria</strong>/<br />
<strong>Stagonospora</strong> spp. on residue from a<br />
previous wheat crop is the most<br />
important means <strong>of</strong> carryover from<br />
one crop to the next. Researchers<br />
have recommended proper crop<br />
rotations in combination with other<br />
practices to reduce the severity <strong>of</strong><br />
<strong>Septoria</strong>/<strong>Stagonospora</strong> diseases.<br />
Plowing <strong>and</strong> burning have also<br />
been used to reduce the amount <strong>of</strong><br />
residue-borne inoculum, but these<br />
practices alone may still leave<br />
sufficient inoculum for the next<br />
wheat crop (Eyal, 1981; King et al.,<br />
1983; Shipton et al., 1971). Although<br />
resistant cultivars are covered by<br />
other authors in these proceedings,<br />
the impact <strong>of</strong> resistant cultivars on<br />
the carryover <strong>of</strong> pathogens should<br />
also be considered. Murray et al.<br />
(1990) pointed out with septoria<br />
tritici blotch (STB) that resistant<br />
cultivars not only reduce crop losses<br />
but the residue can also influence<br />
the next crop. Residue from<br />
resistant crops reduces the potential<br />
disease severity the following year<br />
because less inoculum is provided<br />
to initiate the epidemic.<br />
105<br />
Early researchers, such as Rosen<br />
(1921), included proper crop<br />
rotation in their recommendations<br />
for control <strong>of</strong> stagonospora<br />
nodorum blotch (SNB). Crop<br />
rotations take advantage <strong>of</strong> the fact<br />
that plant pathogens important on<br />
one crop may not cause disease<br />
problems on another crop.<br />
Appropriate crop rotations lengthen<br />
the time between crop types so that<br />
pathogen populations have time to<br />
decline. Although pathogens may<br />
not be completely eliminated, there<br />
is a reduction in the pathogen<br />
population. By rotating among crop<br />
types, the pathogens on residue<br />
from the previous crop will not<br />
infect the crop being grown<br />
(Krupinsky et al., 1997). Crop<br />
rotation allows time for the<br />
decomposition <strong>of</strong> residue on which<br />
pathogens carry over, <strong>and</strong> natural<br />
competitive organisms reduce the<br />
pathogens on the remaining residue<br />
while unrelated crops are being<br />
grown. Crop rotation is a key factor<br />
affecting the health <strong>and</strong> productivity<br />
<strong>of</strong> future wheat crops (Cook <strong>and</strong><br />
Veseth, 1991). Crop rotation <strong>and</strong><br />
residue management are most<br />
effective with pathogens that are<br />
disseminated only short distances<br />
but not as effective for pathogens
106<br />
Session 6A / Session 6B — J.M. Krupinsky<br />
disseminated over long distances.<br />
When inoculum is produced within<br />
a field <strong>and</strong> is disseminated only<br />
short distances, as with<br />
<strong>Stagonospora</strong> nodorum in the<br />
southeastern USA, crop rotation is<br />
an important management practice<br />
(Cunfer, 1998).<br />
When evaluating methods <strong>of</strong><br />
reducing the risk <strong>of</strong> disease<br />
severity with reduced tillage,<br />
Bailey <strong>and</strong> Duczek (1996) indicated<br />
that the most effective means <strong>of</strong><br />
reducing disease is crop rotation. In<br />
Saskatchewan, Canada, Pedersen<br />
<strong>and</strong> Hughes (1992) reported that<br />
crop rotation was effective in<br />
reducing the severity <strong>of</strong> epidemics<br />
caused by a leaf spot disease<br />
complex <strong>of</strong> S. nodorum <strong>and</strong> <strong>Septoria</strong><br />
tritici. In general, <strong>Septoria</strong>/<br />
<strong>Stagonospora</strong> disease severity is<br />
greater when wheat is grown in<br />
monoculture, compared to wheat<br />
following an alternative crop. In<br />
Germany, Käesbohrer <strong>and</strong><br />
H<strong>of</strong>fmann (1989) reported that<br />
winter wheat was infected earlier<br />
<strong>and</strong> more frequently by S. nodorum<br />
in wheat after wheat, compared<br />
with wheat in a crop rotation.<br />
Sutton <strong>and</strong> Vyn (1990) found that<br />
the severity <strong>of</strong> SNB on spikes <strong>of</strong><br />
winter wheat was higher after<br />
wheat compared to wheat after<br />
soybeans.<br />
Disease severity was<br />
significantly higher under<br />
monoculture than under rotation,<br />
particularly with no tillage, with a<br />
leaf-spot disease complex<br />
composed <strong>of</strong> Pyrenophora triticirepentis,<br />
Bipolaris sorokiniana, <strong>and</strong> S.<br />
nodorum (Reis et al., 1992, 1997).<br />
With P. tritici-repentis <strong>and</strong> S. tritici<br />
as the dominant pathogens, STB<br />
was significantly higher on winter<br />
wheat when grown in monoculture<br />
(Odorfer et al., 1994). With P. triticirepentis<br />
<strong>and</strong> S. nodorum as the<br />
dominant pathogens, Fern<strong>and</strong>ez et<br />
al. (1998) observed that wheat after<br />
wheat had a higher disease severity<br />
than wheat grown after flax or<br />
lentil. This was particularly evident<br />
in years with high disease pressure<br />
but not in years with low disease<br />
pressure.<br />
In general, most carry-over<br />
inoculum <strong>of</strong> <strong>Septoria</strong>/<strong>Stagonospora</strong><br />
diseases is minimized by crop<br />
rotations that include wheat or<br />
other cereals every third year<br />
(Wiese, 1987). Considering that<br />
plant residue decays more rapidly<br />
in warm humid conditions than in<br />
drier <strong>and</strong> cooler conditions, the<br />
amount <strong>of</strong> time necessary between<br />
wheat crops may vary depending<br />
on the environment <strong>and</strong> location.<br />
In Israel, Eyal (1981) reported that a<br />
3-5 year rotation is needed to<br />
decrease the incidence <strong>of</strong> STB.<br />
Under favorable disease conditions<br />
in Saskatchewan, Canada, a<br />
rotation <strong>of</strong> two years between<br />
spring wheat crops reduced disease<br />
severity <strong>and</strong> provided adequate<br />
control <strong>of</strong> STB <strong>and</strong> SNB (Pedersen<br />
<strong>and</strong> Hughes, 1992). Also in<br />
Saskatchewan, Fern<strong>and</strong>ez et al.<br />
(1998) recommended two years <strong>of</strong><br />
spring wheat followed by two<br />
years <strong>of</strong> a non-cereal crop, or by a<br />
non-cereal crop <strong>and</strong> summer fallow<br />
to reduce disease severity (P. triticirepentis<br />
<strong>and</strong> S. nodorum).<br />
Since Fern<strong>and</strong>ez et al. (1993)<br />
indicated that S. nodorum, along<br />
with Fusarium graminearum <strong>and</strong> B.<br />
sorokiniana, survived longer on<br />
wheat residue after summer fallow<br />
compared to wheat residue after<br />
corn or soybean crops, summer<br />
fallow may not be as effective as an<br />
alternative crop in reducing<br />
inoculum levels. In the<br />
southeastern USA, S. nodorum<br />
inoculum may be present on wheat<br />
stubble on the soil surface 22<br />
months after a wheat crop is<br />
harvested, leading to<br />
recommendations for crop rotation<br />
<strong>and</strong> tillage (Cunfer, 1998). In<br />
addition to crop rotation,<br />
intercropping can be used to<br />
reduce inoculum movement in the<br />
field. In Irel<strong>and</strong>, Bannon <strong>and</strong> Cooke<br />
(1998) reported that the dispersal <strong>of</strong><br />
pycnidiospores <strong>of</strong> S. tritici was<br />
reduced 33% horizontally <strong>and</strong> 63%<br />
vertically in a wheat-clover<br />
intercrop.<br />
Tillage<br />
Infested residue provides an<br />
important mechanism for the<br />
carryover <strong>of</strong> <strong>Septoria</strong>/<strong>Stagonospora</strong><br />
spp. from one crop to the next.<br />
Tillage promotes residue<br />
decomposition by fracturing the<br />
residue <strong>and</strong> exposing it to residuedecomposing<br />
microorganisms.<br />
Thus, tillage operations such as<br />
plowing have been recommended<br />
as a means to reduce residue (King<br />
et al., 1983; Shipton et al., 1971).<br />
In recent years, conservation<br />
tillage practices have been<br />
promoted to reduce soil erosion<br />
<strong>and</strong> conserve soil moisture. These<br />
minimum or no tillage operations<br />
increase the quantity <strong>of</strong> crop<br />
residue on the soil surface.<br />
Increased levels <strong>of</strong> crop residues<br />
may influence the incidence <strong>and</strong><br />
severity <strong>of</strong> plant diseases,<br />
depending upon the disease <strong>and</strong><br />
region. Conservation tillage or<br />
reduced tillage practices increase,<br />
decrease, or have no effect on plant<br />
diseases (Bailey <strong>and</strong> Duczek, 1996;<br />
Sumner et al., 1981). Conservation<br />
tillage may have different effects on<br />
plant diseases depending on the<br />
soils, region, or prevailing<br />
environment, <strong>and</strong> the biology <strong>of</strong><br />
the disease organism.
Differences in weather cycles in<br />
wheat growing areas are <strong>of</strong>ten<br />
dramatic, which may account for<br />
some <strong>of</strong> the differences in the<br />
impact <strong>of</strong> diseases described in the<br />
literature. When comparing tillage<br />
effects in northeastern North<br />
Dakota, USA, Stover et al. (1996)<br />
observed that different diseases<br />
dominated each year with different<br />
weather conditions. They found<br />
that P. tritici-repentis dominated in<br />
the first two years <strong>of</strong> the study, <strong>and</strong><br />
septoria foliar diseases <strong>and</strong><br />
fusarium head blight dominated in<br />
the third year.<br />
Tillage or lack <strong>of</strong> tillage can also<br />
affect the severity <strong>of</strong> individual<br />
diseases. Sutton <strong>and</strong> Vyn (1990)<br />
reported that P. tritici-repentis <strong>and</strong><br />
S. nodorum were promoted when<br />
wheat was grown after wheat <strong>and</strong><br />
minimum or no tillage was used,<br />
whereas S. tritici was suppressed.<br />
The reverse occurred when wheat<br />
followed alternative crops in all<br />
tillage treatments or following<br />
wheat under conventional tillage.<br />
With airborne ascospores <strong>of</strong> S.<br />
tritici implicated as the source <strong>of</strong><br />
primary inoculum in Oklahoma,<br />
USA, Schuh (1990) concluded that<br />
the amount <strong>of</strong> residue after<br />
different tillage operations did not<br />
have a strong effect on disease<br />
severity.<br />
In western Canada, Bailey <strong>and</strong><br />
Duczek (1996) indicated that foliar<br />
diseases increase under reduced<br />
tillage but not always to damaging<br />
levels. With conditions for low<br />
disease development, Bailey et al.<br />
(1992) did not observe consistent<br />
tillage effects on foliar diseases. In<br />
Kentucky, USA, Ditsch <strong>and</strong> Grove<br />
(1991) reported that SNB was not<br />
affected by tillage but powdery<br />
mildew infection (Erysiphe<br />
graminis) was higher under no<br />
tillage. In North Dakota, USA, with<br />
a leaf-spot disease complex<br />
composed mainly <strong>of</strong> P. triticirepentis<br />
<strong>and</strong> S. nodorum, Krupinsky<br />
et al. (1998) found generally higher<br />
levels <strong>of</strong> necrosis <strong>and</strong> chlorosis<br />
associated with no tillage<br />
compared to minimum or<br />
conventional tillage. However, in<br />
some trials the tillage effect varied<br />
depending on the nitrogen<br />
treatment. With a leaf-spot disease<br />
complex composed <strong>of</strong> P. triticirepentis,<br />
B. sorokiniana, <strong>and</strong> S.<br />
nodorum, Reis et al. (1992, 1997)<br />
reported that disease incidence <strong>and</strong><br />
severity were higher with<br />
minimum <strong>and</strong> no tillage treatments<br />
in Brazil. In the eastern USA, both<br />
tillage <strong>and</strong> crop rotation are<br />
recommended to avoid losses to<br />
SNB (Cunfer, 1998; Milus <strong>and</strong><br />
Chalkley, 1997).<br />
Burning <strong>of</strong> Residues<br />
Although burning crop residue<br />
was recommended in the past<br />
(King et al., 1983; Shipton et al.,<br />
1971), it is no longer recommended<br />
because <strong>of</strong> environmental concerns.<br />
In addition, burning may not be<br />
hot enough to eliminate all residue,<br />
leaving sufficient infested residue<br />
to provide carryover <strong>of</strong> inoculum<br />
for another wheat crop (Eyal, 1981).<br />
In northeastern North Dakota,<br />
USA, Stover et al. (1996) compared<br />
chisel plowing (high residue) to<br />
moldboard plowing <strong>and</strong> burning<br />
followed by moldboard plowing.<br />
The effect <strong>of</strong> chisel plowing on<br />
early season foliar disease did not<br />
consistently carry over to late<br />
season severity ratings. In the first<br />
year, chisel plowing resulted in the<br />
highest late season disease severity,<br />
in the second year, there were no<br />
differences, <strong>and</strong> in the third year,<br />
the chisel plow treatment had the<br />
lowest late season disease severity.<br />
Influence <strong>of</strong> Cultural Practices on <strong>Septoria</strong>/<strong>Stagonospora</strong> <strong>Diseases</strong> 107<br />
Overall, yields were not affected by<br />
a tillage (burning)-related foliar<br />
disease effect.<br />
Plant Nutrition: Nitrogen<br />
Fertilizer<br />
As with other management<br />
practices discussed above,<br />
environmental conditions influence<br />
disease development <strong>and</strong> treatment<br />
responses, leading to variation in<br />
research results among different<br />
geographical regions. The severity <strong>of</strong><br />
<strong>Septoria</strong>/<strong>Stagonospora</strong> diseases may<br />
increase or decrease with increasing<br />
nitrogen rates depending on the<br />
region. Tiedemann (1996) suggested<br />
that inconsistencies <strong>of</strong> <strong>Septoria</strong>/<br />
<strong>Stagonospora</strong> disease response to<br />
nitrogen fertilization may be due to<br />
in part to environmental factors such<br />
as ozone.<br />
High rates <strong>of</strong> nitrogen fertilizer<br />
have been reported to increase the<br />
severity <strong>of</strong> <strong>Septoria</strong>/<strong>Stagonospora</strong><br />
diseases. In addition, increased<br />
nitrogen can increase the danger <strong>of</strong><br />
lodging <strong>and</strong> delayed maturity<br />
(Shipton et al., 1971). In<br />
Pennsylvania, USA, Broscious et al.<br />
(1985) reported that the severity <strong>of</strong><br />
SNB <strong>and</strong> powdery mildew increased<br />
significantly on winter wheat as the<br />
rate <strong>of</strong> spring applied nitrogen<br />
fertilizer increased. Similarly, Ditsch<br />
<strong>and</strong> Grove (1991) in Kentucky, USA,<br />
reported that SNB <strong>and</strong> powdery<br />
mildew were lowest on winter<br />
wheat at the zero nitrogen treatment<br />
but increased as nitrogen rates<br />
increased. In New York, USA, under<br />
conditions <strong>of</strong> low disease severity,<br />
Cox et al. (1989) suggested that high<br />
rates <strong>of</strong> nitrogen have the potential<br />
to increase yield <strong>and</strong> foliar disease<br />
severity on winter wheat in the<br />
northeastern USA. In Tennessee,<br />
USA, Howard et al. (1994) reported<br />
that the severity <strong>of</strong> three foliar
108<br />
Session 6A / Session 6B — J.M. Krupinsky<br />
diseases (S. tritici, S. nodorum, <strong>and</strong><br />
leaf rust [Puccinia recondita])<br />
increased on winter wheat with<br />
higher nitrogen rates, especially<br />
when fungicides were not applied.<br />
In the United Kingdom, Leitch <strong>and</strong><br />
Jenkins (1995) reported that<br />
<strong>Septoria</strong>/<strong>Stagonospora</strong> disease<br />
(principally STB) development on<br />
winter wheat was enhanced with<br />
the application <strong>of</strong> nitrogen<br />
throughout the season. A wide<br />
range <strong>of</strong> timing <strong>and</strong> splits <strong>of</strong><br />
nitrogen application did not<br />
significantly influence the level <strong>of</strong><br />
disease severity after anthesis. Also<br />
in the United Kingdom, Jenkyn <strong>and</strong><br />
King (1988) found an increase in<br />
<strong>Septoria</strong>/<strong>Stagonospora</strong> diseases<br />
(mostly STB) on winter wheat after<br />
fallow compared to winter wheat<br />
after ryegrass. They attributed the<br />
increase in disease severity to an<br />
increased accumulation <strong>of</strong> available<br />
nitrogen during the fallow period.<br />
There have also been reports <strong>of</strong><br />
no effect or a decrease in the<br />
severity <strong>of</strong> <strong>Septoria</strong>/<strong>Stagonospora</strong><br />
diseases on wheat with increased<br />
nitrogen rates. In Germany,<br />
assessing disease damage by the<br />
number <strong>of</strong> pycnidia <strong>and</strong> number <strong>of</strong><br />
latent infections <strong>of</strong> S. nodorum on<br />
winter wheat, Büschbell <strong>and</strong><br />
H<strong>of</strong>fmann (1992) reported that the<br />
influence <strong>of</strong> nitrogen rates was not<br />
significant. Also in Germany,<br />
Tiedemann (1996) reported that<br />
increased nitrogen reduced the<br />
severity <strong>of</strong> SNB on spring wheat,<br />
while increasing the severity <strong>of</strong><br />
powdery mildew <strong>and</strong> leaf rust.<br />
This nitrogen effect on the disease<br />
severity <strong>of</strong> SNB was reversed at<br />
elevated ozone concentrations. In<br />
the United Kingdom, late season<br />
applications <strong>of</strong> urea solution<br />
reduced the severity <strong>of</strong> STB on the<br />
flag leaf <strong>of</strong> winter wheat (Gooding<br />
et al., 1988). Naylor <strong>and</strong> Su (1988)<br />
reported that the severity <strong>of</strong> SNB<br />
on winter wheat was not affected<br />
by increased nitrogen levels early<br />
in the season <strong>and</strong> even decreased<br />
with increasing nitrogen later in the<br />
season.<br />
In Maryl<strong>and</strong>, USA, SNB was<br />
reduced on winter wheat with a<br />
higher nitrogen fertility rate (Orth<br />
<strong>and</strong> Grybauskas, 1994). They<br />
suggested that the reduction <strong>of</strong><br />
SNB was apparently due to<br />
interference <strong>of</strong> splash dispersal <strong>of</strong><br />
spores in a denser canopy <strong>and</strong> the<br />
suppressive effect <strong>of</strong> high nitrogen<br />
fertility. In glasshouse trials, they<br />
also reported that increased<br />
nitrogen fertility decreased the<br />
severity <strong>of</strong> SNB on the same winter<br />
wheat cultivars tested in the field.<br />
In North Dakota, USA, with a leafspot<br />
disease complex composed<br />
mainly <strong>of</strong> P. tritici-repentis <strong>and</strong> S.<br />
nodorum, Krupinsky et al. (1998)<br />
reported that with low nitrogen<br />
levels disease severity was higher<br />
in no tillage compared to<br />
conventional tillage. At higher<br />
nitrogen levels, the difference in<br />
disease severity for tillage<br />
treatments was greatly reduced.<br />
In Saskatchewan, Canada, the<br />
development <strong>of</strong> <strong>Septoria</strong>/<br />
<strong>Stagonospora</strong> diseases on winter<br />
wheat was influenced by nitrogen<br />
fertility in one trial out <strong>of</strong> nine<br />
(Tompkins et al., 1993). Greater<br />
disease severity was associated<br />
with low nitrogen fertility. They<br />
suggested that lesion development<br />
may be promoted by nitrogen<br />
deficiency or a nutrient imbalance.<br />
Also in Saskatchewan, Fern<strong>and</strong>ez<br />
et al. (1998) reported an increase in<br />
disease severity with an increase in<br />
nitrogen deficiency in dry years<br />
with a leaf-spot disease complex<br />
composed mainly <strong>of</strong> P. triticirepentis<br />
<strong>and</strong> S. nodorum. Disease<br />
severity declined with treatments<br />
receiving no phosphorus.<br />
Seeding Operations<br />
A higher disease severity <strong>of</strong> STB was<br />
associated with earlier sowings in New<br />
South Wales, Australia (Murray et al.,<br />
1990). The longer time between sowing<br />
<strong>and</strong> heading probably leads to a higher<br />
disease severity. When studying<br />
seeding rates, row spacing, <strong>and</strong> depth<br />
<strong>of</strong> seeding in Pennsylvania, USA,<br />
Broscious et al. (1985) reported that<br />
increased seeding rates increased SNB<br />
in four out <strong>of</strong> 13 trials <strong>and</strong> decreased<br />
SNB in one trial. They suggested that<br />
growers could reduce row spacing from<br />
18 cm to 13 cm to increase yields<br />
without increasing disease severity. In<br />
Saskatchewan, Tompkins et al. (1993)<br />
reported that the severity <strong>of</strong> <strong>Septoria</strong>/<br />
<strong>Stagonospora</strong> diseases increased with a<br />
higher seeding rate. Disease severity<br />
was not influenced by row spacing.<br />
Narrow row spacing (10 cm) with<br />
increased nitrogen rates reduced SNB<br />
on winter wheat <strong>and</strong> increased yields in<br />
Maryl<strong>and</strong>, USA (Orth <strong>and</strong> Grybauskas,<br />
1994).<br />
Use <strong>of</strong> Disease-Free Seed<br />
Seed infected with S. nodorum can be<br />
a source <strong>of</strong> primary inoculum <strong>and</strong> a<br />
probable source <strong>of</strong> transmission from<br />
one wheat crop to the next. Inoculum<br />
can survive in seed for extended<br />
periods <strong>of</strong> time. <strong>Septoria</strong> tritici can be<br />
seed-borne but is not a significant<br />
source <strong>of</strong> inoculum (Eyal, 1981; King et<br />
al., 1983; Shipton et al., 1971). Although<br />
the use <strong>of</strong> disease-free seed may not be<br />
considered a cultural practice, it should<br />
be evaluated by the producer as a<br />
management practice for reducing<br />
disease severity. Cultural practices such<br />
as crop rotation may not be effective if<br />
seed infected with S. nodorum is used<br />
for planting (Luke et al., 1983). The<br />
beneficial effect <strong>of</strong> disease-free seed can<br />
be negated by sowing into residue from<br />
a previous wheat crop (Luke et al., 1983,<br />
1985; Milus <strong>and</strong> Chalkley, 1997).
In Germany, Rambow (1990)<br />
indicated that infected seed was the<br />
main source <strong>of</strong> primary inoculum. In<br />
Pol<strong>and</strong>, Arseniuk et al. (1998)<br />
reported that higher seed infestation<br />
by S. nodorum resulted in higher<br />
disease levels in the field.<br />
<strong>Stagonospora</strong> nodorum was found in<br />
98% <strong>of</strong> the wheat seed samples<br />
tested in North Carolina, USA, <strong>and</strong><br />
the fungus survived for more than<br />
two years on stored seed (Babadoost<br />
<strong>and</strong> Hebert, 1984). Using seed lots<br />
infected 1% to 40% with S. nodorum,<br />
Luke et al. (1986) reported that 10%<br />
seed infection was adequate to cause<br />
a severe epidemic in the southeastern<br />
USA. In the northeastern USA, Shah<br />
et al. (1995) found that in 1990-91, a<br />
season mildly conductive to disease<br />
development, plots sown to seed<br />
with less than 1% infection by S.<br />
nodorum developed epidemics. In<br />
1991-92, epidemics were initiated<br />
with seed infection levels as low as<br />
0.5%. They demonstrated that the<br />
same isolates in the seed used for<br />
planting were found in the seed<br />
harvested. This indicated that seed<br />
populations <strong>of</strong> S. nodorum could<br />
initiate epidemics <strong>of</strong> SNB in new<br />
locations <strong>and</strong> could provide year-toyear<br />
perpetuation <strong>of</strong> these<br />
populations. In Manitoba, Canada,<br />
plants from shriveled seed caused by<br />
S. nodorum had lower seedling vigor<br />
but with increased number <strong>of</strong> tillers<br />
on plants from the shriveled seed,<br />
the yield from plump or shriveled<br />
seed plots could not be differentiated<br />
(Gilbert et al., 1995).<br />
When disease-free seed is not<br />
available, chemical treatments <strong>and</strong><br />
fungicides can be used to reduce or<br />
eliminate <strong>Septoria</strong>/<strong>Stagonospora</strong><br />
pathogens from seed used for<br />
planting (Eyal, 1981; King et al., 1983;<br />
Shipton et al., 1971). The use <strong>of</strong><br />
fungicides for seed treatment are<br />
covered by other authors in these<br />
proceedings.<br />
Conclusion<br />
With relatively low value crops<br />
such as small cereal grains, there is<br />
a need for cultural management<br />
practices that can reduce the<br />
impact <strong>of</strong> diseases (Howard, 1996).<br />
With the adoption <strong>of</strong> conservation<br />
tillage practices, additional longterm<br />
research is needed to<br />
minimize disease severity with<br />
integrated diverse cropping<br />
systems. Hopefully new<br />
experiments that study the<br />
interaction <strong>of</strong> cultural practices,<br />
including the development <strong>of</strong> new<br />
crop rotation sequences, will<br />
provide additional information in<br />
the future. Adapted resistant<br />
cultivars, which are an important<br />
factor in reducing disease<br />
epidemics, should be used when<br />
available. The best strategy to<br />
minimize the impact <strong>of</strong> <strong>Septoria</strong>/<br />
<strong>Stagonospora</strong> diseases is to integrate<br />
several cultural practices into one<br />
system. The integration <strong>of</strong> several<br />
cultural practices should minimize<br />
disease impacts on the economics<br />
for the grower without increasing<br />
the cost <strong>of</strong> production.<br />
Acknowledgments<br />
I thank B. M. Cunfer <strong>and</strong> D. E.<br />
Mathre for their reviews <strong>and</strong><br />
constructive comments. Mention <strong>of</strong><br />
a trademark, proprietary product,<br />
or company by USDA personnel is<br />
intended for explicit description<br />
only <strong>and</strong> does not constitute a<br />
guarantee or warranty <strong>of</strong> the<br />
product by the USDA <strong>and</strong> does not<br />
imply its approval to the exclusion<br />
<strong>of</strong> other products that may also be<br />
suitable. USDA-ARS, Northern<br />
Plains Area, is an equal<br />
opportunity/affirmative action<br />
employer <strong>and</strong> all agency services<br />
are available without<br />
discrimination.<br />
Influence <strong>of</strong> Cultural Practices on <strong>Septoria</strong>/<strong>Stagonospora</strong> <strong>Diseases</strong> 109<br />
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Disease Management Using<br />
Varietal Mixtures<br />
C.C. Mundt, C. Cowger, <strong>and</strong> M.E. H<strong>of</strong>fer<br />
Department <strong>of</strong> Botany <strong>and</strong> Plant Pathology, Oregon State University, Corvallis, OR, USA<br />
Abstract<br />
Few data are available that evaluate the impact <strong>of</strong> variety mixtures on the <strong>Septoria</strong>/<strong>Stagonospora</strong> diseases <strong>of</strong> cereals.<br />
The splash-dispersed nature <strong>of</strong> the pathogens <strong>and</strong> the predominance <strong>of</strong> quantitative variation for host resistance <strong>and</strong><br />
pathogenicity in the <strong>Septoria</strong>/<strong>Stagonospora</strong> diseases may make interactions more complex than for the rusts <strong>and</strong><br />
mildews, where major gene interactions have been the main object <strong>of</strong> study. We have found that epidemic progression <strong>of</strong><br />
septoria tritici blotch can be substantially suppressed in mixtures <strong>of</strong> a susceptible <strong>and</strong> a moderately resistant variety,<br />
sometimes to below the level <strong>of</strong> the more resistant variety grown in pure st<strong>and</strong>. Mycosphaerella graminicola populations<br />
sampled from variety mixtures have been found to be reduced in pathogenicity in all mixtures that have been investigated<br />
thus far. Disruptive selection may be an important mechanism affecting disease progression <strong>and</strong> evolution <strong>of</strong> M.<br />
graminicola in variety mixtures. Major genes for resistance have the potential to contribute substantially to the use <strong>of</strong><br />
variety mixtures for control <strong>of</strong> <strong>Septoria</strong>/<strong>Stagonospora</strong> diseases, both through their epidemiological impacts on disease<br />
spread, <strong>and</strong> through effects <strong>of</strong> induced resistance between avirulent <strong>and</strong> virulent genotypes <strong>of</strong> the pathogen that may occur<br />
in variety mixtures.<br />
Variety mixtures have been<br />
investigated to the greatest extent<br />
for obligate parasites <strong>of</strong> small<br />
grains (Garrett <strong>and</strong> Mundt, 1999;<br />
Wolfe, 1985), <strong>and</strong> are being utilized<br />
commercially for control <strong>of</strong> these<br />
diseases (Garrett <strong>and</strong> Mundt, 1999;<br />
Mundt, 1994). Much less<br />
information is available regarding<br />
the impact <strong>of</strong> variety mixtures on<br />
diseases caused by non-obligate<br />
pathogens.<br />
The effects <strong>of</strong> variety mixtures<br />
may differ for the <strong>Septoria</strong>/<br />
<strong>Stagonospora</strong> diseases compared to<br />
rusts <strong>and</strong> mildews for at least two<br />
reasons. First, steep dispersal<br />
gradients caused by splash<br />
dispersal <strong>of</strong> conidia may decrease<br />
inoculum exchange among host<br />
genotypes in mixture <strong>and</strong>, hence,<br />
reduce the efficacy <strong>of</strong> mixtures for<br />
disease control (Fitt <strong>and</strong><br />
McCartney, 1986; Mundt <strong>and</strong><br />
Leonard, 1986). Second, qualitative<br />
incompatibility reactions are much<br />
less common for septoria diseases<br />
in current agricultural systems<br />
compared to the rusts <strong>and</strong><br />
mildews.<br />
Though there clearly is much<br />
more host/pathogen specificity<br />
than has been suggested in the past<br />
for Mycosphaerella graminicola<br />
(Ahmed et al., 1995; Ahmed et al.,<br />
1996; Cowger et al., this volume;<br />
Kema et al., 1986; 1987), the general<br />
lack <strong>of</strong> use <strong>of</strong> major genes for<br />
resistance to the <strong>Septoria</strong>/<br />
<strong>Stagonospora</strong> diseases in agriculture<br />
places much greater emphasis on<br />
quantitative interactions. For M.<br />
graminicola, there clearly can be<br />
substantial quantitative variation<br />
for pathogenicity that is under the<br />
influence <strong>of</strong> host selection (Ahmed<br />
et al., 1995; 1996; Mundt et al.,<br />
1999) <strong>and</strong> may be influenced by<br />
host diversity. In addition, Jeger et<br />
al. (1981a) have derived models<br />
111<br />
which indicate, based on<br />
epidemiological considerations,<br />
that variety mixtures can impact<br />
disease severity even in the absence<br />
<strong>of</strong> host/pathogen specificity.<br />
There are a limited number <strong>of</strong><br />
studies that address the impact <strong>of</strong><br />
variety mixtures on <strong>Septoria</strong>/<br />
<strong>Stagonospora</strong> diseases in the field.<br />
Jeger et al. (1981b) found that<br />
mixing wheat varieties with<br />
apparent non-specific resistance<br />
reduced the severity <strong>of</strong><br />
stagonospora nodorum blotch by<br />
more than 50% relative to the<br />
mixture components grown<br />
separately in pure st<strong>and</strong>s, though<br />
overall severity levels were very<br />
low. Mundt et al. (1995) studied all<br />
possible equiproportional mixtures<br />
among two susceptible, one<br />
moderately resistant, <strong>and</strong> one<br />
highly resistant wheat variety.<br />
Mean disease reductions late in the
Session 6A / Session 6B — C.C. Mundt, C. Cowger, <strong>and</strong> M.E. H<strong>of</strong>fer<br />
112<br />
epidemics were 27, 9, <strong>and</strong> 15% for<br />
the three seasons, with substantial<br />
differences among specific<br />
mixtures.<br />
The purpose <strong>of</strong> the work<br />
reported herein was to investigate<br />
in greater detail the effects <strong>of</strong> wheat<br />
variety mixtures on epidemic<br />
progression <strong>of</strong> septoria tritici blotch<br />
<strong>and</strong> to determine the influence <strong>of</strong><br />
host diversity on pathogenicity <strong>of</strong><br />
M. graminicola populations derived<br />
from field plots <strong>of</strong> pure <strong>and</strong> mixed<br />
st<strong>and</strong>s <strong>of</strong> wheat varieties in the<br />
Willamette Valley <strong>of</strong> Oregon, USA.<br />
This location is highly conducive<br />
for development <strong>of</strong> the <strong>Septoria</strong>/<br />
<strong>Stagonospora</strong> blotches <strong>of</strong> wheat, with<br />
frequent rains from November<br />
through June. Naturally occurring<br />
epidemics have varied from<br />
moderate to severe over the past<br />
decade, <strong>and</strong> commercial fields <strong>of</strong><br />
susceptible varieties are sprayed<br />
every year.<br />
In Oregon, septoria tritici blotch<br />
is currently more important than<br />
stagonospora nodorum blotch,<br />
perhaps due to competitive<br />
exclusion caused by earlier<br />
ascospore showers <strong>of</strong> M. graminicola<br />
as compared to Phaeosphaeria<br />
nodorum (DiLeone et al., 1997).<br />
<strong>Septoria</strong> tritici blotch epidemics in<br />
Oregon begin from a very dominant<br />
sexual stage, with primary<br />
infections resulting from heavy<br />
ascospore showers (Mundt et al.,<br />
1999). Ascospore showers <strong>of</strong>ten<br />
peak in November, after fall rains<br />
have begun, but continue at some<br />
level throughout the crop season<br />
(DiLeone et al., 1997), including<br />
secondary cycles <strong>of</strong> sexual<br />
recombination (Zhan et al., 1998).<br />
Materials <strong>and</strong> Methods<br />
Field plots<br />
Plots were grown at the Botany<br />
<strong>and</strong> Plant Pathology Field<br />
Laboratory near Corvallis, OR,<br />
during the 1997-98 winter wheat<br />
season. Plots were sown at a rate <strong>of</strong><br />
322 seeds/m2 on 20 October 1997.<br />
Each plot was 3.0 m (12 rows) x 5.2<br />
m, <strong>and</strong> planted within a<br />
“checkerboard” <strong>of</strong> barley (Hordeum<br />
vulgare) plots <strong>of</strong> equal size.<br />
St<strong>and</strong>ard fertilization <strong>and</strong> weed<br />
control practices were used,<br />
supplemented by h<strong>and</strong>-weeding.<br />
Epidemics were initiated from<br />
naturally occurring ascospore<br />
showers.<br />
Treatments included two<br />
susceptible (Stephens <strong>and</strong> W-301)<br />
<strong>and</strong> two moderately resistant<br />
(Cashup <strong>and</strong> Madsen) varieties,<br />
<strong>and</strong> the four possible<br />
equiproportional mixtures <strong>of</strong> a<br />
susceptible <strong>and</strong> a moderately<br />
resistant variety. Treatments were<br />
replicated four times in a<br />
r<strong>and</strong>omized complete block design.<br />
Percent <strong>of</strong> leaf area covered by<br />
lesions, on a whole-canopy basis,<br />
was recorded from early February<br />
through mid-June. Each plot<br />
received a rating that was the<br />
average <strong>of</strong> two observers.<br />
At the end <strong>of</strong> the season, leaves<br />
were sampled from each plot to<br />
obtain isolates for greenhouse tests<br />
<strong>of</strong> pathogenicity, as described<br />
below. The pathogen was sampled<br />
along a diagonal across the inner 10<br />
rows <strong>of</strong> each plot, with one<br />
sampling point per row. Two or<br />
three flag leaves were collected<br />
from each sampling point. Leaf<br />
samples were collected on 12 June<br />
1998, a time when the pathogen<br />
had been exposed to repeated<br />
generations <strong>of</strong> selection on the host<br />
varieties.<br />
Greenhouse experiment<br />
A greenhouse experiment was<br />
conducted in spring 1999 to<br />
determine pathogenicity <strong>of</strong> the<br />
field-collected isolates. Plants <strong>of</strong><br />
Stephens, W-301, Cashup, <strong>and</strong><br />
Madsen were raised in 10-cm<br />
plastic pots filled with a<br />
greenhouse mix that included a<br />
slow-release fertilizer.<br />
Approximately 15 seeds <strong>of</strong> a<br />
cultivar were sown in each pot.<br />
Before inoculation, plants were<br />
thinned to 10 per pot. Daytime<br />
greenhouse temperature was<br />
maintained from 20-25°C, <strong>and</strong><br />
sodium-halide lights were used to<br />
extend daylength to 16 h.<br />
Cultures were obtained from<br />
field-collected leaves, stored, <strong>and</strong><br />
increased by st<strong>and</strong>ard methods that<br />
have previously been described<br />
(Ahmed et al., 1996). Equal<br />
numbers <strong>of</strong> spores <strong>of</strong> the 10 isolates<br />
(one from each <strong>of</strong> the sampling<br />
points <strong>of</strong> each plot) were combined<br />
<strong>and</strong> the total suspension adjusted<br />
to 105 spores/ml.<br />
At 21 days after seeding, groups<br />
<strong>of</strong> four pots (one <strong>of</strong> each <strong>of</strong> the four<br />
varieties) were inoculated with a<br />
suspension derived from each field<br />
plot by placing them on a turntable<br />
at 16 rpm <strong>and</strong> applying 33.3 ml <strong>of</strong><br />
the suspension with a h<strong>and</strong><br />
sprayer. Surfactant (Tween) was<br />
added at the rate <strong>of</strong> one drop per 50<br />
ml <strong>of</strong> spore suspension. After<br />
inoculation, the plants were kept in
a moist chamber (wooden frame<br />
covered with a polyethylene<br />
sheet) for 72 hours. High<br />
humidity (95% or above) was<br />
maintained using a humidifier<br />
(ultrasonic, cool mist type) inside<br />
the moist chamber. The pots were<br />
subsequently returned to a<br />
greenhouse bench in a<br />
r<strong>and</strong>omized complete block<br />
design.<br />
Percent lesion area on the<br />
second leaf from the base <strong>of</strong> each<br />
<strong>of</strong> the 10 plants in each pot was<br />
estimated visually at three weeks<br />
after inoculation. Each <strong>of</strong> two<br />
observers half <strong>of</strong> the plants in<br />
each pot.<br />
% diseased leaf area<br />
% diseased leaf area<br />
90<br />
80<br />
70<br />
60<br />
50<br />
40<br />
30<br />
20<br />
10<br />
0<br />
90<br />
80<br />
70<br />
60<br />
50<br />
40<br />
30<br />
20<br />
10<br />
0<br />
6-Feb<br />
6-Feb<br />
Madsen (MR)<br />
Stephens (S)<br />
Mixture<br />
27-Feb<br />
20-Mar<br />
Madsen (MR)<br />
W-301 (S)<br />
Mixture<br />
27-Feb<br />
20-Mar<br />
10-Apr<br />
10-Apr<br />
Effects <strong>of</strong> pathogen diversity<br />
on epidemic progression<br />
A field experiment that allowed<br />
us to examine the influence <strong>of</strong><br />
pathogen diversity on epidemic<br />
increase in variety mixtures was<br />
conducted in the 1994-95 season.<br />
These plots were treated in a<br />
manner highly similar to those<br />
described above, <strong>and</strong> have been<br />
discussed in more detail elsewhere<br />
(Zhan et al., 1998). Treatments were<br />
a complete factorial <strong>of</strong> three host<br />
treatments (pure st<strong>and</strong> <strong>of</strong> Madsen,<br />
pure st<strong>and</strong> <strong>of</strong> Stephens, <strong>and</strong> a 1:1<br />
mixture <strong>of</strong> Madsen:Stephens) <strong>and</strong><br />
two inoculation treatments (natural<br />
<strong>and</strong> artificial) in a completely<br />
r<strong>and</strong>om design with three<br />
replications. One set <strong>of</strong> plots was<br />
naturally inoculated, while the<br />
1-May<br />
1-May<br />
22-May<br />
22-May<br />
12-Jun<br />
12-Jun<br />
% diseased leaf area<br />
% diseased leaf area<br />
90<br />
80<br />
70<br />
60<br />
50<br />
40<br />
30<br />
20<br />
10<br />
0<br />
90<br />
80<br />
70<br />
60<br />
50<br />
40<br />
30<br />
20<br />
10<br />
0<br />
6-Feb<br />
6-Feb<br />
Cashup (MR)<br />
Stephens (S)<br />
Mixture<br />
27-Feb<br />
20-Mar<br />
Cashup (MR)<br />
W-301 (S)<br />
Mixture<br />
27-Feb<br />
20-Mar<br />
Disease Management Using Varietal Mixtures 113<br />
other was sprayed in November<br />
1994 with a mixture <strong>of</strong> 10 isolates to<br />
competitively exclude the highly<br />
diverse inoculum provided by<br />
outside ascospore showers.<br />
Results<br />
The Madsen/Stephens <strong>and</strong><br />
Cashup/Stephens mixtures<br />
suppressed epidemic development<br />
below that <strong>of</strong> the more resistant<br />
component in each mixture (Figure<br />
1). In contrast, the mixtures<br />
containing the variety W-301 as the<br />
susceptible component performed<br />
less well, with the Cashup/W-301<br />
mixture showing no epidemic<br />
suppression relative to the mean <strong>of</strong><br />
the component pure st<strong>and</strong>s<br />
(Figure 1).<br />
Figure 1. Disease progress <strong>of</strong> septoria tritici blotch in naturally-occurring epidemics <strong>of</strong> pure st<strong>and</strong> varieties<br />
<strong>and</strong> 1:1 variety mixtures. MR = moderately resistant <strong>and</strong> S = susceptible.<br />
10-Apr<br />
10-Apr<br />
1-May<br />
1-May<br />
22-May<br />
22-May<br />
12-Jun<br />
12-Jun
Session 6A / Session 6B — C.C. Mundt, C. Cowger, <strong>and</strong> M.E. H<strong>of</strong>fer<br />
114<br />
Greenhouse tests indicated that non-inoculated genotypes<br />
host diversity had a substantial comprising 35-40% <strong>of</strong> the<br />
impact on pathogenicity <strong>of</strong> M. population later in the season. In<br />
graminicola (Table 1). All populations contrast, natural inoculum has been<br />
derived from the mixtures produced estimated to result in hundreds <strong>of</strong><br />
less disease on its component unique genotypes per m<br />
varieties in the greenhouse than the<br />
mean <strong>of</strong> the populations derived<br />
from the pure st<strong>and</strong> components in<br />
the field.<br />
Molecular analyses (Zhan et al.,<br />
1998) have previously shown that the<br />
artificial inoculation used in the<br />
1994/95 season competitively<br />
excluded 97% <strong>of</strong> potential ascospore<br />
infections early in the season, though<br />
immigration <strong>and</strong> sexual<br />
recombination among inoculated<br />
<strong>and</strong>/or naturally occurring<br />
genotypes within plots resulted in<br />
2<br />
(McDonald et al., 1996). For the<br />
artificially-inoculated plots,<br />
epidemic progression for the<br />
Madsen/Stephens mixture was<br />
approximately midway between<br />
the two pure st<strong>and</strong>s throughout the<br />
epidemic (Figure 2). By contrast, in<br />
the naturally-inoculated plots,<br />
epidemic progression in the<br />
mixture began midway between<br />
the pure st<strong>and</strong>s, but became<br />
suppressed to near that <strong>of</strong> the<br />
moderately resistant variety as the<br />
season progressed (Figure 2).<br />
Table 1. Disease severity (percent <strong>of</strong> second leaf<br />
area covered by septoria tritici blotch lesions)<br />
caused by Mycosphaerella graminicola populations<br />
derived from single wheat varieties <strong>and</strong> variety<br />
mixtures in replicated field plots.<br />
Source <strong>of</strong> population<br />
Variety mixture Mixturea Pure st<strong>and</strong>sb Madsen/Stephens 9.5 15.9<br />
Madsen/W-301 9.3 11.6<br />
Cashup/Stephens 10.8 13.4<br />
Cashup/W-301 6.6 9.0<br />
a Mean disease severity for populations derived from a<br />
given mixture when tested separately on each <strong>of</strong> the<br />
component varieties. For example, populations<br />
collected from the Madsen/Stephens mixture in the<br />
field were tested separately on Madsen <strong>and</strong> Stephens<br />
in the greenhouse.<br />
b Mean disease severity for populations derived from the<br />
component pure st<strong>and</strong>s <strong>and</strong> tested on the same variety.<br />
For example, to compare with populations derived from<br />
the Madsen/Stephens mixture, populations collected<br />
from pure st<strong>and</strong>s <strong>of</strong> Madsen in the field were tested on<br />
Madsen in the greenhouse <strong>and</strong> populations derived<br />
from pure st<strong>and</strong>s <strong>of</strong> Stephens in the field were tested<br />
on Stephens in the greenhouse.<br />
percent disease<br />
percent disease<br />
100<br />
90<br />
80<br />
70<br />
60<br />
50<br />
40<br />
30<br />
20<br />
10<br />
0<br />
25-Jan<br />
100<br />
90<br />
80<br />
70<br />
60<br />
50<br />
40<br />
Discussion<br />
Our results indicate that some<br />
variety mixtures suppress epidemic<br />
progression <strong>of</strong> septoria tritici blotch<br />
even in the absence <strong>of</strong> major genes for<br />
resistance. There are at least two<br />
plausible explanations for this result.<br />
First, the models <strong>of</strong> Jeger et al. (1981a)<br />
have shown that, in absence <strong>of</strong> host/<br />
pathogen specificity, mixtures can<br />
decrease, increase, or have no effect<br />
on epidemic progression, depending<br />
on the relative levels <strong>of</strong> sporulation<br />
<strong>and</strong> infection frequency in the<br />
component varieties. Thus,<br />
differences between mixtures <strong>and</strong><br />
pure st<strong>and</strong>s in our experiment might<br />
be explained by differences in<br />
resistance components between<br />
cultivars, though we have not<br />
measured these components.<br />
Artificially inoculated plots<br />
Madsen<br />
Stephens<br />
Stephens/Madsen mixture<br />
15-Mar 4-May 23-Jun<br />
Naturally inoculated plots<br />
Madsen<br />
Stephens<br />
Stephens/Madsen mixture<br />
30<br />
20<br />
10<br />
0<br />
25-Jan 15-Mar 4-May 23-Jun<br />
Figure 2. Disease progress <strong>of</strong> septoria tritici blotch on a<br />
moderately resistant variety (Madsen), a susceptible<br />
variety (Stephens) <strong>and</strong> a 1:1 mixture <strong>of</strong> the two varieties<br />
in epidemics initiated from artificial inoculation with 10<br />
isolates or from naturally occurring inoculum.
A second explanation is that<br />
disruptive selection may reduce the<br />
fitness <strong>of</strong> pathogen populations<br />
that cycle between host genotypes<br />
in mixtures. The disruptive<br />
selection hypothesis is supported<br />
by the reduced fitness <strong>of</strong> M.<br />
graminicola populations derived<br />
from the variety mixtures as<br />
compared to the pure st<strong>and</strong>s. It<br />
should be noted that the mean 27%<br />
fitness reduction <strong>of</strong> populations<br />
from mixtures that was found in<br />
our study is likely an<br />
underestimation, as disease<br />
severity in a monocyclic test<br />
accounts for only infection<br />
efficiency <strong>and</strong> lesion expansion, but<br />
not other fitness components such<br />
as sporulation rate or latent period.<br />
Additional support for the<br />
disruptive selection hypothesis is<br />
that variety mixtures suppressed<br />
epidemic development under<br />
conditions <strong>of</strong> natural inoculation,<br />
but not when artificially inoculated<br />
with a limited number <strong>of</strong> isolates.<br />
This result would be expected<br />
under the disruptive selection<br />
hypothesis, as there would be<br />
insufficient pathogen variation<br />
within the artificially inoculated<br />
plots for disruptive selection to<br />
occur. Such disruptive selection<br />
was earlier suggested to increase<br />
the efficacy <strong>of</strong> barley variety<br />
mixtures for control <strong>of</strong> powdery<br />
mildew (caused by Erysiphe<br />
graminis) (Wolfe et al., 1981) <strong>and</strong> to<br />
reduce the fitness <strong>of</strong> mildew<br />
genotypes that were able to<br />
overcome more than one major<br />
resistance gene in the mixtures<br />
(Chin <strong>and</strong> Wolfe, 1984).<br />
The levels <strong>of</strong> disease<br />
suppression contributed by our<br />
variety mixtures may not be<br />
sufficient as the sole control<br />
practice, but would certainly be<br />
useful in an integrated<br />
management program. In addition,<br />
we are currently more interested in<br />
the potential for varietal<br />
diversification to slow pathogen<br />
evolution towards increased<br />
virulence <strong>and</strong>/or aggressiveness<br />
(sensu V<strong>and</strong>erplank, 1968) than we<br />
are for the immediate effects on<br />
epidemic progression. This is<br />
crucial in the Willamette Valley <strong>of</strong><br />
Oregon, as there is evidence that<br />
even quantitative resistance to M.<br />
graminicola may erode in our<br />
environment (Mundt et al., 1999).<br />
Though not addressed in the<br />
research reported here, major genes<br />
for resistance may contribute<br />
substantially to control <strong>of</strong> septoria<br />
diseases in wheat variety mixtures.<br />
Our earlier studies showed that<br />
mixtures containing both a<br />
susceptible <strong>and</strong> a highly resistant<br />
variety provided greater disease<br />
control than other types <strong>of</strong><br />
mixtures (Mundt et al. 1995). The<br />
subsequent “breakdown” <strong>of</strong> the<br />
high-level resistance in the variety<br />
Gene (Cowger et al., this volume)<br />
might be seen to increase the<br />
overall level <strong>of</strong> disease in such<br />
mixtures. On the other h<strong>and</strong>,<br />
mixtures <strong>of</strong> a virulent <strong>and</strong> avirulent<br />
pathogen isolate have been shown<br />
to reduce pycnidial production <strong>of</strong><br />
M. graminicola very substantially<br />
(Halperin et al., 1996), which could<br />
greatly increase the value <strong>of</strong><br />
“defeated” major genes in variety<br />
mixtures.<br />
Disease Management Using Varietal Mixtures 115<br />
In summary, there appears to be<br />
some potential for variety mixtures<br />
to suppress septoria blotch<br />
epidemics <strong>and</strong> to slow pathogen<br />
evolution. Currently available data<br />
are very scant, however, <strong>and</strong><br />
substantially greater field<br />
evaluation is required to determine<br />
the short- <strong>and</strong> long-term value <strong>of</strong><br />
the diversity approach for control<br />
<strong>of</strong> <strong>Septoria</strong>/<strong>Stagonospora</strong> diseases.<br />
References<br />
Ahmed, H.U., Mundt, C.C., <strong>and</strong><br />
Coakley, S.M. 1995. Host-pathogen<br />
relationship <strong>of</strong> geographically<br />
diverse isolates <strong>of</strong> <strong>Septoria</strong> tritici<br />
<strong>and</strong> wheat cultivars. Plant Pathol.<br />
44:838-847.<br />
Ahmed, H.U., Mundt, C.C., H<strong>of</strong>fer,<br />
M.E., <strong>and</strong> Coakley, S.M. 1996.<br />
Selective influence <strong>of</strong> wheat<br />
cultivars on pathogenicity <strong>of</strong><br />
Mycosphaerella graminicola<br />
(anamorph <strong>Septoria</strong> tritici).<br />
Phytopathology 86:454-458.<br />
Chin, K.M., <strong>and</strong> Wolfe, M.S. 1984.<br />
Selection on Erysiphe graminis in<br />
pure <strong>and</strong> mixed st<strong>and</strong>s <strong>of</strong> barley.<br />
Plant Pathol. 33:535-546.<br />
DiLeone, J.A., Karow, R.S., Coakley,<br />
S.M., <strong>and</strong> Mundt, C.C. 1997.<br />
Biology <strong>and</strong> Control <strong>of</strong> <strong>Septoria</strong><br />
<strong>Diseases</strong> <strong>of</strong> Winter Wheat in Western<br />
Oregon. Oregon State Univ. Ext.<br />
Serv. Crop Sci. Bull. 109. 17 pp.<br />
Fitt, B.D.L., <strong>and</strong> McCartney, H.A.<br />
1986. Spore dispersal in relation to<br />
epidemic models. Pages 311-345 in:<br />
Plant Disease Epidemiology, Vol. 1.<br />
K.J. Leonard <strong>and</strong> W.E. Fry, eds.<br />
McGraw-Hill, New York.<br />
Garrett, K.A., <strong>and</strong> Mundt, C.C. 1999.<br />
Epidemiology in mixed host<br />
populations (mini-review).<br />
Phytopathology 89:accepted<br />
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Halperin, T., Schuster, S., Pnini-<br />
Cohen, S., Zilberstein, A., <strong>and</strong><br />
Eyal, Z. 1996. The suppression <strong>of</strong><br />
pycnidial production on wheat<br />
seedlings following sequential<br />
inoculation by isolates <strong>of</strong> <strong>Septoria</strong><br />
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Session 6A / Session 6B — C.C. Mundt, C. Cowger, <strong>and</strong> M.E. H<strong>of</strong>fer<br />
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Jeger, M.J., Griffiths, E., <strong>and</strong> Jones,<br />
D.G. 1981a. Disease progress <strong>of</strong><br />
non-specialised fungal pathogens<br />
in intraspecific mixed st<strong>and</strong>s <strong>of</strong><br />
cereal cultivars. I. Models. Ann.<br />
Appl. Biol. 98:187-198.<br />
Jeger, M.J., Jones, D.G., <strong>and</strong> Griffiths,<br />
E. 1981b. Disease progress <strong>of</strong> nonspecialised<br />
fungal pathogens in<br />
intraspecific mixed st<strong>and</strong>s <strong>of</strong> cereal<br />
cultivars. II. Field experiments.<br />
Ann. Appl. Biol. 98:199-210.<br />
Kema, G.H.J., Sayoud, R., <strong>and</strong> van<br />
Silfhout, C.H. 1996. Genetic<br />
variation for virulence <strong>and</strong><br />
resistance in the wheat-<br />
Mycosphaerella graminicola<br />
pathosystem. II. Analysis <strong>of</strong><br />
interactions between pathogen<br />
isolates <strong>and</strong> host cultivars.<br />
Phytopathology 86:213-220.<br />
Kema, G.H.J., <strong>and</strong> van Silfhout, C.H.<br />
1997. Genetic variation for<br />
virulence <strong>and</strong> resistance in the<br />
wheat-Mycosphaerella graminicola<br />
pathosystem. III. Comparative<br />
seedling <strong>and</strong> adult plant<br />
experiments. Phytopathology<br />
86:213-220.<br />
McDonald, B.A., Mundt, C.C., <strong>and</strong><br />
Chen, R.S. 1996. The role <strong>of</strong><br />
selection on the genetic structure<br />
<strong>of</strong> pathogen populations: Evidence<br />
from field experiments with<br />
Mycosphaerella graminicola.<br />
Euphytica 92:73-80.<br />
Mundt, C.C. 1994. Use <strong>of</strong> host genetic<br />
diversity to control cereal diseases:<br />
Implications for rice blast. Pages<br />
293-307 in: Rice Blast Disease. S.A.<br />
Leong, R.S. Zeigler, <strong>and</strong> P.S. Teng,<br />
eds. CAB International,<br />
Cambridge, <strong>and</strong> the International<br />
Rice Research Institute, Manila.<br />
Mundt, C.C., <strong>and</strong> Leonard, K.J. 1986.<br />
Analysis <strong>of</strong> factors affecting<br />
disease increase <strong>and</strong> spread in<br />
mixtures <strong>of</strong> immune <strong>and</strong><br />
susceptible plants in computersimulated<br />
epidemics.<br />
Phytopathology 76:832-840.<br />
Mundt, C.C., H<strong>of</strong>fer, M.E., Ahmed,<br />
H.U., Coakley, S.M., DiLeone, J.A.,<br />
<strong>and</strong> Cowger, C. 1999. Population<br />
genetics <strong>and</strong> host resistance. Pages<br />
115-130 in: <strong>Septoria</strong> in <strong>Cereals</strong>: a<br />
Study <strong>of</strong> Pathosystems. Lucas, J.A.,<br />
Bowyer, P., Anderson, H.M., eds.<br />
CABI Publishing, Wallingford, UK.<br />
Mundt, C.C., Brophy, L.S., <strong>and</strong><br />
Schmitt, M.E. 1995. Choosing crop<br />
cultivars <strong>and</strong> mixtures under high<br />
versus low disease pressure: A<br />
case study with wheat. Crop Prot.<br />
14:509-515.<br />
V<strong>and</strong>erplank, J.E. 1968. Disease<br />
Resistance in Plants. Academic<br />
Press, New York. 206 pp.<br />
Wolfe, M.S. 1985. The current status<br />
<strong>and</strong> prospects <strong>of</strong> multiline<br />
cultivars <strong>and</strong> variety mixtures for<br />
disease resistance. Annu. Rev.<br />
Phytopathol. 23:251-273.<br />
Wolfe, M.S., Barrett, J.A., <strong>and</strong> Jenkins,<br />
J.E.E. 1981. The use <strong>of</strong> cultivar<br />
mixtures for disease control. Pages<br />
73-80 in: J.F. Jenkyn <strong>and</strong> R.T.<br />
Plumb, eds. Strategies for the<br />
Control <strong>of</strong> Cereal Disease. Blackwell,<br />
Oxford.<br />
Zhan, J., Mundt, C.C., <strong>and</strong> McDonald,<br />
B.A. 1998. Measuring immigration<br />
<strong>and</strong> sexual reproduction in field<br />
populations <strong>of</strong> Mycosphaerella<br />
graminicola. Phytopathology<br />
88:1330-1337.
Session 6C: Breeding for Disease Resistance<br />
Breeding for Resistance to the <strong>Septoria</strong>/<strong>Stagonospora</strong><br />
Blights <strong>of</strong> Wheat<br />
M. van Ginkel <strong>and</strong> S. Rajaram<br />
Wheat Program, <strong>CIMMYT</strong>, El Batan, Mexico<br />
Abstract<br />
Genetic resistance remains the first line <strong>of</strong> defense against the septoria foliar blights, especially in developing countries.<br />
Resistance genes with major or minor effects may be either recessive or dominant. A few genes may be enough to confer<br />
resistance, as in the case <strong>of</strong> partial resistance. Additive gene effects contribute more to resistance than dominance effects.<br />
Among normally maturing semidwarf wheats possessing resistance, those carrying Rht2 may be more resistant to <strong>Septoria</strong><br />
tritici than those with Rht1. In the case <strong>of</strong> <strong>Stagonospora</strong> nodorum, resistance <strong>of</strong> the flag leaf <strong>and</strong> <strong>of</strong> the spike may be at least<br />
partly under separate genetic control.<br />
Five confounding factors complicate selection for resistance: 1) maturity <strong>and</strong> plant height affect the expression <strong>of</strong><br />
resistance; 2) the relationship between seedling <strong>and</strong> adult plant responses is highly inconsistent; 3) the correlation between<br />
disease response <strong>and</strong> yield loss is very variable; 4) there is interaction among fungal isolates on the leaf surface; <strong>and</strong> 5) it is<br />
essential to determine the implications <strong>of</strong> the existence <strong>of</strong> races for resistance breeding.<br />
Durability <strong>of</strong> resistance across years <strong>and</strong> locations is a particular concern, <strong>and</strong> examples <strong>of</strong> both erosion <strong>of</strong> resistance <strong>and</strong><br />
stable resistance have been put forward. There is clear pro<strong>of</strong> <strong>of</strong> differential variety by isolate interactions at the seedling stage<br />
<strong>and</strong> on adult plants in the field. However, almost without exception, the size <strong>of</strong> the interaction component is about a<br />
magnitude smaller than that <strong>of</strong> the main effects due to varieties <strong>and</strong> isolates. Recent molecular experiments on the genetic<br />
structure <strong>of</strong> S. tritici populations have found a lack <strong>of</strong> adaptation to the host genotype.<br />
The methodology applied at <strong>CIMMYT</strong> for pyramiding resistance genes in wheat includes utilizing proven resistance<br />
sources as parents, selecting for resistance using a shuttle breeding program between contrasting locations, <strong>and</strong> confirming<br />
resistance through global multilocation testing. The best resulting lines are fed back into the crossing program as parents. The<br />
first genepool exploited for resistance to S. tritici by <strong>CIMMYT</strong> breeders originated in Brazil <strong>and</strong> Argentina, followed by<br />
winter wheats <strong>and</strong>, subsequently, Chinese materials. New sources <strong>of</strong> resistance, such as highly promising synthetic wheats,<br />
are currently being exploited in combination with the previous sources.<br />
Globally the septoria diseases<br />
have increased in importance over<br />
the past decades, <strong>and</strong> as Walter<br />
Spurgeon Beach wrote in 1919:<br />
“The genus is the more worthy <strong>of</strong><br />
study on account <strong>of</strong> its high<br />
economic importance.” The two<br />
main septoria blights addressed in<br />
this paper are caused by <strong>Septoria</strong><br />
tritici <strong>and</strong> <strong>Stagonospora</strong> nodorum.<br />
However, inferences can be drawn<br />
for the septoria blights in other<br />
cereals, such as <strong>Septoria</strong> passerinii on<br />
barley. Several excellent overviews<br />
have been written on the genetics <strong>of</strong><br />
resistance to the septoria diseases <strong>of</strong><br />
cereals <strong>and</strong> on how to breed for<br />
such resistance (Shipton et al., 1971;<br />
King et al., 1983; Nelson <strong>and</strong><br />
Marshall, 1990; Lucas et al., 1999;<br />
Eyal, 1999). Most <strong>of</strong> those reviews<br />
were published 10 years or more<br />
ago, but a great deal <strong>of</strong> the<br />
information they contain is still<br />
correct <strong>and</strong> relevant today.<br />
117<br />
This paper emphasizes some <strong>of</strong><br />
the more recent literature (post 1990)<br />
on the genetics <strong>and</strong> breeding <strong>of</strong><br />
resistance, <strong>and</strong> how these new<br />
insights may be applied in developing<br />
varieties that express durable<br />
resistance to the septoria blights.
118<br />
Session 6C — M. van Ginkel <strong>and</strong> S. Rajaram<br />
The Need for Resistance<br />
In most wheat production<br />
environments, although not in all,<br />
genetic resistance is the most<br />
economical approach to control<br />
fungal diseases. There are, however,<br />
two other control methods—cultural<br />
<strong>and</strong> chemical—that may be utilized.<br />
In the case <strong>of</strong> the septoria foliar<br />
blights, crop residues play a critical<br />
role in the over-seasoning <strong>of</strong> the<br />
pathogen <strong>and</strong> in supplying the<br />
primary inoculum for the<br />
subsequent cropping cycle. In recent<br />
years zero <strong>and</strong> reduced tillage<br />
practices have been gaining<br />
popularity for reasons ranging from<br />
the economics <strong>of</strong> production to<br />
protection <strong>of</strong> the environment, <strong>and</strong><br />
the trend is likely to become<br />
stronger in the near term. Hence<br />
cultural control methods involving<br />
specific residue removal seem less <strong>of</strong><br />
an option than a few decades ago.<br />
However, in the more distant future,<br />
we may once again see a<br />
contraction, particularly in the use<br />
<strong>of</strong> the zero tillage option, as the<br />
problems <strong>of</strong> excessive fungicide use<br />
<strong>and</strong> disease buildup both above <strong>and</strong><br />
below the soil become more<br />
intractable.<br />
Chemical control will always<br />
need to be available as an option. In<br />
particular when conditions are<br />
unexpectedly conducive to<br />
disease—for example, due to<br />
excessive rainfall—a wellresearched<br />
chemical option must be<br />
close at h<strong>and</strong>. Under such<br />
circumstances chemical control may<br />
<strong>of</strong>ten be justified because it can<br />
avoid production losses <strong>of</strong> great<br />
economic value.<br />
Considering the above<br />
mentioned control options,<br />
breeding for resistance must<br />
remain the first line <strong>of</strong> defense,<br />
especially in developing<br />
countries. In such countries other<br />
options, while attractive in<br />
theory, <strong>of</strong>ten cannot be<br />
implemented in a timely fashion<br />
<strong>and</strong>/or the resources needed for<br />
their development <strong>and</strong> largescale<br />
application are lacking,<br />
even in an emergency.<br />
Where resistance is not<br />
effective, tolerance can be sought<br />
(McKendry <strong>and</strong> Henke, 1994a).<br />
Despite more than 25 years <strong>of</strong><br />
interest in tolerance to the<br />
septoria blights, its mechanism(s)<br />
in general remains vague<br />
(Zuckerman et al., 1997). There is,<br />
however, little doubt that<br />
tolerance is widely available <strong>and</strong><br />
operational in modern-day<br />
germplasm.<br />
Sources <strong>of</strong> Resistance<br />
Many sources <strong>of</strong> resistance to<br />
the septoria foliar blights,<br />
including some wild relatives <strong>of</strong><br />
wheat, have been studied over<br />
the years. The interest in “alien<br />
sources” is not new; in the early<br />
part <strong>of</strong> this century, wild relatives<br />
resistant to the <strong>Septoria</strong> spp. were<br />
already being tested <strong>and</strong><br />
identified (Beach, 1919; Mackie,<br />
1929). Some <strong>of</strong> those sources were<br />
reported in the <strong>of</strong>ficial literature,<br />
while others were used in<br />
breeding programs <strong>and</strong> reported<br />
less in written form.<br />
Although grouping the<br />
sources <strong>of</strong> resistance based on<br />
origin or wheat class is to a<br />
certain extent arbitrary, commonly<br />
used classifications are: derived<br />
from South American (in particular<br />
Brazilian) sources, winter wheats,<br />
Chinese origin, <strong>and</strong> wild relatives<br />
<strong>of</strong> wheat. Some <strong>of</strong> the major<br />
sources confirmed by several<br />
authors are listed in Table 1.<br />
Table 1. Sources <strong>of</strong> resistance to <strong>Septoria</strong> tritici<br />
<strong>and</strong> <strong>Stagonospora</strong> nodorum confirmed by several<br />
authors. Generally only one key reference is given.<br />
<strong>Septoria</strong> tritici Reference<br />
Anza Wilson (1994)<br />
Bezostaya 1 Danon et al. (1982)<br />
Bobwhite Gilchrist et al. (1995)<br />
Bulgaria 88 Rillo <strong>and</strong> Caldwell (1966)<br />
Carifen Lee & Gough (1984)<br />
Colotana Danon et al. (1982)<br />
Fortaleza 1 Danon et al. (1982)<br />
Israel 493 Wilson (1979)<br />
Milan Gilchrist et al. (1995)<br />
Nabob Narvaez & Caldwell (1957)<br />
Oasis Danon et al. (1982)<br />
Seabreeze Rosielle & Brown (1979)<br />
Sheridan<br />
Synthetic wheats<br />
Danon et al. (1982)<br />
(some) May & Lagudah (1992)<br />
Tadinia Somasco et al. (1996)<br />
Tinamou Gilchrist et al. (1995)<br />
T. dicoccon Gilchrist & Skovm<strong>and</strong> (1995)<br />
T. speltoides McKendry & Henke (1994)<br />
T. tauschii Appels & Lagudah (1990); May<br />
& Lagudah (1992); McKendry<br />
& Henke (1994b)<br />
Veranopolis Rosielle & Brown (1979)<br />
Vilmorin<br />
<strong>Stagonospora</strong><br />
nodorum<br />
Gough & Smith (1985)<br />
Aegilops longissima Ecker et al. (1990a)<br />
Atlas 66 Kleijer et al. (1977)<br />
Blueboy II Nelson (1980)<br />
Cotipora Bostwick et al. (1993)<br />
Frondoso Mullaney et al. (1982)<br />
Fronthatch Mullaney et al. (1982)<br />
Oasis Nelson (1980)<br />
T. monococcum Ma & Hughes (1993)<br />
T. speltoides Ecker et al. (1990b)<br />
T. tauschii Ma & Hughes (1993)<br />
T. timopheevii Ma & Hughes (1993)<br />
Inheritance <strong>of</strong><br />
Resistance<br />
Resistance genes having major<br />
effects have been identified in<br />
wheat (Nelson <strong>and</strong> Marshall, 1990).<br />
They have been found to transmit
their resistance in either a recessive<br />
or dominant fashion (Eyal, 1999).<br />
However, in most cases, resistance<br />
appears dominant, with the F1<br />
from a cross between a resistant<br />
<strong>and</strong> a susceptible parent expressing<br />
an intermediate level <strong>of</strong> resistance<br />
that is more similar to that <strong>of</strong> the<br />
resistant parent. A few genes may<br />
be enough to confer resistance that<br />
will hold up in farmers’ fields<br />
(Dubin <strong>and</strong> Rajaram, 1996). While<br />
heritabilities tend to be only <strong>of</strong><br />
moderate magnitude, progress in<br />
breeding for resistance is evident.<br />
Although the number <strong>of</strong> genes<br />
available may be quite high,<br />
accumulating a few key ones may<br />
be sufficient to achieve resistance.<br />
As has been shown for durable<br />
resistance to leaf rust (Singh et al.,<br />
1991), several <strong>of</strong> the alleged<br />
components <strong>of</strong> partial resistance to<br />
S. tritici may also be controlled by<br />
only one or a just a few genes<br />
(Jlibene <strong>and</strong> El Bouami, 1995). It<br />
would seem that those components<br />
that are genetically different could<br />
be combined into the same genetic<br />
background by crossing. Once<br />
available, molecular markers will<br />
be <strong>of</strong> tremendous use for<br />
accumulating resistance to such<br />
environmentally sensitive diseases.<br />
The expression <strong>of</strong> S. tritici<br />
resistance was studied in T. tauschii<br />
accessions, synthetic wheats<br />
derived from them, plus<br />
derivatives <strong>of</strong> synthetic wheats<br />
crossed to common wheats. The<br />
results obtained all pointed to<br />
inheritance based on one or a few<br />
dominant genes (Appels <strong>and</strong><br />
Lagudah, 1990; May <strong>and</strong><br />
Lagudah, 1992).<br />
Chromosome 5D <strong>of</strong> T. tauschii<br />
contributed a high level <strong>of</strong> resistance<br />
to S. nodorum in a synthetic cross<br />
with a T. dicoccum line <strong>and</strong> in<br />
derived substitution lines<br />
(Nicholson et al., 1993).<br />
Chromosomes 5D, 3D, <strong>and</strong> 7D<br />
contributed resistance in decreasing<br />
order <strong>of</strong> importance. Aegilops<br />
longissima <strong>and</strong> Ae. speltoides were<br />
shown to contribute S. nodorum<br />
resistance based on two to four<br />
partially dominant to overdominant<br />
genes (Ecker et al., 1990a<br />
<strong>and</strong> b). Triticum timopheevii<br />
transmitted one S. nodorum<br />
resistance gene located on<br />
chromosome 3A to its progeny from<br />
a cross with a susceptible durum<br />
parent (Ma <strong>and</strong> Hughes, 1995).<br />
In most published accounts <strong>of</strong><br />
quantitative analyses, additive gene<br />
effects or general combining ability<br />
(GCA) effects contributed more to<br />
resistance than dominance or special<br />
combining ability (SCA) effects (van<br />
Ginkel <strong>and</strong> Scharen, 1987; Bruno<br />
<strong>and</strong> Nelson, 1990; Danon <strong>and</strong> Eyal,<br />
1990; Wilkinson et al., 1990; Jonsson,<br />
1991; Jlibene et al., 1994). However,<br />
significant non-additive effects were<br />
<strong>of</strong>ten identified in the same studies.<br />
SCA effects tend to be an order <strong>of</strong><br />
magnitude weaker than GCA<br />
effects. Indirectly Nelson <strong>and</strong> Fang<br />
(1994) confirmed these conclusions<br />
when they observed an absence <strong>of</strong><br />
heterosis for components <strong>of</strong> S.<br />
nodorum resistance, indicating a<br />
general lack <strong>of</strong> over-dominance<br />
effects that enhance resistance.<br />
Jlibene et al. (1994) found a small<br />
cytoplasmic effect in a few <strong>of</strong><br />
their crosses.<br />
Breeding for Resistance to the <strong>Septoria</strong>/<strong>Stagonospora</strong> Blights <strong>of</strong> Wheat 119<br />
Triticales were found not to be<br />
any more resistant than most<br />
wheats, <strong>and</strong> a range <strong>of</strong> reactions to<br />
both S. tritici <strong>and</strong> S. nodorum was<br />
observed (Scharen et al., 1990).<br />
Tritordeum amphiploids (doubled<br />
derivatives from crosses between<br />
Hordeum chilense <strong>and</strong> Triticum spp.)<br />
expressed the S. tritici resistance <strong>of</strong><br />
the barley parent both as seedlings<br />
in greenhouse tests <strong>and</strong> adult<br />
plants in field trials (Rubiales et<br />
al., 1992).<br />
Tolerance to S. nodorum was<br />
shown to be a mechanism that is<br />
genetically different from (partial)<br />
resistance <strong>and</strong> involves several<br />
chromosomes (Rapilly et al., 1988).<br />
Distinct characters were identified<br />
within each <strong>of</strong> these two defense<br />
mechanisms.<br />
Although the introduction <strong>of</strong><br />
alien cytoplasm from wild wheat<br />
relatives reduced partial resistance<br />
somewhat, it did increase tolerance<br />
as measured by reduced yield<br />
losses (Keane <strong>and</strong> Jones, 1990).<br />
Agronomic Traits<br />
<strong>and</strong> Resistance<br />
After a period in the 1960s <strong>and</strong><br />
1970s <strong>of</strong> seemingly increased levels<br />
<strong>of</strong> disease susceptibility in the<br />
semidwarf wheats (Santiago,<br />
1970), modern semidwarf<br />
germplasm carrying high levels <strong>of</strong><br />
resistance was subsequently<br />
identified. Many are now widely<br />
grown as commercial varieties.<br />
As in previous studies,<br />
Camacho-Casas et al. (1995)<br />
showed that it is possible to breed<br />
normally maturing semidwarf<br />
wheats if proper disease conditions
120<br />
Session 6C — M. van Ginkel <strong>and</strong> S. Rajaram<br />
are created during the selection<br />
process <strong>and</strong> attention is paid to<br />
achieving the desired maturity.<br />
Nevertheless, tall <strong>and</strong>/or late<br />
versions <strong>of</strong> similar isogenic lines<br />
tend to be less affected by the<br />
septoria/stagonospora blights than<br />
their shorter, earlier sister lines.<br />
The roles in conferring S. tritici<br />
resistance <strong>of</strong> the two most common<br />
dwarfing genes may not be the<br />
same (Baltazar et al., 1990). In the<br />
four crosses studied, the Rht2<br />
dwarfing gene tended to be<br />
associated more <strong>of</strong>ten with<br />
increased levels <strong>of</strong> resistance.<br />
Plants carrying Rht1 were more<br />
susceptible.<br />
In the case <strong>of</strong> S. nodorum,<br />
resistance <strong>of</strong> the flag leaf <strong>and</strong> <strong>of</strong> the<br />
spike may be at least partly under<br />
separate genetic control (Rosielle<br />
<strong>and</strong> Brown, 1980; Bostwick et al.,<br />
1993; Hu et al., 1996). Resistance <strong>of</strong><br />
the flag leaf was found to be coded<br />
for by genes on chromosomes 3A,<br />
4A, <strong>and</strong> 3B, while spike resistance<br />
was located on the same<br />
chromosomes <strong>and</strong> on 7A (Hu et al.,<br />
1996).<br />
Determination<br />
<strong>of</strong> Resistance<br />
One <strong>of</strong> the most confounding<br />
factors in determining resistance to<br />
the septoria blights has been <strong>and</strong><br />
continues to be the interaction<br />
between resistance <strong>and</strong> maturity<br />
<strong>and</strong> plant height, as mentioned<br />
above. Parlevliet (1990) describes<br />
how the ranking <strong>of</strong> resistance may<br />
even change dramatically, once<br />
these factors have been<br />
corrected for.<br />
Deviation from regression was<br />
proposed by van Beuningen <strong>and</strong><br />
Kohli (1990) to correct for the<br />
confounding factors <strong>of</strong> heading <strong>and</strong><br />
plant height. Loughman et al.<br />
(1994a) proposed a numerical<br />
classification approach that creates<br />
clusters <strong>of</strong> similar entries based on<br />
response patterns including disease<br />
reaction, maturity, <strong>and</strong> height. If<br />
proper checks are included, cluster<br />
analysis helps to identify resistant<br />
material.<br />
A second confounding factor in<br />
quantifying resistance is the<br />
relationship between seedling <strong>and</strong><br />
adult plant responses. Somasco et<br />
al. (1996) showed that long-term<br />
resistance to S. tritici, such as that<br />
found in Tadinia (derived from the<br />
winter wheat Tadorna), was<br />
strongly expressed in both<br />
seedlings <strong>and</strong> adult plants. Kema<br />
<strong>and</strong> van Silfhout (1997) showed<br />
that not all isolates respond<br />
similarly to seedling <strong>and</strong> adult<br />
plant infection.<br />
Several studies have found that<br />
resistance to S. nodorum is<br />
expressed differently in seedlings<br />
than in adult plants (Koric, 1988;<br />
Arseniuk et al., 1991). These studies<br />
concluded that while only<br />
preliminary seedling data should<br />
be considered, subsequent adult<br />
plant screening is imperative.<br />
In a set <strong>of</strong> bread wheats <strong>and</strong><br />
durum wheats, seedling response<br />
to a crude toxic extract <strong>of</strong> S. tritici<br />
produced a varietal ranking<br />
different from the adult plant<br />
reaction to field inoculation with a<br />
spore mixture <strong>of</strong> the same fungus<br />
(Harrabi et al., 1995).<br />
However, media containing<br />
crude extracts from grain inoculated<br />
with S. nodorum elicited a differential<br />
response that correlated well with<br />
field resistance or susceptibility <strong>of</strong><br />
the spike (Keller et al., 1994).<br />
Resistant lines showed a higher<br />
percentage <strong>of</strong> embryo-forming callus<br />
in the presence <strong>of</strong> the extract.<br />
A third major confounding factor<br />
is the correlation between disease<br />
response <strong>and</strong> yield loss. Under field<br />
conditions visually observed<br />
symptoms <strong>of</strong> S. nodorum on the flag<br />
leaf or spike were not always well<br />
correlated to yield losses (Tvaruzek<br />
<strong>and</strong> Klem, 1994). Lack <strong>of</strong><br />
consistently high correlations<br />
between S. nodorum disease scores<br />
<strong>and</strong> final yield loss motivated<br />
Walther (1990) to study a more<br />
extensive scoring method. When<br />
flag leaves <strong>of</strong> protected <strong>and</strong> nonprotected<br />
plots were scored on three<br />
dates around the middle <strong>of</strong> the<br />
epidemic, correlations rose to 0.73.<br />
<strong>Stagonospora</strong> nodorum severity<br />
scores on the flag leaf had the<br />
highest correlations with yield loss,<br />
followed by those on the flag leaf -1<br />
(Walther <strong>and</strong> Bohmer, 1992). Spike<br />
infection was poorly correlated with<br />
yield. Heritability <strong>of</strong> disease<br />
variables could be further increased<br />
if plant height <strong>and</strong> maturity were<br />
corrected for, with heritability values<br />
for weighted assessments<br />
reaching 0.89.<br />
The practice <strong>of</strong> using detached<br />
leaves to determine resistance<br />
appears to have diminished in the<br />
past decade. Most work is now<br />
carried out on seedlings <strong>and</strong> adult<br />
plants in the greenhouse or the field.
Durability <strong>of</strong> Resistance<br />
Durability <strong>of</strong> S. nodorum<br />
resistance within a crop cycle<br />
decreased with leaf age, <strong>and</strong> the<br />
older lower leaves proved more<br />
susceptible than the younger upper<br />
leaves (Jonsson, 1991). Due to this<br />
effect, late maturing germplasm is<br />
<strong>of</strong>ten wrongly considered more<br />
resistant than early maturing lines.<br />
Thus it is crucial to measure<br />
resistance at the same adult<br />
growth stage.<br />
More debated than durability<br />
within a crop cycle is the issue <strong>of</strong><br />
durability <strong>of</strong> resistance across years<br />
<strong>and</strong> locations. Johnson in his 1992<br />
general review <strong>of</strong> breeding for<br />
disease resistance highlights the<br />
difficulty <strong>of</strong> establishing the<br />
presence <strong>of</strong> differential hostpathogen<br />
interaction in the septoria<br />
foliar blights. He compares data<br />
gathered by the Eyal group (Eyal et<br />
al., 1973) with those <strong>of</strong> van Ginkel<br />
(van Ginkel <strong>and</strong> Scharen, 1985) <strong>and</strong><br />
concludes that even the<br />
interpretation <strong>of</strong> quite similar data<br />
may differ. In practice, he<br />
concludes, no unequivocal demise<br />
<strong>of</strong> varieties due to truly differential<br />
races <strong>of</strong> the septoria foliar blights<br />
has been reported.<br />
The increase <strong>of</strong> disease severity<br />
over time on the same variety has<br />
been noted in the Netherl<strong>and</strong>s,<br />
Australia, Israel (Jorgensen <strong>and</strong><br />
Smedegaard-Petersen, 1999), <strong>and</strong><br />
the USA (Mundt et al., 1999).<br />
However, it was not unequivocally<br />
shown whether this was due to an<br />
evolution <strong>of</strong> aggressiveness in the<br />
pathogen population or due to<br />
differential host-pathogen<br />
interaction based on the evolution<br />
<strong>of</strong> new virulence patterns. Mundt et<br />
al. (1999) conclude that the<br />
cultivation <strong>of</strong> susceptible hosts will<br />
result in selection for<br />
aggressiveness. In Germany several<br />
varieties were shown to retain their<br />
resistance to isolates <strong>of</strong> S. nodorum<br />
collected annually from all over the<br />
former East Germany for more than<br />
10 years, except for small variations<br />
over years due to specific weather<br />
conditions (Walther, 1993).<br />
Clear pro<strong>of</strong> <strong>of</strong> differential<br />
variety by isolate interaction at the<br />
seedling stage was presented by<br />
Kema et al. (1996). However, as in<br />
previous studies by this group <strong>and</strong><br />
others, the size <strong>of</strong> the interaction<br />
component was about a magnitude<br />
smaller than that <strong>of</strong> the main effects<br />
due to varieties <strong>and</strong> isolates. In a<br />
later monocyclic field experiment,<br />
Kema <strong>and</strong> van Silfhout (1997)<br />
applied isolates specifically selected<br />
for their virulence differences at the<br />
seedling level, to adult plant plots.<br />
Again, small but significant<br />
interactions between isolates <strong>and</strong><br />
varieties were apparent in this<br />
monocyclic situation. The<br />
significant interaction effect was<br />
largely due to two <strong>of</strong> the three<br />
isolates interacting differentially<br />
with two <strong>of</strong> the 22 varieties tested.<br />
Recent work on Kenyan isolates<br />
indicated that variety by isolate<br />
interaction in the field, while<br />
present, was small, with the<br />
interaction component accounting<br />
for only about 5% <strong>of</strong> the main<br />
effects (Arama, 1996).<br />
Breeding for Resistance to the <strong>Septoria</strong>/<strong>Stagonospora</strong> Blights <strong>of</strong> Wheat 121<br />
Recent experiments on the<br />
genetic structure <strong>of</strong> S. tritici<br />
populations in response to host<br />
differences found a total lack <strong>of</strong><br />
evidence for adaptation to the host<br />
genotype (McDonald et al., 1996).<br />
Different hosts had no differential<br />
effects on the dynamics <strong>of</strong> the<br />
pathogen population. On the other<br />
h<strong>and</strong>, Ahmed et al. (1996) found<br />
that susceptible varieties selected<br />
for more aggressive pathogen<br />
populations. However, the<br />
virulence levels <strong>of</strong> the pathogen<br />
isolates were associated with the<br />
variety <strong>of</strong> origin.<br />
Varietal mixtures <strong>of</strong> resistant<br />
<strong>and</strong> susceptible varieties have met<br />
with mixed success. Recent<br />
comparisons by Loughman et al.<br />
(1994b) <strong>of</strong> pure lines <strong>and</strong> mixtures<br />
did not find that mixtures have any<br />
advantage when it comes to<br />
durability <strong>of</strong> resistance.<br />
Interaction among isolates on<br />
the leaf, which can enhance or<br />
reduce expected disease severities<br />
(Eyal, 1992; Gilchrist <strong>and</strong><br />
Velazquez, 1994), adds another<br />
component to the mix <strong>of</strong> virulence<br />
<strong>and</strong> aggressiveness.<br />
The implications for resistance<br />
breeding <strong>of</strong> the above findings on<br />
variety by isolate interactions<br />
remain to be tested further in real<br />
agricultural settings. However, it<br />
does appear that the response <strong>of</strong><br />
the septoria/stagonospora foliar<br />
blights to their hosts cannot<br />
compare to the highly volatile<br />
population dynamics we see in the<br />
wheat rusts.
122<br />
Session 6C — M. van Ginkel <strong>and</strong> S. Rajaram<br />
It is essential to determine the<br />
practical relevance <strong>of</strong> the presence <strong>of</strong><br />
races <strong>of</strong> the septoria foliar blights<br />
when applying a resistance breeding<br />
strategy. Indeed, the importance <strong>of</strong><br />
this topic cannot be overstated. New<br />
insights into the genetic structure <strong>of</strong><br />
the pathogen population <strong>and</strong> its<br />
dynamics, especially as supported<br />
by biotechnological tools, may<br />
increase our underst<strong>and</strong>ing<br />
significantly in the near term.<br />
Breeding for Resistance<br />
at <strong>CIMMYT</strong><br />
The methodology applied at<br />
<strong>CIMMYT</strong> for breeding for disease<br />
resistance in wheat has been<br />
outlined in previous publications,<br />
including the proceedings <strong>of</strong> several<br />
international septoria workshops<br />
(Mann et al. 1985; van Ginkel <strong>and</strong><br />
Rajaram, 1989; van Ginkel <strong>and</strong><br />
Rajaram, 1993; Gilchrist et al., 1995;<br />
van Ginkel <strong>and</strong> Rajaram, 1995). The<br />
methodology consists <strong>of</strong> three key<br />
components: utilizing proven<br />
resistance sources as parents,<br />
selecting for increased resistance in<br />
a shuttle breeding program, <strong>and</strong><br />
confirming resistance by<br />
multilocation testing. The best<br />
identified lines are fed back into the<br />
crossing program as parents to<br />
further accumulate resistance,<br />
bringing the process full circle.<br />
The materials that provide the<br />
best resistance are made available<br />
by <strong>CIMMYT</strong> to all collaborators<br />
around the world who request<br />
them. Lists <strong>of</strong> such materials are<br />
published periodically (Gilchrist,<br />
1994). Gilchrist et al. (this volume)<br />
have provided a new overview <strong>of</strong><br />
lines found to be consistently<br />
resistant by <strong>CIMMYT</strong> researchers.<br />
Every year <strong>CIMMYT</strong><br />
distributes to collaborators the<br />
newest wheat lines that combine S.<br />
tritici resistance with a full<br />
“agronomic package,” including<br />
adaptation to high rainfall<br />
environments, superior yield, a<br />
certain level <strong>of</strong> resistance to other<br />
prevalent diseases such as the rusts<br />
<strong>and</strong> scab, <strong>and</strong> acceptable grain<br />
quality.<br />
The parental stocks emphasized<br />
in developing these lines have a<br />
relatively long history <strong>of</strong> proven<br />
resistance in many locations<br />
around the world. The first<br />
genepool exploited for resistance to<br />
S. tritici by <strong>CIMMYT</strong> breeders was<br />
that represented by such lines as<br />
IAS20 from Brazil <strong>and</strong> Klein Atlas<br />
from Argentina. The Tinamou line<br />
is an example <strong>of</strong> a widely adapted,<br />
resistant line that was developed<br />
from those resistance sources. Then<br />
followed the winter wheats, in<br />
particular those from the former<br />
USSR, such as Aurora. Examples <strong>of</strong><br />
elite lines emanating from that<br />
work are the group <strong>of</strong> Bobwhite<br />
lines, such as the cultivar<br />
PROINTA Federal <strong>and</strong> the Milan<br />
lines, which have shown strong<br />
disease resistance throughout the<br />
world, including in the hotspots <strong>of</strong><br />
eastern Africa <strong>and</strong> the Southern<br />
Cone <strong>of</strong> South America (Kohli,<br />
1995; Dubin <strong>and</strong> Rajaram, 1996).<br />
Subsequently, Chinese materials,<br />
such as the Ning, Shanghai, <strong>and</strong><br />
Suzhoe series, plus other varieties<br />
from China, were heavily crossed<br />
by <strong>CIMMYT</strong> breeders. A key<br />
resistant line that resulted from<br />
that effort was Catbird. This<br />
history is discussed in more detail<br />
by Gilchrist et al. (this volume).<br />
New sources <strong>of</strong> resistance, such<br />
as highly promising synthetic<br />
wheats, are currently being<br />
exploited. <strong>CIMMYT</strong>’s Wide Crosses<br />
Program produces synthetic wheats<br />
by crossing <strong>CIMMYT</strong> elite durum<br />
wheats to accessions <strong>of</strong> Aegilops<br />
squarrosa (syn. Triticum tauschii <strong>and</strong><br />
Ae. tauschii). The resulting triploid<br />
seedlings are treated with colchicine<br />
to double the number <strong>of</strong><br />
chromosomes, resulting in manmade<br />
(hence “synthetic”) hexaploid<br />
bread wheats. Certain <strong>of</strong> those<br />
wheats have shown remarkable<br />
levels <strong>of</strong> S. tritici resistance, <strong>and</strong> low<br />
severities bordering on immunity<br />
have also been observed. Since<br />
synthetic wheats are relatively easy<br />
to cross with common wheats, their<br />
resistance can be readily<br />
introgressed into agronomically<br />
acceptable plant types, <strong>and</strong><br />
combined with other resistances.<br />
<strong>CIMMYT</strong>’s breeding<br />
methodology emphasizes regularly<br />
exposing segregating materials to<br />
disease epidemics. Since the<br />
breeding program also targets<br />
resistance to several other diseases<br />
besides S. tritici, straw is used as the<br />
source <strong>of</strong> primary inoculum. Straw<br />
residues may carry inoculum for tan<br />
spot, fusarium head scab, fusarium<br />
foliar blight, <strong>and</strong> different species <strong>of</strong><br />
bacteria in addition to the septorias.<br />
In special cases <strong>and</strong> in specific<br />
studies, pure liquid S. tritici spores<br />
are applied.<br />
<strong>CIMMYT</strong> applies a shuttle<br />
breeding methodology in which<br />
materials are alternately grown in a<br />
high rainfall site in the Mexican<br />
highl<strong>and</strong>s (Toluca) <strong>and</strong> an irrigated<br />
site in northwestern Mexico (Cd.<br />
Obregon). As a result, the material
targeted for S. tritici-prone regions<br />
is exposed to the disease 3-4 times<br />
during the segregating phase when<br />
grown in Toluca. During the<br />
selection process, selection<br />
intensity is increased as the<br />
segregating populations are<br />
advanced. At the end <strong>of</strong> the shuttle<br />
breeding process, all homozygous<br />
lines are exposed for two<br />
additional cycles at three sites in<br />
the high rainfall Mexican<br />
highl<strong>and</strong>s: Toluca, Patzcuaro<br />
(Gomez <strong>and</strong> Gonzalez, 1987), <strong>and</strong><br />
El Tigre.<br />
Shuttle breeding is followed by<br />
multilocation testing at key<br />
locations around the world through<br />
a network <strong>of</strong> cooperators. The<br />
global data allow truly outst<strong>and</strong>ing<br />
material (see Gilchrist et al., these<br />
proceedings) to be identified <strong>and</strong><br />
used as new parents in the ongoing<br />
process <strong>of</strong> recombining genetically<br />
different sources <strong>of</strong> resistance, <strong>and</strong><br />
they also <strong>of</strong>fer the prospect <strong>of</strong><br />
combining different resistance<br />
mechanisms.<br />
Future work will concentrate on<br />
combining accumulated resistances<br />
that are based on different genes<br />
<strong>and</strong>/or different resistance<br />
mechanisms, with superior yield,<br />
scab resistance, shattering<br />
tolerance, <strong>and</strong> industrial quality.<br />
Conclusions<br />
Several conclusions in regard to<br />
the impact <strong>of</strong> our increased<br />
underst<strong>and</strong>ing <strong>of</strong> resistance on<br />
related breeding aspects can be<br />
drawn from the combined research<br />
<strong>of</strong> the past decade:<br />
1. There are many sources <strong>of</strong> genetic<br />
resistance available that have<br />
either been shown to be different<br />
or seem to behave differently.<br />
Many <strong>of</strong> those resistances have<br />
proven to be quite stable.<br />
2. Seedling data at best are an<br />
indication <strong>of</strong> adult plant<br />
resistance, <strong>and</strong> testing adult<br />
plants in a field situation appears<br />
crucial.<br />
3. A variety is rarely replaced<br />
solely or primarily because it<br />
succumbs to one <strong>of</strong> the<br />
septoria/stagonospora blights.<br />
4. The relative role <strong>of</strong> virulence <strong>and</strong><br />
aggressiveness in the field may<br />
soon be elucidated using<br />
molecular tools in combination<br />
with the ability to cross among<br />
isolates. The outcome should<br />
have direct impact on breeding<br />
strategy.<br />
5. The diversity <strong>of</strong> isolates at the<br />
field level makes gaining a<br />
deeper underst<strong>and</strong>ing <strong>of</strong><br />
interactions among these isolates<br />
<strong>of</strong> paramount importance. The<br />
breeder needs to take this into<br />
consideration when planning to<br />
artificially inoculate, or rely on<br />
multisite testing.<br />
6. Multilocation confirmation <strong>of</strong><br />
advanced lines remains a<br />
necessity, while the debate on<br />
possible specificity continues.<br />
Breeding for Resistance to the <strong>Septoria</strong>/<strong>Stagonospora</strong> Blights <strong>of</strong> Wheat 123<br />
While several <strong>of</strong> these exciting<br />
issues remain in debate, advances<br />
in breeding for resistance to the<br />
septoria/stagonospora pathogens<br />
have not ceased. Breeders have<br />
continued to produce varieties with<br />
higher yields, better industrial<br />
quality, <strong>and</strong> improved resistance to<br />
multiple diseases.<br />
References<br />
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Mycosphaerella graminicola<br />
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Phytopathology 86:454-458.<br />
Arama, P.F. 1996. Effects <strong>of</strong> Cultivar,<br />
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Arseniuk, E., P.M. Fried, H. Winzeler,<br />
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Kronstad. 1990. Association<br />
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Dubin, H.J., <strong>and</strong> S. Rajaram. 1996.<br />
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Rachis 7(1,2): 31-32.<br />
Lee, T.S., <strong>and</strong> F.J. Gough. 1984.<br />
Inheritance <strong>of</strong> <strong>Septoria</strong> leaf blotch<br />
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Triticum aestivum cv. Carifen 12.<br />
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Basford, R.F. Gilmour, <strong>and</strong> I.H.<br />
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Villareal. 1985 progress in<br />
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Mundt, C.C., M.E. H<strong>of</strong>fer, H.U.<br />
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Breeding for Resistance to the <strong>Septoria</strong>/<strong>Stagonospora</strong> Blights <strong>of</strong> Wheat 125<br />
Nelson, L.R. 1980. Inheritance <strong>of</strong><br />
resistance to <strong>Septoria</strong> nodorum in<br />
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Nelson, L.R., <strong>and</strong> Xiaobing Fang.<br />
1994. Effect <strong>of</strong> heterosis on <strong>Septoria</strong><br />
nodorum disease level <strong>and</strong> plant<br />
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<strong>Septoria</strong> <strong>of</strong> <strong>Cereals</strong>. E. Arseniuk, T.<br />
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IHAR, Radzikow, Pol<strong>and</strong>.<br />
Hodowla Roslin Aklimatyzacja I<br />
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38(3-4):213-218.<br />
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Breeding wheat for resistance to<br />
<strong>Septoria</strong> nodorum <strong>and</strong> <strong>Septoria</strong><br />
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1980. Selection for resistance to<br />
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Euphytica 29:337-346.
126<br />
Session 6C — M. van Ginkel <strong>and</strong> S. Rajaram<br />
Rubiales, D., J. Ballesteros, <strong>and</strong> A.<br />
Martin. 1992. Resistance to <strong>Septoria</strong><br />
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Santiago, J.C. 1970. Resultado das<br />
observacoes effectuadas em<br />
Marrocos na Tunisia respeifantes<br />
as doencas e pragas doe cereais,<br />
principalmente em recalcao aos<br />
trigos Mexicanos em 1969 a<br />
expensas das Missoes Americanas<br />
de auxilio technico a Marrocos e<br />
Tunisia. 12. Junho de 1970. Elvas.<br />
Portugal, estacao de<br />
Melhoramento.<br />
Scharen, A.L., E. Arseniuk, W. Sowa,<br />
J. Zimmy, <strong>and</strong> W. Podyma. 1990.<br />
Seedling resistance <strong>of</strong> triticale <strong>and</strong><br />
Triticum spp. germplasm to<br />
<strong>Septoria</strong> nodorum <strong>and</strong> S. tritici. In:<br />
Proceedings <strong>of</strong> the 2 nd<br />
International Triticale Symposium,<br />
1-5 October, 1990, Passo Fundo,<br />
Rio Gr<strong>and</strong>e do Sul, Brazil. pp 256-<br />
259.<br />
Shipton, W.A., W.R.J. Boyd, A.A.<br />
Rosielle, <strong>and</strong> B.I. Shearer. 1971. The<br />
common septoria diseases <strong>of</strong><br />
wheat. Bot. Rev. 37:231-262.<br />
Singh, R.P., T.S. Payne, <strong>and</strong> S.<br />
Rajaram. 1991. Characterization <strong>of</strong><br />
variability <strong>and</strong> relationships<br />
among components <strong>of</strong> partial<br />
resistance to leaf rust in <strong>CIMMYT</strong><br />
bread wheats. Theor. Appl. Genet.<br />
82:674-680.<br />
Somasco, O.A., C.O. Qualset, <strong>and</strong><br />
D.G. Gilchrist. 1996. Single-gene<br />
resistance to <strong>Septoria</strong> tritici blotch<br />
in the spring wheat cultivar<br />
‘Tadinia’. Plant Breeding 115:261-<br />
267.<br />
Tvaruzek, L., <strong>and</strong> K. Klem. 1994.<br />
Varieties <strong>and</strong> lines <strong>of</strong> winter wheat<br />
with stable tolerance <strong>and</strong> low yield<br />
loss to <strong>Septoria</strong> nodorum (Berk). Cer.<br />
Res. Comm. 22(4): 369-374.<br />
Van Beuningen, L.T., <strong>and</strong> M.M. Kohli.<br />
1990. Deviation from the<br />
regression <strong>of</strong> infection on heading<br />
<strong>and</strong> height as a measure <strong>of</strong><br />
resistance to septoria tritici blotch<br />
on wheat. Plant Disease 74:488-<br />
493.<br />
Van Ginkel, M., <strong>and</strong> A.L. Scharen.<br />
1987. Generation mean analysis<br />
<strong>and</strong> heritabilities <strong>of</strong> resistance to<br />
<strong>Septoria</strong> tritici in durum wheat.<br />
Phytopathology 77:1629-1633.<br />
Van Ginkel, M., <strong>and</strong> S. Rajaram. 1989.<br />
Breeding for global resistance to<br />
<strong>Septoria</strong> tritici in wheat. In: <strong>Septoria</strong><br />
<strong>of</strong> <strong>Cereals</strong>. P.M. Fried (ed.). July 4-<br />
7, Swiss Federal Research Station<br />
for Agronomy, Zurich,<br />
Switzerl<strong>and</strong>. pp 174-176.<br />
Van Ginkel, M., <strong>and</strong> S. Rajaram. 1993.<br />
Breeding for Durable Resistance to<br />
<strong>Diseases</strong> in Wheat: An<br />
International Perspective. In:<br />
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Th. Jacobs <strong>and</strong> J.E. Parlevliet (eds.).<br />
Kluwer Academic Publishers, The<br />
Netherl<strong>and</strong>s. pp. 259-272.<br />
Van Ginkel, M., <strong>and</strong> S. Rajaram. 1995.<br />
Breeding for Resistance to <strong>Septoria</strong><br />
tritici at <strong>CIMMYT</strong>. In: Proceedings<br />
<strong>of</strong> a <strong>Septoria</strong> tritici Workshop. L.<br />
Gilchrist, M. van Ginkel, A.<br />
McNab, <strong>and</strong> G.H.J. Kema (eds.).<br />
Mexico, D.F.: <strong>CIMMYT</strong>. pp. 55-61.<br />
Walther, H. 1990. An improved<br />
assessment procedure for breeding<br />
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in wheat. Plant Breeding 105:<br />
53-61.<br />
Walther, H. Durability <strong>and</strong> stability <strong>of</strong><br />
resistance <strong>of</strong> wheat to <strong>Septoria</strong><br />
nodorum (glume blotch) as<br />
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Durability <strong>of</strong> Disease Resistance.<br />
Th. Jacobs <strong>and</strong> J.E. Parlevliet (eds.).<br />
Kluwer Academic Publishers, The<br />
Netherl<strong>and</strong>s. p. 354.<br />
Walther, H., <strong>and</strong> M. Bohmer. 1992.<br />
Improved quantitative-genetic<br />
selection in breeding for resistance<br />
to <strong>Septoria</strong> nodorum (Berk.) in<br />
wheat. J. <strong>of</strong> Plant Dis. <strong>and</strong> Prot.<br />
99:371-380.<br />
Wilkinson, C.A., J.P. Murphy, <strong>and</strong><br />
R.C. Rufty. 1990. Diallel analysis <strong>of</strong><br />
components <strong>of</strong> partial resistance to<br />
<strong>Septoria</strong> nodorum in wheat. Plant<br />
Dis. 74:47-50.<br />
Wilson, R.E. 1979. Resistance to<br />
<strong>Septoria</strong> tritici in two wheat<br />
cultivars, determined by<br />
independent, single dominant<br />
genes. Aust. Plant Pathol. 8:16-18.<br />
Wilson, R.E. 1994. Progress toward<br />
breeding for resistance to the two<br />
septoria diseases <strong>of</strong> wheat in<br />
Australia. In: Proceedings <strong>of</strong> the<br />
4 th International Workshop on:<br />
<strong>Septoria</strong> <strong>of</strong> <strong>Cereals</strong>. E. Arseniuk, T.<br />
Goral, <strong>and</strong> P. Czembor (eds.).<br />
IHAR, Radzikow, Pol<strong>and</strong>.<br />
Hodowla Roslin Aklimatyzacja I<br />
Nasiennictwo (Special edition)<br />
38(3-4):149-152.<br />
Zuckerman, E., A. Eshel, <strong>and</strong> Z Eyal.<br />
1997. Physiological aspects related<br />
to tolerance <strong>of</strong> spring wheat<br />
cultivars to septoria tritici blotch.<br />
Phytopathology 87:60-65.
Breeding for Resistance to <strong>Septoria</strong> <strong>and</strong><br />
<strong>Stagonospora</strong> Blotches in Winter Wheat in<br />
the United States<br />
G. Shaner<br />
Department <strong>of</strong> Botany <strong>and</strong> Plant Pathology, Purdue University,<br />
West Lafayette, IN, USA<br />
This report will concentrate on<br />
resistance breeding efforts in the<br />
winter wheat region <strong>of</strong> the United<br />
States, where leaf blotch, caused by<br />
either <strong>Septoria</strong> tritici or <strong>Stagonospora</strong><br />
nodorum, has been a chronic<br />
disease. Leaf blotch has been a<br />
recognized problem in the eastern<br />
s<strong>of</strong>t winter wheat region <strong>of</strong> the US<br />
for more than 50 years. Historically,<br />
<strong>Stagonospora</strong> nodorum was the major<br />
pathogen in the southeastern US<br />
<strong>and</strong> <strong>Septoria</strong> tritici was the major<br />
pathogen in the north-central<br />
regions. Since the mid 1980s,<br />
however, S. nodorum has been at<br />
least as damaging in the northcentral<br />
region as S. tritici.<br />
The Purdue University-USDA<br />
small grain improvement program<br />
provides an example <strong>of</strong> efforts to<br />
manage leaf blotch by use <strong>of</strong><br />
genetic resistance. During the mid<br />
1950s, wheat breeders began to<br />
incorporate resistance to S. tritici<br />
into adapted s<strong>of</strong>t red winter wheat<br />
cultivars. At that time, S. tritici was<br />
clearly the dominant leaf blotch<br />
pathogen. The primary sources <strong>of</strong><br />
resistance were wheat cultivars<br />
from South America, e.g. Bulgaria<br />
88, Sao Sepe, <strong>and</strong> Sudeste.<br />
Eventually, most effort<br />
concentrated on Bulgaria 88 as the<br />
source <strong>of</strong> resistance. Cultivars<br />
Oasis <strong>and</strong> Sullivan, released in 1973<br />
<strong>and</strong> 1977, respectively, carried this<br />
resistance (Patterson et al., 1975;<br />
1979). When Oasis or Sullivan are<br />
inoculated at the adult plant stage<br />
in the greenhouse with spores <strong>of</strong> S.<br />
tritici, they are highly resistant.<br />
Infection results in small chlorotic<br />
flecks, with little or no chlorosis<br />
<strong>and</strong> no formation <strong>of</strong> pycnidia.<br />
Resistance in these cultivars<br />
segregates as a single, dominant<br />
Mendelian factor when they are<br />
crossed to a susceptible cultivar.<br />
Although Oasis derived its<br />
resistance to S. tritici mainly from<br />
Bulgaria 88, it evidently carried<br />
additional factors for resistance to<br />
S. nodorum because it exhibited a<br />
degree <strong>of</strong> resistance <strong>and</strong> performed<br />
well in areas <strong>of</strong> the southeastern US<br />
where this species was the<br />
dominant leaf spotting pathogen.<br />
This additional resistance may<br />
have resulted from the fact that<br />
selection for resistance in Indiana<br />
was entirely in the field under<br />
conditions <strong>of</strong> natural infection, <strong>and</strong><br />
there may have been more S.<br />
nodorum present than was<br />
suspected.<br />
Since the latter half <strong>of</strong> the 1980s,<br />
S. nodorum has emerged as the<br />
dominant leaf spotting pathogen in<br />
Indiana (Shaner <strong>and</strong> Buechley,<br />
1995). This shift in pathogen<br />
populations has evidently occurred<br />
throughout the north central region<br />
127<br />
<strong>of</strong> the US, <strong>and</strong> leaf blotch must now<br />
be regarded as a complex involving<br />
both S. tritici <strong>and</strong> S. nodorum. In<br />
more southern regions, S. nodorum<br />
continues to be the major leaf<br />
spotting pathogen.<br />
Management <strong>of</strong> this disease<br />
complex by genetic resistance<br />
requires resistance to both<br />
pathogens. Despite at least 50 years<br />
<strong>of</strong> effort in breeding for resistance<br />
to leaf blotch, progress has been<br />
modest. Evidence for this<br />
conclusion comes from descriptions<br />
<strong>of</strong> cultivars developed in this<br />
region <strong>and</strong> from direct observation<br />
<strong>of</strong> many cultivars in field trials.<br />
An estimation <strong>of</strong> the degree <strong>of</strong><br />
resistance available in currently<br />
available cultivars <strong>of</strong> s<strong>of</strong>t red<br />
winter wheat can be obtained from<br />
data collected in statewide wheat<br />
performance trials conducted each<br />
year in Indiana. For many years,<br />
entries in this trial have been<br />
evaluated for disease. Leaf blotch is<br />
encountered every year to some<br />
degree in Indiana wheat fields, but<br />
at varying severity. For the period<br />
1973–91, leaf blotch symptoms<br />
reached the flag leaf <strong>of</strong> susceptible<br />
cultivars within 26 days <strong>of</strong> heading<br />
in 10 <strong>of</strong> the 19 years (Shaner <strong>and</strong><br />
Buechley, 1995).
Session 6C — G. Shaner<br />
128<br />
An examination <strong>of</strong> leaf blotch<br />
severity data from 1986 through<br />
1996, collected from two locations<br />
in Indiana (Tippecanoe <strong>and</strong><br />
Daviess Counties), can be used to<br />
estimate progress in development<br />
<strong>of</strong> cultivars with resistance to this<br />
disease complex. The number <strong>and</strong><br />
identity <strong>of</strong> entries in these trials<br />
varied from year to year. Typically,<br />
a cultivar would be included for<br />
several consecutive years, but not<br />
for the full period covered by these<br />
trials. Each year, the trial included<br />
most cultivars <strong>of</strong> s<strong>of</strong>t red winter<br />
wheat <strong>of</strong>fered for sale in Indiana<br />
<strong>and</strong> adjacent states. The cultivars<br />
Arthur <strong>and</strong> Caldwell were<br />
included in every trial as long-term<br />
checks. Severity <strong>of</strong> leaf blotch in<br />
these trials was estimated using a 0<br />
to 9.5 scale, modified from one<br />
developed at <strong>CIMMYT</strong> (Shaner <strong>and</strong><br />
Buechley, 1995). This scale is<br />
reproduced here as Table 1 for<br />
convenient reference. The ratings <strong>of</strong><br />
leaf blotch severity discussed here<br />
were made when wheat was in the<br />
early to mid dough stage <strong>of</strong><br />
development.<br />
Table 1. Leaf blotch severity scale for wheat.<br />
There are several ways<br />
that the question <strong>of</strong><br />
improvement in resistance to<br />
leaf blotch can be<br />
investigated using data from<br />
these trials. If the general<br />
level <strong>of</strong> resistance among<br />
cultivars increased over the<br />
14-year period, then the trial<br />
mean leaf blotch severity,<br />
based on all cultivars,<br />
should decline relative to the<br />
severities for the check<br />
cultivars. Among the 16<br />
trials, trial means ranged<br />
from 5.4 to 9.2, reflecting<br />
variation among years in the<br />
suitability <strong>of</strong> weather for leaf<br />
blotch development. This<br />
variation in trial means was<br />
closely followed by the<br />
values for the two long-term<br />
check cultivars, Arthur <strong>and</strong><br />
Caldwell. The trial mean<br />
was always less than the<br />
severity for Caldwell <strong>and</strong><br />
usually less than the severity<br />
for Arthur, but there was no<br />
evidence for a greater<br />
disparity between the trial<br />
mean <strong>and</strong> the checks over<br />
Severity<br />
10<br />
9<br />
8<br />
7<br />
6<br />
5<br />
4<br />
Mean<br />
Arthur<br />
Caldwell<br />
86 PAF<br />
87 PAF<br />
89 PAF<br />
90 PAF<br />
90 DAV<br />
91 PAF<br />
9 DAV<br />
93 PAF<br />
93 DAV<br />
94 PAF<br />
94 DAV<br />
96 PAF<br />
96 DAV<br />
97 DAV<br />
98 DAV<br />
99 PAF<br />
Trial<br />
Figure 1. Trial means <strong>and</strong> means for check cultivars<br />
Arthur <strong>and</strong> Caldwell for leaf blotch ratings, 1986<br />
through 1999, at two locations in Indiana. PAF =<br />
Purdue Agronomy Farm (Tippecanoe County); DAV =<br />
Daviess County.<br />
time (Figure 1). The conclusion<br />
from this is that the newer cultivars<br />
are not more resistant collectively<br />
than those included in trials during<br />
earlier years, as represented by the<br />
two check cultivars.<br />
If only a few cultivars in a trial<br />
had substantially improved<br />
resistance, then the mean <strong>of</strong> all<br />
entries in that trial might not reflect<br />
this improvement, but the st<strong>and</strong>ard<br />
deviation <strong>of</strong> cultivar means should<br />
be greater than in trials lacking<br />
exceptionally resistant cultivars. If<br />
there has been progress in<br />
Range in percent severity on indicated leaf<br />
Scale value Flag Flag-1 Flag-2 Flag-3 Mean severityx 1 0-5 0.1<br />
2 5-20 2.9<br />
3 20-40 8.1<br />
4 1-10 40-70 15.6<br />
5 0-1 10-25 70-90 25.5<br />
6 1-10 25-75 90-100 37.8<br />
7 10-50 75-100 100 52.3<br />
8 1-20 50-90 100 100 69.3<br />
9 20-90 90-100 100 100 88.5<br />
9.5 90-100 100 100 100 99.1<br />
x Mean severity is the average for the four leaves, based on midpoint values for each range. Mean severity (P) can be<br />
calculated from the scale value (S) according to: P = -0.38253 – 0.69435 S + 1.17499 S2 (R2 = 0.999).
development <strong>of</strong> cultivars resistant to<br />
leaf blotch, then the variance <strong>of</strong><br />
cultivar means should be seen to<br />
increase over the years. Plotting trial<br />
means <strong>and</strong> st<strong>and</strong>ard deviations over<br />
time revealed no tendency for the<br />
st<strong>and</strong>ard deviation to increase with<br />
time or to vary with the trial mean<br />
(Figure 2).<br />
Examination <strong>of</strong> data from<br />
individual trials may reveal to what<br />
extent resistance is expressed under<br />
conditions <strong>of</strong> severe, moderate, or<br />
mild disease pressure. Results from<br />
three trials are presented here for<br />
illustration. The trial with the<br />
greatest severity <strong>of</strong> leaf blotch was<br />
in Daviess County during 1990. The<br />
trial mean severity was 9.2. The<br />
cultivars with the least disease in<br />
this trial had a rating <strong>of</strong> 8. Thus,<br />
Severity<br />
10<br />
8<br />
6<br />
4<br />
2<br />
0<br />
Breeding for Resistance to <strong>Septoria</strong> <strong>and</strong> <strong>Stagonospora</strong> Blotches in Winter Wheat in the United States 129<br />
Mean<br />
St dev<br />
86 PAF<br />
87 PAF<br />
89 PAF<br />
90 PAF<br />
90 DAV<br />
91 PAF<br />
9 DAV<br />
93 PAF<br />
93 DAV<br />
94 PAF<br />
94 DAV<br />
96 PAF<br />
96 DAV<br />
97 DAV<br />
98 DAV<br />
99 PAF<br />
Trial<br />
Figure 2. Mean <strong>and</strong> st<strong>and</strong>ard deviation <strong>of</strong> leaf blotch<br />
severity on wheat in cultivar trials from 1986 through<br />
1999 at two locations in Indiana.<br />
8<br />
;; yy<br />
6<br />
;; yy ;; yy<br />
4<br />
;; yy ;; yy ;; yy ;; yy ;; yy ;; yy ;; yy<br />
2<br />
;; yy<br />
0<br />
;; yy ;; yy ;; yy ;; yy ;; yy ;; yy<br />
4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5<br />
Severity class<br />
Figure 4. Frequency distribution <strong>of</strong> mean leaf blotch<br />
severities on wheat cultivars evaluated during 1993<br />
in Tippecanoe County, Indiana.<br />
Number <strong>of</strong> entries<br />
under conditions <strong>of</strong> great disease<br />
pressure, symptoms progressed to<br />
the flag leaves <strong>of</strong> all cultivars<br />
(Figure 3). The entire range in<br />
resistance was confined to the<br />
degree <strong>of</strong> flag leaf area that was<br />
blighted. All lower leaves were<br />
completed destroyed by leaf blotch.<br />
Mean leaf blotch severity was<br />
very low in the trial conducted in<br />
Tippecanoe County during 1993<br />
(Figure 4). The range among<br />
cultivar severity means was<br />
somewhat greater than in the<br />
previous example, because severity<br />
values were not truncated by the<br />
maximum possible value. The most<br />
severely affected cultivars showed<br />
only a few lesions on the flag leaf.<br />
On most <strong>of</strong> the cultivars, leaf blotch<br />
was at most only 10% on leaf F-1.<br />
Number <strong>of</strong> entries<br />
Number <strong>of</strong> entries<br />
15<br />
The trial conducted during 1997<br />
in Daviess County represents<br />
moderately severe disease pressure.<br />
The trial mean was 7.8 (Figure 5).<br />
Blotch symptoms reached the flag<br />
leaf on fewer than half <strong>of</strong> the<br />
cultivars in this trial, but the<br />
cultivars with the least amount <strong>of</strong><br />
disease had symptoms on leaf F-1.<br />
Regardless <strong>of</strong> overall disease<br />
pressure, the range in leaf blotch<br />
severity among cultivars within a<br />
trial was not great.<br />
Analysis <strong>of</strong> these field trial data<br />
provides no evidence for substantial<br />
increases in resistance in a region <strong>of</strong><br />
the United States where leaf blotch<br />
is a chronic <strong>and</strong> <strong>of</strong>ten severe disease<br />
<strong>of</strong> wheat. At best, a resistant cultivar<br />
will hold symptom development<br />
back by a few days on the flag <strong>and</strong><br />
flag-1 leaves.<br />
10<br />
5<br />
0<br />
4 4.5 5 5.5<br />
;; yy ; y ;<br />
;; yy;<br />
y; 6 6.5 7 7.5 8 8.5 9 9.5<br />
Severity class<br />
; ;; yy<br />
;; yy<br />
;; yy<br />
Figure 3. Frequency distribution <strong>of</strong> mean leaf blotch<br />
severities on wheat cultivars evaluated during 1990<br />
in Daviess County, Indiana.<br />
20 ;; yy<br />
15<br />
10 ;; yy ;; yy<br />
5 ;; yy ;; yy ;; yy<br />
0<br />
;; yy ;; yy ;; yy ;; yy<br />
4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5<br />
Severity class;;<br />
Figure 5. Frequency distribution <strong>of</strong> mean leaf blotch<br />
severities on wheat cultivars evaluated during 1997<br />
in Daviess County, Indiana.<br />
y<br />
y<br />
y
Session 6C — G. Shaner<br />
130<br />
Another approach to<br />
determining progress in breeding<br />
winter wheat for resistance to leaf<br />
blotch is to examine cultivar<br />
registrations in the journal Crop<br />
Science over a period <strong>of</strong> years.<br />
Registrations for 131 wheat<br />
cultivars were published in<br />
volumes 28-39, <strong>of</strong> which 96 were<br />
for winter wheat. Of these winter<br />
wheat cultivars, 25 were reported<br />
to have some degree <strong>of</strong> resistance<br />
to S. tritici or S. nodorum <strong>and</strong> 2 were<br />
reported to be susceptible. For the<br />
other 69 cultivars there was no<br />
mention <strong>of</strong> reaction to either <strong>of</strong><br />
these pathogens. The summary for<br />
winter wheat cultivars includes<br />
both hard <strong>and</strong> s<strong>of</strong>t, <strong>and</strong> red <strong>and</strong><br />
white classes, covering the eastern<br />
portion <strong>of</strong> the US, the Great Plains,<br />
<strong>and</strong> the Pacific Coast states.<br />
Because leaf blotch has historically<br />
been a greater problem in the<br />
eastern region <strong>of</strong> the US, data were<br />
summarized for this region. Of 39<br />
wheat cultivars developed in the<br />
eastern s<strong>of</strong>t wheat region <strong>of</strong> the US,<br />
15 were described as having some<br />
resistance, none were described as<br />
susceptible, <strong>and</strong> there was no<br />
mention <strong>of</strong> reaction for 24 cultivars.<br />
The absence <strong>of</strong> any comment about<br />
reaction to leaf blotch is interesting,<br />
because for many other wheat<br />
pathogens <strong>and</strong> pests (e.g. rusts,<br />
powdery mildew, viruses, Hessian<br />
fly), the registration articles<br />
commonly documented<br />
susceptibility as well as resistance.<br />
Of the 15 cultivars described as<br />
being resistant, 12 were described<br />
as having resistance to S. nodorum,<br />
7 <strong>of</strong> which were described as<br />
having resistance to S. tritici as<br />
well. The other three cultivars were<br />
described as being resistant only to<br />
S. tritici.<br />
An association has long been<br />
noted between tall stature, late<br />
maturity, <strong>and</strong> resistance to leaf<br />
blotch caused by either S. tritici or<br />
S. nodorum (Camacho-Casas et al.,<br />
1995; Scott et al., 1982). Late<br />
maturity <strong>and</strong> tall stature per se may<br />
confer a significant degree <strong>of</strong> leaf<br />
blotch in such cultivars. Where leaf<br />
blotch is the greatest threat in<br />
North America, the eastern s<strong>of</strong>t<br />
wheat region, breeders have<br />
emphasized development <strong>of</strong> short<br />
(90 to 100 cm), early maturing<br />
cultivars, as a way to reduce risk<br />
from rust infection, to permit grain<br />
filling before the excessive hot <strong>and</strong><br />
humid conditions <strong>of</strong> summer, <strong>and</strong><br />
to permit double-cropping with<br />
soybeans. The desired st<strong>and</strong>ards <strong>of</strong><br />
early maturity <strong>and</strong> short stature<br />
may be an important reason for the<br />
lack <strong>of</strong> progress in achieving<br />
adequate levels <strong>of</strong> resistance to the<br />
leaf blotch pathogens.<br />
Hectares <strong>of</strong> s<strong>of</strong>t wheat<br />
production in the eastern US have<br />
been declining for many years.<br />
Poor yields <strong>and</strong> poor grain quality<br />
because <strong>of</strong> disease have been a<br />
major reason for the ab<strong>and</strong>onment<br />
<strong>of</strong> wheat production by many<br />
farmers. Unless greater degrees <strong>of</strong><br />
resistance can be bred into highyielding,<br />
early-maturing, shortstatured<br />
wheat cultivars, this<br />
downward trend in production will<br />
likely continue.<br />
References<br />
Camacho-Casas, M.A., W.E.<br />
Kronstad, <strong>and</strong> A.L. Scharen. 1995.<br />
<strong>Septoria</strong> tritici resistance <strong>and</strong><br />
associations with agronomic traits<br />
in a wheat cross. Crop Sci. 35:971-<br />
976.<br />
Patterson, F.L., Roberts, J.J., Finney,<br />
R.E., Shaner, G.E., Gallun, R.L.,<br />
<strong>and</strong> Ohm, H.W. 1975. Registration<br />
<strong>of</strong> Oasis wheat. Crop Sci. 15:736-<br />
737.<br />
Patterson, F.L., Shaner, G.E., Huber,<br />
D.M., Ohm, H.W., Finney, R.E.,<br />
Gallun, R.L., <strong>and</strong> Roberts, J.J. 1979.<br />
Registration <strong>of</strong> Sullivan wheat<br />
Crop Sci. 19:297.<br />
Scott, P.R., Benedikz, P.M., <strong>and</strong> Cox,<br />
C.J. 1982. A genetic study <strong>of</strong> the<br />
relationship between height, time<br />
<strong>of</strong> ear emergence <strong>and</strong> resistance to<br />
<strong>Septoria</strong> nodorum in wheat. Plant<br />
Pathology 31:45-60.<br />
Shaner, G., <strong>and</strong> Buechley, G. 1995.<br />
Epidemiology <strong>of</strong> leaf blotch <strong>of</strong> s<strong>of</strong>t<br />
red winter wheat caused by<br />
<strong>Septoria</strong> tritici <strong>and</strong> <strong>Stagonospora</strong><br />
nodorum. Plant Disease 79:928-938.
<strong>Septoria</strong> tritici Resistance <strong>of</strong> Wheat<br />
Cultivars at Different Growth Stages<br />
M. Díaz de Ackermann, 1 M.M. Kohli, 2 <strong>and</strong> V. Ibañez1 1 INIA La Estanzuela, Colonia, Uruguay<br />
2 <strong>CIMMYT</strong> Regional Wheat Program, Uruguay<br />
Abstract<br />
Nine wheat cultivars planted in the greenhouse were inoculated with a mixture <strong>of</strong> three isolates <strong>of</strong> <strong>Septoria</strong> tritici at<br />
four different growth stages. The first evaluation, done six weeks after inoculation, showed that the facultative wheats at<br />
growth stage (GS) 39 (flag leaf ligule just visible) <strong>and</strong> spring wheats at GS 65 (flowering halfway complete) reached the<br />
highest levels <strong>of</strong> infection, with two exceptions, cvs E. Pelón 90 <strong>and</strong> P. Oasis. In the second evaluation a week later, the<br />
highest level <strong>of</strong> infection was reached when facultative wheats were inoculated at GS 32-33 (stem elongation-first node<br />
detectable) <strong>and</strong> spring wheats at GS 39 (flag leaf ligule just visible). In both evaluations, the effects <strong>of</strong> cultivars, seeding<br />
date/GS at inoculation, <strong>and</strong> the interaction between cultivars <strong>and</strong> GS at inoculation were significant. <strong>Septoria</strong> tritici<br />
susceptibility increased with the age <strong>of</strong> the plants. The increase in the level <strong>of</strong> infection was significantly lower in cv P.<br />
Superior (Bobwhite), indicating a certain level <strong>of</strong> adult plant resistance.<br />
Wheat is an important crop in the<br />
western region <strong>of</strong> Uruguay, where it<br />
is grown for grain <strong>and</strong> forage<br />
(double purpose) in the mixed<br />
farming system, or solely for grain<br />
production. Among diseases present<br />
in the country, <strong>Septoria</strong> tritici leaf<br />
blotch is one <strong>of</strong> the most important,<br />
<strong>and</strong> it can be particularly severe<br />
during the wheat-growing season in<br />
this temperate high rainfall<br />
environment. The wide range <strong>of</strong><br />
planting dates from May (or even<br />
earlier for double purpose crops) to<br />
August determines the growth stage<br />
(GS) at the time <strong>of</strong> primary infection,<br />
later in the fall or in early spring.<br />
Facultative wheats, sown in the fall,<br />
are infected very early (seedling<br />
stage) or at the beginning <strong>of</strong> spring<br />
(boot stage), depending on the year.<br />
Spring wheats, sown later than the<br />
facultative types, are exposed mainly<br />
to spring infections.<br />
Reduced disease severity<br />
associated with tall plant stature <strong>and</strong><br />
late maturity has been observed<br />
(Danon et al., 1982; Eyal <strong>and</strong> Talpaz,<br />
1990; van Beuningen <strong>and</strong> Kohli,<br />
1990; Tavella, 1978). However, the<br />
relationship between the resistance<br />
expressed at the seedling stage <strong>and</strong><br />
the adult plant stage, or vice versa,<br />
is not fully understood. Kema <strong>and</strong><br />
van Silfhout (1995) found<br />
significant correlations for one <strong>of</strong><br />
three isolates inoculated on 22<br />
wheat cultivars at the seedling <strong>and</strong><br />
adult plant stages. On the other<br />
h<strong>and</strong>, Arama, Parlevliet, <strong>and</strong> van<br />
Silfhout (1994) described three<br />
types <strong>of</strong> resistance: resistance only<br />
in seedling (seedling resistance),<br />
resistance only in adult plant (adult<br />
plant resistance), <strong>and</strong> resistance in<br />
both seedling <strong>and</strong> adult plant<br />
(overall resistance).<br />
The objective <strong>of</strong> this study was<br />
to determine the level <strong>of</strong> resistance<br />
in nine wheat cultivars,<br />
representing different reactions to S.<br />
tritici in the field, at different<br />
growth stages <strong>and</strong> under controlled<br />
conditions.<br />
Materials <strong>and</strong> Methods<br />
131<br />
Nine wheat cultivars with<br />
different growth habits <strong>and</strong> field<br />
reactions to <strong>Septoria</strong> tritici were<br />
planted in three pots each, five<br />
plants per pot, on four seeding<br />
dates: 28 April . , 16 May . , 31 May . ,<br />
<strong>and</strong> 27 June 1995. The pots<br />
measured 29 <strong>and</strong> 20 cm in their<br />
upper <strong>and</strong> lower diameters,<br />
respectively, <strong>and</strong> 20 cm in height.<br />
The cultivars’ growth habit,<br />
progenitors, <strong>and</strong> field reaction to S.<br />
tritici are presented in Table 1. The<br />
concentration <strong>of</strong> S. tritici spore<br />
suspension was adjusted to 106 conidia per ml, with the aid <strong>of</strong> a<br />
hemacytometer. The cultivars were<br />
inoculated with a mixture <strong>of</strong> three<br />
isolates (26S, 4407, <strong>and</strong> E. Federal)<br />
on 17 July, at the growth stages<br />
(Zadoks et al., 1974) indicated in<br />
Table 2. The selected isolates were<br />
characterized by Diaz de<br />
Ackermann et al. (1994). The plants<br />
were kept in a humid chamber until<br />
20 July. Due to the delayed
Session 6C — M. Díaz de Ackermann, M.M. Kohli, <strong>and</strong> V. Ibañez<br />
132<br />
appearance <strong>of</strong> symptoms, two<br />
evaluations were done, the first<br />
on 28 August . <strong>and</strong> the second on<br />
5 September.<br />
The infection was scored as<br />
percent <strong>of</strong> leaf area affected by<br />
the disease, on two tillers per<br />
plant <strong>and</strong> five leaves per tiller.<br />
Transformed data (Loge +0.5) for<br />
the flag leaf, F-1, <strong>and</strong> F-2, as<br />
well as the average <strong>of</strong> the three<br />
leaves, were analyzed using<br />
SAS GLM procedure (Version<br />
6.12; SAS Institute, Cary, NC).<br />
Infection on the lower two<br />
leaves was not included in this<br />
analysis due to many missing<br />
values.<br />
Results <strong>and</strong><br />
Discussion<br />
The analysis <strong>of</strong> variance<br />
(ANOVA) for each leaf <strong>and</strong> the<br />
average <strong>of</strong> the three leaves<br />
showed significant differences<br />
between cultivars <strong>and</strong> seeding<br />
dates, as well as significant<br />
interaction between seeding dates<br />
<strong>and</strong> cultivars in both evaluations. As<br />
the results were similar for<br />
individual leaves <strong>and</strong> the average <strong>of</strong><br />
the three top leaves, only data for<br />
the latter are presented (Table 3).<br />
Differences among cultivars<br />
were found on the first two seeding<br />
dates (28/04 <strong>and</strong> 16/05) during the<br />
first evaluation, <strong>and</strong> on the<br />
intermediate dates (16/05 <strong>and</strong> 31/<br />
05) during the second evaluation.<br />
All the cultivars except B.<br />
Charrúa showed different infection<br />
levels on different seeding dates at<br />
first evaluation. This interaction is<br />
explained by the cultivars’ different<br />
Table 1. Cross, growth habit <strong>and</strong> field reaction to <strong>Septoria</strong> tritici <strong>of</strong> nine wheat cultivars.<br />
Cultivar Cross Growth habit Reaction<br />
E. Cardenal Veery#3 Spring S1 INIA Mirlo Car 853/Coc//Vee#5/3/Ures Spring R<br />
E Pelón 90 Kavkaz/Torim73 Spring R-MR<br />
ProINTA Superior Bobwhite Spring MR<br />
ProINTA Oasis Oasis/Torim Spring S<br />
E. Federal E.Hornero/CNT8 Facultative MR<br />
E Halcón Buck6/MR74507 Facultative MR<br />
Buck Charrúa RAP/RE//IRAP/3/LOV/4/RAP/RE//IRAP Facultative R<br />
LE 2196 E. Jilguero/ND526 Facultative R<br />
1 S: susceptible, R: resistant, MR: moderately resistant.<br />
Table 2. Growth stages <strong>of</strong> spring <strong>and</strong> facultative wheat varieties at time <strong>of</strong> inoculation.<br />
Seeding date 28/04 16/05 31/05 27/06<br />
Spring wheats 65 39 30-31 22-23<br />
Facultative wheats 39 32-33 30 24-25<br />
Table 3. Average level <strong>of</strong> infection <strong>of</strong> top three leaves, comparison among date <strong>of</strong> seeding (small<br />
letter) <strong>and</strong> cultivars (capital letter). Evaluation August 28th <strong>and</strong> September 5th .<br />
Sowing date 28/04 16/05 31/05 27/06<br />
GS Facultative 39 32/33 30 24/25<br />
GS Spring 65 39 30/31 22/23<br />
Cultivar 1st . eval 1st . eval 2nd . eval 1st . eval 2nd . eval 1st . eval 2nd . eval<br />
E.Cardenal 11.65 aA 3.81 bA 46.24 aA 0.16 c 3.57 bA 0.00 c 0.45 c<br />
I. Mirlo 1.98 aCD 2.17 aAB 23.93 aB 0.00 b 1.96 bB 0.00 b 0.66 c<br />
E. Pelón 90 1.32 aDE 2.20 aAB 16.86 aC 0.02 b 1.76 bB 0.00 b 0.19 c<br />
P. Superior 2.92 aC 1.50 bB 8.14 aD 0.00 c 1.86 bB 0.00 c 0.11 c<br />
P. Oasis 1.79 bCD 4.25 aA 27.22 aB 0.26 c 2.18 bB 0.02 c 0.44 c<br />
E. Federal 7.64 aB 3.17 bA 19.15 aC 0.00 c 0.50 bD 0.00 c 0.43 b<br />
E. Halcón 1.25 aE 0.23 bC 3.61 aE 0.03 b 1.13 bC 0.00 b 0.17 c<br />
B. Charrúa 0.14 aE 0.03 aC 2.91 aE 0.00 a 0.56 bCD 0.00 a 0.44 b<br />
LE 2196 1.66 aDE 0.22 bC 6.65 aD 0.03 b 1.23 bB 0.00 b 0.33 c<br />
Average 3.37 1.95 17.19 0.06 1.64 0.00 0.36<br />
behavior on the different seeding<br />
dates/GS at inoculation. Early<br />
seedling infection demonstrated<br />
higher sensitivity for detecting<br />
differences among cultivars.<br />
During this evaluation, the<br />
facultative wheat varieties were<br />
found to have higher <strong>Septoria</strong><br />
susceptibility at GS 39 (flag leaf<br />
ligule just visible) <strong>and</strong> spring<br />
varieties at GS 65 (flowering<br />
halfway complete). These were<br />
followed by GS 32/33 (stem<br />
elongation-first node detectable)<br />
<strong>and</strong> GS 39 for facultative <strong>and</strong><br />
spring varieties, respectively.<br />
Inoculations at GS 22 to GS 31<br />
did not produce adequate<br />
infection levels.<br />
At the second evaluation on 5<br />
September, the group seeded on the<br />
first date was already dry. For the<br />
remaining three sowing dates, the<br />
highest infection level was shown by<br />
facultative wheat varieties inoculated<br />
at GS 32-33 <strong>and</strong> by spring wheat<br />
varieties inoculated at GS 39.<br />
Comparison <strong>of</strong> the cultivars’ field<br />
reactions to S. tritici with infection<br />
under controlled conditions showed<br />
differences in I. Mirlo, P. Superior, E.<br />
Halcón, <strong>and</strong> P. Oasis. These cultivars<br />
are considered resistant (R),<br />
moderately resistant (MR), moderately<br />
resistant (MR), <strong>and</strong> susceptible (S),<br />
respectively, under field conditions;<br />
under controlled conditions they were
MS, R, R, <strong>and</strong> MS, respectively. The<br />
interaction between cultivar <strong>and</strong><br />
growth stage at inoculation<br />
indicated the lack <strong>of</strong> precision that<br />
may occur while evaluating<br />
resistance <strong>of</strong> a variety.<br />
In the first evaluation, with<br />
relatively low levels <strong>of</strong> infection,<br />
cultivars such as P. Oasis showed<br />
significantly lower infection levels<br />
at GS 65 than at GS 39. E. Pelón 90<br />
showed similar behavior without<br />
being significantly different at<br />
these two growth stages. However,<br />
in contrast to P. Oasis <strong>and</strong> E. Pelón<br />
90, P. Superior presented a<br />
significantly higher infection level<br />
at GS 65 than at GS 39 (Table 3;<br />
Figure 1). Although the infection<br />
level <strong>of</strong> P. Superior at the initial<br />
stages was almost equal to that <strong>of</strong><br />
% <strong>of</strong> infection<br />
0<br />
Sowing date 27/06 31/05 16/05 28/04<br />
ZGS 22-23 30-31 39 65<br />
Figure 1. Level <strong>of</strong> infection, average <strong>of</strong> three<br />
% <strong>of</strong> infection<br />
12<br />
9<br />
6<br />
3<br />
50<br />
40<br />
30<br />
20<br />
10<br />
Leaves, August 28th.<br />
Cardenal<br />
Oasis<br />
Mirlo<br />
Pelón<br />
Superior<br />
Leaves, September 5th.<br />
Charrúa<br />
Halcón<br />
LE2196<br />
Federal<br />
Superior<br />
Pelón<br />
Mirlo<br />
Oasis<br />
Cardenal<br />
0<br />
Sowing date 27/06 31/05 16/05<br />
S-ZGS 22-23 30-31 39<br />
F-ZGS 24-25 30 32-33<br />
Figure 2. Level <strong>of</strong> infection, average <strong>of</strong> three<br />
susceptible varieties P. Oasis <strong>and</strong> E.<br />
Cardenal at the second evaluation, P.<br />
Superior showed the slowest<br />
increase over time to become the<br />
most resistant genotype at the adult<br />
plant stage. This suggests that P.<br />
Superior (Bobwhite) may have a<br />
certain degree <strong>of</strong> adult plant<br />
resistance.<br />
In general, spring habit, high<br />
yielding, early maturing semidwarf<br />
wheats showed higher levels <strong>of</strong><br />
<strong>Septoria</strong> infection than the facultative<br />
wheats (tall <strong>and</strong> late maturing). The<br />
highest infection level observed in a<br />
facultative wheat (E. Federal) is<br />
equivalent to the level demonstrated<br />
by a moderately resistant spring<br />
variety such as E. Pelón 90 (Figure 2).<br />
As per the definition put forward by<br />
Arama, Parleviet, <strong>and</strong> van Silfhout<br />
(1994), Buck Charrúa seems to<br />
possess overall resistance. A high<br />
degree <strong>of</strong> resistance was observed at<br />
the juvenile stage in all varieties.<br />
Only P. Superior demonstrated<br />
adult-plant resistance. Further<br />
studies are required to evaluate the<br />
level <strong>of</strong> resistance in stages earlier<br />
than GS 22-25.<br />
Conclusions<br />
The S. tritici susceptibility <strong>of</strong><br />
different wheat varieties seems to<br />
increase with the age <strong>of</strong> the plants.<br />
However, the rate <strong>of</strong> this increase<br />
(the slope <strong>of</strong> the curve) is different<br />
for each cultivar. As a result<br />
interactions were observed between<br />
the cultivars’ susceptibility (infection<br />
level) <strong>and</strong> growth stage at the time <strong>of</strong><br />
infection (inoculation). This suggests<br />
that several evaluations may be<br />
required to determine a cultivar’s<br />
potential as a parent in a crop<br />
improvement program.<br />
<strong>Septoria</strong> tritici Resistance <strong>of</strong> Wheat Cultivars at Different Growth Stages 133<br />
References<br />
Arama, P.F., Parlevliet, J.E., <strong>and</strong> van<br />
Silfhout, C.H.1994. Effect <strong>of</strong><br />
plant height <strong>and</strong> days to heading<br />
on the expression <strong>of</strong> resistance in<br />
Triticum aestivum to <strong>Septoria</strong> tritici<br />
in Kenya. In: Proceedings <strong>of</strong> the<br />
4 th . International Workshop on:<br />
<strong>Septoria</strong> <strong>of</strong> cereals. E. Arseniuk,<br />
T. Goral, <strong>and</strong> P. Czembor (eds.).<br />
July 4-7, 1994. IHAR Radzikow,<br />
Pol<strong>and</strong>. pp. 153-157.<br />
Danon, T., Sacks, J.M., <strong>and</strong> Eyal, Z.<br />
1982. The relationship among<br />
plant stature, maturity class, <strong>and</strong><br />
susceptibility to septoria leaf<br />
blotch <strong>of</strong> wheat. Phytopathology<br />
72:1037-1042.<br />
Díaz de Ackermann, M., Stewart, S.,<br />
Ibañez,V., Capdeville, F., <strong>and</strong><br />
Stoll, M. 1994. Pathogenic<br />
variability <strong>of</strong> <strong>Septoria</strong> tritici in<br />
isolates from South America. In:<br />
Proceedings <strong>of</strong> the 4 th<br />
International Workshop on:<br />
<strong>Septoria</strong> <strong>of</strong> cereals. July 4-7, 1994.<br />
Ihar Radzikow, Pol<strong>and</strong>.<br />
pp. 335-338.<br />
Eyal, Z., <strong>and</strong> Talpaz, H. 1990. The<br />
combined effect <strong>of</strong> plant stature<br />
<strong>and</strong> maturity on the response <strong>of</strong><br />
wheat <strong>and</strong> triticale accessions to<br />
<strong>Septoria</strong> tritici. Euphytica<br />
46:133-141.<br />
Kema, G.H.J., <strong>and</strong> van Silfhout,<br />
C.H. 1995. Comparative<br />
virulence analysis <strong>of</strong> <strong>Septoria</strong><br />
tritici to seedling <strong>and</strong> adult plant<br />
resistance. In: Breeding for<br />
disease resistance with emphasis<br />
on durability. Regional<br />
Workshop for Eastern, Central<br />
<strong>and</strong> Southern Africa. D.I. Danial<br />
(ed.). Njoro, Kenya, October 4-7,<br />
1994. p. 221.<br />
Tavella, C.M. 1978. Date <strong>of</strong> heading<br />
<strong>and</strong> plant height <strong>of</strong> wheat<br />
cultivars, as related to septoria<br />
leaf blotch damage. Euphytica<br />
27:577-580.<br />
Van Beuningen, L.T., <strong>and</strong> Kohli,<br />
M.M. 1990. Deviation from the<br />
regression <strong>of</strong> infection on<br />
heading <strong>and</strong> height as a measure<br />
<strong>of</strong> resistance to septoria tritici<br />
blotch in wheat. Plant Disease<br />
74:488-493.<br />
Zadoks, J.C., Chang, T.T., <strong>and</strong><br />
Konzak, C.F. 1974. A decimal<br />
code for the growth stages <strong>of</strong><br />
cereals. Weed Res. 14:415-4214.
134<br />
<strong>Septoria</strong> tritici Resistance Sources <strong>and</strong> Breeding<br />
Progress at <strong>CIMMYT</strong>, 1970-99<br />
L. Gilchrist, 1 B. Gomez, 2 R. Gonzalez, 2 S. Fuentes, 3 A. Mujeeb-Kazi, 1 W. Pfeiffer, 1 S. Rajaram, 1 R. Rodriguez, 3 B.<br />
Skovm<strong>and</strong>, 1 M. van Ginkel, 1 <strong>and</strong> C. Velazquez1 (Field presentation)<br />
1 <strong>CIMMYT</strong> Wheat Program, El Batan, Mexico<br />
2 National Livestock <strong>and</strong> Agricultural Research Institute (INIFAP), Mexico<br />
3 Retired <strong>CIMMYT</strong> Staff<br />
Abstract<br />
An overview is provided <strong>of</strong> the sources <strong>of</strong> <strong>Septoria</strong> tritici resistance used in the <strong>CIMMYT</strong> bread wheat program, <strong>and</strong> <strong>of</strong><br />
progress in breeding for resistance for wheat mega-environment 2 (high rainfall) in 1970-99. Several phases can be<br />
distinguished: use <strong>of</strong> Russian winter wheat, lines from the Southern Cone <strong>of</strong> South America (Brazil, Chile <strong>and</strong> Argentina)<br />
<strong>and</strong> USA as resistance sources; introduction <strong>of</strong> disease resistance from Brazilian sources into semi-dwarf <strong>and</strong> early<br />
backgrounds; introduction <strong>of</strong> genes from triticale <strong>and</strong> Triticum diccocon into common wheat; introgression <strong>of</strong> resistance<br />
from synthetic hexaploids <strong>and</strong> derivatives into susceptible bread wheat stocks; pyramiding <strong>of</strong> Chinese resistance sources with<br />
the sources mentioned above, <strong>and</strong> combining them into adapted agronomic types. Some preliminary work on resistant durum<br />
wheat sources, including some <strong>of</strong> the best ones from Tunisia, is presented.<br />
Bread Wheat Program<br />
The semidwarf wheats<br />
developed in Mexico in the 1960s<br />
were widely distributed. The<br />
modern wheat varieties that<br />
replaced local wheats were<br />
successful because <strong>of</strong> their wide<br />
adaptation, high yield potential,<br />
<strong>and</strong> disease resistance (mainly to<br />
stem <strong>and</strong> leaf rust) for favorable,<br />
irrigated production conditions<br />
(Rajaram et al., 1994).<br />
The emphasis <strong>of</strong> <strong>CIMMYT</strong>’s<br />
bread wheat breeding program on<br />
disease resistance was exp<strong>and</strong>ed in<br />
the 1970s to include resistance to<br />
Puccinia striiformis <strong>and</strong> <strong>Septoria</strong><br />
tritici for the high rainfall wheat<br />
growing areas <strong>of</strong> the world (megaenvironment<br />
2). These are<br />
temperate environments with an<br />
average precipitation <strong>of</strong> >500 mm.<br />
The search for S. tritici resistance,<br />
the accumulation <strong>of</strong> resistance<br />
genes, <strong>and</strong> the incorporation <strong>of</strong><br />
resistance into high yielding<br />
advanced lines have been very<br />
successful in the <strong>CIMMYT</strong> bread<br />
wheat program.<br />
Three main groups <strong>of</strong> sources<br />
were used to introduce resistance:<br />
a) Russian winter wheat, b) lines<br />
from the wheat growing areas <strong>of</strong><br />
Brazil, Chile, <strong>and</strong> Argentina<br />
(Southern Cone <strong>of</strong> South America),<br />
<strong>and</strong> c) to a lesser extent, lines from<br />
the USA (Mann et al., 1985)<br />
(Table 1). The resistance <strong>of</strong> these<br />
lines was identified <strong>and</strong>/or<br />
confirmed in different countries<br />
through the International <strong>Septoria</strong><br />
Nursery (ISEPTON), beginning<br />
in 1971.<br />
The breeding program focused<br />
on combining the semidwarf plant<br />
type with high yield potential <strong>and</strong><br />
resistance to leaf <strong>and</strong> stripe rust<br />
plus septoria leaf blotch.<br />
Germplasm was screened <strong>and</strong><br />
selected for septoria leaf blotch at<br />
two locations in Mexico: Patzcuaro<br />
<strong>and</strong> Toluca. Patzcuaro is located at<br />
2,200 m altitude in the western<br />
Mexican highl<strong>and</strong>s (Michoacan<br />
state). It proved suitable for testing<br />
advanced lines, since epidemics <strong>of</strong><br />
S. tritici occur naturally every year.<br />
In some years we can also select for<br />
resistance to Pyrenophora triticirepentis<br />
<strong>and</strong> <strong>Stagonospora</strong> nodorum.<br />
The Atizapan experiment station in<br />
Toluca (Mexico state) at 2,600 m<br />
altitude has an intense rainy season<br />
during the summer months (800-<br />
900 mm). Artificial epidemics are<br />
created by inoculating with<br />
infected straw or a spore<br />
suspension involving a mixture <strong>of</strong><br />
isolates.<br />
Numerous lines with acceptable<br />
resistance levels were derived from<br />
crosses using the above mentioned<br />
sources <strong>and</strong> subsequent testing in<br />
Toluca <strong>and</strong> Patzcuaro (Table 2).
Another set <strong>of</strong> resistant lines<br />
was created by the wheat<br />
germplasm enhancement<br />
section. The S. tritici resistance <strong>of</strong><br />
lines from Brazil (IAS 20, Pelotas<br />
Arthur, Colotana, IAS 58, <strong>and</strong><br />
Maringa), which are tall <strong>and</strong><br />
late-maturing with weak straw,<br />
was incorporated into earlymaturing,<br />
high yielding<br />
semidwarf lines. Thus earlymaturing,<br />
short, high yielding<br />
lines were developed that had a<br />
level <strong>of</strong> resistance similar to that<br />
<strong>of</strong> the original resistant parent<br />
(Table 3).<br />
Bread wheat lines were also<br />
crossed with triticale, <strong>and</strong> the<br />
resulting lines showed high<br />
levels <strong>of</strong> S. tritici resistance<br />
(Table 4). Subsequently, some <strong>of</strong><br />
the lines listed in Table 3 were<br />
combined with triticale-derived<br />
lines (Table 5) in an attempt to<br />
pyramid several resistance<br />
genes.<br />
<strong>CIMMYT</strong>’s wheat wide<br />
crosses program has generated a<br />
wide range <strong>of</strong> resistant<br />
germplasm from D genome<br />
synthetics <strong>and</strong> their synthetic<br />
derivatives (Mujeeb-Kazi et al.,<br />
1996; 1998; 1999). These<br />
materials express high levels <strong>of</strong><br />
resistance to several leaf<br />
pathogens, including Bipolaris<br />
sorokiniana, Pyrenophora triticirepentis,<br />
<strong>and</strong> S. tritici. Their<br />
resistance may be to one or a<br />
combination <strong>of</strong> these pathogens.<br />
Table 6 shows a group <strong>of</strong><br />
representative synthetic wheats<br />
with high levels <strong>of</strong> resistance to<br />
S. tritici.<br />
<strong>Septoria</strong> tritici Resistance Sources <strong>and</strong> Breeding Progress at <strong>CIMMYT</strong>, 1970-99 135<br />
Table 1. Main sources <strong>of</strong> <strong>Septoria</strong> tritici resistance used by the <strong>CIMMYT</strong> wheat breeding<br />
program in the 1970s.<br />
Ex-USSR Southern Cone <strong>of</strong> SA USA spring Other<br />
winter wheat spring wheat wheat sources<br />
Aurora IAS 55 Chris Enkoy<br />
Kavkaz IAS 58 Era Salomoni Seafoam<br />
Bezostaja 1 PF 70254 Frontana-Kenya Kvz/K4500 L.6A.6<br />
Maringa x Newthatch<br />
Carazinho<br />
IAS 63<br />
Lagoa Vermelha<br />
Tizano Pinto Precoz<br />
IAS 62<br />
CTN 7<br />
CTN 8<br />
Gaboto<br />
IAS 20<br />
Table 2. <strong>CIMMYT</strong>-derived lines with acceptable <strong>Septoria</strong> tritici resistance from the ex-USSR,<br />
USA, France, Brazil, Chile, <strong>and</strong> Romania.<br />
Advanced line Pedigree cross Number S. tritici score a Origin<br />
Veery Kvz/Buho//Kal/Bb CM 33027 76-86 Ex-USSR<br />
Bobwhite Au//Kal/Bb/3/Wop CM 33203 32-76 Ex-USSR<br />
Musala Lee/Kvz/3/Cc//Ron/Cha CM16780 Ex-USSR<br />
Sunbird Gll/Cuc//Kvz/Sx CM34630 42-77 Ex-USSR<br />
Milan VS73.600/Mirlo/3/ CM75113 21-74 France/Ex<br />
Bow//Ye/Trf USSR<br />
Attila ND/VG9144//Kal/Bb/ CM85836 73-76 USA/Ex-<br />
3/Yaco/4/Veery USSR<br />
Bagula Ore F1 158/Fdl// CM59123 53-75 USA/Romania<br />
Mfn/2*Tiba63/3/Coc<br />
/4/Bb/GLl//Carp/3/Pvn<br />
Prinia Prl/Vee#6/Myna/Vul CM90722 74-76 Ex-USSR<br />
Chilero 4777*2//Fkn/Gb54/3/ CM66684 74-76 Ex-USSR<br />
Vee#5/4/Buc/Pvn<br />
Corydon Car 853/Coc/Vee/3/ CM81074 41-75 Chile/ Brazil/<br />
E7408/Pam//Hork/PF73226 Ex - USSR<br />
Tinamu IAS58/4/Kal/Bb// CM81812 32-75 Brazil/Ex-<br />
Cjm71/Ald/5/Au// USSR<br />
Kal/Bb/3/Wop<br />
a Double digit scale (Saari <strong>and</strong> Prescott, 1975; Eyal et al., 1987) indicating the range <strong>of</strong> damage during two<br />
screening cycles in Toluca.<br />
Table 3. Short, early-maturing, <strong>Septoria</strong> tritici resistant lines derived from tall, late-maturing,<br />
resistant Brazilian varieties, with a resistance similar to that <strong>of</strong> the original resistant parents.<br />
Lines Cross number S. tritici score a<br />
IAS 20/H567.71//4*IAS 20 CMH 79.243 35-52<br />
IAS 20/H567.71//IAS 20/3/IAS 58 CMH 78.390 31-42<br />
IAS 20/H567.71//IAS20/3/3*MRNG CMH 78.443 32-63<br />
CLT/H471.71A//4*CLT CMH 83.2277 32-53<br />
H.567.71/3*P.Ar CMH 77.308 32-74<br />
IAS 20 – 11-62<br />
IAS 58 – 31-54<br />
Maringa – 31-54<br />
Colotona – 11-51<br />
Pelotas Arthur – 21-71<br />
a Double digit scale (Saari <strong>and</strong> Prescott, 1975; Eyal et al., 1987) indicating the range <strong>of</strong> damage during two<br />
screening cycles in Toluca.
Session 6C — L. Gilchrist et al.<br />
136<br />
Table 4. <strong>Septoria</strong> tritici resistant lines derived from bread wheat x triticale crosses.<br />
Lines Cross number S. tritici score a<br />
INIA 66/RYE*2//ARM/3/H277.69 CMH 72.A576 21-42**<br />
M2A/CML//2*NYUBAY CMH 80.A1267 22-52<br />
M2A/CML//Nyubay/3/ CMH72A576/MRG CMH 82.961 32-43<br />
a Double digit scale (Saari <strong>and</strong> Prescott, 1975; Eyal et al., 1987) indicating the range <strong>of</strong> damage during two<br />
screening cycles in Toluca.<br />
Table 5. <strong>Septoria</strong> tritici resistant lines combining resistance from Brazilian sources <strong>and</strong> bread<br />
wheat x triticale derived lines.<br />
Lines Cross number S. tritici score a<br />
H.567.71/3*P. Ar/MRNG//IAS20/H567.71/<br />
IAS20/3/3*MRNG/79.243/4/M2A/CML//<br />
NYUBAY/3/INIA66/Rye*2//ARM/3/H277.69<br />
CMH 85.3215 31-42<br />
EAGLE/H567.71//4*EAGLE/3/2*IAS20<br />
/H567.71//4*IAS20<br />
CMH 79.243 11-43<br />
a Double digit scale (Saari <strong>and</strong> Prescott, 1975; Eyal et al., 1987) indicating the range <strong>of</strong> damage during two<br />
screening cycles in Toluca.<br />
Table 6. Synthetic hexaploid wheats (Triticum turgidum x Aegilops tauschii; 2n=6x=42) with high<br />
levels <strong>of</strong> resistance to <strong>Septoria</strong> tritici.<br />
Synthetic hexaploids Cross number S. tritici score a<br />
Aco 89/Ae. tauschii (309) b CIGM90.595 11-11<br />
Croc1/Ae. tauschii (879) CIGM89.479 11-21<br />
Doy1/Ae. tauschii (372) CIGM93.229 11-21<br />
Sty-US/Celta//Pals/3/SRN-5/4/Ae. tauschii (502) CIGM93.261 11-11<br />
Altar 84/Ae. tauschii (502) CIGM93.395 11-11<br />
a Double digit scale (Saari <strong>and</strong> Prescott, 1975; Eyal et al., 1987) indicating the range <strong>of</strong> damage during two<br />
screening cycles in Toluca.<br />
b Accession number in the wide cross working collection in <strong>CIMMYT</strong>’s wheat genebank.<br />
Table 7. Synthetic hexaploids crossed with susceptible <strong>and</strong> moderately susceptible bread<br />
wheats to develop advanced derivatives resistant to <strong>Septoria</strong> tritici.<br />
Advanced derivative Cross number S. tritici score a<br />
Altar 84// Aegilops tauschii (191) b CIGM92.337 21-52<br />
//Yaco/3/2*Bau<br />
Croc 1/ Aegilops tauschii (205)//Kauz CIGM90.248 11-31<br />
Croc 1/ Aegilops tauschii (205)/5/Br12*3/4/ CIGM90.252 11-32<br />
Ias 55*4…..<br />
Altar 84/Aegilops tauschii (219)//Opata CIGM90.429 11-32<br />
Altar 84/Aegilops tauschii (219)//2*Seri CMSS92YO1855M 21-32<br />
Altar 84/Aegilops tauschii (224)//2*Yaco CIGM91.191 11-32<br />
Bcn/3/Fgo/USA2111//Aegilops tauschii (658) CASS94Y00146S 11-22<br />
Opata/6/68111/Rgb-V//Ward/3/Fgo/4/ CASS94Y00247S 11-22<br />
Rabi/5/Aegilops tauschii (878)<br />
Filin/4/Snip/Yan79//Dack/Teal/3/ CASS94Y00072S 32-32<br />
Aegilops tauschii (633)<br />
Altar 84/Aegilops tauschii (191)// Opata/3/ CIGM93.566 11-22<br />
Altar 84/Aegilops tauschii (224)//Yaco(1)…<br />
Mayoor//TkSN1081/Aegilops tauschii (222) CASS94Y00009S 11-22<br />
a Double digit scale (Saari <strong>and</strong> Prescott, 1975; Eyal et al., 1987) indicating the range <strong>of</strong> damage during two<br />
screening cycles in Toluca.<br />
b Accession number in the wide cross working collection in <strong>CIMMYT</strong>’s wheat genebank.<br />
The high levels <strong>of</strong> resistance<br />
present in the synthetic hexaploids<br />
were incorporated into susceptible<br />
bread wheat advanced lines <strong>and</strong> in<br />
some cases were used to reinforce<br />
the intermediate susceptibility<br />
present in some lines (Table 7). To<br />
pyramid the resistance genes,<br />
resistant alien germplasm<br />
possessing tertiary gene pool<br />
diversity was crossed with resistant<br />
Chinese germplasm (Table 8).<br />
Another effort to diversify the<br />
sources <strong>of</strong> resistance focused on the<br />
Triticum dicoccon collection in the<br />
wheat genebank (Gilchrist <strong>and</strong><br />
Skovm<strong>and</strong>, 1995). Early tall<br />
accessions with resistance to P.<br />
striiformis <strong>and</strong> S. tritici, <strong>and</strong> weak<br />
straw were identified from<br />
genebank collections. One <strong>of</strong> these<br />
accessions was selected <strong>and</strong><br />
crossed to a very susceptible<br />
selection <strong>of</strong> the line Kauz. The<br />
resulting lines combine high levels<br />
<strong>of</strong> resistance to P. striiformis <strong>and</strong> S.<br />
tritici with good agronomic type<br />
(Table 9).<br />
In a second effort <strong>of</strong> the bread<br />
wheat breeding program (1986-87)<br />
to increase variability <strong>of</strong> the<br />
resistance base, a group <strong>of</strong><br />
advanced bread wheat lines<br />
consisting <strong>of</strong> resistance sources in<br />
an adapted background (Table 10)<br />
was crossed with a group <strong>of</strong><br />
Chinese lines having acceptable<br />
levels <strong>of</strong> resistance (Sumai #3,<br />
Suzhoe #8, Suzhoe #6 Ningmai #4,<br />
Yangmai #4, Ning 8401, YMI #6,<br />
Shangai #5, Shangai #, Nanjing<br />
7840, Wuhan 2, <strong>and</strong> Wuhan 3).<br />
Once the resistance <strong>of</strong> the<br />
Chinese materials had been<br />
introduced, a new effort was
initiated to combine high yield<br />
potential <strong>and</strong> pyramid resistance<br />
genes (Table 11).<br />
Shuttle breeding between<br />
different sites is very important<br />
for selection <strong>and</strong> helps to increase<br />
effective resistance across<br />
different locations where S. tritici<br />
is a problem. A large number <strong>of</strong><br />
advanced lines showing very high<br />
levels <strong>of</strong> resistance have been<br />
selected through shuttle breeding.<br />
New sources have also been<br />
selected with this methodology. A<br />
representative example <strong>of</strong> new<br />
sources coming from the Southern<br />
Cone <strong>of</strong> South America that were<br />
identified in the region through<br />
the LACOS (Advanced Lines for<br />
the Southern Cone) nursery is<br />
given in Table 12.<br />
Durum Wheat Program<br />
In the countries <strong>of</strong> West Asia<br />
<strong>and</strong> North Africa (WANA), severe<br />
epidemics <strong>of</strong> septoria leaf blotch<br />
have occurred, especially in<br />
Morocco, Tunisia, <strong>and</strong> Turkey<br />
(Saari, 1974). Isolates <strong>of</strong> the<br />
pathogen have been identified in<br />
different regions, along with their<br />
specificity for durum <strong>and</strong> bread<br />
wheat. Breeding efforts have<br />
increased resistance levels, but<br />
selection for resistance is<br />
complicated by different disease<br />
reactions at different locations<br />
<strong>and</strong> in different years. This may<br />
be due to different environmental<br />
conditions or to the variability <strong>of</strong><br />
S. tritici populations (Arama et al.,<br />
1988); van Silfhout et al. (1989)<br />
have reported specificity in some<br />
durum <strong>and</strong> bread wheat isolates.<br />
Complementary information was<br />
<strong>Septoria</strong> tritici Resistance Sources <strong>and</strong> Breeding Progress at <strong>CIMMYT</strong>, 1970-99 137<br />
Table 8. Advanced wheat lines resistant to <strong>Septoria</strong> tritici <strong>and</strong> derived from crosses <strong>of</strong> perennial<br />
Triticeae species <strong>and</strong> Chinese germplasm.<br />
Lines Crosses S. tritici score a<br />
CS/Thinopyrum curvifolium//Glenn.81/3/Ald/Pvn CIGM84295 31-43<br />
Cno79/4/CS/Thinopyrum curvifolium// CIGM88829 32-62<br />
Glenn.81/3/Ald/Pvn<br />
Cno 79/4/CS/Thinopyrum curvifolium//Gen/3/ CIGM8876 32-42<br />
Ald/Pvn/4/CS/Leymus racemosus//2*CS/3/Cno 79<br />
Inia66/Thinopyrum distichum//Inia66/3//4/Gen/ CIGM88734 31-62<br />
CS/Thinopyrum curvifolium//Glenn.81/3/Ald/Pvn<br />
CS/Thinopyrum curvifolium//Glenn 81/3/Ald/Pvn/ CIGM87.116 51-72<br />
4/Ningmai#4/Oleson//Ald/Yangmai#4<br />
CS/Thinopyrum curvifolium/Glenn 81/3/ CIGM87.123 31-42<br />
Ald/Pvn/4/Suzhoe#8<br />
CS/Thinopyrum curvifolium//Glenn 81/3/ CIGM87.1109 41-53<br />
Ald/Pvn/4/Ningmai 8401<br />
Chir3/5/CS/Thinopyrum curvifolium//Glenn 81 CIGM93.612 21-22<br />
/3/Ald/Pvn/4/Cs/Leymus racemosus//2*CS/3/Cno79<br />
a Double digit scale (Saari <strong>and</strong> Prescott, 1975; Eyal et al., 1987) indicating the range <strong>of</strong> damage during two<br />
screening cycles in Toluca.<br />
Table 9. Triticum dicoccon/Kauz advanced lines with resistance to <strong>Septoria</strong> tritici.<br />
Advanced lines <strong>and</strong> pedigree Cross number S. tritici score a<br />
T. dicoccon PI 1254156/2*Kauz GRSS93SH18-0M-12SH-2M-1Y 11-51<br />
T. dicoccon PI 1254156/2*Kauz GRSS93SH18-0M-13SH-3M-1Y 11-21<br />
T. dicoccon PI 1254156/2*Kauz GRSS93SH18-0M-13SH-3M-1Y 11-21<br />
T. dicoccon PI 1254156/2*Kauz GRSS93SH27-OM-10SH-3M-1Y 11-72<br />
T. dicoccon PI 1254156/2*Kauz GRSS93SH29-0M-5SH-3M-1Y 32-73<br />
Kauz (susceptible parent) 89-99<br />
Bobwhite (moderately resistance check) 41-46<br />
T. dicoccon PI 1254156 (resistant parent) 11-71<br />
a Double digit scale (Saari <strong>and</strong> Prescott, 1975; Eyal et al., 1987) indicating the range <strong>of</strong> damage during two<br />
screening cycles in Toluca.<br />
Table 10. Advanced lines consisting <strong>of</strong> sources <strong>of</strong> <strong>Septoria</strong> tritici resistance in an adapted<br />
background, crossed with a group <strong>of</strong> resistant Chinese lines.<br />
Advanced line Cross S. tritici score a<br />
Shangai#5/Bow CM91100 32-72<br />
Ald/Pvn//YM#6 CM91065 21-72<br />
Suzhoe#6//Ald/Pvn CM91128 32-71<br />
YM#6/Thb*2 CMH87.2698 42-72<br />
Ald/Pvn//Ning#7840 CMH91325 11-47<br />
Sha#5/Weaver CM95103 11-32<br />
Sha#8/Gen CM95124 11-32<br />
Milan/Sha#7 CM97550 52-63<br />
Sabuf CM95073 53-32<br />
Catbird CM91045 42-76<br />
a Double digit scale (Saari <strong>and</strong> Prescott, 1975; Eyal et al., 1987) indicating the range <strong>of</strong> damage during two<br />
screening cycles in Toluca.
Session 6C — L. Gilchrist et al.<br />
138<br />
Table 11. Bread Wheat Program advanced lines with high yield potential containing different<br />
combinations <strong>of</strong> <strong>Septoria</strong> tritici resistance sources in their pedigrees.<br />
Advanced lines Cross S. tritici score a<br />
Trap#1/Bow CM 84548 21-35<br />
PF70354/Bow CM 67910 21-54<br />
Gov/Az//Mus/3/Dodo/4/Bow CM 79515 31-64<br />
Ald/Pvn//YMI#6/3/Ald/Pvn CM 96723 11-41<br />
Ias20*3/H567.71//Sara CM 81021 11-31<br />
Ning8675/Catbird CMSS92Y00639S 32-52<br />
Thb/CEP7780//Suz#9/Weaver/3/Ng8675 CMSS92Y02302T 52-63<br />
Cbrd/5/Cs/Thinopyrum curvifolium//<br />
Glen/3/Gen/4/L2266/1406.101//Buc/<br />
3/Vpm/Mos83.11.4.8//Nac<br />
CMBW91MO3723 11-21<br />
Sha3/Seri//Kauz/3/Sha4/Chil CMBW91M03723 11-21<br />
Bau/Milan CM103873 11-21<br />
Lfn/II58.57//PRL/3/Hahn CM77224 31-42<br />
Sha #3/Seri//Psn/Bow CMBW90M2470 31-42<br />
Suz #6/Weaver//Tui CMBW90M2474 11-21<br />
a Double digit scale (Saari <strong>and</strong> Prescott, 1975; Eyal et al., 1987) indicating the range <strong>of</strong> damage during two<br />
screening cycles in Toluca.<br />
Table 12. Bread wheat germplasm resistant to <strong>Septoria</strong> tritici that was selected through shuttle<br />
breeding <strong>and</strong> collaborative nurseries (<strong>CIMMYT</strong>/national agricultural research programs).<br />
S. tritici<br />
Lines <strong>and</strong> crosses score a Origin<br />
T00011/T00007<br />
A8972-1T-2N-1B-2T-2T-0T<br />
53 Argentina<br />
L.A.CIAT(Santa Cruz) 63 Bolivia<br />
Talhuen INIA 31 Chile<br />
Br14/CEP847<br />
B31615-0A-0Z-1A-15A-0A<br />
21 Brazil<br />
Iapi/Fink<br />
CP3409-1E-0Y-0E-18Y-0E<br />
31 Paraguay<br />
E Fed/F.5.83.7792 (Bajas) 42 Uruguay<br />
a Double digit scale (Saari <strong>and</strong> Prescott, 1975; Eyal et al., 1987) indicating the range <strong>of</strong> damage during two<br />
screening cycles in Toluca.<br />
found by Eyal et al. (1985), whose<br />
studies revealed that isolates from<br />
Syria <strong>and</strong> Tunisia were more<br />
virulent on tetraploid than on<br />
hexaploid varieties.<br />
Based on this information, elite<br />
durum wheat lines were selected in<br />
Toluca <strong>and</strong> Patzcuaro, as was<br />
described earlier for bread wheat. A<br />
representative group <strong>of</strong> these<br />
selections is listed in Table 13. This<br />
germplasm is under observation in<br />
the WANA region, but the dry<br />
conditions that have prevailed in<br />
recent years have not permitted<br />
accurate information to be obtained<br />
on the resistance <strong>of</strong> germplasm preselected<br />
in Mexico. However,<br />
information from Tunisia (Table 14)<br />
has contributed greatly to<br />
enhancing the effective resistance<br />
in durum wheat germplasm, <strong>and</strong><br />
the disease data from that location<br />
have been used to design crosses.<br />
Conclusion<br />
In conclusion, there are<br />
numerous sources <strong>of</strong> resistance to<br />
S. tritici. For the most part, the<br />
genetic constitution <strong>of</strong> these<br />
sources remains unclear, but the<br />
information contained in this paper<br />
suggests that they are genetically<br />
different. Resistance sources in<br />
<strong>CIMMYT</strong>’s genebank collections<br />
are available on request.<br />
References<br />
Arama, P.F., van Silfhout, C.H., <strong>and</strong><br />
Kema, G.H.J. 1988. Report on the<br />
Cooperative research project<br />
between IPO, Tel Aviv University<br />
<strong>and</strong> <strong>CIMMYT</strong>. 31 pp.<br />
Eyal, Z., Scharen, A.L., H<strong>of</strong>fman,<br />
M.D., <strong>and</strong> J.M. Prescott. 1985.<br />
Global insights into virulence<br />
frequencies <strong>of</strong> Mycosphaerella<br />
graminicola. Phytopathology<br />
75(12):1456-1462.<br />
Eyal, Z., A.L. Sharen, J.M. Prescott,<br />
<strong>and</strong> M. van Ginkel. 1987. The<br />
<strong>Septoria</strong> <strong>Diseases</strong> <strong>of</strong> Wheat:<br />
Concepts <strong>and</strong> Methods <strong>of</strong> Disease<br />
Management. Mexico, D.F.:<br />
<strong>CIMMYT</strong>. 46 pp.<br />
Gilchrist, L., <strong>and</strong> Skovm<strong>and</strong>, B. 1995.<br />
Evaluation <strong>of</strong> emmer wheat<br />
(Triticum dicoccon) for resistance to<br />
<strong>Septoria</strong> tritici . In: Gilchrist, L., van<br />
Ginkel, M., McNab, A., <strong>and</strong> Kema,<br />
G.H.J., eds. Proceedings <strong>of</strong> a<br />
<strong>Septoria</strong> tritici workshop. Mexico,<br />
D.F.: <strong>CIMMYT</strong>. Pp 130-134.<br />
Mann, C.E., Rajaram, S., <strong>and</strong> R.L.<br />
Villarreal. 1985. Progress in<br />
breeding for <strong>Septoria</strong> tritici<br />
resistance in semidwarf spring<br />
wheat at <strong>CIMMYT</strong>. In: Sharen, A.L.<br />
<strong>Septoria</strong> <strong>of</strong> <strong>Cereals</strong>: Proceedings <strong>of</strong><br />
the workshop, held August 2-4,<br />
1983, at Montana State University,<br />
Bozeman, Montana. Pp. 22-26.
Mujeeb-Kazi, A., V. Rosas, <strong>and</strong> S.<br />
Roldan. 1996. Conservation <strong>of</strong> the<br />
genetic variation <strong>of</strong> Triticum<br />
tauschii (Coss) Schmalh. (Aegilops<br />
squarrosa auct. Non L.) in synthetic<br />
hexaploid wheats (T. turgidum L.s.<br />
lat X T. tauschii; 2n=6x=42,<br />
AABBDD) <strong>and</strong> its utilization for<br />
wheat improvement. Genetic<br />
Resources <strong>and</strong> Crop Evolution<br />
43:129-134.<br />
Mujeeb-Kazi, A., Gilchrist, L.,<br />
Villareal, R.L., <strong>and</strong> Delgado, R.<br />
1998. D-genome synthetic<br />
hexaploids: Production <strong>and</strong><br />
utilization in wheat improvement<br />
In: Triticeae III, Jaradat, A.A.(ed.),<br />
ICARDA, Aleppo. Pp. 369-374.<br />
Mujeeb-Kazi, A., Gilchrist, L.,<br />
Villareal, R.L., <strong>and</strong> Delgado, R.<br />
1999. Registration <strong>of</strong> ten wheat<br />
germplasm lines resistant to<br />
<strong>Septoria</strong> tritici leaf blotch. Crop<br />
Science (in press).<br />
Rajaram, S., van Ginkel, M., <strong>and</strong><br />
Fischer, R.A. 1994. <strong>CIMMYT</strong>’s<br />
wheat breeding megaenvironments<br />
(ME). In:<br />
Proceedings <strong>of</strong> the 8 th International<br />
Wheat Genetics Symposium, July<br />
19-24, Beijing, China. Pp 1101-1106.<br />
Saari, E.E. 1974. Results <strong>of</strong> studies on<br />
<strong>Septoria</strong> in the near East <strong>and</strong><br />
Africa. Proc. <strong>of</strong> Fourth FAO<br />
Rockefeller Foundation Wheat<br />
Seminar. Tehran, Iran, May 21-June<br />
2, 1973. Pp. 275-283.<br />
Saari, E.E., <strong>and</strong> Prescott, J.M. 1975. A<br />
scale for appraising the foliar<br />
intensity <strong>of</strong> wheat diseases. Plant.<br />
Dis. Rep. 59:377-380.<br />
van Silfhout, C.H., Arama, P.F., <strong>and</strong><br />
Kema, G.H.J. 1989. International<br />
survey <strong>of</strong> factors <strong>of</strong> virulence <strong>of</strong><br />
<strong>Septoria</strong> tritici. In: Fried, M.P., ed.,<br />
Proceedings, <strong>Septoria</strong> <strong>of</strong> <strong>Cereals</strong>.<br />
Zurich, Switzerl<strong>and</strong>, July 4-7, 1989.<br />
<strong>Septoria</strong> tritici Resistance Sources <strong>and</strong> Breeding Progress at <strong>CIMMYT</strong>, 1970-99 139<br />
Table 13. Elite durum wheat lines with resistance to <strong>Septoria</strong> tritici selected at Toluca <strong>and</strong> Patzcuaro.<br />
Lines Cross S. tritici score a<br />
Aaz77 3/Olus CD 94270 21-22<br />
Ajaia 12/F3Local<br />
(Sel.Ethio.135.85)//Plata 13<br />
CD 98331 21-32<br />
Eco/CMH76A.722//Yav/3/<br />
Altar 84/4/Ajaia 2/5/Kjove 1<br />
CD 91B2636 32-42<br />
Gs/Cra//Sba81/3/Ho/4/Mex 1/<br />
5/ Memo/6/2*Altar 84<br />
CDSS 92B1193 32-33<br />
Himan9/ Bejah 6 CD91B2096 21-21<br />
Kucuk CD 91B2620 T-11<br />
Lhnke//Gs/Str/3/Altar 84/4/Focha 1 CDSS92B1076 21-22<br />
Liro 2 CD 93352 31-31<br />
Lotus 1/Espe CD97015 11-21<br />
Lymno 8 CD92314 21-42<br />
Nus/Silver//CMH82A.1062/<br />
Rissa/3/Chen/Altar84/4/Don<br />
CD91B2664 21-32<br />
Patka 3 CD 78995 11-21<br />
Plata1 Smn//Plata 9 CD97899 T- 11<br />
Plata10/6/Mque/4/Usda573//Qfn/<br />
Aa 7/3/Alba-D/5/Avohui/7/ Plata 1<br />
CD98581 32-43<br />
Plata 6/ Green17 CD96789 11-21<br />
Plata7/Fillo 9//Sbak CD99545 31-32<br />
Plata 8/4/Garza/Afn//Cra/<br />
3/Gta/5/Rascon<br />
CD91B2061 32-42<br />
Rascon 21/Long CDWS91M377 32-42<br />
Rascon 37/Green 2 CD91B1975 31-32<br />
Rascon 37/Tarro 2//Rascon 37 CDSS92B1022 31-32<br />
Rascon 39/Tilo 1 CDSS92B61 21-22<br />
Sn Turrk Mi 83-84 503/<br />
Lotus 4//Lotus 5<br />
CD97775 T - 11<br />
Sooty 9/Rascon 37 CD91B1938 31-32<br />
Sora/2*Plata 12 CD92011 T - 11<br />
Topdy 18/Focha 1//Altar 84 CDSS92B1034 22-32<br />
a Double digit scale (Saari <strong>and</strong> Prescott, 1975; Eyal et al., 1987) indicating the range <strong>of</strong> damage during two<br />
screening cycles in Toluca.<br />
Table 14. Durum wheat germplasm with resistance to <strong>Septoria</strong> tritici from Tunisia.<br />
Lines Cross S. tritici score a<br />
Karim (Check S ) CM9799 98-97<br />
Khiar (check S) CD57.005 97-97<br />
BD 2337 – 53-42<br />
BD 2338 – 64<br />
BD 2339 – 64<br />
Src2/Src1 ICD88.66 75-74<br />
Gdfl/ T. dicoccoides-<br />
SY20013/Bcr<br />
– 64-53<br />
Bcr/Guerou 1 ICD87.0572 75-76<br />
Zeina 2 ICD88.1233 44-32<br />
Zeina 4 ICD88.1233 64-32<br />
Aus 1/5/Cndo/4/Bry*2/<br />
Tace//II27655/3/Tme/Zb/2*W<br />
ICD88.1120 53-76<br />
Srn 3/Ajaia 15//Don 87 CD96855 73-42<br />
Porron 1 CD85328 54<br />
Poho/Ajaia CD99818 64<br />
Cali/ship 2// Fillo 7 CD98278 42<br />
Plata 6/Green 17 CD96789 52<br />
CMH82A.1062/3/Ggovz394//Sba81/<br />
Plc/4/Aaz 1/Crex/5/Hui//Cit71/CII<br />
CD97395 43<br />
a Double digit scale (Saari <strong>and</strong> Prescott, 1975; Eyal et al., 1987) indicating the range <strong>of</strong> damage during two<br />
screening cycles in Toluca.
140<br />
Selecting Wheat for Resistance to<br />
<strong>Septoria</strong>/<strong>Stagonospora</strong> in Patzcuaro, Michoacan, Mexico<br />
R.M. Gonzalez I., 1 S. Rajaram, 2 <strong>and</strong> M. van Ginkel2 1 Campo Morelia, National Livestock <strong>and</strong> Agricultural Research Institute, Mexico<br />
2 <strong>CIMMYT</strong> Bread Wheat Program<br />
Abstract<br />
The research reported in this paper was conducted in the humid, temperate area <strong>of</strong> Patzcuaro in the state <strong>of</strong> Michoacan, in<br />
Mexico, where there is a high natural incidence <strong>of</strong> <strong>Septoria</strong>/<strong>Stagonospora</strong> spp. Nine wheat genotypes that had previously been<br />
selected for resistance to <strong>Septoria</strong>/<strong>Stagonospora</strong> spp. were included in the study. They were compared to two check varieties, one<br />
tolerant <strong>and</strong> one susceptible. The materials were tested with <strong>and</strong> without chemical protection. Three years’ data were analyzed <strong>and</strong><br />
relative yield losses <strong>of</strong> 10-32% were found. Two different responses were observed among the outst<strong>and</strong>ing genotypes. On the one<br />
h<strong>and</strong>, several lines expressed little reduction in yield when challenged by the septoria pathogens. Depending on their yield potential,<br />
final yield could be moderate to quite good. These could be classified as resistant or tolerant to <strong>Septoria</strong>/<strong>Stagonospora</strong> spp. Some <strong>of</strong><br />
these same materials did show a considerable level <strong>of</strong> severity; their response would thus be better classified as tolerance rather than<br />
resistance. On the other h<strong>and</strong>, certain materials with high yield potential did lose yield following the attack by the septoria foliar<br />
blights, but retained sufficient expression <strong>of</strong> yield potential to compete well with the local check variety. These should not be classified<br />
as resistant nor tolerant, but may well be desirable from a production st<strong>and</strong>point. The line that best combined these traits is IAS20/<br />
H567.71/5*IAS 20. It is being considered for release under the name Patzcuaro. Severity <strong>of</strong> foliar disease on the flag leaf proved to be<br />
well correlated with yield loss.<br />
Over the past 15 years, Mexico’s<br />
National Livestock <strong>and</strong><br />
Agricultural Research Institute<br />
(INIFAP) <strong>and</strong> <strong>CIMMYT</strong> have<br />
worked together on breeding bread<br />
wheats for resistance to <strong>Septoria</strong><br />
tritici <strong>and</strong> <strong>Stagonospora</strong> nodorum in<br />
the Patzcuaro, Michoacan, region <strong>of</strong><br />
Mexico. Natural conditions in this<br />
area favor the yearly development<br />
<strong>of</strong> these two <strong>Septoria</strong> species<br />
(Gomez <strong>and</strong> Gonzalez, 1987). The<br />
area enjoys a temperate climate,<br />
with mean temperatures <strong>of</strong> 8-21ºC,<br />
> 800 mm annual rainfall, <strong>and</strong> ><br />
85% relative humidity. The isolates<br />
<strong>of</strong> <strong>Septoria</strong>/<strong>Stagonospora</strong> spp. that<br />
thrive under these conditions are<br />
very aggressive, which makes this<br />
a stress environment for wheat.<br />
According to Eyal et al. (1985), the<br />
<strong>Septoria</strong> isolates present in the<br />
Patzcuaro area are among the most<br />
virulent in the world.<br />
Materials <strong>and</strong> Methods<br />
In this study, 11 advanced<br />
wheat lines from <strong>CIMMYT</strong><br />
nurseries (Table 1) targeted for high<br />
rainfall production environments<br />
(e.g., HRWSN <strong>and</strong> ASWSN) were<br />
evaluated with <strong>and</strong> without<br />
chemical protection during 1994-96.<br />
The variety Curinda M-87,<br />
which has functioned as reference<br />
in our work for the past 10 years,<br />
was used as the resistant/tolerant<br />
check variety in the trials (Table 1).<br />
The variety Batan was the<br />
susceptible check. Although both S.<br />
tritici <strong>and</strong> S. nodorum were present,<br />
the former was found in a higher<br />
proportion in most years.<br />
Fungicides Terbuconazole <strong>and</strong><br />
Tecto were used for chemical<br />
control. Terbuconazole was applied<br />
every 10 days starting from<br />
tillering to grain milk stage at a<br />
dosage <strong>of</strong> 0.5 l/ha. Tecto was<br />
applied in the same dosage every<br />
eight days, starting at the end <strong>of</strong><br />
the booting stage.<br />
Days to flowering, percentage<br />
flag leaf area affected by <strong>Septoria</strong>/<br />
<strong>Stagonospora</strong> spp. (% flag leaf<br />
severity, FLS) at grain milk stage,<br />
plant height, grain yield, <strong>and</strong> test<br />
weight were measured. Yield losses<br />
<strong>and</strong> correlations were calculated for<br />
individual years.<br />
Results <strong>and</strong> Discussion<br />
Several lines expressed higher<br />
levels <strong>of</strong> resistance than the<br />
resistant check variety Curinda<br />
(29% FLS) (Table 1). Severities as<br />
low as 1-3% were noted in<br />
individual years. Across the three
Selecting Wheat for Resistance to <strong>Septoria</strong>/<strong>Stagonospora</strong> in Patzcuaro, Michoacan, Mexico 141<br />
Table 1. Response <strong>of</strong> 11 wheat lines to natural infection by <strong>Septoria</strong>/<strong>Stagonospora</strong>, in regard to flag leaf severity<br />
(FLS in %), yield, percentage loss, in field plots either protected or unprotected with fungicide, in Pátzcuaro,<br />
Michoacan. México, during 1994, 1995, <strong>and</strong> 1996.<br />
Yield (kg/ha) %<br />
Entry Cross/Selection history CC * % ** UP % Yield loss % Flag leaf<br />
1 CURINDA<br />
Tolerant check<br />
2875 e *** 100 2042 d 100 29 28<br />
2 BATAN<br />
Susceptible check<br />
3521 b 122 2271 c 111 36 23<br />
3 IAS20/H567.71/5* IAS20<br />
CM78A-544-7B-1Y-1B-1Y-2B-1Y-0B<br />
3826 b 133 3694 a 181 4 1<br />
4 CHIL//ALD/PVN<br />
CM92801-65Y-0M-0Y-4M-0RES<br />
2820 e 98 1590 e 78 44 36<br />
5 THB/CNT 7<br />
CM 830057-0Z-0A-1A-1A-1A-0Y<br />
2632 f 92 2243 c 110 15 10<br />
6 LIRA/TAN//SPB<br />
CM96824-U-0Y-0H-0SY-4M-0RES<br />
2792 f 97 1847 d 90 34 18<br />
7 ALD/PVN//YMI 6<br />
CM 91065-2M-0M-0Y-0M-2Y-0B-5PZ<br />
3681 b 128 2681 b 131 27 3<br />
8 BAU/MILAN<br />
CM103873-2M-030Y-020Y-010M-4Y-0M<br />
3312 c 115 1986 d 97 40 15<br />
9 ISEPTON89-82E/BR6//PF83144<br />
F32938-2M-0AL-0AL-0AL-4Y-0M<br />
3049 d 106 3014 b 148 1 1<br />
10 BOW/MII//RES/NAC/3/PFAU<br />
CM1033644-2FC-0M-1FC-0C<br />
4432 a 154 2688 d 132 39 8<br />
11 CAR853/COC//VEE/3/E7408/PAM//HORK/PF73226<br />
CM80174-32Y-04M-0Y-3M-1M-0M<br />
3854 b 134 2292 d 112 41 24<br />
LSD 498 545<br />
Mean 3344 2396<br />
C.V. 10 16<br />
Yield (kg/ha) %<br />
Entry Cross/Selection history CC * % ** UP % Yield loss % Flag leaf<br />
1 CURINDA<br />
Tolerant check<br />
4153 c 100 3125 a 100 25 18<br />
2 BATAN<br />
Susceptible check<br />
3492 e 84 2285 c 73 35 53<br />
3 IAS20/H567.71/5* IAS20<br />
CM78A-544-7B-1Y-1B-1Y-2B-1Y-0B<br />
4521 b 108 3375 a 108 35 26<br />
4 CHIL//ALD/PVN<br />
CM92801-65Y-0M-0Y-4M-0RES<br />
4007 d 96 2722 b 87 32 45<br />
5 THB/CNT 7<br />
CM 830057-0Z-0A-1A-1A-1A-0Y<br />
4035 d 97 3153 a 101 22 21<br />
6 LIRA/TAN//SPB<br />
CM96824-U-0Y-0H-0SY-4M-0RES<br />
3854 d 92 2986 a 96 22 30<br />
7 ALD/PVN//YMI 6<br />
CM 91065-2M-0M-0Y-0M-2Y-0B-5PZ<br />
4722 b 114 2875 a 92 39 16<br />
8 BAU/MILAN<br />
CM103873-2M-030Y-020Y-010M-4Y-0M<br />
3952 d 95 3333 a 102 20 13<br />
9 ISEPTON89-82E/BR6//PF83144<br />
F32938-2M-0AL-0AL-0AL-4Y-0M<br />
4007 d 97 3368 a 108 17 5<br />
10 BOW/MII//RES/NAC/3/PFAU<br />
CM1033644-2FC-0M-1FC-0C<br />
5292 a 127 3236 a 104 39 13<br />
11 CAR853/COC//VEE/3/E7408/PAM//HORK/PF73226<br />
CM80174-32Y-04M-0Y-3M-1M-0M<br />
4549 b 110 3222 a 103 29 21<br />
LSD 484 592<br />
Mean 4235 3045<br />
C.V. 8 13<br />
* (CC) Chemical control <strong>and</strong> (UP) Unprotected.<br />
** Percentage relative to the tolerant check, Curinda M87.<br />
*** Values followed by the same letter are not significantly different at the 0.05 probability level.<br />
1994<br />
1995
Session 6C — R.M. Gonzalez I., S. Rajaram, <strong>and</strong> M. van Ginkel<br />
142<br />
Table 1. Continued...<br />
Yield (kg/ha) %<br />
Entry Cross/Selection history CC * % ** UP % Yield loss % Flag leaf<br />
1 CURINDA<br />
Tolerant check<br />
2896 c 100 2826 c 100 3 40<br />
2 BATAN<br />
Suscepible check<br />
2038 e 70 1637 f 57 19 61<br />
3 IAS20/H567.71/5* IAS20<br />
CM78A-544-7B-1Y-1B-1Y-2B-1Y-0B<br />
3359 b 116 3173 a 112 6 27<br />
4 CHIL//ALD/PVN<br />
CM92801-65Y-0M-0Y-4M-0RES<br />
2644 d 91 2289 e 81 13 75<br />
5 THB/CNT 7<br />
CM 830057-0Z-0A-1A-1A-1A-0Y<br />
3206 b 111 2766 c 98 14 31<br />
6 LIRA/TAN//SPB<br />
CM96824-U-0Y-0H-0SY-4M-0RES<br />
2710 d 94 2655 c 94 2 62<br />
7 ALD/PVN//YMI 6<br />
CM 91065-2M-0M-0Y-0M-2Y-0B-5PZ<br />
3961 a 137 3291 a 116 17 28<br />
8 BAU/MILAN<br />
CM103873-2M-030Y-020Y-010M-4Y-0M<br />
3028 c 105 2326 d 82 23 10<br />
9 ISEPTON89-82E/BR6//PF83144<br />
F32938-2M-0AL-0AL-0AL-4Y-0M<br />
3146 b 109 2847 b 101 10 22<br />
10 BOW/MII//RES/NAC/3/PFAU<br />
CM1033644-2FC-0M-1FC-0C<br />
3456 b 119 3185 a 113 26 38<br />
11 CAR853/COC//VEE/3/E7408/PAM//HORK/PF73226<br />
CM80174-32Y-04M-0Y-3M-1M-0M<br />
3156 b 110 2852 b 101 28 33<br />
LSD 412 345<br />
Mean 3094 2713<br />
C.V. 9 11<br />
* (CC) Chemical control <strong>and</strong> (UP) Unprotected.<br />
** Percentage relative to the tolerant check, Curinda M87.<br />
*** Values followed by the same letter are not significantly different at the 0.05 probability level.<br />
years Curinda, when unprotected,<br />
yielded on average 2688 kg/ha,<br />
expressing a 19% loss (Table 2).<br />
Several lines were also superior to<br />
the check in yield, <strong>and</strong> suffered less<br />
yield loss.<br />
In this study percent flag leaf<br />
area affected correlated negatively<br />
with yield loss (-0.71 to -0.83;<br />
Table 3). This negative correlation<br />
appears strong <strong>and</strong> reliable over<br />
years.<br />
Although grain yield <strong>of</strong> most<br />
lines tested varied over years<br />
depending on climatic <strong>and</strong><br />
production conditions (Figure 1), in<br />
general their performance relative<br />
to the resistant check Curinda<br />
showed impressive consistency<br />
over time. In contrast, the<br />
susceptible check Batan yielded<br />
less every year as a percentage <strong>of</strong><br />
the check (Tables 1 <strong>and</strong> 2; Figure 1),<br />
<strong>and</strong> seemed to be losing its<br />
resistance progressively.<br />
The plots protected with<br />
fungicide provide a partial<br />
expression <strong>of</strong> each line’s yield<br />
potential in the absence <strong>of</strong> disease<br />
stress. However, other abiotic<br />
stresses, such as soil acidity <strong>and</strong><br />
nutrient imbalances related to<br />
leaching following excessive rain,<br />
also reduce yield in this high<br />
rainfall environment. The<br />
difference between the protected<br />
<strong>and</strong> unprotected plots provides a<br />
measure <strong>of</strong> yield loss due to<br />
disease. Mean yield losses were in<br />
the 10-32% range. The line<br />
ISEPTON89-82E/BR6//PF83144<br />
experienced the lowest loss (10%),<br />
while the susceptible check Batan<br />
suffered the highest (32%).<br />
1996<br />
Comparing protected <strong>and</strong><br />
unprotected plots provides a measure<br />
<strong>of</strong> the contribution <strong>of</strong> resistance or<br />
tolerance to yield. If the yield<br />
difference is small (i.e. the loss is<br />
small), the genotype tested<br />
apparently has a certain level <strong>of</strong><br />
resistance/tolerance that enables it to<br />
express most, if not all, <strong>of</strong> its yield<br />
potential. However, useful<br />
information can also be gained from<br />
direct comparison <strong>of</strong> lines in a<br />
disease-prone setting, in companion<br />
plots without the use <strong>of</strong> fungicides, in<br />
particular when screening germplasm<br />
to identify potential varieties. This is<br />
less expensive than also growing<br />
protected plots, <strong>and</strong> hence allows<br />
larger numbers <strong>of</strong> entries to be tested.<br />
To be acceptable to farmers, a<br />
variety must yield the same or better<br />
than the commercial check variety.<br />
C<strong>and</strong>idate varieties can be acceptable
<strong>and</strong> appear well adapted to the<br />
point <strong>of</strong> being releasable to farmers<br />
for two reasons, or a combination <strong>of</strong><br />
both. On the one h<strong>and</strong>, a line may<br />
express true resistance or tolerance.<br />
In that case challenge by the<br />
pathogen does not result in great<br />
reductions in yield (Tables 1 <strong>and</strong> 2).<br />
Yield potential is roughly<br />
maintained due to resistance, with<br />
only minimum disease severity, or<br />
due to tolerance when considerable<br />
disease severity may be noted but<br />
losses are little. The line<br />
ISEPTON89-82E/BR6//PF83144 is a<br />
good example <strong>of</strong> a resistant<br />
Selecting Wheat for Resistance to <strong>Septoria</strong>/<strong>Stagonospora</strong> in Patzcuaro, Michoacan, Mexico 143<br />
genotype (9% FLS) with minimum<br />
yield loss (10%) <strong>and</strong> a high mean<br />
yield <strong>of</strong> 3034 kg/ha, 13% more<br />
than the tolerant check, Curinda,<br />
in the presence <strong>of</strong> disease. Most<br />
lines suffered yield losses which<br />
percentage-wise were roughly<br />
equivalent to about two-thirds <strong>of</strong><br />
their percentage-wise flag leaf<br />
severity scores. Good examples <strong>of</strong><br />
very sensitive lines are ALD/<br />
PVN//YMI 6 <strong>and</strong> BAU/MILAN,<br />
whose mean % yield losses (27%-<br />
28%) were almost twice that <strong>of</strong><br />
their FLS scores (13%-16%).<br />
Table 2. Mean response <strong>of</strong> 11 wheat lines to natural infection by <strong>Septoria</strong>/<strong>Stagonospora</strong>, in regard to flag leaf<br />
severity, yield, percentage loss, in field plots either protected or unprotected with fungicide, in Patzcuaro,<br />
Michoacan, Mexico, over 1994, 1995, <strong>and</strong> 1996.<br />
Mean values<br />
% Overall<br />
Yield mean yield<br />
Entry Cross/Selection history CC * % ** UP % loss % Flag leaf kg/ha %<br />
1 CURINDA<br />
Tolerant check<br />
3376 c *** 100 2688 d 100 19 29 3005 c 100<br />
2 BATAN<br />
Susceptible check<br />
3016 e 89 2003 h 75 32 46 2470 f 82<br />
3 IAS20/H567.71/5* IAS20<br />
CM78A-544-7B-1Y-1B-1Y-2B-1Y-0B<br />
4094 b 121 3380 a 126 13 18 3709 a 123<br />
4 CHIL//ALD/PVN<br />
CM92801-65Y-0M-0Y-4M-0RES<br />
3062 d 91 2216 g 82 30 52 2606 d 87<br />
5 THB/CNT 7<br />
CM 830057-0Z-0A-1A-1A-1A-0Y<br />
3298 c 98 2727 c 101 17 21 2991 c 99<br />
6 LIRA/TAN//SPB<br />
CM96824-U-0Y-0H-0SY-4M-0RES<br />
3098 d 91 2519 e 94 20 37 2786 d 93<br />
7 ALD/PVN//YMI 6<br />
CM 91065-2M-0M-0Y-0M-2Y-0B-5PZ<br />
4160 a 123 2998 b 112 28 16 3534 a 118<br />
8 BAU/MILAN<br />
CM103873-2M-030Y-020Y-010M-4Y-0M<br />
3449 c 102 2473 f 92 27 13 2924 d 97<br />
9 ISEPTON89-82E/BR6//PF83144<br />
F32938-2M-0AL-0AL-0AL-4Y-0M<br />
3486 c 103 3034 b 113 10 9 3242 b 108<br />
10 BOW/MII//RES/NAC/3/PFAU<br />
CM1033644-2FC-0M-1FC-0C<br />
4441 a 132 3058 b 114 31 20 3696 a 123<br />
11 CAR853/COC//VEE/3/E7408/PAM//HORK/PF73226<br />
CM80174-32Y-04M-0Y-3M-1M-0M<br />
3870 b 115 2798 b 104 28 26 3292 b 109<br />
LSD 346 312 314<br />
Mean 3577 2717 3114<br />
C.V. 12 15 18<br />
Chemical control (CC) 3577 a<br />
Unprotected (UP) 2717 b<br />
1994 3344 b ns<br />
1995 4234 a ns<br />
1996 3153 c ns<br />
LSD 181<br />
* (CC) Chemical control <strong>and</strong> (UP) Unprotected.<br />
** Percentage relative to the tolerant check, Curinda M87.<br />
*** Values followed by the same letter are not significantly different at the 0.05 probability level.<br />
On the other h<strong>and</strong>, a line may<br />
have a very high intrinsic yield<br />
potential in the absence <strong>of</strong> biotic<br />
stresses, such as attacks by <strong>Septoria</strong>/<br />
<strong>Stagonospora</strong> spp. Even if<br />
susceptible to the fungus, such<br />
lines may express a residual yield<br />
that competes well with that <strong>of</strong> the<br />
check variety (<strong>and</strong> may even<br />
outyield it in certain cases). These<br />
lines are not resistant—on the<br />
contrary, they may be quite<br />
susceptible. Their virtue lies in<br />
their high initial yield potential,<br />
which allows a considerable loss<br />
due to disease, <strong>and</strong> still produces
Session 6C — R.M. Gonzalez I., S. Rajaram, <strong>and</strong> M. van Ginkel<br />
144<br />
an impressive final yield when<br />
challenged by the fungus. In this<br />
study BOW/MII//RES/NAC/3/<br />
PFAU is an example <strong>of</strong> a line with<br />
high yield potential, which despite<br />
having 20% <strong>of</strong> its flag leaf area<br />
damaged by <strong>Septoria</strong>/<strong>Stagonospora</strong><br />
spp., resulting in a 31% mean yield<br />
loss, still yielded 14% more than<br />
the commercial check.<br />
The most desirable genotype is<br />
one that combines high yield<br />
potential with resistance/tolerance<br />
to the fungus; this results in<br />
relatively low losses in the presence<br />
;<br />
200<br />
;;;<br />
% yield <strong>of</strong> check Curinda<br />
150<br />
100<br />
CC 1994<br />
CC 1995<br />
CC 1996<br />
UP 1994<br />
UP 1995<br />
UP 1996<br />
<strong>of</strong> disease, <strong>and</strong> outst<strong>and</strong>ing yields<br />
in its absence. The line IAS20/<br />
H567.71/5*IAS 20 combines these<br />
traits; only 18% <strong>of</strong> its foliage was<br />
affected, which translated into a<br />
minimal yield loss <strong>of</strong> 13% <strong>and</strong><br />
resulted in the trial’s top mean<br />
yield <strong>of</strong> 3380 kg/ha, 26% over the<br />
commercial check, Curinda, in the<br />
presence <strong>of</strong> disease (Table 2 <strong>and</strong><br />
Figure 1). It produced a very<br />
respectable yield <strong>of</strong> 4094 kg/ha, the<br />
third highest <strong>of</strong> the group, in the<br />
absence <strong>of</strong> disease. This line is now<br />
being considered for release under<br />
the name Patzcuaro.<br />
References<br />
Table 3. Correlation coefficients <strong>of</strong> flag leaf severity <strong>and</strong> yield with various other characters, Patzcuaro,<br />
Michoacan, Mexico, during 1994, 1995, <strong>and</strong> 1996.<br />
Eyal, Z., A.L. Scharen, M.D. Huffman,<br />
<strong>and</strong> J.M. Prescott. 1985. Global<br />
insights into virulence frequencies<br />
<strong>of</strong> Mycosphaerella graminicola.<br />
Phytopathology 75:1456-1462.<br />
Gomez, B.L., <strong>and</strong> R.M. Gonzalez I.<br />
1987. Mejoramiento genético de<br />
trigos harineros para resistencia a<br />
<strong>Septoria</strong> tritici en el área de<br />
temporal húmedo en Mexico. In<br />
Conferencia regional sobre la<br />
septoriosis del trigo, M.M. Kohli<br />
<strong>and</strong> L.T. van Beuningen, eds.<br />
Mexico, D.F.: <strong>CIMMYT</strong>. pp 42-57.<br />
Variables Variables 1994 1995 1996<br />
Flag leaf severity due to infection Yield -0.81 -0.83 -0.71<br />
by <strong>Septoria</strong>/<strong>Stagonospora</strong> Test weight -0.72 -0.34 -0.44<br />
Plant height -0.48 -0.55 -0.62<br />
Yield loss (%) 0.71 0.33 0.71<br />
Days to flowering -0.42 -0.71 -0.84<br />
Yield Test weight 0.72 0.85 0.76<br />
Plant height 0.62 0.44 0.55<br />
Yield loss (%) -0.75 -0.49 -0.86<br />
Days to flowering 0.46 0.86 0.79<br />
;<br />
; ; ;<br />
; ; ; ; ;<br />
;<br />
; ; ; ; ;; ; ; ;<br />
; ;; ; ; ; ; ;; ; ; ;<br />
; ; ; ; ;; ; ; ;<br />
; ;; ; ;; ; ; ; ; ; ; ; ; ;<br />
; ;;<br />
; ;; ;; ; ;<br />
; ;; ;; ; ; ;<br />
; ; ; ; ; ; ;<br />
;<br />
; ; ; ; ; ; ;<br />
; ;;;<br />
; ; ; ; ; ;<br />
;;; ; ;<br />
; ; ;<br />
;;<br />
;; ; ; ;<br />
; ;; ; ; ; ;<br />
; ;; ; ; ; ;<br />
; ;; ; ; ; ;<br />
50 Curinda Batan 3 4 5 6 7 8 9 10 11<br />
Figure 1. Yield as percentage <strong>of</strong> the tolerant check Curinda, in chemically controlled plots (CC) <strong>and</strong><br />
unprotected plots (UP) <strong>of</strong> nine wheat lines, during 1994, 1995 <strong>and</strong> 1996. Patzcuaro, Michoacan, Mexico.
Varieties <strong>and</strong> Advanced Lines Resistant to <strong>Septoria</strong><br />
<strong>Diseases</strong> <strong>of</strong> Wheat in Western Australia<br />
R. Loughman, 1 R.E. Wilson, 1 I.M. Goss, 1 D.T. Foster, 1 <strong>and</strong> N.E.A. Murphy2 1 Agriculture Western Australia, Bentley Delivery Centre, Western Australia<br />
2 WA State Agriculture Biotechnology Centre, Division <strong>of</strong> Science <strong>and</strong> Engineering, Murdoch University,<br />
Murdoch, Australia<br />
Abstract<br />
The development <strong>of</strong> bread wheats in Western Australia has resulted in the release <strong>of</strong> several varieties with improved<br />
response to the commonly occurring leaf spot diseases caused by Phaeosphaeria nodorum, Mycosphaerella graminicola,<br />
<strong>and</strong> Pyrenophora tritici-repentis. In a yield trial evaluating resistance to M. graminicola, susceptible varieties suffered 20-<br />
50% yield loss, while yield loss <strong>of</strong> resistant crossbreds <strong>and</strong> varieties was 0-15%. Similar responses were observed in most lines<br />
for grain weight when assessed in M. graminicola-infected row plots. In P. nodorum-infected row plots, susceptible<br />
varieties suffered 30-50% grain weight loss, while resistant lines suffered 0-30% loss in grain weight depending on year<br />
<strong>of</strong> testing.<br />
In Western Australia resistance<br />
to Phaeosphaeria nodorum is sought<br />
in combination with Mycosphaerella<br />
graminicola <strong>and</strong> Pyrenophora triticirepentis<br />
resistance, as these<br />
pathogens frequently occur in<br />
combination (Loughman et al.,<br />
1994). Improved resistance to these<br />
diseases is sought by continually<br />
crossing the most resistant, best<br />
adapted lines with resistance<br />
donors. A selection <strong>of</strong> varieties,<br />
advanced lines, <strong>and</strong> potential<br />
resistance sources were assessed for<br />
resistance in replicated field<br />
experiments using susceptible<br />
adapted wheats with comparable<br />
maturities as benchmarks.<br />
Materials <strong>and</strong> Methods<br />
Lines were evaluated for<br />
response to infection with M.<br />
graminicola or P. nodorum in<br />
separate experiments in 1995-96<br />
using split plot designs <strong>of</strong> three<br />
replicates. At Mt. Barker lines were<br />
sown as 5-row, 5-m subplots. Main<br />
plots were established in early<br />
spring as either fungicide<br />
protected (three applications <strong>of</strong><br />
125 g tebuconazole/ha) or<br />
inoculated twice with aqueous<br />
pycnidiospore suspensions <strong>of</strong> the<br />
pathogen. Growth stage (Zadoks<br />
et al., 1974) <strong>and</strong> % infection <strong>of</strong> the<br />
flag-3 leaves were assessed in late<br />
spring. Yields were assessed. At<br />
South Perth lines were sown as<br />
40-cm, 2-row subplots with 18-cm<br />
row spacings, each subplot<br />
separated from its neighbor by a<br />
single row <strong>of</strong> barley. Main plots<br />
were established with fungicide<br />
or inoculation as above. Separate<br />
experiments were established for<br />
M. graminicola <strong>and</strong> P. nodorum.<br />
Heading date (as day <strong>of</strong> the year)<br />
<strong>and</strong> disease severity as percent<br />
infection <strong>of</strong> the flag or flag <strong>and</strong><br />
second top leaves were recorded<br />
in spring. Grain weights were<br />
assessed from a r<strong>and</strong>om<br />
subsample <strong>of</strong> harvested grain.<br />
Relative grain weights <strong>of</strong> infected<br />
plots were calculated as a<br />
percentage <strong>of</strong> fungicide<br />
protected plots.<br />
Results <strong>and</strong><br />
Discussion<br />
Resistance to M.<br />
graminicola<br />
Cascades (Tadorna.Inia /<br />
3*Aroona), Tammin (Bod/Eradu<br />
sib.xbvt//Atlas66.2Madden),<br />
<strong>and</strong> Nyabing (Ar-<br />
3Ag3(WT329)/<br />
(IW753)Jab.Ag4C) developed<br />
low to moderate infection <strong>and</strong><br />
yielded 5.4, 5.2, <strong>and</strong> 4.1 t/ha,<br />
respectively in the presence <strong>of</strong><br />
disease. These yields were not<br />
significantly different from<br />
fungicide sprayed plots (yield<br />
diseased was 89-99% relative to<br />
the fungicide protected plots). In<br />
contrast, two susceptible<br />
varieties <strong>of</strong> similar maturity,<br />
Gamenya <strong>and</strong> Datatine,<br />
developed high infection scores<br />
<strong>and</strong> yielded 1.5 <strong>and</strong> 3.2 t/ha,<br />
respectively when diseased.<br />
These yields were significantly<br />
different from fungicide sprayed<br />
plots (yield diseased was 48-72%<br />
relative to fungicide protected).<br />
145
Session 6C — R. Loughman, R.E. Wilson, I.M. Goss, D.T. Foster, <strong>and</strong> N.E.A. Murphy<br />
146<br />
Table 1. Maturity (growth stage), percent leaf disease (Dis), yield, hundred-grain weight (HGW), <strong>and</strong> relative<br />
yield (RY) <strong>and</strong> relative grain weight (RGW) <strong>of</strong> wheat varieties infected with Mycosphaerella graminicola in a<br />
yield trial at Mt. Barker <strong>and</strong> small row plots at South Perth, 1995. Comparisons for percent flag leaf disease<br />
should be made between varieties <strong>of</strong> similar maturity.<br />
Mt. Barker South Perth<br />
Growth Dis Yield(t/ha) RY Dis HGW (g) RGW<br />
stage Flag-3 Fung Inoc % Flag Fung Inoc %<br />
Resistant <strong>and</strong> moderately resistant lines<br />
77Z893 44 2 5.4 5.3 98 86 3.6 2.5 70<br />
Nyabing 42 30 4.1 4.1 99 38 4.2 3.4 81<br />
Cascades 41 23 6.0 5.4 89 86 4.2 2.5 57<br />
Tammin 41 3 5.8 5.2 90 23 4.2 3.1 76<br />
483W3 40 0 5.1 5.2 101 21 3.2 3.4 105<br />
486W189 39 0 4.6 4.3 92 32 3.9 4.2 106<br />
78Z976 39 5 5.7 5.0 89 40 3.1 2.7 87<br />
Calingiri 38 1 5.9 5.0 85 43 4.0 3.7 94<br />
Resistant <strong>and</strong> moderately resistant control varieties<br />
Millewa 43 9 4.5 3.9 86 78 3.3 2.2 69<br />
Aroona 40 13 3.9 4.4 114 99 3.6 2.5 72<br />
Corrigin 40 2 5.7 5.2 91 70 3.1 2.5 80<br />
Susceptible control varieties<br />
Kulin 55 85 4.0 2.2 55 99 2.7 1.6 63<br />
Tincurrin 42 90 4.5 3.6 80 99 3.6 2.2 68<br />
Datatine 41 50 4.5 3.2 72 73 3.4 2.5 72<br />
Gamenya 41 88 3.2 1.5 48 99 3.6 1.9 59<br />
Stiletto 38 23 4.5 3.2 70 98 4.1 2.8 69<br />
lsd 5% 2.5 23 1.0 32 0.7 23<br />
cv % 4 41 13 44 13 19<br />
Table 2. Maturity (heading day <strong>of</strong> year, Hd), percent leaf disease (Dis), hundred-grain weight (HGW), <strong>and</strong><br />
relative grain weight (RGW) <strong>of</strong> wheat varieties infected with Phaeosphaeria nodorum in short row plot<br />
experiments in South Perth, 1995-96 (- denotes testing discontinued). Comparisons for percent flag leaf<br />
disease should be made between varieties <strong>of</strong> similar maturity.<br />
1995 1996<br />
Hd Dis HGW (g) RGW Dis HGW(g) RGW<br />
d.o.y.<br />
Moderately resistant lines<br />
Flag Fung Inoc % F,F-1 Fung Inoc %<br />
Brookton 225 64 3.6 3.7 101 52 3.5 2.8 80<br />
W48795 228 46 3.6 3.6 101 4 3.8 2.9 76<br />
81W1137 230 43 3.8 3.6 97 - - - -<br />
Tammin 231 32 3.9 3.9 103 8 4.3 3.0 71<br />
W48433 232 33 3.2 3.1 98 30 3.8 3.0 81<br />
Resistant <strong>and</strong> moderately resistant controls<br />
ALMRES-83-18 225 72 4.3 3.6 83 56 3.9 2.8 74<br />
CNT2 236 21 4.6 4.2 93 45 4.1 3.9 103<br />
Aus20917 236 40 3.1 2.5 81 27 2.8 2.4 87<br />
Qualset601-68<br />
Intermediate lines<br />
238 18 3.6 3.0 85 20 4.0 2.9 73<br />
W486110 220 50 4.9 4.2 87 39 4.0 3.3 83<br />
Westonia 220 83 4.1 3.3 80 76 3.2 2.2 69<br />
Cascades 226 83 4.1 3.2 80 83 3.7 2.0 54<br />
Carnamah 226 68 3.4 2.9 86 32 4.1 2.1 51<br />
Cunderdin 228 50 3.7 3.0 82 41 2.9 2.4 81<br />
Susceptible control varieties<br />
Kulin 221 99 3.4 2.2 66 91 3.2 1.5 46<br />
Aroona 225 87 4.6 2.2 49 92 3.2 1.6 50<br />
Millewa 225 96 2.6 2.0 77 97 3.3 1.3 44<br />
Gamenya 228 77 3.4 2.1 64 51 3.2 2.0 63<br />
Cranbrook 236 71 3.1 2.5 80 48 3.4 2.0 61<br />
Spear 242 59 4.2 2.6 63 1 4.1 3.0 75<br />
lsd 5% 23 0.6 24 18 0.8 28<br />
cv % 26 10 19 23 16 26
Calingiri (Chino/Kulin//<br />
Reeves) also exhibited resistance<br />
<strong>and</strong> while some <strong>of</strong> this low disease<br />
expression may have been due to<br />
later maturity, the performance was<br />
measurably superior to the similar<br />
maturing but susceptible variety<br />
Stiletto. Crossbreds 483W3, 78Z976<br />
<strong>and</strong> 77Z893 (Corrigin sib) also<br />
expressed high levels <strong>of</strong> resistance,<br />
as indicated by low disease scores,<br />
the non-significant yield losses, <strong>and</strong><br />
the resulting high relative yields<br />
(yield diseased was 89-101%<br />
relative to fungicide protected)<br />
(Table 1).<br />
Resistance to P. nodorum<br />
Five moderately resistant lines<br />
achieved high grain weights in<br />
diseased plots relative to fungicide<br />
protected plots. Variety Brookton<br />
(Torres/Cranbrook//76W596/<br />
Cranbrook) developed significantly<br />
less disease <strong>and</strong> no significant<br />
grain weight reduction when<br />
compared with susceptible<br />
varieties <strong>of</strong> equivalent maturity<br />
such as Aroona or Millewa.<br />
W48795, Tammin, <strong>and</strong> sister line<br />
81W1137 had similar maturity to<br />
susceptible variety Gamenya.<br />
These lines developed significantly<br />
less disease than Gamenya <strong>and</strong> had<br />
smaller grain weight reductions in<br />
the presence <strong>of</strong> disease. Line<br />
W48433, derived from CNT2, was<br />
similar to Tammin <strong>and</strong> developed<br />
low disease in 1995 <strong>and</strong> high<br />
relative grain weights in 1995 <strong>and</strong><br />
1996. This line was comparable<br />
with the resistant parent CNT2 for<br />
disease severity <strong>and</strong> relative grain<br />
weight in 1995, though relative<br />
grain weight appeared less robust<br />
than that <strong>of</strong> CNT2 in 1996 (Table 2).<br />
Varieties <strong>and</strong> Advanced Lines Resistant to <strong>Septoria</strong> <strong>Diseases</strong> <strong>of</strong> Wheat in Western Australia 147<br />
Westonia (SpicaTimg.Tosc/<br />
Cr:J2.Bob), Cunderdin (Cranbrook<br />
sib/Sunfield sib), Carnamah<br />
(Bolsena-1ch(RAC529)/77W660),<br />
<strong>and</strong> Cascades gave responses that<br />
were intermediate between the<br />
resistant <strong>and</strong> susceptible lines.<br />
Although hundred grain weight<br />
reductions in the presence <strong>of</strong><br />
disease were generally significant,<br />
the grain weight reductions were<br />
less than for susceptible lines.<br />
Although excellent levels <strong>of</strong> M.<br />
graminicola resistance from diverse<br />
sources have been observed in lines<br />
such as 483W3 <strong>and</strong> 78Z976,<br />
moderate resistance derived from<br />
WW15 via Aroona <strong>and</strong> Condor<br />
(Wilson, 1994) has had the greatest<br />
impact in the development <strong>of</strong><br />
commercial varieties. While this<br />
moderate resistance can be more<br />
difficult to identify in intensive<br />
disease nurseries, its effect has been<br />
observed <strong>of</strong>ten in practice <strong>and</strong><br />
previously reported (Loughman<br />
<strong>and</strong> Thomas, 1992).<br />
Significant progress has been<br />
made in developing moderate<br />
resistance to P. nodorum.<br />
Commercial varieties currently<br />
exhibit improved responses<br />
compared with their susceptible<br />
predecessors, <strong>and</strong> some varieties<br />
<strong>and</strong> advanced lines perform at<br />
similar levels to resistant control<br />
varieties. In some cases moderate<br />
resistance to different leaf spot<br />
diseases has been combined.<br />
Cascades combines moderate<br />
resistance to both M. graminicola<br />
<strong>and</strong> P. tritici-repentis (Loughman et<br />
al., 1998). Brookton, Cunderdin,<br />
<strong>and</strong> Westonia have moderate<br />
resistance to P. tritici-repentis<br />
(Loughman et al., 1998), in<br />
combination with partial resistance<br />
to P. nodorum. Because P. nodorum,<br />
M. graminicola, <strong>and</strong> P. tritici-repentis<br />
frequently occur as disease<br />
complexes in Western Australia,<br />
combinations <strong>of</strong> resistance are<br />
important for effective disease<br />
management.<br />
References<br />
Loughman, R., <strong>and</strong> Thomas, G.J. 1992.<br />
Fungicides <strong>and</strong> cultivar control <strong>of</strong><br />
<strong>Septoria</strong> diseases <strong>of</strong> wheat. Crop<br />
Protection 11: 349-54.<br />
Loughman, R., Wilson, R.E., <strong>and</strong><br />
Thomas, G.J. 1994. Influence <strong>of</strong><br />
disease complexes involving<br />
Leptosphaeria (<strong>Septoria</strong>) nodorum on<br />
detection <strong>of</strong> resistance to three leaf<br />
spot diseases in wheat. Euphytica<br />
72: 31-42.<br />
Loughman, R., Wilson, R.E., Roake,<br />
J.E., Platz, G.J., Rees, R.G., <strong>and</strong><br />
Ellison, F.W. 1998. Crop<br />
management <strong>and</strong> breeding for<br />
control <strong>of</strong> Pyrenophora triticirepentis<br />
causing yellow spot <strong>of</strong><br />
wheat in Australia. In Duveiller, E.,<br />
H.J. Dubin, J. Reeves, <strong>and</strong> A.<br />
McNab (eds.). Helminthosporium<br />
Blights <strong>of</strong> Wheat: Spot Blotch <strong>and</strong><br />
Tan Spot. Mexico, D.F.: <strong>CIMMYT</strong>.<br />
Wilson, R.E. 1994. Progress toward<br />
breeding for resisance to the two<br />
septoria disease <strong>of</strong> wheat in<br />
Australia. Pp. 149-152 In: E.<br />
Arseniuk (ed.). <strong>Septoria</strong> <strong>of</strong> <strong>Cereals</strong>,<br />
Proc. Fourth International<br />
Workshop on <strong>Septoria</strong> <strong>Diseases</strong> <strong>of</strong><br />
<strong>Cereals</strong>. IHAR, Radzikow, Pol<strong>and</strong>.<br />
Zadoks, J.C., Chang, T.T., <strong>and</strong><br />
Konzak, C.F. 1974. A decimal code<br />
for the growth stages <strong>of</strong> cereals.<br />
Weed Research 14: 415-21.
148<br />
Field Resistance <strong>of</strong> Wheat to <strong>Septoria</strong> Tritici Leaf Blotch,<br />
<strong>and</strong> Interactions with Mycosphaerella graminicola<br />
Isolates<br />
J.K.M. Brown, 1 G.H.J. Kema, 2 H.-R. Forrer, 3 E.C.P. Verstappen, 2 L.S. Arraiano, 1 P.A. Brading, 1 E.M. Foster, 1 A.<br />
Hecker, 3 <strong>and</strong> E. Jenny3 1 John Innes Centre, Norwich, UK<br />
2 DLO-Research Institute for Plant Protection, Wageningen, The Netherl<strong>and</strong>s<br />
3 Swiss Federal Research Station for Agroecology <strong>and</strong> Agriculture, Zürich, Switzerl<strong>and</strong><br />
Abstract<br />
The resistance <strong>of</strong> 71 varieties <strong>of</strong> bread wheat to six isolates <strong>of</strong> Mycosphaerella graminicola was studied in field trials in<br />
the Netherl<strong>and</strong>s, Switzerl<strong>and</strong>, <strong>and</strong> the UK, carried out over three years. There was a wide range <strong>of</strong> <strong>Septoria</strong> tritici infection.<br />
Some varieties had especially good resistance, including lines from Europe (especially Switzerl<strong>and</strong>) <strong>and</strong> Latin America. Many<br />
interactions between varieties <strong>and</strong> isolates were detected. In particular, 27 varieties <strong>of</strong> diverse origins were specifically<br />
resistant to the isolate IPO323. Variety-by-isolate interactions were stable over years <strong>and</strong> locations. The existence <strong>of</strong> these<br />
interactions <strong>and</strong> the fact that they are stable over environments implies that certain widely used resistances to <strong>Septoria</strong> tritici<br />
might break down through the evolution <strong>of</strong> specific virulence in the fungus. Breeders should take these interactions into<br />
account in their efforts to develop varieties with durable resistance.<br />
<strong>Septoria</strong> tritici leaf blotch is<br />
now the most economically<br />
important foliar disease <strong>of</strong> wheat in<br />
Europe, <strong>and</strong> controlling it with<br />
fungicides costs several hundred<br />
million dollars a year. Resistance to<br />
septoria tritici leaf blotch is<br />
therefore a major target for most<br />
European wheat breeders.<br />
However, if resistant varieties are<br />
to be economically worthwhile,<br />
resistance must be both effective<br />
<strong>and</strong> durable.<br />
Strong, specific interactions<br />
between wheat varieties <strong>and</strong><br />
isolates <strong>of</strong> the pathogen,<br />
Mycosphaerella graminicola, have<br />
been found in both seedlings <strong>and</strong><br />
adult plants (Kema <strong>and</strong> van<br />
Silfhout, 1997), while the resistance<br />
<strong>of</strong> at least one variety, Gene, has<br />
“broken down” through the<br />
evolution <strong>of</strong> a virulent pathogen<br />
population (Mundt et al., 1999).<br />
This means that breeders need to<br />
know not only which varieties may<br />
be good sources <strong>of</strong> resistance in<br />
breeding programs, but also<br />
whether or not resistance genes in<br />
these varieties are at risk from<br />
virulent isolates <strong>of</strong> M. graminicola.<br />
This paper reports a series <strong>of</strong><br />
field trials carried out in Engl<strong>and</strong>,<br />
The Netherl<strong>and</strong>s, <strong>and</strong> Switzerl<strong>and</strong><br />
between 1994 <strong>and</strong> 1997. The aims<br />
were to investigate resistance to<br />
septoria tritici leaf blotch in a<br />
representative set <strong>of</strong> European<br />
varieties, to evaluate potential new<br />
sources <strong>of</strong> resistance, to study the<br />
responses <strong>of</strong> varieties to different<br />
isolates <strong>of</strong> M. graminicola <strong>and</strong> to test<br />
the stability <strong>of</strong> variety-by-isolate<br />
interactions over a range <strong>of</strong><br />
environments.<br />
Materials <strong>and</strong> Methods<br />
A total <strong>of</strong> 71 wheat varieties <strong>of</strong><br />
diverse origins were included in<br />
the field trials. The majority were<br />
cultivars or breeding lines<br />
developed by European breeders.<br />
Most <strong>of</strong> these were winter wheats.<br />
Several varieties that are potential<br />
sources <strong>of</strong> septoria tritici leaf blotch<br />
resistance were also included, as<br />
were a number <strong>of</strong> varieties that are<br />
parents <strong>of</strong> precise genetic stocks<br />
held by the John Innes Centre.<br />
Trials were inoculated with six<br />
monospore isolates <strong>of</strong> M.<br />
graminicola from the IPO-DLO<br />
collection. Six trials were<br />
conducted, three in the<br />
Netherl<strong>and</strong>s, one in Switzerl<strong>and</strong>,<br />
<strong>and</strong> two in Engl<strong>and</strong>. Each trial was<br />
sown in a split-plot design with<br />
two replicate blocks. Within each<br />
block, there were five or six main
plots, each inoculated with one<br />
isolate, such that isolates were<br />
r<strong>and</strong>omized within blocks, while<br />
varieties were r<strong>and</strong>omized within<br />
main plots. The percentage <strong>of</strong> the<br />
leaf area that was necrotic or<br />
covered by lesions bearing<br />
pycnidia was estimated visually.<br />
Results<br />
Levels <strong>of</strong> necrosis <strong>and</strong> pycnidia<br />
were highly correlated, so further<br />
analysis was based on pycnidia<br />
scores. There was highly significant<br />
variation between varieties in the<br />
level <strong>of</strong> disease <strong>and</strong> highly<br />
significant interactions between<br />
varieties <strong>and</strong> isolates. However,<br />
there was no significant variation<br />
in variety-by-isolate interactions<br />
among the six trials. This implies<br />
that variety-by-isolate interactions<br />
were stable over the range <strong>of</strong><br />
environments used in this series <strong>of</strong><br />
trials.<br />
There was a wide range <strong>of</strong><br />
resistance among the varieties<br />
grown in the trials. The existence <strong>of</strong><br />
strong variety-by-isolate<br />
interactions <strong>and</strong> the small number<br />
<strong>of</strong> isolates used means that one<br />
cannot be certain which varieties<br />
have good, overall resistance to the<br />
European population <strong>of</strong> M.<br />
graminicola. However, some lines<br />
were identified that should be<br />
tested further, preferably with more<br />
isolates. The most resistant was the<br />
Brazilian cultivar Veranopolis, a<br />
well-known source <strong>of</strong> resistance.<br />
Four <strong>of</strong> the top ten varieties were<br />
from the FAP breeding program in<br />
Switzerl<strong>and</strong>, including the popular<br />
cultivar Arina. Other varieties<br />
which had very good resistance to<br />
Field Resistance <strong>of</strong> Wheat to <strong>Septoria</strong> Tritici Leaf Blotch, <strong>and</strong> Interactions with Mycosphaerella graminicola Isolates 149<br />
the isolates used in these trials<br />
included cultivars <strong>and</strong> breeding<br />
lines from several European<br />
countries, as well as another<br />
well-known source <strong>of</strong> resistance,<br />
Kavkaz-K4500 l.6.a.4, <strong>and</strong><br />
Frontana, one <strong>of</strong> the parents <strong>of</strong><br />
Veranopolis.<br />
Variety-by-isolate interactions<br />
were most pronounced in tests<br />
with the isolate IPO323. A total <strong>of</strong><br />
27 cultivars <strong>and</strong> breeding lines,<br />
from several European countries,<br />
China, Israel, <strong>and</strong> the USA,<br />
showed specific resistance to this<br />
isolate. One or more varieties<br />
showed specific resistance to four<br />
<strong>of</strong> the other five isolates, while<br />
specific susceptibility to four<br />
isolates was also detected.<br />
Discussion<br />
There is good resistance to<br />
septoria tritici in a wide range <strong>of</strong><br />
European wheat germplasm<br />
from several countries. Plant<br />
breeders may be able to exploit<br />
this resistance by recombining<br />
quantitative resistance from<br />
different sources. There may be<br />
some additional value in<br />
introducing resistance from<br />
sources outside Europe. The<br />
striking success <strong>of</strong> the Swiss<br />
breeding program may be<br />
attributed to a long tradition <strong>of</strong><br />
selection for foliar disease<br />
resistance, using both artificial<br />
<strong>and</strong> natural inoculation.<br />
Furthermore, trials are conducted<br />
without fungicides in several<br />
locations with diverse climatic<br />
conditions.<br />
The existence <strong>of</strong> strong<br />
variety-by-isolate interactions, as<br />
well as the fact that they are<br />
stable over environments, means<br />
that breeders must take account<br />
<strong>of</strong> the possibility that certain<br />
resistances may not be durable.<br />
This is particularly the case with<br />
resistance to IPO323, although it<br />
is not known if this resistance is<br />
controlled by the same genes in<br />
different varieties. One strategy<br />
for breeding might be to use<br />
several specific resistance genes<br />
in a breeding program <strong>and</strong> aim to<br />
select varieties with combinations<br />
<strong>of</strong> resistances. An alternative<br />
would be to avoid selecting for<br />
specific resistances altogether,<br />
aiming instead for a high level <strong>of</strong><br />
quantitative resistance.<br />
Acknowledgments<br />
This research was supported<br />
in part by the EU Framework 4<br />
Biotechnology program, the UK<br />
Ministry <strong>of</strong> Agriculture, Fisheries<br />
<strong>and</strong> Food, <strong>and</strong> Praxis XXI<br />
(Portugal).<br />
References<br />
Kema, G.H.J., <strong>and</strong> C.H. van<br />
Silfhout. 1997. Genetic variation<br />
for virulence <strong>and</strong> resistance in<br />
the wheat-Mycosphaerella<br />
graminicola pathosystem III.<br />
Comparative seedling <strong>and</strong> adult<br />
plant experiments.<br />
Phytopathology 87:266-272.<br />
Mundt, C.C., M.E. H<strong>of</strong>fer, H.U.<br />
Ahmed, S.M. Coakley, J.A.<br />
DiLeone, <strong>and</strong> C. Cowger. 1999.<br />
Population genetics <strong>and</strong> host<br />
resistance. In: <strong>Septoria</strong> on<br />
<strong>Cereals</strong>: a Study <strong>of</strong><br />
Pathosystems. J.A. Lucas, P.<br />
Bowyer, <strong>and</strong> H.A. Anderson<br />
(eds.). CAB Publishing,<br />
Wallingford, UK. pp. 115-130.
150<br />
Using Precise Genetic Stocks to Investigate the Control <strong>of</strong><br />
<strong>Stagonospora</strong> nodorum Resistance in Wheat<br />
C.M. Ellerbrook, 1 V. Korzun, 2 <strong>and</strong> A.J. Worl<strong>and</strong> 1 (Poster)<br />
1 John Innes Centre, Colney, Norwich, UK (Poster)<br />
2 IPK, Gatersleben, Germany<br />
Abstract<br />
A substitution series <strong>of</strong> ‘Synthetic 6x’ into ‘Chinese Spring’ had previously been studied to determine which <strong>of</strong> the 21<br />
chromosomes <strong>of</strong> ‘Synthetic 6x’ conferred resistance to <strong>Stagonospora</strong> nodorum; from this single chromosome recombinant<br />
lines (SCRs) have been developed for substitution lines that improve the resistance <strong>of</strong> ‘Chinese Spring’ to the pathogen. The<br />
SCRs were screened for their response to infection. Marker assisted screening was also employed on the SCR populations;<br />
micro-satellite, RFLP <strong>and</strong> iso-electric focusing, coupled with conventional QTL analysis <strong>and</strong> morphological character scoring<br />
(vernalization response). As these markers had been previously mapped, it would be possible to pinpoint the resistance gene(s)<br />
<strong>and</strong> find a linked marker. Chromosome 5D had been shown to be the most effective against <strong>Stagonospora</strong> nodorum <strong>and</strong> is<br />
also known to carry the isozyme genes, Ibf-1 <strong>and</strong> Mdh-3, a range <strong>of</strong> micro-satellite markers, a sparse selection <strong>of</strong> RFLP<br />
markers, <strong>and</strong> a vernalization response gene (Vrn 3). Screening revealed that the resistance gene (Srb3) was located on the<br />
long arm between Ibf-1 <strong>and</strong> the RFLP probe Xpsr 912, but being 15.6 <strong>and</strong> 12.0 cM, respectively, away from Srb3, they could<br />
not be used as markers for resistance. SCRs have been developed for the remaining chromosomes, <strong>and</strong> screening continues to<br />
precisely map the resistance gene(s).<br />
<strong>Stagonospora</strong> nodorum (<strong>Septoria</strong><br />
nodorum Berk.) is the causal agent<br />
<strong>of</strong> leaf <strong>and</strong> glume blotch in wheat<br />
with infection reducing yield by up<br />
to 40%. Conventional fungicide<br />
control has significant<br />
environmental <strong>and</strong> economic<br />
consequence, thus alternative<br />
methods <strong>of</strong> control are being<br />
sought. Several Aegilops species<br />
including Ae. squarrosa exhibit<br />
partial resistance to the pathogen.<br />
This resistance is expressed in a<br />
synthetic amphiploid ‘Synthetic 6x’<br />
derived from a cross between Ae.<br />
squarrosa <strong>and</strong> Triticum dicoccum<br />
(McFadden <strong>and</strong> Sears, 1946; Sears,<br />
1976). A substitution series <strong>of</strong><br />
‘Synthetic 6x’ chromosomes into<br />
‘Chinese Spring’ was developed<br />
<strong>and</strong> is maintained at the John Innes<br />
Centre. This series was utilized in<br />
disease tests to determine the<br />
chromosomal location <strong>of</strong> the<br />
resistance gene(s) (Nicholson et al.,<br />
1993) as ‘Chinese Spring’ was<br />
shown to be susceptible to<br />
<strong>Stagonospora</strong> nodorum (Scott <strong>and</strong><br />
Benedikz, 1977).<br />
From the D genome Ae.<br />
squarrosa parent, chromosome 5D<br />
conferred a high level <strong>of</strong> resistance,<br />
as did 3D <strong>and</strong> 7D. Resistance was<br />
most pronounced in 5D where the<br />
percentage leaf infection was near<br />
to that <strong>of</strong> the amphiploid. Three<br />
chromosomes from the T. dicoccum<br />
parent (AB genome) 2A, 3B, <strong>and</strong> 5A<br />
conferred resistance but to a lesser<br />
extent than that promoted by the D<br />
genome chromosomes. SCRs were<br />
then developed for these<br />
chromosomes. This paper reports<br />
the results <strong>of</strong> experiments to<br />
localize the resistance genes <strong>and</strong><br />
assign markers linked to the genes.<br />
Materials <strong>and</strong> Methods<br />
Single chromosome<br />
recombinant lines were developed<br />
by crossing the individual<br />
chromosome substitution lines,<br />
previously identified as conferring<br />
resistance, to ‘Chinese Spring’<br />
euploid (or ditelocentric), <strong>and</strong> then<br />
backcrossing the F 1 with the<br />
corresponding monosomic from<br />
the ‘Chinese Spring’ monosomic<br />
series (Sears, 1954). Monosomic<br />
plants were then selected in the<br />
backcrossed F 1 progeny <strong>and</strong> selfed<br />
for disomic extraction (Figure 1).<br />
Infection response was scored<br />
on juvenile plants. Seeds had been<br />
placed on moist filter paper in petri<br />
dishes <strong>and</strong> incubated at 25 o C for 2<br />
days (with 2 days cold shock at 4 o C<br />
to promote germination if<br />
necessary) <strong>and</strong> then planted<br />
individually in pots <strong>of</strong> John Innes<br />
no. 2 compost. In all experiments<br />
the plantlets were grown fully<br />
r<strong>and</strong>omized over 10 replications<br />
with an additional uninfected<br />
replicate as a control.
The seedlings were inoculated<br />
when the second leaves were fully<br />
exp<strong>and</strong>ed with a 1 x 10 6 spore/ml<br />
suspension <strong>of</strong> an aggressive isolate<br />
(S353/88). The plantlets were then<br />
placed in propagators <strong>and</strong><br />
transferred to growth chambers for<br />
72 h at 15 o C in complete darkness<br />
to establish infection. Once this had<br />
been completed, the plants were<br />
moved to a containment glasshouse<br />
<strong>and</strong> removed from their<br />
propagators. Disease was scored at<br />
10 <strong>and</strong> 17 days post-inoculation<br />
<strong>and</strong> expressed as the percentage <strong>of</strong><br />
leaf area lesioned (at the intervals 1,<br />
5, 10, 25, 40, 60, 75, 90, <strong>and</strong> 100%).<br />
The scores were transformed using<br />
the angular transformation <strong>and</strong> an<br />
analysis <strong>of</strong> variance carried out<br />
using the GENSTAT 5 statistical<br />
package. The significance <strong>of</strong><br />
Chinese Spring ditelocentric for the long arm<br />
(2n = 42 tt)<br />
Background<br />
Chinese Spring monosomic (2n = 41)<br />
Selfing <strong>of</strong> each monosomic plant<br />
Using Precise Genetic Stocks to Investigate the Control <strong>of</strong> <strong>Stagonospora</strong> nodorum Resistance in Wheat 151<br />
differences in the mean scores<br />
relative to ‘Chinese Spring’ <strong>and</strong><br />
‘Synthetic 6x’ was estimated using<br />
a two-tailed t-test.<br />
Morphological characters were<br />
scored on field plots, with three<br />
replicates <strong>of</strong> 11 plants in<br />
r<strong>and</strong>omized blocks to enable QTL<br />
analysis <strong>and</strong> screening for Vrn3.<br />
Biochemical analysis involved<br />
the isoelectric focusing technique,<br />
using seed embryo or endosperm<br />
on flat bed electrophoresis<br />
apparatus, showing separation <strong>of</strong><br />
the proteins at their relevant<br />
isoelectric points (pI) (Liu <strong>and</strong><br />
Gale, 1989).<br />
X<br />
X<br />
Cs/Syn 5D (2n = 42)<br />
Selection <strong>of</strong> monosomic plants (2n = 41 ) or monotelosomics (2n = 41 t)<br />
Selection <strong>of</strong> disomic (2n = 42) single chromosome recombinant lines (SCRs)<br />
Figure 1. Development <strong>of</strong> single chromosome recombinant lines (SCRs) for the long arm <strong>of</strong> chromosome<br />
5D (18).<br />
F1<br />
Molecular marker techniques<br />
required extraction <strong>of</strong> DNA (phenol<br />
chlor<strong>of</strong>orm method) <strong>and</strong> in the case<br />
<strong>of</strong> RFLPs digesting with enzymes<br />
that have a 4 base-pair recognition<br />
sequence (Bam HI, Eco RI) <strong>and</strong><br />
then hybridizing with P 32 labeled<br />
CTP (Southern, 1975). The microsatellite<br />
technique was PCR-based<br />
<strong>and</strong> used micro-satellite probes<br />
developed at IPK-Gatersleben. Both<br />
techniques clearly revealed the SCR<br />
progeny as either ‘Chinese Spring’<br />
or ‘Synthetic 6x’ type.<br />
Marker results were input into<br />
Mapmaker (Lincoln et al., 1992) to<br />
compare the segregation <strong>of</strong><br />
markers in the progeny, <strong>and</strong> a map<br />
<strong>of</strong> resistance gene(s) location<br />
5D
Session 6C — C.M. Ellerbrook, V. Korzun, <strong>and</strong> A.J. Worl<strong>and</strong><br />
152<br />
related to the markers analyzed<br />
was compiled. QTLs were also<br />
assigned to markers on the<br />
chromosome using the Students ttest.<br />
To date only the 5D SCR<br />
population has been analyzed for<br />
disease resistance <strong>and</strong> associated<br />
markers.<br />
6.8 cM<br />
8.2 cM<br />
2.1 cM<br />
3.2 cM<br />
12.0 cM<br />
12.0 cM<br />
15.6 cM<br />
7.8 cM<br />
7.4 cM<br />
16.1 cM<br />
7.4 cM<br />
10.3 cM<br />
Results <strong>and</strong> Discussion<br />
Following analysis <strong>of</strong> the 5D<br />
population (85 lines), it was found<br />
that S. nodorum resistance was<br />
under the control <strong>of</strong> a single gene<br />
Srb3, which was located between<br />
Ibf-1 <strong>and</strong> Xpsr 912, with a QTL for<br />
plant height (Qph) being linked to<br />
Ibf-1 (Figure 2). However, these<br />
genes are too remote from Srb3 to<br />
Mdh 3<br />
Wms 205<br />
Dms 3<br />
Xpsr 628<br />
Xpsr 906<br />
Xpsr 574<br />
Xpsr 912<br />
Srb 3<br />
Ibf-1<br />
Wms 182<br />
Wms 174<br />
Wms 292<br />
Xpsr 819<br />
Vrn 3<br />
Qsy (QTL for plant yield)<br />
Qey (QTL for ear yield)<br />
Qph (QTL for plant height)<br />
Qph (QTL for plant height)<br />
be useful as flanking markers.<br />
Work is continuing to locate more<br />
markers on chromosome 5D, as the<br />
region in which Srb3 is located<br />
currently has a low marker<br />
concentration. RFLP screening is<br />
also underway with recently<br />
developed markers <strong>and</strong> with the<br />
collaboration at IPK-Germany; any<br />
new 5D micro-satellites produced<br />
will also be screened. It is hoped<br />
Qsn (QTL for spikelet number)<br />
Qph (QTL for plant height)<br />
Figure 2. Relative position <strong>of</strong> the Stagnospora nodorum resistance gene Srb3 <strong>and</strong> previously mapped markers.
that this continuing work will help<br />
to complete the map more<br />
successfully.<br />
Mapping, field trials, <strong>and</strong><br />
disease screening are now in<br />
progress on the SCR populations<br />
for chromosomes 2A, 3D, <strong>and</strong> 7D.<br />
Our results have shown that S.<br />
nodorum resistance in wheat is<br />
inherited in a complex manner<br />
involving several genes. By<br />
studying them in single<br />
chromosome recombinant lines, we<br />
should be more able to fully<br />
underst<strong>and</strong> their heritability as<br />
well as highlighting linked<br />
markers.<br />
Using Precise Genetic Stocks to Investigate the Control <strong>of</strong> <strong>Stagonospora</strong> nodorum Resistance in Wheat 153<br />
References<br />
Lincoln, S.E., Daly, M., <strong>and</strong> L<strong>and</strong>er,<br />
E.S. 1992. Constructing genetic<br />
maps with MAPMAKER/EXP 3.0.<br />
Whitehead Institute Technical<br />
Report (Second edition).<br />
Lui, C.J., <strong>and</strong> Gale, M.D. 1989. Ibf-1, a<br />
highly variable marker system in<br />
Triticeae. TAG 77:233-240.<br />
McFadden, E.S., <strong>and</strong> Sears, E.R. 1946.<br />
The origin <strong>of</strong> Triticum spelta <strong>and</strong> its<br />
free-threshing hexaploid relatives.<br />
J. Heredity 37:81-89 <strong>and</strong> 107-116.<br />
Nicholson, P., Rezanoor, H.N., <strong>and</strong><br />
Worl<strong>and</strong>, A.J. 1993. Chromosomal<br />
location <strong>of</strong> resistance to <strong>Septoria</strong><br />
nodorum in a synthetic hexaploid<br />
wheat determined by the study <strong>of</strong><br />
chromosomal substitution lines in<br />
‘Chinese Spring’ wheat. Plant<br />
Breeding 110:177-184.<br />
Scott, P.R., <strong>and</strong> Benedikz, P.W. 1977.<br />
<strong>Septoria</strong> Plant Breeding Institute<br />
Annual Report 128-129.<br />
Sears, E.R. 1954. The aneuploids <strong>of</strong><br />
common wheat. Missouri<br />
Agricultural Experimental Station<br />
Research Bulletin 572, pp. 1-58.<br />
Sears, E.R. 1976. A synthetic<br />
hexaploid wheat with fragile<br />
rachis. Wheat Information Service<br />
41-42, 31-32.<br />
Southern, E.M. 1975. Detection <strong>of</strong><br />
specific sequences among DNA<br />
fragments separated by gel<br />
electrophoresis. J. Mol. Biol.<br />
98:503-517.
154<br />
Evaluating Triticum durum x Triticum tauschii Germplasm<br />
for Resistance to <strong>Stagonospora</strong> nodorum<br />
L.R. Nelson 1 <strong>and</strong> M.E. Sorrells 2 (Poster)<br />
1 Agricultural Research & Extension Center, Texas A&M University, Overton, TX, USA<br />
2 Dept. <strong>of</strong> Plant Breeding, Cornell University, Ithaca, NY, USA<br />
Abstract<br />
This study was conducted to determine resistance <strong>of</strong> 50 <strong>CIMMYT</strong> Triticum durum x Triticum tauschii populations to<br />
septoria glume blotch. Seedlings were inoculated at the 2-leaf stage, <strong>and</strong> data were recorded on disease severity <strong>and</strong> latent<br />
period. Several lines from this population demonstrated good resistance compared to check entries <strong>and</strong> are being used as<br />
sources <strong>of</strong> resistance to septoria glume blotch in the wheat breeding program.<br />
<strong>Septoria</strong> glume blotch caused<br />
by <strong>Stagonospora</strong> nodorum is a<br />
significant disease <strong>of</strong> wheat in the<br />
southern <strong>and</strong> eastern USA.<br />
Immunity <strong>of</strong> wheat to S. nodorum<br />
has not been found (Dantuma,<br />
1955), but variation in resistance is<br />
apparent. New sources <strong>of</strong> genetic<br />
resistance are needed in order to<br />
reduce yield losses cause by this<br />
fungal disease. In our research we<br />
have studied components <strong>of</strong> partial<br />
resistance (Nelson <strong>and</strong> Marshall,<br />
1990). A relatively simple test to<br />
evaluate resistance is based on<br />
measuring incubation period <strong>and</strong><br />
disease severity at a different<br />
length(s) <strong>of</strong> time after inoculation.<br />
In this study, we utilized these two<br />
components to evaluate resistance<br />
<strong>of</strong> wheat germplasm to septoria<br />
glume blotch. The objective <strong>of</strong> this<br />
study was to screen a collection <strong>of</strong><br />
50 exotic <strong>CIMMYT</strong> germplasm<br />
accessions for resistance to septoria<br />
glume blotch, <strong>and</strong> compare any<br />
resistance found with that <strong>of</strong><br />
moderately resistant checks to<br />
determine the potential <strong>of</strong> exotic<br />
germplasm.<br />
Materials <strong>and</strong> Methods<br />
Seed <strong>of</strong> 50 <strong>CIMMYT</strong><br />
populations <strong>and</strong> a susceptible<br />
(TAM 107) <strong>and</strong> a resistant (TX76-<br />
40-2) check (Nelson et al., 1994)<br />
were planted in rows in soil in flats.<br />
The <strong>CIMMYT</strong> germplasm consisted<br />
<strong>of</strong> 50 amphiploids from crosses<br />
between Triticum durum <strong>and</strong><br />
Triticum tauschii obtained from Dr.<br />
A. Mujeeb Kazi, <strong>CIMMYT</strong>, via Dr.<br />
Mark Sorrells at Cornell University.<br />
The wheat was germinated <strong>and</strong><br />
grown in the greenhouse until the<br />
2-leaf stage. At that time spores <strong>of</strong><br />
S. nodorum were sprayed on the<br />
wheat, which was then placed in a<br />
humidity chamber. The plants were<br />
removed from the humidity<br />
chamber after 50 hours. During this<br />
50 hour period, a cool mist blower<br />
operated for 15 minutes every 2<br />
hours during the day, <strong>and</strong> for 15<br />
minutes every 4 hours during the<br />
night, which kept the leaves moist,<br />
but did not cause run-<strong>of</strong>f. After the<br />
50-hour period, the flats were<br />
removed from the chamber <strong>and</strong><br />
allowed to continue growing in the<br />
greenhouse. Five days after being<br />
removed from the humidity<br />
chamber, some plants began to<br />
show leaf necrosis. Disease ratings<br />
were recorded for each row, or<br />
entry, on a 0 to 9 disease rating (0 =<br />
no symptoms; 9 = severe necrosis)<br />
on day 5, 8, <strong>and</strong> 11, to obtain an<br />
estimate <strong>of</strong> incubation period <strong>and</strong><br />
disease severity. A mean rating<br />
over the above three days was also<br />
calculated.<br />
Results<br />
Data are presented in Table 1<br />
for each day’s rating <strong>and</strong> the mean<br />
disease rating. The susceptible <strong>and</strong><br />
resistant checks provided a<br />
benchmark or st<strong>and</strong>ard upon<br />
which to compare these lines. Any<br />
lines that have disease ratings<br />
similar or less than the resistant<br />
check (TX76-40-2) may be useful in<br />
a wheat-breeding program <strong>and</strong><br />
could provide new resistance. Any<br />
lines similar to the susceptible<br />
check TAM 107 will not be useful<br />
for increasing resistance to septoria<br />
glume blotch. TAM 107 was<br />
already highly diseased by day 5,<br />
indicating a very short incubation<br />
period. Some lines had low disease<br />
ratings on day 5, but by day 11, had<br />
high disease ratings, similar to<br />
TAM 107. This indicates a longer<br />
incubation period, <strong>and</strong> this<br />
resistance may be useful in a<br />
breeding program to delay the<br />
buildup <strong>of</strong> the disease.
Table 1. Disease rating <strong>of</strong> <strong>CIMMYT</strong> durum x T.<br />
tauschii wheat lines for septoria glume blotch on<br />
a 0 to 9 scale where 0 = no disease <strong>and</strong> 9 = very<br />
high disease rating.<br />
Rating Rating Rating Mean<br />
Entry number day 5 day 8 day 11 rating<br />
TAM 107 Susc. Ck 7 7 7 7<br />
Syn-1 Altar 21 3 7 7 6<br />
Syn-2 Altar 219 1 4 6 4<br />
Syn-3 Altar 219 4 6 6 5<br />
Syn-4 Altar 219 7 6 6 6<br />
Syn-5 Altar 224 7 7 7 7<br />
Tx76-40-2 Res. Ck 0 1 1 1<br />
Syn-6 Altar 224 4 3 4 4<br />
Syn-7 Altar 224 3 3 6 4<br />
Syn-8 Altar 221 3 4 6 4<br />
Syn-9 Altar 223 1 2 3 2<br />
Syn-10 Altar 223 2 3 4 3<br />
Syn-11 Altar 192 1 3 4 3<br />
Syn-12 Altar 198 1 3 2 2<br />
Syn-13 Altar 198 0 0 1 1<br />
Syn-14 Altar 198 1 2 2 2<br />
Syn-15 Altar 211 1 3 2 2<br />
Syn-16 Chen 205 7 6 7 7<br />
Syn-17 Chen 205 8 8 8 8<br />
Syn-18 Chen 205 6 7 7 7<br />
Syn-19 Chen 205 5 5 5 5<br />
Syn-20 Chen 205 3 3 5 4<br />
Syn-21 Chen 205 2 3 5 3<br />
Syn-22 Chen 205 5 5 7 6<br />
Syn-23 Chen 205 4 4 6 5<br />
Syn-24 Chen 205 3 3 3 3<br />
Syn-25 Chen 205 5 5 5 5<br />
Syn-26 Chen 215 3 3 3 3<br />
Syn-27 Chen 215 0 2 2 1<br />
Syn-28 Chen 224 7 6 6 6<br />
Syn-29 Chen 224 7 7 4 6<br />
Syn-30 Chen 224 7 7 4 6<br />
Syn-31 Chen 224 4 5 5 5<br />
Syn-32 Chen 224 3 3 4 3<br />
Syn-33 Chen 224 5 5 5 5<br />
Syn-34 Chen 224 3 5 5 4<br />
Syn-35 Chen 224 6 6 6 6<br />
Syn-36 Chen 224 6 6 4 5<br />
Syn-37 Chen 224 7 7 7 7<br />
Syn-38 Chen 224 5 6 6 6<br />
Syn-39 Cndo/R143//<br />
Ent “S”/Mex 214 7 6 6 6<br />
Syn-40 “ “ 5 6 5 5<br />
Syn 41 “ “ 3 5 5 4<br />
Syn-43 Cndo/R143//<br />
Ent”S”/Mex 221 5 5 5 5<br />
Syn-44 Cndo/R143//<br />
Ent”S”/Mex 221 5 4 4 4<br />
Syn-45 “ “ 4 7 7 6<br />
Syn-45 “ “ 3 4 5 4<br />
Syn-46 “ “ 6 7 6 6<br />
Syn-45 Laru’s 309 3 5 5 4<br />
Syn-48 5 6 6 6<br />
Syn-49 “ “ 8 7 8 8<br />
Syn-50 “ “ 2 3 3 3<br />
Evaluating Triticum durum x Triticum tauschii Germplasm for Resistance to <strong>Stagonospora</strong> nodorum 155<br />
Highly resistant lines were<br />
Syn 9, Syn 12, Syn 13, Syn 14,<br />
Syn 15, <strong>and</strong> Syn 27. Their<br />
resistance was similar to TX76-<br />
40-2. Moderately resistant lines<br />
were Syn 10, Syn 11, Syn 32, <strong>and</strong><br />
Syn 50. Several other lines also<br />
demonstrated a moderate<br />
degree <strong>of</strong> partial resistance.<br />
Lines Syn 1 through 15 may<br />
have a similar genetic<br />
background for the A <strong>and</strong> B<br />
genomes because they all have<br />
Altar as the durum parent. Of<br />
the resistant lines, the T. tauschii<br />
parents were 223 for Syn 9 <strong>and</strong><br />
10, 192 for Syn 11, 198 for Syn<br />
12-14 <strong>and</strong> 211 for Syn 15. ‘Chen’<br />
<strong>and</strong> ‘Laru’ “S” were the durum<br />
parents for Syn 27 <strong>and</strong> 50 <strong>and</strong><br />
the T. tauschii lines were 215 <strong>and</strong><br />
309, respectively. It appears that<br />
at least some <strong>of</strong> the resistance in<br />
this germplasm originated in the<br />
T. tauschii lines used to make the<br />
amphiploids.<br />
Discussion<br />
It appears that we have<br />
several promising new sources<br />
<strong>of</strong> resistance in a wheat<br />
background that can be crossed<br />
with our adapted winter wheat.<br />
We are presently increasing seed<br />
<strong>of</strong> these 50 lines in our<br />
greenhouse. We have crossed<br />
several <strong>of</strong> the best lines for<br />
resistance to septoria glume blotch<br />
with both hard <strong>and</strong> s<strong>of</strong>t red winter<br />
wheat in our crossing program<br />
during 1998/99. Since this<br />
germplasm contains sources <strong>of</strong><br />
resistance to septoria glume blotch<br />
which are likely not present in our<br />
s<strong>of</strong>t red winter wheat, there may be<br />
an opportunity to pyramid genes<br />
<strong>and</strong> make significant improvement<br />
in resistance to this disease.<br />
Acknowledgment<br />
Appreciation is expressed to<br />
<strong>CIMMYT</strong> for developing <strong>and</strong><br />
sharing the germplasm.<br />
References<br />
Dantuma, G. 1955. The heavy attack<br />
<strong>of</strong> diseases during the ripening <strong>of</strong><br />
wheat in 1954. Euphytica 5:94-95.<br />
Nelson, L.R., R.D. Barnett, D.<br />
Marshall, C.A. Erickson, M.E.<br />
McDaniel, W.D. Worrall, N.A.<br />
Tuleen, <strong>and</strong> M.D. Lazar. 1994.<br />
Registration <strong>of</strong> TX76-40-2 wheat<br />
germplasm. Crop Sci. 34:1137.<br />
Nelson, L.R., <strong>and</strong> D. Marshall. 1990.<br />
Breeding wheat for resistance to<br />
<strong>Septoria</strong> nodorum <strong>and</strong> <strong>Septoria</strong><br />
tritici. Advances in Agronomy<br />
44:257-277.
156<br />
Sources <strong>of</strong> Resistance to <strong>Septoria</strong> passerinii in Hordeum<br />
vulgare <strong>and</strong> H. vulgare subsp. spontaneum<br />
H. Toubia-Rahme <strong>and</strong> B.J. Steffenson (Poster)<br />
North Dakota State University, Department <strong>of</strong> Plant Pathology, Fargo, ND, USA<br />
Abstract<br />
<strong>Septoria</strong> speckled leaf blotch (SSLB), incited by <strong>Septoria</strong> passerinii <strong>and</strong> <strong>Stagonospora</strong> avenae f. sp. triticea, has<br />
become one <strong>of</strong> the most serious diseases <strong>of</strong> barley in the Upper Midwest region <strong>of</strong> the USA. In barley SSLB can cause<br />
significant losses in both the yield <strong>and</strong> quality. The major malting <strong>and</strong> feed barley cultivars in the Upper Midwest are very<br />
susceptible to SSLB. Resistance breeding is the most effective strategy for controlling this disease. A diverse group <strong>of</strong> barley<br />
germplasm including Hordeum vulgare subsp. spontaneum accessions, advanced midwestern breeding lines, <strong>and</strong><br />
commercial cultivars was evaluated at the seedling stage in the greenhouse for reaction to S. passerinii. Of 200 accessions<br />
tested, 79 were found resistant. Most <strong>of</strong> H. vulgare subsp. spontaneum accessions (17 <strong>of</strong> 24) were resistant to S. passerinii.<br />
These accessions all originated from the Middle East, except one that was from Tibet. Most <strong>of</strong> the advanced midwestern<br />
breeding lines found resistant to this pathogen have Gloria”S”/Copal”S” (an ICARDA/<strong>CIMMYT</strong> barley line) in their<br />
pedigree, which is believed to be a source <strong>of</strong> resistance to S. passerinii. From this study, it is evident that many barley<br />
accessions possess resistance to S. passerinii. Additional evaluations will be made on this germplasm to S. avenae f. sp.<br />
triticea to identify accessions that possess effective resistance to both SSLB pathogens.<br />
<strong>Septoria</strong> speckled leaf blotch<br />
(SSLB), caused by <strong>Septoria</strong> passerinii<br />
<strong>and</strong> <strong>Stagonospora</strong> avenae f. sp.<br />
triticea, has become an increasingly<br />
important disease <strong>of</strong> barley<br />
(Hordeum vulgare L.) over the past<br />
decade in the Upper Midwest<br />
region <strong>of</strong> the USA. In surveys <strong>of</strong><br />
North Dakota barley fields in 1998,<br />
S. passerinii <strong>and</strong> S. avenae f. sp.<br />
triticea were recovered from 45%<br />
<strong>and</strong> 37% <strong>of</strong> leaves exhibiting leaf<br />
spot symptoms, respectively<br />
(Krupinsky <strong>and</strong> Steffenson, this<br />
volume). Yield losses <strong>of</strong> 20% have<br />
been recorded in barley due to S.<br />
passerinii infection (Green <strong>and</strong><br />
Bendelow, 1961). In recent trials<br />
conducted in North Dakota, yield<br />
losses <strong>of</strong> 23% to 38% were<br />
observed in Robust barley infected<br />
with SSLB (J. Lukach <strong>and</strong> B.<br />
Steffenson, unpublished data). In<br />
addition to reducing yield, SSLB<br />
also reduces kernel plumpness <strong>and</strong><br />
malt extract, which are important<br />
malt quality characters (Green <strong>and</strong><br />
Bendelow, 1961).<br />
Host plant resistance provides<br />
the most practical <strong>and</strong><br />
environmentally safe method <strong>of</strong><br />
disease control. Unfortunately, all<br />
major malting <strong>and</strong> feed barley<br />
cultivars in the Upper Midwest<br />
region are susceptible to SSLB. The<br />
objectives <strong>of</strong> this study were to 1)<br />
identify sources <strong>of</strong> resistance to S.<br />
passerinii in commercial barley<br />
cultivars, agronomically advanced<br />
midwestern breeding lines, <strong>and</strong><br />
Hordeum vulgare subsp. spontaneum<br />
accessions; <strong>and</strong> 2) evaluate<br />
previously reported sources <strong>of</strong> S.<br />
passerinii resistance to a<br />
midwestern isolate <strong>of</strong> this<br />
pathogen.<br />
Materials <strong>and</strong> Methods<br />
In total, 200 barley entries<br />
including 24 H. vulgare subsp.<br />
spontaneum accessions were<br />
evaluated at the seedling stage in<br />
the greenhouse. Five seeds <strong>of</strong> each<br />
entry were planted in pots (10 x 10<br />
cm) filled with a potting mix<br />
consisting <strong>of</strong> peat moss (75%) <strong>and</strong><br />
perlite (25%). Slow-release (14-14-<br />
14, N-P-K, 2 g/pot) <strong>and</strong> watersoluble<br />
(15-0-15, N-P-K, 2 g/pot)<br />
fertilizers were added at the time <strong>of</strong><br />
planting. All seedlings were grown<br />
in the greenhouse at 20±3ºC with a<br />
14-h photoperiod. The fungal<br />
isolate (ND97-15) used in this<br />
study was obtained from naturally<br />
infected barley leaves collected<br />
from a commercial field in<br />
Bottineau county, North Dakota, in<br />
1997. For inoculum production, the<br />
isolate was grown on yeast malt<br />
agar (YMA) (Eyal et al., 1987) in<br />
plastic Petri dishes at 21ºC with a<br />
12-h photoperiod (cool-white<br />
fluorescent tubes). When pycnidia<br />
developed <strong>and</strong> sporulated, mass<br />
spore transfers were made by<br />
removing with a sterile needle<br />
cirrhi from pycnidia <strong>and</strong><br />
transferring them to other YMA<br />
plates. After 4-5 days <strong>of</strong> incubation<br />
under the same conditions,<br />
pycnidia were harvested by<br />
flooding the surface <strong>of</strong> the plates
with double-distilled water <strong>and</strong><br />
scraping the agar surface with a<br />
rubber spatula. This suspension <strong>of</strong><br />
pycnidia was blended for 30 s in a<br />
blender to release pycnidiospores<br />
<strong>and</strong> then filtered through four<br />
layers <strong>of</strong> cheesecloth. Tween-20<br />
(polyoxyethylene-20-sorbitan<br />
monolaurate) was added to the<br />
pycnidiospore suspension at a rate<br />
<strong>of</strong> 100 µl/l to facilitate the uniform<br />
distribution <strong>and</strong> adsorption <strong>of</strong><br />
inoculum onto the leaf surfaces.<br />
Barley seedlings were<br />
inoculated with the pycnidiospore<br />
suspension (5 x 10 5<br />
pycnidiospores/ml) at the two-leaf<br />
stage (10-12 days old). Inoculated<br />
seedlings were incubated at 21ºC/<br />
dark <strong>and</strong> 25ºC/light for 72 h in<br />
mist chambers, where the relative<br />
humidity was maintained near<br />
100%. The first 40 h <strong>of</strong> incubation<br />
was in darkness, followed by a<br />
photoperiod <strong>of</strong> 5 h for next two<br />
days. Plants were allowed to dry<br />
<strong>of</strong>f slowly before being transferred<br />
to the greenhouse under the same<br />
conditions previously described.<br />
The reaction <strong>of</strong> the entries to S.<br />
passerinii was assessed on the<br />
second leaves <strong>of</strong> seedlings 17 days<br />
after inoculation using a 0-5 rating<br />
scale where 0, 1, <strong>and</strong> 2 are<br />
indicative <strong>of</strong> resistance <strong>and</strong> 3, 4,<br />
<strong>and</strong> 5 <strong>of</strong> susceptibility. Resistant<br />
(cv. Atlas) <strong>and</strong> susceptible (cv.<br />
Betzes) checks were included in the<br />
experiment.<br />
Results <strong>and</strong> Discussion<br />
Hordeum vulgare <strong>and</strong> H. vulgare<br />
subsp. spontaneum accessions were<br />
classified as susceptible or resistant<br />
based on their reaction to S.<br />
passerinii at the seedling stage.<br />
Sources <strong>of</strong> Resistance to <strong>Septoria</strong> passerinii in Hordeum vulgare <strong>and</strong> H. vulgare subsp. spontaneum 157<br />
Marked differences were observed<br />
in the reaction <strong>of</strong> barley accessions<br />
to S. passerinii infection. In total, 79<br />
lines were found resistant to S.<br />
passerinii. Of the 24 H. vulgare<br />
subsp. spontaneum accessions<br />
tested, 17 were resistant. These<br />
accessions all originated from the<br />
Middle East, except one, which was<br />
from Tibet. Similar results were<br />
obtained by Metcalfe et al. (1977)<br />
who found that all H. vulgare<br />
subsp. spontaneum accessions<br />
collected in the Middle East were<br />
resistant to S. passerinii.<br />
All <strong>of</strong> the major 6-rowed<br />
malting (Foster, St<strong>and</strong>er, <strong>and</strong><br />
Robust), <strong>and</strong> 2-rowed feed<br />
(Bowman, Conlon, <strong>and</strong> Logan)<br />
cultivars grown in the Upper<br />
Midwest region were susceptible.<br />
Of 120 advanced midwestern 6<strong>and</strong><br />
2-rowed breeding lines, 41<br />
were resistant. Most <strong>of</strong> these<br />
breeding lines have Gloria”S”/<br />
Copal”S” (an ICARDA/<strong>CIMMYT</strong><br />
barley line) in their pedigree. This<br />
line exhibited S. passerinii resistance<br />
under field conditions in North<br />
Dakota <strong>and</strong> is presumed to be the<br />
source <strong>of</strong> resistance to S. passerinii<br />
in these breeding lines (J.<br />
Franckowiak, personal<br />
communication).<br />
Nine accessions (AC Hamilton,<br />
Atlas, Bolron, CIho 4439, CIho<br />
4780, CIho 10644, Feebar, Nomini,<br />
<strong>and</strong> Starling) previously reported<br />
to have resistance to S. passerinii<br />
were also resistant to the North<br />
Dakota isolate (ND97-15) used in<br />
this study. Other barley accessions<br />
resistant to this isolate were: Atlas<br />
54, Baronesse, Belford, CIho 4428,<br />
CIho 4940, Flynn 1, Glacier, <strong>and</strong><br />
Vaughn. Only a few studies have<br />
been advanced on the genetics <strong>of</strong><br />
resistance in barley to S. passerinii.<br />
Peterson (1956) indicated that the<br />
variety Atlas possesses dominant<br />
resistance genes to S. passerinii.<br />
Buchannon (1961) found two<br />
recessive genes conferring<br />
resistance to S. passerinii in the<br />
cultivar Feebar, <strong>and</strong> Rasmusson<br />
<strong>and</strong> Rogers (1963) reported two<br />
different dominant resistant genes,<br />
Sep2 <strong>and</strong> Sep3, in the accessions<br />
CIho 4780 <strong>and</strong> CIho 10644,<br />
respectively. Metcalfe et al. (1970)<br />
found that a single dominant gene<br />
governs resistance to S. passerinii in<br />
CIho 4439. It is evident that many<br />
barley accessions possess resistance<br />
to this pathogen; however, none<br />
has been exploited in midwest<br />
barley breeding programs, as all <strong>of</strong><br />
the major malting <strong>and</strong> feed<br />
cultivars are susceptible to S.<br />
passerinii.<br />
Segregating populations<br />
derived from some <strong>of</strong> the described<br />
sources <strong>of</strong> resistance <strong>and</strong><br />
susceptible commercial cultivars<br />
are under evaluation in our<br />
laboratory to study the genetics <strong>of</strong><br />
resistance in barley to S. passerinii<br />
<strong>and</strong> to identify molecular markers<br />
linked to S. passerinii resistance<br />
gene(s). All accessions found<br />
resistant to S. passerinii in this study<br />
will also be evaluated to S. avenae f.<br />
sp. triticea. This test will enable us<br />
to identify sources <strong>of</strong> resistance to<br />
both S. passerinii <strong>and</strong> S. avenae f. sp.<br />
triticea <strong>and</strong> to determine whether<br />
resistance to both pathogens is<br />
governed by the same or by<br />
different gene(s). The development<br />
<strong>of</strong> barley cultivars with resistance<br />
to both S. passerinii <strong>and</strong> S. avenae f.<br />
sp. triticea is necessary, as both<br />
pathogens are common in the<br />
Upper Midwest production area.
Session 6C — H. Toubia-Rahme <strong>and</strong> B.J. Steffenson<br />
158<br />
References<br />
Buchannon, K.W. 1961. Inheritance <strong>of</strong><br />
reaction to <strong>Septoria</strong> passerinii Sacc.<br />
<strong>and</strong> Pyrenophora teres (Died.)<br />
Drechsl., <strong>and</strong> <strong>of</strong> row number, in<br />
barley. Ph.D. diss. University <strong>of</strong><br />
Saskatchewan, Saskatoon. 40 pp.<br />
Eyal, Z., Scharen, A.L., Prescott, J.M.,<br />
<strong>and</strong> M. van Ginkel. 1987. The<br />
<strong>Septoria</strong> <strong>Diseases</strong> <strong>of</strong> Wheat:<br />
Concepts <strong>and</strong> Methods <strong>of</strong> Disease<br />
Management. Mexico, D.F.:<br />
<strong>CIMMYT</strong>. 46 pp.<br />
Green, G.J., <strong>and</strong> V.M. Bendelow. 1961.<br />
Effect <strong>of</strong> speckled leaf blotch,<br />
<strong>Septoria</strong> passerinii Sacc., on the<br />
yield <strong>and</strong> malting quality <strong>of</strong> barley.<br />
Can. J. Plant Sci 41:431-435.<br />
Metcalfe, D.R., Buchannon, K.W.,<br />
McDonald, W.C., <strong>and</strong> E. Reinbergs.<br />
1970. Relationships between the<br />
‘Jet’ <strong>and</strong> ‘Milton’ genes for<br />
resistance to loose smut <strong>and</strong> genes<br />
for resistance to other barley<br />
diseases. Can. J. Plant Sci 50:423-<br />
427.<br />
Metcalfe, D.R., Chiko, A.W., Martens,<br />
J.W., <strong>and</strong> A. Tekauz. 1977. Reaction<br />
<strong>of</strong> barleys from the Middle East to<br />
Canadian pathogens. Can. J. Plant<br />
Sci 57: 995-999.<br />
Peterson, R.F. 1956. Progress report,<br />
Cereal Breeding Laboratory,<br />
Winnipeg, Manitoba, 1949-54. 36<br />
pp.<br />
Rasmusson, D.C., <strong>and</strong> W.E. Rogers.<br />
1963. Inheritance <strong>of</strong> resistance to<br />
<strong>Septoria</strong> in barley. Crop Sci 3:161-<br />
162.
Partial Resistance to <strong>Stagonospora</strong> nodorum in Wheat 159<br />
S<strong>of</strong>t Red Winter Wheat with Resistance to <strong>Stagonospora</strong><br />
nodorum <strong>and</strong> Other Foliar Pathogens<br />
B.M. Cunfer1 <strong>and</strong> J.W. Johnson2 (Poster)<br />
1 Department <strong>of</strong> Plant Pathology, <strong>and</strong> 2 Department <strong>of</strong> Crops <strong>and</strong> Soils, Griffin Campus, University <strong>of</strong> Georgia,<br />
Griffin, GA, USA<br />
S<strong>of</strong>t red winter wheat<br />
germplasm adapted to the<br />
southeastern USA that has a high<br />
level <strong>of</strong> resistance to <strong>Stagonospora</strong><br />
nodorum has been difficult to<br />
identify. Some partially resistant<br />
cultivars with long-lasting<br />
resistance have been developed,<br />
but these are replaced frequently<br />
due to loss <strong>of</strong> resistance to<br />
powdery mildew (Blumeria<br />
graminis), leaf rust (Puccinia<br />
recondita), <strong>and</strong> Hessian fly<br />
(Mayetiola destructor). Four lines,<br />
GA 84202, GA 85240, GA 85410AB,<br />
<strong>and</strong> GA 861460, with good<br />
agronomic traits have been selected<br />
<strong>and</strong> included among elite lines in<br />
the Georgia wheat breeding<br />
program in the past eight years.<br />
These lines have resistance to S.<br />
nodorum which is equal or better<br />
than that <strong>of</strong> older cultivars such as<br />
Oasis. They are also resistant to<br />
current populations <strong>of</strong> leaf rust<br />
present in the southeastern USA,<br />
most races <strong>of</strong> powdery mildew, <strong>and</strong><br />
Hessian fly.<br />
The lines selected were<br />
evaluated in the greenhouse <strong>and</strong><br />
field over a six-year period against<br />
100 or more advanced <strong>and</strong> elite<br />
lines <strong>and</strong> st<strong>and</strong>ard check cultivars<br />
each year. Seedling plants were<br />
inoculated in the greenhouse each<br />
year. Adult plants were inoculated<br />
in the field with S. nodorum <strong>and</strong><br />
exposed to natural infection. Data<br />
were collected in field trials at<br />
Griffin <strong>and</strong> Plains, GA, under<br />
moderate to severe disease<br />
pressure from powdery mildew,<br />
leaf rust, <strong>and</strong> leaf <strong>and</strong> glume<br />
blotch. Results from the<br />
greenhouse <strong>and</strong> field were<br />
generally in good agreement.<br />
Seeds <strong>of</strong> each line have been<br />
deposited in the USDA Small<br />
Grains Collection, Aberdeen, ID.<br />
These lines may be useful in other<br />
regions where they are adapted.<br />
For example, in addition to being<br />
agronomically adapted, 85410AB<br />
<strong>and</strong> 84202 were found to be<br />
resistant to stripe rust (P. striiformis)<br />
<strong>and</strong> leaf rust in a field trial at<br />
Colonia, Uruguay, in 1995. These<br />
lines are facultative wheats<br />
adapted to winter wheat culture in<br />
regions with a mild to moderate<br />
winter climate. They have been<br />
used as parents in several<br />
advanced <strong>and</strong> elite breeding lines<br />
currently being evaluated in the<br />
Georgia breeding program.
160<br />
Partial Resistance to <strong>Stagonospora</strong> nodorum in Wheat<br />
C.G. Du, 1 L.R. Nelson, 2 <strong>and</strong> M.E. McDaniel 3 (Poster)<br />
1 Dept. <strong>of</strong> Computer Science, Texas A&M Univ., College Station, TX. USA<br />
2 Texas A&M Univ. Agri. Res. & Ext. Center, Overton, TX, USA<br />
3 Soil <strong>and</strong> Crop Sciences Dept., Texas A&M Univ., College Station, TX, USA<br />
Abstract<br />
Seedling plants <strong>of</strong> parents, F 1 crosses, <strong>and</strong> F 2 populations were inoculated at the two-leaf stage with spores <strong>of</strong><br />
<strong>Stagonospora</strong> nodorum in a humidity chamber to determine incubation period (IP), latent period (LP) <strong>and</strong> necrosis<br />
percentage (NP). Incubation period, LP, <strong>and</strong> NP exhibited polygenic inheritance <strong>and</strong> were controlled by 2-3, 3, <strong>and</strong> 1-4 genes,<br />
respectively. Each <strong>of</strong> the components <strong>of</strong> partial resistance showed moderate to high heritabilities.<br />
Breeding wheat for resistance to<br />
<strong>Stagonospora</strong> nodorum, common<br />
name septoria glume blotch (SGB),<br />
has been difficult because no genes<br />
for immunity exist. There are<br />
numerous sources <strong>of</strong> genetic<br />
resistance; however, most have<br />
been labeled as partial resistance<br />
<strong>and</strong> in some manner delay the<br />
growth <strong>of</strong> the pathogen. In this<br />
study our objectives were to<br />
conduct a quantitative genetic<br />
analysis <strong>of</strong> three components <strong>of</strong><br />
resistance to SGB in wheat. Second,<br />
to compare 10 wheat parents, 21 F 1 ,<br />
<strong>and</strong> 21 F 2 crosses for foliar disease<br />
reaction induced by inoculation,<br />
<strong>and</strong> third, to investigate the<br />
heritability estimates <strong>and</strong> gene<br />
numbers governing SGB resistance.<br />
Materials <strong>and</strong> Methods<br />
Six s<strong>of</strong>t winter wheat <strong>and</strong> four<br />
hard wheat genotypes that varied<br />
greatly in resistance to SGB were<br />
used in two incomplete diallel<br />
crosses. S<strong>of</strong>t wheat lines were<br />
TX92D7374, TX82-11, L890682,<br />
18NT, ‘Coker 9803’, <strong>and</strong> ‘Coker<br />
9543’. Hard wheat lines were<br />
TX91V3308, TX84V344, ‘TAM 300’,<br />
<strong>and</strong> SWM14240. SWM14240 is a<br />
wheat line selected in the<br />
northwestern US from <strong>CIMMYT</strong><br />
germplasm <strong>and</strong> therefore is likely<br />
to have different genes for SGB<br />
resistance. Seeds were germinated<br />
on moist filter paper for two days<br />
<strong>and</strong> then transplanted into pots<br />
containing a soil/peat moss<br />
mixture. Two plants in each pot<br />
were treated as two samples <strong>of</strong> the<br />
particular hybrid or parent. Plants<br />
were inoculated <strong>and</strong> placed in a<br />
humidity chamber for 50 h, as<br />
previously described (Nelson,<br />
1980). Hayman’s approach (1954)<br />
was used to calculate components<br />
<strong>of</strong> genetic variations.<br />
Results<br />
Estimation <strong>of</strong> components<br />
<strong>of</strong> variation<br />
Hard winter wheat: Incubation<br />
period. Parents with the highest<br />
frequency <strong>of</strong> dominant alleles have<br />
the smallest Vr (variance) <strong>and</strong> Wr<br />
(parent-<strong>of</strong>fspring covariance); thus<br />
their relative position along the Vr/<br />
Wr regression line reflects the<br />
frequency <strong>of</strong> dominant alleles.<br />
Sorting the parents used in these<br />
crosses in order <strong>of</strong> decreasing<br />
dominance gave the follow<br />
sequence: TX91V3308, TAM 300,<br />
TX84V344, <strong>and</strong> SWM14240.<br />
SWM14240 had least dominant<br />
effect (Figure 1). The fixable<br />
variation D <strong>and</strong> the dominance<br />
component H were calculated<br />
(Table 1). Narrow sense heritability<br />
was 66% <strong>and</strong> 61% in F 1 in F 2<br />
generations, respectively. Gene<br />
number governing the incubation<br />
period was 2-3 (Table 1).<br />
Latent period. The analysis <strong>of</strong> Wr,<br />
Vr, <strong>and</strong> Wr/Vr graphical statistics<br />
in both F 1 <strong>and</strong> F 2 provided detail<br />
information on the interrelation<br />
between the parents. The order <strong>of</strong><br />
decreasing dominance was<br />
TX84V344, TAM 300, TX91V3308,<br />
<strong>and</strong> SWM14240 for F 1 (Figure 1);<br />
TAM 300, TX91V3308, TX84V344,<br />
<strong>and</strong> SWM14240 for F 2 . Average<br />
degree <strong>of</strong> dominance was partial<br />
dominance because <strong>of</strong> the positive<br />
intercepts. SWM14240 had the least<br />
dominant effect. TAM 300 had the<br />
2.0<br />
(F1 LP) 4<br />
4<br />
(F2 LP)<br />
Wr<br />
2<br />
1<br />
4<br />
(F1 LP)<br />
0.5<br />
0<br />
3<br />
2<br />
1<br />
3<br />
1<br />
3<br />
1<br />
2<br />
3<br />
2<br />
4<br />
(F1 LP)<br />
0.5 Vr 2.0<br />
Figure 1. Parent-<strong>of</strong>fspring covariance (Wr)/<br />
variance (Vr) for SNB incubation period <strong>and</strong><br />
latent period <strong>of</strong> F1 <strong>and</strong> F2 hard winter wheat<br />
crosses.<br />
1 = TX91V3308 2 = TX84V344<br />
3 = TAM 300 4 = SWM14240
largest dominant effect. Narrow<br />
sense heritability was 64% <strong>and</strong> 68%<br />
in F 1 <strong>and</strong> F 2 generations,<br />
respectively (Table 1). There were<br />
about three genes governing the<br />
resistance <strong>of</strong> latent period. The<br />
additive effect D <strong>and</strong> dominance<br />
component H1 <strong>and</strong> H2 were also<br />
calculated. Additive D was 3.74 for<br />
the F 1 <strong>and</strong> 3.75 for the F 2<br />
generation.<br />
S<strong>of</strong>t winter wheat: Incubation<br />
period. The order <strong>of</strong> decreasing<br />
dominance was TX82-11,<br />
TX92D7374, 18NT, Coker 9803,<br />
Coker 9543, <strong>and</strong> L890682 for F 1 ,<br />
<strong>and</strong> TX82-11, 18NT, Coker 9803,<br />
Coker 9543, L890682, <strong>and</strong><br />
TX92D7374 for F 2 generations.<br />
Mean square <strong>of</strong> Wr-Vr was not<br />
significant. Dominance seems to<br />
account for the major proportion <strong>of</strong><br />
the non-additive variation. The<br />
additive variation D <strong>and</strong> the<br />
dominant component H were<br />
estimated in Table 2. The<br />
dominance ratio H1/D, an estimate<br />
<strong>of</strong> the average level <strong>of</strong> dominance,<br />
was 0.7 <strong>and</strong> 0.6 for F 1 <strong>and</strong> F 2<br />
generations, respectively. This<br />
indicates that resistance for<br />
incubation period was partially<br />
dominant. There were 2-3 genes<br />
controlling IP in the s<strong>of</strong>t wheat.<br />
Percent necrosis. The order <strong>of</strong><br />
decreasing dominance was TX82-<br />
11, 18NT, Coker 9803, TX92D7374,<br />
Coker 9543, <strong>and</strong> L890682.<br />
Dominance <strong>and</strong> non-allelic<br />
interaction was important in<br />
necrosis. H/D>1 indicates that<br />
necrosis had over-dominance<br />
effects (Table 2). Heritability<br />
estimated for necrosis was similar<br />
by different methods <strong>of</strong> calculation.<br />
Narrow sense heritability for<br />
necrosis was relatively low. Only<br />
one gene controlled % necrosis in<br />
s<strong>of</strong>t winter wheat. It was different<br />
from hard winter wheat <strong>and</strong> may<br />
have resulted from the genotype X<br />
environment interaction-over<br />
dominance effects.<br />
Discussion<br />
Nelson <strong>and</strong> Marshall (1990)<br />
stated that resistance that reduced<br />
the infection rate had polygenic<br />
inheritance. Jeger (1980) indicated<br />
that resistance to SGB may involve<br />
four independent polygenes. The<br />
results <strong>of</strong> this study agree with<br />
these previous studies. IP, LP <strong>and</strong><br />
PN had polygenic inheritance.<br />
Incubation period, LP, <strong>and</strong> NP were<br />
controlled by 2 to 3, 3, <strong>and</strong> 1 to 4<br />
genes in the hard <strong>and</strong> s<strong>of</strong>t wheat<br />
studies, respectively. Previous<br />
studies (Nelson 1980; Mullaney et<br />
al., 1982; Scott et al., 1982) also<br />
provided evidence for polygenic<br />
control <strong>of</strong> host reaction to S.<br />
nodorum both at the seedling plant<br />
stage <strong>and</strong> the mature plant stage.<br />
The most accepted hypothesis is<br />
that these polygenes are<br />
Table 1. Genetic variation <strong>of</strong> components <strong>of</strong> partial resistance for hard winter wheat crosses.<br />
Incubation period Latent period Necrosis<br />
Component F1 F2 F1 F2 F1 F2<br />
D 2.51 2.38 3.74 3.75 365.76 360.37<br />
H1 2.03 2.51 2.16 2.34 459.48 441.15<br />
H2 0.88 1.34 1.33 0.52 265.07 59.52<br />
F 2.40 1.87 3.10 3.24 475.17 350.18<br />
E 0.32 0.51 0.76 0.70 37.12 42.52<br />
H narrow 66% 61% 64% 68% 30% 77%<br />
Number <strong>of</strong> genes 3 2 3 3 4 3<br />
Partial Resistance to <strong>Stagonospora</strong> nodorum in Wheat 161<br />
independently inherited. Our<br />
research may indicate some <strong>of</strong> the<br />
polygenes may have multi-effects.<br />
For example, one <strong>of</strong> the latent<br />
period resistant genes may also be<br />
a resistant gene in incubation<br />
period polygenes.<br />
Analysis <strong>of</strong> components <strong>of</strong><br />
variation. The H1/D value <strong>of</strong> F 1<br />
hard wheat, <strong>and</strong> F 1 <strong>and</strong> F 2 s<strong>of</strong>t<br />
wheat were less than one with the<br />
exception <strong>of</strong> F 2 hard winter wheat<br />
(1.1). This means that additive gene<br />
action was <strong>of</strong> greater importance in<br />
the components in this study.<br />
Therefore, incubation period could<br />
be a very useful selection criterion<br />
for SGB resistant breeding. Both F 1<br />
<strong>and</strong> F 2 hard winter wheat crosses<br />
had larger additive value D than<br />
dominant value H1. It is likely that<br />
the non-additive genetic effects<br />
were due predominantly to<br />
contribution from additive x<br />
additive epistatic gene action rather<br />
than dominance.<br />
Additive gene action <strong>and</strong><br />
additive x additive epistatic gene<br />
action are most easily utilized <strong>and</strong><br />
exploited in homozygous<br />
genotypes. Latent period would<br />
also be a good selection criterion<br />
for SGB resistant breeding. H1/D<br />
values for hard <strong>and</strong> s<strong>of</strong>t wheat<br />
crosses were greater than 1. Over<br />
dominance may have been present<br />
for necrosis. Nelson (1980),<br />
Table 2. Genetic variation <strong>of</strong> components <strong>of</strong><br />
partial resistance for s<strong>of</strong>t winter wheat crosses.<br />
Component Incubation period Necrosis<br />
F1 F2 F1<br />
D 1.19 1.60 3.66<br />
H 1 0.83 0.92 148.27<br />
H 2 0.50 1.44 123.54<br />
F 0.55 1.24 -4.00<br />
E 0.91 0.72 26.99<br />
H narrow 32% 39% 22%<br />
Number <strong>of</strong> genes 2 3 1
Session 6C — C.G. Du, L.R. Nelson, <strong>and</strong> M.E. McDaniel<br />
162<br />
Wilkinson et al. (1990), <strong>and</strong><br />
Bostwick et al. (1993) had similar<br />
results. It is reasonable for the<br />
components <strong>of</strong> resistance to show<br />
dominant gene action in the F 1<br />
generation because <strong>of</strong> gene<br />
interaction. From the breeder’s<br />
viewpoint, necrosis should not be<br />
an early generation selection<br />
criterion for SGB resistant breeding.<br />
Heritability. Estimates <strong>of</strong><br />
heritability in all crosses studied<br />
were moderate to high. Relatively<br />
high heritability for spike <strong>and</strong> flag<br />
leaf reaction to SGB have been<br />
estimated in previous studies<br />
(Rosielle <strong>and</strong> Brown, 1980; Fried<br />
<strong>and</strong> Meister, 1987; Bostwick et al.<br />
1993). Therefore, early generation<br />
selection for SGB should be<br />
effective.<br />
References<br />
Bostwick, D.E., H.W. Ohm, <strong>and</strong> G.<br />
Shaner. 1993. Inheritance <strong>of</strong><br />
septoria glume blotch resistance in<br />
wheat. Crop Sci. 33:493-443.<br />
Fried, P.M., <strong>and</strong> E. Meister. 1987.<br />
Inheritance <strong>of</strong> leaf <strong>and</strong> head<br />
resistance <strong>of</strong> winter wheat to<br />
<strong>Septoria</strong> nodorum in a diallel cross.<br />
Genetics 77:1371-1375.<br />
Hayman, B.I. 1954. The theory <strong>and</strong><br />
analysis <strong>of</strong> diallel crosses. Genetics<br />
39:789-809.<br />
Jeger, M.J. 1980. Ultivariate models <strong>of</strong><br />
the components <strong>of</strong> partial<br />
resistance. Protection Ecology<br />
2:265-269.<br />
Mullaney, E.J., J.M. Martin, <strong>and</strong> A.L.<br />
Scharen. 1982. Generation mean<br />
analysis to identify <strong>and</strong> partition<br />
the components <strong>of</strong> genetic<br />
resistance to <strong>Septoria</strong> nodorum in<br />
wheat. Euphytica 31:539-545.<br />
Nelson, L.R. 1980. Inheritance <strong>of</strong><br />
resistance to <strong>Septoria</strong> nodorum in<br />
wheat. Crop Sci. 20:447-449.<br />
Nelson, L.R., <strong>and</strong> D. Marshall. 1990.<br />
Breeding wheat for resistance to<br />
<strong>Septoria</strong> nodorum <strong>and</strong> S. tritici.<br />
Advances in Agronomy 44:257-<br />
277.<br />
Rosielle, A.A., <strong>and</strong> A.G.P. Brown.<br />
1980. Selection for resistance to<br />
<strong>Septoria</strong> nodorum Berk. in wheat.<br />
Euphytica 29:337-346.<br />
Scott, P.R., P.W. Benedikz, <strong>and</strong> C.J.<br />
Cox. 1982. A genetic study on the<br />
relationship between height, time<br />
<strong>of</strong> ear emergence, <strong>and</strong> resistance to<br />
<strong>Septoria</strong> nodorum in wheat. Plant<br />
Pathol. 31:45-60.<br />
Wilkinson, C.A., J.P. Murphy, <strong>and</strong><br />
R.R. Rufty. 1990. Diallel analysis <strong>of</strong><br />
components <strong>of</strong> partial resistance to<br />
<strong>Septoria</strong> nodorum in wheat. Plant<br />
Dis. 74:47-50.
Comparison <strong>of</strong> Methods <strong>of</strong> Screening for <strong>Stagonospora</strong><br />
nodorum Resistance in Winter Wheat<br />
D.E. Fraser, 1 J.P. Murphy, 1 <strong>and</strong> S. Leath 2 (Poster)<br />
1 Department <strong>of</strong> Plant Pathology, North Carolina State Univ., Raleigh, NC, USA<br />
2 Department <strong>of</strong> Plant Pathology, USDA-ARS, NCSU, Raleigh, NC, USA<br />
Abstract<br />
Isolates <strong>of</strong> <strong>Stagonospora</strong> nodorum that varied in levels <strong>of</strong> aggressiveness were used in both controlled environment <strong>and</strong><br />
field tests to determine whether isolate aggressiveness enhanced selection <strong>of</strong> resistant wheat genotypes. Two segregating wheat<br />
populations that differed in mean levels <strong>of</strong> resistance to S. nodorum were developed. Components <strong>of</strong> resistance measured in<br />
controlled environments indicated significant differences among isolate treatments. Measurement <strong>of</strong> disease severity in field<br />
studies also indicated significant differences among the isolate treatments. For the breeding population (r<strong>and</strong>om selections <strong>of</strong><br />
genotypes in F 3 , F 4 , F 5 , <strong>and</strong> F 6 generations), incubation period measured in both juvenile <strong>and</strong> adult plant tests were most<br />
highly correlated with resistance measured in the field. For the biparental population (progeny <strong>of</strong> a resistant by susceptible<br />
cross), incubation period <strong>and</strong> percent leaf area diseased on the flag leaf for adult plant tests were most highly correlated with<br />
resistance measured in the field. Rankings <strong>of</strong> genotypes based on their resistance in both field <strong>and</strong> controlled environment<br />
tests indicated that five <strong>of</strong> the ten most resistant genotypes measured under controlled conditions were also identified as<br />
resistant in field tests. Similarly, four <strong>of</strong> the ten most susceptible genotypes measured under controlled conditions were also<br />
identified as susceptible in field tests. Combinations <strong>of</strong> adult <strong>and</strong> juvenile screening tests may allow for the identification <strong>of</strong> up<br />
to eight <strong>of</strong> ten genotypes resistant in controlled environments that are also resistant in field tests.<br />
Glume blotch, caused by the<br />
ascomycete fungus <strong>Stagonospora</strong><br />
nodorum (teleomorph Leptosphaeria<br />
nodorum) is an economically<br />
important disease <strong>of</strong> wheat.<br />
Resistance to S. nodorum is<br />
controlled by multiple genes (Jeger<br />
et al., 1983; Mullaney et al., 1982;<br />
Nelson <strong>and</strong> Gates, 1982; Wilkinson<br />
et al., 1990) <strong>and</strong> expression <strong>of</strong><br />
resistance is strongly affected by<br />
the environment.<br />
Previous studies attempting to<br />
correlate components <strong>of</strong> host<br />
resistance measured in controlled<br />
environments with host resistance<br />
under field conditions have<br />
reported variable results<br />
(Wilkinson et al., 1990; Rufty et al.,<br />
1981). A better underst<strong>and</strong>ing <strong>of</strong><br />
the relationship between isolate<br />
aggressiveness <strong>and</strong> host response<br />
could provide more effective<br />
selection methods for resistance to<br />
S. nodorum. The objective <strong>of</strong> this<br />
study was 1) to use isolates with<br />
different levels <strong>of</strong> aggressiveness to<br />
measure differences in components<br />
<strong>of</strong> resistance among genotypes in<br />
two segregating wheat<br />
populations, under both field <strong>and</strong><br />
controlled environments <strong>and</strong> 2) to<br />
determine which components are<br />
the most effective in selection <strong>of</strong><br />
resistant genotypes.<br />
Materials <strong>and</strong> Methods<br />
Two wheat populations were<br />
used in all studies. Population 1<br />
(biparental) consisted <strong>of</strong> the F3:5<br />
progeny <strong>of</strong> NKC 8427 (resistant) by<br />
Caldwell (susceptible) cross.<br />
Population 2 (breeding) was<br />
composed <strong>of</strong> r<strong>and</strong>om F3, F4, F5,<br />
<strong>and</strong> F6 lines from a southern US<br />
s<strong>of</strong>t red winter wheat nursery in<br />
North Carolina. A total <strong>of</strong> 50 lines<br />
from each population were used in<br />
all studies. A collection <strong>of</strong> 60 NC<br />
field isolates was screened in a<br />
seedling assay <strong>and</strong> the most<br />
163<br />
aggressive isolate <strong>and</strong> least<br />
aggressive isolate in the collection<br />
were selected for further study. The<br />
same isolate treatments were used<br />
in all subsequent studies <strong>and</strong><br />
consisted <strong>of</strong> the most <strong>and</strong> least<br />
aggressive isolates (A <strong>and</strong> D,<br />
respectively) applied singly, in<br />
combination (AD), <strong>and</strong> a noninoculated<br />
control.<br />
Field tests<br />
Hill-plots were planted on a 0.3m<br />
square grid in three replications<br />
at Laurel Springs, NC, in 1997 <strong>and</strong><br />
at both Laurel Springs <strong>and</strong> Kinston,<br />
NC, in 1998. Each population <strong>of</strong> 50<br />
genotypes was inoculated with a<br />
spore suspension <strong>of</strong> a different<br />
isolate treatment at either Feekes<br />
growth stage (GS) 10.1 in 1997 <strong>and</strong><br />
Feekes GS 9 in 1998. Percent leaf<br />
area diseased (% LAD) was<br />
recorded at four bi-weekly<br />
intervals after inoculation using the<br />
Saari-Prescott scale.
Session 6C — D.E. Fraser, J.P. Murphy, <strong>and</strong> S. Leath<br />
164<br />
Greenhouse tests<br />
Seeds <strong>of</strong> each genotype were<br />
over-planted <strong>and</strong> thinned to two<br />
plants per pot <strong>and</strong> allowed to grow<br />
in alternating 12-hour light <strong>and</strong><br />
dark periods at 16 to 26 o C.<br />
Experimental design was a<br />
r<strong>and</strong>omized complete block with<br />
four replications in time.<br />
At GS 10 the penultimate leaf <strong>of</strong><br />
one plant/pot was marked <strong>and</strong> a 2<br />
ml droplet <strong>of</strong> a single isolate spore<br />
suspension st<strong>and</strong>ardized to 1 x 10 6<br />
spores ml -1 applied to the leaf.<br />
Presence/absence <strong>of</strong> a lesion at 5<br />
<strong>and</strong> 10 days was recorded.<br />
Following the last rating on the<br />
penultimate leaf <strong>of</strong> one plant, the<br />
flag leaf <strong>of</strong> the other plant/pot was<br />
marked. Spore suspensions <strong>of</strong> each<br />
isolate treatment were applied to 50<br />
genotypes per population using<br />
h<strong>and</strong> atomizers until run-<strong>of</strong>f<br />
occurred. Glass slides placed in the<br />
plant canopy during inoculation<br />
indicated that 50-75 spores were<br />
deposited per mm -2 . Incubation<br />
period <strong>and</strong> % LAD on each plant<br />
were recorded.<br />
Growth chamber<br />
Seeds <strong>of</strong> each genotype were<br />
planted <strong>and</strong> grown for 21 days in<br />
12 h light <strong>and</strong> darkness periods in<br />
day/night temperatures <strong>of</strong> 22/<br />
18 o C. At the 2-leaf stage, spore<br />
suspensions <strong>of</strong> the same four<br />
isolate treatments as described<br />
previously were applied to 50<br />
genotypes per population using<br />
h<strong>and</strong> atomizers until run-<strong>of</strong>f<br />
occurred, with approximately 65<br />
spores deposited per mm -2 <strong>of</strong> leaf<br />
tissue. Incubation period <strong>and</strong> %<br />
LAD on each plant were recorded.<br />
Experimental design was a<br />
r<strong>and</strong>omized complete block with<br />
four replications.<br />
Detached leaf test–adult <strong>and</strong><br />
juvenile<br />
Seeds <strong>of</strong> each genotype were<br />
planted <strong>and</strong> grown in the<br />
greenhouse for three weeks<br />
(juvenile) <strong>and</strong> ten weeks (adult),<br />
respectively. For the juvenile test,<br />
fully exp<strong>and</strong>ed second leaves were<br />
cut from each genotype in both<br />
populations. For the adult test,<br />
plants were grown to Feeke’s GS<br />
10.1, <strong>and</strong> the flag leaf was excised<br />
from each plant. Each single leaf<br />
was cut into four equal sized pieces<br />
<strong>and</strong> placed on 150 ppm<br />
benzimidazole agar in a box<br />
divided in 4-cm grid squares. Each<br />
grid square represented each <strong>of</strong> the<br />
isolate treatments on a single<br />
genotype. A 2 ml droplet <strong>of</strong> a single<br />
isolate spore suspension was<br />
placed in the center <strong>of</strong> each leaf<br />
piece. Incubation period, lesion<br />
expansion rate, <strong>and</strong> final lesion size<br />
was recorded. Both the adult <strong>and</strong><br />
juvenile tests were replicated four<br />
times.<br />
Results <strong>and</strong> Discussion<br />
The level <strong>of</strong> resistance in the<br />
breeding population was generally<br />
higher than the biparental<br />
population in all tests. The AUDPC<br />
values indicated significant<br />
differences among isolate<br />
treatments in the field at Laurel<br />
Springs in 1997 <strong>and</strong> at both<br />
locations in 1998 (Table 1).<br />
Genotype by environment<br />
interaction was significant.<br />
Isolate treatments in<br />
greenhouse evaluations were<br />
significantly different for most <strong>of</strong><br />
the components <strong>of</strong> resistance<br />
measured (Tables 2 <strong>and</strong> 3). Results<br />
<strong>of</strong> inoculations with the most <strong>and</strong><br />
least aggressive isolates in<br />
combination (AD) were similar to<br />
inoculations with the most<br />
aggressive isolate alone <strong>and</strong> both <strong>of</strong><br />
these treatments resulted in higher<br />
numbers <strong>of</strong> lesions, more rapid<br />
rates <strong>of</strong> lesion expansion <strong>and</strong><br />
higher % LAD than inoculation the<br />
least aggressive isolate. Shorter<br />
incubation periods were not<br />
consistently associated with the<br />
most aggressive isolate or the most<br />
<strong>and</strong> least aggressive isolate<br />
together. Components <strong>of</strong> resistance<br />
measured in controlled<br />
environments were highly<br />
correlated with each other (Table 4).<br />
Few components <strong>of</strong> resistance<br />
measured in controlled<br />
environments correlated with field<br />
measured disease severity. A<br />
comparison <strong>of</strong> the controlled<br />
environment studies with field data<br />
from Kinston in 1998 indicated that<br />
adult plant incubation period <strong>and</strong><br />
% LAD on the flag leaf for adult<br />
plant spray inoculation were highly<br />
correlated (P>0.01) with field<br />
Table 1. Area under the disease progress curve values for disease severities measured on hill<br />
plots inoculated with isolates <strong>of</strong> <strong>Stagonospora</strong> nodorum.<br />
Laurel Springs 1997 Laurel Springs 1998 Kinston 1998<br />
Isolate treatments Breeding Biparental Breeding Biparental Breeding<br />
A (most) a 2724b 1669b 2196a 1678a 1587a<br />
D (least) 2783a 1667b 1972c 1557c 1561c<br />
AD (both) 2770a 1806a 1733d 1608b 1628a<br />
NI (control) 2694c 1829a 2087b 1367d 1452d<br />
a ‘Most’ <strong>and</strong> ‘least’ aggressive refer to isolate reaction measured in the isolate screening assay done on<br />
seedlings in controlled conditions.
disease severity in the biparental<br />
population. Adult <strong>and</strong> juvenile<br />
plant incubation period <strong>and</strong> lesion<br />
expansion rate measured in the<br />
detached leaf test were highly<br />
correlated with field disease<br />
severity measured at Kinston in<br />
1998 in the breeding population.<br />
Similar results were obtained<br />
for disease measured at Laurel<br />
Springs in 1997. Components <strong>of</strong><br />
resistance were not highly<br />
correlated with field disease<br />
measured at Laurel Springs in 1998.<br />
Comparison <strong>of</strong> Methods <strong>of</strong> Screening for <strong>Stagonospora</strong> nodorum Resistance in Winter Wheat 165<br />
Resistant genotypes<br />
A comparison <strong>of</strong> genotype<br />
rankings measured in the field with<br />
those measured in controlled<br />
environments indicate that for the<br />
biparental population at Kinston in<br />
1998, five <strong>of</strong> the ten most resistant<br />
genotypes in the field were also<br />
identified by the measurements <strong>of</strong><br />
the incubation period <strong>and</strong> % LAD<br />
on the flag leaf in the adult plant<br />
tests. Similarly, rankings based on<br />
% LAD measured in the adult plant<br />
spray test ranked five <strong>of</strong> the same<br />
ten most resistant genotypes<br />
identified by the field disease<br />
severity values at Laurel Springs in<br />
1998. The numbers were slightly<br />
lower for the breeding population.<br />
At Kinston in 1998, four <strong>of</strong> the ten<br />
most resistant genotypes in the<br />
field were also identified as<br />
resistant by measurements <strong>of</strong> %<br />
LAD for adult plant spray<br />
inoculation. Combining adult plant<br />
greenhouse tests <strong>and</strong> juvenile<br />
detached leaf tests could increase<br />
this number to seven or eight <strong>of</strong> the<br />
top ten resistant genotypes<br />
identified in controlled<br />
Table 2. Components <strong>of</strong> resistance to <strong>Stagonospora</strong> nodorum measured on F 3:5 lines <strong>of</strong> NKC 8433 x Caldwell cross in controlled environments.<br />
Dlt a Juvenile Dlt Adult Phytotron(Juvenile) Greenhouse<br />
Isolate Incubation # <strong>of</strong> Incubation Lesion Incubation # <strong>of</strong> Droplet Spray<br />
treatments b (days) lesions (days) expansion ( days) lesions incubation %LAD<br />
A (most) 7.9 a 2.4 b 12.7 c 0.14 b 12.9 a 2.4 bc 3.8 a 5.5 ab<br />
D (least) 8.1 b 2.3 b 10.4 b 0.13 a 13.1 a 1.7 a 4.3 a 3.9 a<br />
AD (both) 7.6 a 2.7 c 9.4 a 0.20 c 12.9 a 1.8 b 5.4 b 4.8 a<br />
NI(control) 20.0 c 0.0 a - - 17.1 b 1.2 a 20.0 c -<br />
a Detached leaf test.<br />
‘b ‘Most’ <strong>and</strong> ‘least’ aggressive refer to isolate reaction measured in the isolate screening assay done on seedlings in controlled conditions.<br />
Table 3. Components <strong>of</strong> resistance to <strong>Stagonospora</strong> nodorum measured on r<strong>and</strong>om F 3 , F 4 , F 5 <strong>and</strong> F 6 lines from a southern US winter wheat nursery<br />
in controlled environments.<br />
Dlt a Juvenile Dlt Adult Phytotron(Juvenile) Greenhouse<br />
Isolate Incubation # <strong>of</strong> Incubation Lesion Incubation # <strong>of</strong> Droplet Spray<br />
treatments b (days) lesions (days) expansion ( days) lesions incubation %LAD<br />
A (most) 8.4 a 2.5 b 12.7 b 0.11 a 13.5 a 2.1 c 5.1 b 3.7 a<br />
D (least) 9.0 b 2.4 b 10.8 a 0.12 b 12.8 a 1.6 b 3.4 a 2.8 a<br />
AD (both) 8.3 a 2.4 b 11.0 a 0.19 c 13.7 a 2.2 c 4.9 b 4.1 ab<br />
NI (control) 20.0 c 0.0 a - - 17.5 b 0.9 a 20.0 c -<br />
a Detached leaf test.<br />
b ‘ Most’ <strong>and</strong> ‘least’ aggressive refer to isolate reaction measured in the isolate screening assay done on seedlings in controlled conditions.<br />
Table 4. Correlations among resistance components measured in controlled environments <strong>and</strong> in field tests.<br />
Treatment A (most) a Treatment AD(most/least) Treatment D(least)<br />
Resistance component Biparental Breeding Biparental Breeding Biparental Breeding<br />
Adult dlt – incubation 0.23 * 0.30 * NS NS NS 0.25 *<br />
Adult dlt- lesion expansion NS -0.34 ** NS NS 0.2 0* 0.20 *<br />
Juvenile dlt – incubation NS -0.40 ** NS NS NS NS<br />
Juvenile dlt – lesion size NS NS 0.20 * 0.20* 0.27 * NS<br />
Adult spray test - %LAD NS NS 0.23 * NS 0.33** 0.14<br />
a ‘Most’ <strong>and</strong> ‘least’ aggressive refer to isolate reaction measured in the isolate screening assay done on seedlings in controlled conditions.
Session 6C — C.G. Du, L.R. Nelson, <strong>and</strong> M.E. McDaniel<br />
166<br />
environment tests also being<br />
identified as resistant under field<br />
conditions.<br />
Susceptible genotypes<br />
Of the ten most susceptible<br />
genotypes in each population, four<br />
were also identified as susceptible<br />
by measurement <strong>of</strong> juvenile plant<br />
incubation period. Combining<br />
results from both the adult plant<br />
<strong>and</strong> juvenile plant detached leaf<br />
tests resulted in as high as seven <strong>of</strong><br />
the ten most resistant genotypes in<br />
the field also being identified as<br />
resistant under controlled<br />
conditions.<br />
In general, evaluation <strong>of</strong><br />
components <strong>of</strong> resistance may<br />
provide a more efficient selection<br />
method by allowing for the<br />
removal <strong>of</strong> extremely susceptible<br />
types <strong>and</strong> identification <strong>of</strong> resistant<br />
types before large-scale field<br />
selection. Based on our results <strong>and</strong><br />
practicability to the plant breeder, a<br />
combination <strong>of</strong> juvenile detached<br />
leaf tests <strong>and</strong> adult plant<br />
inoculations (droplet or spray) in<br />
the greenhouse could aid in the<br />
selection <strong>of</strong> parental materials with<br />
resistance to S. nodorum. These<br />
methods could also be used for<br />
screening the progeny <strong>of</strong><br />
segregating populations. Highly<br />
aggressive isolates or combinations<br />
<strong>of</strong> isolates with different levels <strong>of</strong><br />
aggressiveness help to maximize<br />
differences among genotypes in<br />
both field <strong>and</strong> controlled<br />
environments<br />
Epidemiology<br />
In addition to measuring<br />
components <strong>of</strong> resistance to S.<br />
nodorum in the field <strong>and</strong> in<br />
controlled conditions, molecular<br />
analyses <strong>of</strong> the development <strong>of</strong> the<br />
epidemics in the field were also<br />
studied using these same isolate<br />
treatments <strong>and</strong> populations. The<br />
information provided by the<br />
molecular analyses may explain<br />
differences among treatments in<br />
the field <strong>and</strong> help elucidate the role<br />
<strong>of</strong> the natural inoculum in<br />
epidemic development.<br />
References<br />
Jeger, M.J., D.G. Jones, <strong>and</strong> H.<br />
Griffiths. 1983. Components <strong>of</strong><br />
partial resistance <strong>of</strong> wheat<br />
seedlings to <strong>Septoria</strong> nodorum.<br />
Euphytica 32:575-584.<br />
Mullaney, E.J., J.M. Martin, <strong>and</strong> A.L.<br />
Scharen. 1982. Generation mean<br />
analysis to identify <strong>and</strong> partition<br />
the components <strong>of</strong> genetic<br />
resistance to <strong>Septoria</strong> nodorum in<br />
wheat. Euphytica 31:539-545.<br />
Nelson, L.R., <strong>and</strong> C.E. Gates. 1982.<br />
Genetics <strong>of</strong> host plant resistance <strong>of</strong><br />
wheat to <strong>Septoria</strong> nodorum. Crop<br />
Sci. 22:771-773.<br />
Rufty, R.C., T.T. Hebert, <strong>and</strong> C.F.<br />
Murphy. 1981. Evaluation <strong>of</strong><br />
resistance to <strong>Septoria</strong> nodorum in<br />
wheat. Plant Dis. 65:406-409.<br />
Wilkinson, C.A., J.P. Murphy, <strong>and</strong><br />
R.C. Rufty. 1990. Diallel analysis <strong>of</strong><br />
components <strong>of</strong> partial resistance to<br />
<strong>Septoria</strong> nodorum in wheat. Plant<br />
Dis. 74:47-50.
Response <strong>of</strong> Winter Wheat Genotypes to Artificial<br />
Inoculation with Several <strong>Septoria</strong> tritici Populations<br />
M. Mincu (Poster)<br />
Research Institute for <strong>Cereals</strong> <strong>and</strong> Industrial Crops, Fundulea, Romania<br />
Abstract<br />
A study was conducted to identify genotypic differences in the response <strong>of</strong> winter wheat to artificial inoculation with<br />
several <strong>Septoria</strong> tritici populations. Twenty-two genotypes were grown in two locations <strong>and</strong> artificially inoculated with four<br />
<strong>Septoria</strong> tritici populations. Significant effects <strong>of</strong> host genotype, pathogen population, <strong>and</strong> host-pathogen interaction were<br />
found. Most released cultivars were susceptible. Several entries, including some Aegilops spp. derivatives, were resistant to<br />
all <strong>Septoria</strong> populations in both locations. <strong>Septoria</strong> tritici populations differed both in aggressiveness <strong>and</strong> virulence. A<br />
strong host-pathogen interaction was found in several cultivars with medium average resistance.<br />
<strong>Septoria</strong> tritici is a major foliar<br />
disease <strong>of</strong> wheat in Romania. Yield<br />
losses caused by septoria leaf<br />
blotch can reach 25% in years that<br />
favor the disease. Most currently<br />
grown wheat cultivars are more or<br />
less susceptible to <strong>Septoria</strong> tritici.<br />
Therefore, resistance to this<br />
pathogen is a high-priority<br />
breeding goal.<br />
Material <strong>and</strong> Methods<br />
Twenty-two winter wheat<br />
genotypes were grown on 2-m+<br />
plots using three replications in<br />
each <strong>of</strong> two locations (Fundulea in<br />
the south <strong>and</strong> Brasov in the central<br />
part, near the mountains). Artificial<br />
inoculation was conducted by<br />
spraying the plants at heading with<br />
a spore suspension diluted to 10<br />
spores/ml, from four <strong>Septoria</strong> tritici<br />
populations, collected from several<br />
regions <strong>of</strong> Romania. After spraying,<br />
the plants were covered with<br />
plastic sacks to maintain the<br />
humidity necessary for infection.<br />
The intensity <strong>of</strong> the attack was read<br />
30 days after inoculation, using the<br />
0 to 9 scale developed by Saari <strong>and</strong><br />
Prescott at <strong>CIMMYT</strong> (1995). For<br />
statistical analysis, the arc sin<br />
transformation was used.<br />
Significance <strong>of</strong> differences among<br />
genotypes was determined using<br />
the Duncan test.<br />
Results <strong>and</strong> Discussion<br />
The analysis <strong>of</strong> variance<br />
(ANOVA) shows there are<br />
significant effects <strong>of</strong> wheat<br />
genotypes, pathogen populations,<br />
<strong>and</strong> host x pathogen interaction<br />
(Table 1). Genotype classifications<br />
for each pathogen population in<br />
the two locations are presented in<br />
Tables 2 <strong>and</strong> 3. Most released<br />
cultivars were susceptible to all<br />
populations <strong>and</strong> in both locations.<br />
On the other h<strong>and</strong>, several entries<br />
showed low intensities <strong>of</strong> attack<br />
with all populations <strong>and</strong> in both<br />
locations (e.g. F91552G8-01, Admis,<br />
ZE16547, KS93U134, G1662-51,<br />
G557-5, G557-6). The last four are<br />
derivatives from crosses with<br />
Aegilops spp. Finally, some entries<br />
(such as Turda 81) showed a very<br />
variable response to different<br />
<strong>Septoria</strong> populations.<br />
167<br />
The differences between host<br />
genotypes are more pronounced<br />
than between pathogen<br />
populations. The pathogen<br />
populations produced different<br />
attack intensities in the two<br />
locations. In Fundulea, all <strong>Septoria</strong><br />
populations had a higher<br />
pathogenicity than in Brasov. In<br />
Brasov, the pathogenicity <strong>of</strong> all<br />
populations was approximately<br />
equal, but in Fundulea Population<br />
4 was less pathogenic, compared<br />
with the other populations. The<br />
host-pathogen interactions in the<br />
two locations are exemplified in<br />
Figures 1-4. A strong host-pathogen<br />
interaction was noticed for several<br />
cultivars in both locations.<br />
Reference<br />
Saari, E.E., <strong>and</strong> Prescott, M. 1975. A<br />
scale for appraising the foliar<br />
intensity <strong>of</strong> wheat diseases. Plant<br />
Dis. Rep. 59:377-380.<br />
Table 1. Analysis <strong>of</strong> variance (MS) for the<br />
intensity <strong>of</strong> <strong>Septoria</strong> tritici attack on the leaf in<br />
Fundulea <strong>and</strong> Brasov.<br />
Source <strong>of</strong> variation Fundulea Brasov<br />
Genotypes (G) 1479.276*** 696.308***<br />
Error (a) 81.600 5.582<br />
Populations (P) 3041.026*** 688.315***<br />
G x P 339.740*** 69.820***<br />
Error (b) 30.020 3.300
Session 6C — M. Mincu<br />
168<br />
Arcsin (sqrt (intensity %))<br />
90<br />
80<br />
70<br />
60<br />
50<br />
40<br />
30<br />
ZE-16547<br />
G 1662-51<br />
G 557-5<br />
KS93U134<br />
20<br />
P4 P2 P1 P3<br />
<strong>Septoria</strong> tritici populations<br />
Figure 1. Interaction between genotypes <strong>and</strong><br />
population (intensity <strong>of</strong> the attack on the leaf),<br />
Fundulea - sources <strong>of</strong> resistance.<br />
Arcsin (sqrt (intensity %))<br />
90<br />
80<br />
70<br />
60<br />
50<br />
40<br />
30<br />
Admis<br />
Dropia<br />
Lovrin 34<br />
Turda 81<br />
Apullum<br />
20<br />
P4 P2 P1 P3<br />
<strong>Septoria</strong> tritici populations<br />
Figure 3. Interaction between genotypes <strong>and</strong><br />
population (intensity <strong>of</strong> the attack on the leaf),<br />
Fundulea - cultivars.<br />
Arcsin (sqrt (intensity %))<br />
Arcsin (sqrt (intensity %))<br />
90<br />
80<br />
70<br />
60<br />
50<br />
40<br />
30<br />
20<br />
P1 P4 P3 P2<br />
<strong>Septoria</strong> tritici populations<br />
Figure 2. Interaction between genotypes <strong>and</strong><br />
population (intensity <strong>of</strong> the attack on the leaf),<br />
Brasov - sources <strong>of</strong> resistance.<br />
90<br />
80<br />
70<br />
60<br />
50<br />
40<br />
30<br />
ZE-16547<br />
G 1662-51<br />
G 557-5<br />
KS93U134<br />
Admis<br />
Dropia<br />
Lovrin 34<br />
Turda 81<br />
Apullum<br />
20<br />
P1 P4 P3 P2<br />
<strong>Septoria</strong> tritici populations<br />
Figure 4. Interaction between genotypes <strong>and</strong><br />
population (intensity <strong>of</strong> the attack on the leaf),<br />
Brasov - cultivars.<br />
Table 2. The reaction <strong>of</strong> genotypes to <strong>Septoria</strong> tritici infection–intensity <strong>of</strong> attack on the leaf in Fundulea.<br />
Genotypes Population 1 Population 2 Population 3 Population 4 Mean<br />
ZE16547 26.6 a † 26.6 a 26.6 a 26.6 a 26.6<br />
G557-5 26.6 a 26.6 a 26.6 a 26.6 a 26.6<br />
G557-6 26.6 a 26.6 a 26.6 a 26.6 a 26.6<br />
KS93U134 26.6 a 26.6 a 26.6 a 26.6 a 26.6<br />
91552G8-01 28.8 a 28.8 a 30.8 a 26.6 a 28.7<br />
G1662-51 26.6 a 26.6 a 37.2 b 26.6 a 29.2<br />
649T2-1 28.8 a 33.0 ab 39.2 bc 26.6 a 31.9<br />
Lovrin 34 51.2 bc 43.1 cde 38.9 bc 34.9 ab 32.3<br />
Rapid 45.0 b 28.8 a 31.0 ab 26.6 a 32.8<br />
Admis 26.6 a 49.2 ef 43.3 cd 26.6 a 36.4<br />
577U1-106 46.9 b 48.9 ef 50.8 d 26.6 a 36.6<br />
Dropia 44.9 b 36.9 bc 42.7 c 33.0 a 39.4<br />
93396G1-1 26.6 a 52.8 ef 59.0 ef 26.6 a 41.2<br />
92033G2-1 55.0 bcd 28.8 a 51.2 de 33.2 ab 42.0<br />
91375GA-13 57.0 cd 31.0 b 63.9 fg 26.6 a 44.6<br />
93009G11-12 59.0 de 35.2 abc 61.2 ef 26.6 a 45.5<br />
Fundulea – 4 61.2 def 46.9 de 55.0 e 35.2 ab 49.6<br />
93122G6-2 26.6 a 61.2 gh 63.5 f 48.9 d 50.0<br />
Apullum 68.9 f 30.8 ab 59.7 ef 43.0 bc 50.6<br />
508U3-201 26.6 a 56.6 fg 72.8 g 46.9 cd 50.7<br />
Turda 81 83.9 g 43.0 cd 83.9 h 37.2 b 62.0<br />
Cadril 249 66.2 de 63.9 h 72.3 g 48.9 d 62.8<br />
† Means in columns followed by the same letter are not significantly different by Duncan’s test at the 0.05% level.
Response <strong>of</strong> Winter Wheat Genotypes to Artificial Inoculation with Several <strong>Septoria</strong> tritici Populations 169<br />
Table 3. The reaction <strong>of</strong> genotypes to <strong>Septoria</strong> tritici infection–intensity <strong>of</strong> attack on the leaf in Brasov.<br />
Genotypes Population 1 Population 2 Population 3 Population 4 Mean<br />
ZE16547 26.6 a † 26.6 a 26.6 a 26.6 a 26.6<br />
G557-6 26.6 a 26.6 a 26.6 a 26.6 a 26.6<br />
KS93U134 26.6 a 26.6 a 26.6 a 26.6 a 26.6<br />
91552G8-01 26.6 a 26.6 a 26.6 a 26.6 a 26.6<br />
Admis 26.6 a 26.6 a 26.6 a 26.6 a 26.6<br />
649T2-1 26.6 a 28.8 a 26.6 a 26.6 a 27.1<br />
G557-5 26.6 a 26.6 a 26.6 a 28.8 a 27.1<br />
Cadril 249 26.6 a 31.0 b 26.6 a 26.6 a 27.7<br />
G1662-51 26.6 a 33.2 b 26.6 a 26.6 a 28.2<br />
Rapid 26.6 a 28.8 a 37.2 c 28.8 a 30.3<br />
93396G1-1 26.6 a 37.2 c 33.2 b 26.6 a 30.9<br />
93009G11-12 26.6 a 39.2 c 33.2 b 26.6 a 31.4<br />
508U3-201 26.6 a 37.2 c 35.2 b 26.6 a 31.4<br />
Apullum 26.6 a 33.2 b 37.2 c 31.0 b 32.0<br />
Turda 81 33.2 b 26.6 a 43.1 d 31.0 b 33.5<br />
91375GA-13 31.0 b 45.0 d 35.2 b 26.6 a 34.5<br />
93122G6-2 26.6 a 45.0 d 37.2 c 31.0 b 35.0<br />
577U1-106 33.2 b 39.2 c 37.2 c 33.2 c 35.7<br />
92033G2-1 37.2 d 48.9 e 45.0 d 37.2 d 42.1<br />
Dropia 33.2 b 56.8 g 43.1 d 50.8 e 46.0<br />
Fundulea – 4 50.8 e 52.8 f 52.8 e 39.2 d 48.90<br />
Lovrin – 34 35.2 c 52.8 f 68.9 f 50.8 e 51.92<br />
† Means in columns followed by the same letter are not significantly different by Duncan’s test at the 0.05% level.
170<br />
Comparison <strong>of</strong> Greenhouse <strong>and</strong> Field Levels <strong>of</strong> Resistance<br />
to <strong>Stagonospora</strong> nodorum<br />
S.L. Walker, 1,2 S. Leath, 1,3 <strong>and</strong> J.P. Murphy 2 (Poster)<br />
1 Department <strong>of</strong> Plant Pathology, North Carolina State University, Raleigh, USA<br />
2 Department <strong>of</strong> Crop Science, North Carolina State University, Raleigh, USA<br />
3 USDA-ARS, North Carolina State University, Raleigh, USA<br />
Abstract<br />
A biparental population <strong>of</strong> 150 recombinant wheat inbred (RI) lines segregating for reaction to <strong>Stagonospora</strong> nodorum<br />
was developed <strong>and</strong> tested in the greenhouse <strong>and</strong> field for levels <strong>of</strong> resistance. The F 3:4 lines were tested in the greenhouse for<br />
incubation period <strong>and</strong> percent leaf infection 5 <strong>and</strong> 10 days after initial symptoms <strong>of</strong> disease. Plants were tested at both the<br />
juvenile <strong>and</strong> adult stages. Adult plants were also tested for percent disease on the spike. The F 3:5 lines were tested in the field<br />
over four locations for level <strong>of</strong> adult leaf <strong>and</strong> head resistance. Heritability <strong>of</strong> leaf resistance to S. nodorum was calculated to<br />
be 0.56 on an entry means basis <strong>and</strong> 0.17 on a per plot basis. Heritability <strong>of</strong> resistance <strong>of</strong> the spike was calculated to be 0.57 on<br />
an entry means basis <strong>and</strong> 0.20 on a per plot basis. Both heritability estimates are based on field data. Correlations between<br />
field data <strong>and</strong> greenhouse data were generally weak, notably between field data <strong>and</strong> greenhouse performance <strong>of</strong> juvenile plants.<br />
However, resistance <strong>of</strong> the spike in the field <strong>and</strong> the greenhouse were highly correlated (r = 0.74).<br />
Resistance to <strong>Stagonospora</strong><br />
nodorum is an economical way to<br />
control glume blotch as well as a<br />
component <strong>of</strong> integrated<br />
management <strong>of</strong> the disease.<br />
Resistance to S. nodorum is complex<br />
<strong>and</strong> polygenic, particularly in adult<br />
plants. There is some evidence that<br />
resistance in the spike <strong>and</strong> at the<br />
juvenile growth stage are under<br />
different genetic control than adult<br />
leaf resistance (Eyal et al., 1987). By<br />
underst<strong>and</strong>ing the relationships<br />
between juvenile, adult leaf, <strong>and</strong><br />
resistance <strong>of</strong> the spike as well as<br />
field <strong>and</strong> greenhouse resistance,<br />
one could improve progress in<br />
selecting the most resistant lines.<br />
Also, an underst<strong>and</strong>ing <strong>of</strong> the<br />
various components <strong>of</strong> resistance<br />
<strong>and</strong> the epidemiology <strong>of</strong> glume<br />
blotch in a given area would allow<br />
deploying the most useful<br />
component <strong>of</strong> resistance in that<br />
environment.<br />
Materials <strong>and</strong> Methods<br />
A cross <strong>of</strong> the s<strong>of</strong>t red winter<br />
wheat cultivars Caldwell= (CItr<br />
17897) ( mod. susceptible) x Coker<br />
8427 (9766) (PI601429) (mod.<br />
resistant) was made, <strong>and</strong> progeny<br />
were advanced in bulk to the F 3<br />
generation. Individual spikes were<br />
taken from the F 3 generation <strong>and</strong><br />
used to form 150 F 3:4 derived lines.<br />
The F 3:4 lines were tested in the<br />
greenhouse for incubation period<br />
<strong>and</strong> percent leaf area diseased at<br />
both the juvenile <strong>and</strong> adult plant<br />
growth stages. Percent diseased<br />
tissue on the spike was measured<br />
at the adult growth stage.<br />
A test consisted <strong>of</strong> a single plant<br />
from each F 3:4 line using a<br />
completely r<strong>and</strong>omized design. A<br />
total <strong>of</strong> four tests were replicated in<br />
time during the winter <strong>of</strong> 1997-98.<br />
Plants were inoculated with a<br />
mixture <strong>of</strong> three S. nodorum isolates<br />
at a concentration <strong>of</strong> 1.0 x 10 6<br />
conidia/ml plus 0.01% Tween 80.<br />
Inoculations on juvenile plants<br />
were performed at the three-leaf<br />
stage, while adult inoculations<br />
occurred at anthesis. After<br />
inoculation, plants were kept in<br />
high humidity for 72 hours. Plants<br />
were rated each day for incubation<br />
period, <strong>and</strong> then at five <strong>and</strong> ten<br />
days after the incubation period<br />
was determined for each individual<br />
line. Leaf <strong>and</strong> spike ratings in the<br />
greenhouse were recorded as a<br />
percentage <strong>of</strong> the total leaf or head<br />
tissue displaying symptoms. Spike<br />
ratings underwent square root<br />
transformation prior to statistical<br />
analysis.<br />
Field testing was performed<br />
during the 1998-99 growing season<br />
at three locations: Johnston Co.,<br />
Lenoir Co., <strong>and</strong> Ashe Co., North<br />
Carolina, USA. F 3:5 lines were<br />
planted as hillplots, consisting <strong>of</strong> 25<br />
seeds planted together as a group.<br />
Hillplots were planted on a grid,<br />
with 0.3 m separating each plot.<br />
Three repetitions <strong>of</strong> each line were<br />
planted in a r<strong>and</strong>omized complete
lock design within each location.<br />
Dried wheat straw cut the previous<br />
season from a field heavily infected<br />
with S. nodorum was distributed at<br />
each location to induce disease.<br />
Overhead irrigation was used at<br />
the Lenoir <strong>and</strong> Ashe County<br />
locations, but was unavailable at<br />
the Johnston County location.<br />
Heritability estimates were<br />
calculated as described by Fehr<br />
(1991).<br />
Each hillplot was rated for level<br />
<strong>of</strong> disease on the leaf <strong>and</strong> on the<br />
spike. Leaf disease ratings were<br />
done using the double digit scale<br />
(Eyal et al., 1987), indicating both<br />
the highest point <strong>of</strong> disease in a<br />
hillplot <strong>and</strong> the percentage <strong>of</strong><br />
disease at that point. Disease on the<br />
spike was recorded as the<br />
percentage <strong>of</strong> diseased tissue <strong>and</strong><br />
underwent square root<br />
transformation prior to statistical<br />
analysis. Statistical analysis was<br />
performed using SAS release 6.12.<br />
Correlation analysis used the<br />
Spearman ranked test.<br />
Results <strong>and</strong> Discussion<br />
A range <strong>of</strong> values for each<br />
measured trait was produced<br />
among lines in both the greenhouse<br />
<strong>and</strong> in the field (Table 1). The<br />
Comparison <strong>of</strong> Greenhouse <strong>and</strong> Field Levels <strong>of</strong> Resistance to <strong>Stagonospora</strong> nodorum 171<br />
analysis <strong>of</strong> variance demonstrated<br />
significant differences among lines<br />
(p < 0.001) for each trait in both the<br />
greenhouse <strong>and</strong> the field (data not<br />
shown). Using field data,<br />
heritability <strong>of</strong> adult leaf resistance<br />
was calculated <strong>and</strong> found to have a<br />
value <strong>of</strong> 0.56 on an entry means<br />
basis <strong>and</strong> a value <strong>of</strong> 0.17 on a per<br />
hillplot basis. Heritability <strong>of</strong><br />
resistance <strong>of</strong> the spike was 0.57 on<br />
an entry means basis <strong>and</strong> 0.20 on a<br />
per plot basis. These data indicate<br />
the genetic component <strong>of</strong> resistance<br />
in this population has a large effect<br />
on disease expression. Ranking <strong>of</strong><br />
mean resistance scores among lines<br />
was consistent over three locations.<br />
Correlation analysis<br />
demonstrated a negative<br />
correlation between adult<br />
incubation period <strong>and</strong> leaf<br />
resistance in the greenhouse <strong>and</strong><br />
field, as well as a small positive<br />
correlation between adult<br />
greenhouse leaf resistance <strong>and</strong> field<br />
leaf resistance (Table 2). The largest<br />
<strong>and</strong> most significant correlation<br />
was between greenhouse <strong>and</strong> field<br />
resistance <strong>of</strong> the spike (0.74). This<br />
indicates screening for resistance <strong>of</strong><br />
the spike in the greenhouse may<br />
provide an alternative to field<br />
testing. In the greenhouse, juvenile<br />
plant reactions demonstrated little<br />
Table 1. Means, st<strong>and</strong>ard deviations, <strong>and</strong> ranges <strong>of</strong> <strong>Stagonospora</strong> nodorum traits measured in the greenhouse <strong>and</strong> field.<br />
correlation with adult plant<br />
resistance traits either in the<br />
greenhouse or in the field,<br />
indicating a possible separate<br />
genetic mechanism for juvenile<br />
resistance. Resistance <strong>of</strong> the spike<br />
<strong>and</strong> leaf resistance in the field<br />
produced a correlation value <strong>of</strong><br />
0.55, indicating some relationship<br />
between the traits, but a large<br />
degree <strong>of</strong> variation between the<br />
traits remained unexplained.<br />
We are currently using AFLP<br />
<strong>and</strong> RAPD markers in an attempt<br />
to match polymorphisms at the<br />
molecular level with traits<br />
measured in the field <strong>and</strong><br />
greenhouse to gain some insight<br />
into the genetics <strong>of</strong> resistance to<br />
this disease.<br />
References<br />
Eyal, Z., A.L. Scharen, J.M. Prescott,<br />
<strong>and</strong> M. van Ginkel. 1987. The<br />
<strong>Septoria</strong> <strong>Diseases</strong> <strong>of</strong> Wheat:<br />
Concepts <strong>and</strong> Methods <strong>of</strong> Disease<br />
Management. Mexico, D.F.:<br />
<strong>CIMMYT</strong>. pp. 33-35.<br />
Fehr, W.R. 1991. Principles <strong>of</strong> Cultivar<br />
Development. Vol 1. MacMillan<br />
Publishing. USA. p. 99.<br />
Trait Growth Stage <strong>and</strong> Environment Mean Std. Dev. Min. Max.<br />
Incubation period Juvenile, greenhouse 5.8 days 0.94 days 4.0 days 10.0 days<br />
Leaf area disease<br />
after 5 days Juvenile, greenhouse 5.5% 4.7% 0.5% 30.0%<br />
Leaf area disease<br />
after 10 days Juvenile, greenhouse 10.2% 7.8% 0.4% 42.5%<br />
Incubation period Adult, greenhouse 5.4 days 0.86 days 3.5 days 9.5 days<br />
Leaf area disease<br />
after 5 days Adult, greenhouse 8.4% 7.2% 1.0% 48.8%<br />
Leaf area disease<br />
after 10 days Adult, greenhouse 27.8% 19.0% 3.0% 90.0%<br />
Head area disease Adult, greenhouse 3.2% 0.8% 0% 23.0%<br />
Leaf rating Adult, field 72.1% 7.7% 48.0% 88.8%<br />
Head rating Adult, field 25.0% 1.0% 0% 100%
Session 6C — S.L. Walker, S. Leath, <strong>and</strong> J.P. Murphy<br />
172<br />
Table 2. Spearman correlation coefficients between <strong>Stagonospora</strong> nodorum traits measured in the greenhouse <strong>and</strong> field.<br />
Adult a Adult b Adult c Adult d Juv. e Juv. f Juv. g Field h Field i<br />
incub. day 5 day 10 head incub. day 5 day 10 leaf head<br />
Adult incub. 1.0 -0.40*** -0.36*** -0.11 0.04 -0.12 -0.16* -0.26** -0.13<br />
Adult day5 -0.40*** 1.0 0.75*** 0.12 0.00 0.16* 0.20** 0.18* 0.21**<br />
Adult day10 -0.36*** 0.75*** 1.0 0.13 -0.03 0.14 0.18* 0.23** 0.19*<br />
Adult head -0.11 0.12 0.13 1.0 0.11 0.03 0.06 0.48*** 0.74***<br />
Juv. incub. 0.04 0.00 -0.03 0.11 1.0 -0.05 -0.15 -0.08 0.00<br />
Juv. day5 -0.12 0.16* 0.14 0.03 -0.05 1.0 0.69*** 0.10 0.08<br />
Juv. day10 -0.16* 0.20* 0.18* 0.06 -0.15 0.69*** 1.0 0.09 0.08<br />
Field leaf -0.26** 0.18* 0.23** 0.48*** -0.08 0.10 0.09 1.0 0.55***<br />
Field head -0.13 0.21** 0.19* 0.74*** 0.00 0.08 0.08 0.55*** 1.0<br />
*, **, *** p < 0.05, 0.01, 0.0001, respectively.<br />
a. Incubation period <strong>of</strong> adult plants in greenhouse test.<br />
b. Percent leaf area disease <strong>of</strong> adult plants five days after first sign <strong>of</strong> disease in greenhouse test.<br />
c. Percent leaf area disease <strong>of</strong> adult plants ten days after first sign <strong>of</strong> disease in greenhouse test.<br />
d. Percent area head disease <strong>of</strong> adult plants in greenhouse test.<br />
e. Incubation period <strong>of</strong> juvenile plants in greenhouse test.<br />
f. Percent leaf area disease <strong>of</strong> juvenile plants five days after first sign <strong>of</strong> disease in greenhouse test.<br />
g. Percent leaf area disease <strong>of</strong> juvenile plants five days after first sign <strong>of</strong> disease in greenhouse test.<br />
h. Leaf disease rating in field tests.<br />
i. Head disease rating in field tests.
Session 6D: Chemical Control<br />
Adjusting Thresholds for <strong>Septoria</strong> Control in Winter Wheat<br />
Using Strobilurins<br />
L.N. Jørgensen, 1 K.E. Henriksen, 1 <strong>and</strong> G.C. Nielsen2 1 Department <strong>of</strong> Crop Protection, Danish Institute <strong>of</strong> Agricultural Sciences, Slagelse, DK<br />
2 The Danish Agricultural Advisory Centre, Skejby, DK<br />
Abstract<br />
In semi-field trials in spring wheat, azoxystrobin has shown a longer residual <strong>and</strong> preventive effect on <strong>Stagonospora</strong><br />
nodorum than the triazole propiconazole. Three weeks after application, more than 90% control was still obtained with one<br />
quarter <strong>of</strong> the recommended rate <strong>of</strong> azoxystrobin. Propiconazole gave in comparison only 50% control. In another semi-field<br />
trial using <strong>Septoria</strong> tritici, azoxystrobin <strong>and</strong> propiconazole had a similar curative effect, both being significantly better than<br />
chlorothalonil. Different dosages (12-100% <strong>of</strong> normal rate <strong>of</strong> azoxystrobin) were tested in field trials. When taking into<br />
account the different timing, the most pr<strong>of</strong>itable dose has varied between 50 <strong>and</strong> 100% <strong>of</strong> normal rate, depending on growth<br />
stage <strong>and</strong> disease pressure. If optimal timing is used, 25-50% <strong>of</strong> normal rate has generally been sufficient. In 1998 in field<br />
trials azoxystrobin <strong>and</strong> the co-formulation propiconazole + fenpropimorph or tebuconazole provided similar control <strong>of</strong> S.<br />
tritici when applied after 4 or 8 days <strong>of</strong> precipitation, respectively. The test model for azoxystrobin recommended between 36<br />
<strong>and</strong> 45% <strong>of</strong> the normal rates. Azoxystrobin gave an increase in yield <strong>of</strong> 100-600 kg ha -1 above the co-formulation or<br />
tebuconazole. So far azoxystrobin has shown pr<strong>of</strong>itable yields in all trials carried out in Denmark since 1994. However, it is<br />
still not known how many days <strong>of</strong> precipitation are required to make spraying with azoxystrobin pr<strong>of</strong>itable.<br />
Based on historical trial data, it<br />
has been shown that in Denmark an<br />
economically important attack <strong>of</strong><br />
<strong>Septoria</strong> tritici or <strong>Stagonospora</strong><br />
nodorum requires more than 7-8<br />
days <strong>of</strong> precipitation (>1 mm rain)<br />
between GSZ 32 <strong>and</strong> 30 days<br />
thereafter (Hansen et al., 1994). This<br />
model has been incorporated into<br />
the decision support system PC-<br />
Plant Protection <strong>and</strong> is widely used<br />
by Danish farmers as a guide for<br />
septoria control (Secher et al., 1995).<br />
Under validation the model has<br />
given correct answers in 75% <strong>of</strong> the<br />
events studied (Jørgensen et al.,<br />
1999). The experience using<br />
triazoles for control <strong>of</strong> septoria<br />
diseases in Denmark has been that<br />
their application is justified in<br />
approximately half <strong>of</strong> the years.<br />
Adjusting the model for<br />
strobilurins requires knowledge <strong>of</strong><br />
their preventive <strong>and</strong> curative effect,<br />
the residual effect, the dose<br />
response at different timing, <strong>and</strong> the<br />
yield responses measured in relation<br />
to fungicides. Several <strong>of</strong> these<br />
aspects are under investigation<br />
using azoxystrobin as representative<br />
<strong>of</strong> the strobilurins.<br />
Materials <strong>and</strong> Methods<br />
In outdoor semi-field trials,<br />
spring wheat (cv. Dragon) was<br />
grown in 8-liter pots using 20 seeds<br />
per pot <strong>and</strong> 4 replicates. The plants<br />
were exposed to normal weather<br />
conditions (May, June, <strong>and</strong> July).<br />
Two types <strong>of</strong> trials were carried out<br />
using either artificial inoculation <strong>of</strong><br />
S. nodorum or S. tritici at growth<br />
stage 39-45. The plants were<br />
inoculated by spraying each pot<br />
with 25 ml <strong>of</strong> a suspension<br />
containing 2.5 x 10 6 spores ml -1 <strong>of</strong><br />
either S. nodorum or S. tritici. After<br />
inoculation the pots were covered<br />
with polyethylene for two days to<br />
provide high humidity conditions<br />
173<br />
for infection. Plants were treated<br />
with fungicide either after<br />
inoculation to investigate the<br />
curative effect, or before inoculation<br />
to examine the preventive <strong>and</strong><br />
residual effect. Azoxystrobin,<br />
propiconazole, <strong>and</strong> chlorothalonil<br />
were tested at 100-25% <strong>of</strong> the<br />
normal rate using a laboratory pot<br />
sprayer with flat-fan nozzles (Hardi<br />
4110-14) <strong>and</strong> a water volume <strong>of</strong> 167 l<br />
ha -1 . Percent total coverage <strong>of</strong> the<br />
plants by disease as well as percent<br />
severity on individual leaves were<br />
assessed.<br />
Dose response field trials were<br />
carried out by the Danish Institute<br />
<strong>of</strong> Agricultural Sciences (DIAS), <strong>and</strong><br />
the trials testing the septoria model<br />
using either 4 or 8 days <strong>of</strong><br />
precipitation were conducted by<br />
DIAS <strong>and</strong> the Danish Agricultural<br />
Advisory Centre (DAAC). The<br />
design <strong>of</strong> the trials was in the first<br />
case split plot <strong>and</strong> in the second
Session 6D — L.N. Jørgensen, K.E. Henriksen, <strong>and</strong> G.C. Nielsen<br />
174<br />
systematic complete block design<br />
with 5 replicates <strong>and</strong> a plot size <strong>of</strong><br />
20-32 m 2 . The fungicides were<br />
applied with knapsack sprayers at<br />
low pressure (2-3 bar) using flat<br />
fan nozzles in a volume <strong>of</strong> 200-300<br />
l ha -1 . The following products<br />
were tested: Tilt top (125 g<br />
propiconazole + 375 g<br />
fenpropimorph per liter), Folicur<br />
(250 g/l tebuconazole per liter),<br />
<strong>and</strong> Amistar (250 g azoxystrobin<br />
per liter). <strong>Septoria</strong> attacks were<br />
observed in all trials, with S. tritici<br />
being dominant, but mixed<br />
infections with S. nodorum were<br />
also seen. Plots were harvested<br />
with a plot combine harvester,<br />
<strong>and</strong> grain yield was corrected to<br />
15% moisture content. When<br />
calculating net yields (pr<strong>of</strong>it), the<br />
prices used were: Tilt top 375<br />
% control<br />
10<br />
8<br />
6<br />
4<br />
2<br />
- 1 1 1<br />
Days before <strong>and</strong> after application<br />
Figure 1. Curative control <strong>of</strong> <strong>Septoria</strong> tritici using 1 / 2<br />
rate <strong>of</strong> three different fungicides at different timing.<br />
12% attack in untreated.<br />
% control<br />
50<br />
40<br />
30<br />
20<br />
10<br />
vs. 65vs.55-<br />
Chlorothalonil<br />
vs.39- DKkr per liter <strong>and</strong> Folicur 400<br />
DKkr per liter, Amistar 583 dkr per<br />
liter, <strong>and</strong> 60 dkr per application;<br />
grain price, 750 dkr per ton. Danish<br />
fungicide prices include a 33% tax.<br />
Results <strong>and</strong> Discussion<br />
Semi-field trials have shown<br />
that azoxystrobin has a curative<br />
effect on S. tritici similar to<br />
propiconazole <strong>and</strong> much better<br />
than chlorothalonil (Figure 1).<br />
Compared to very effective<br />
triazoles like epoxiconazole, the<br />
effect <strong>of</strong> azoxystrobin is, however,<br />
lower (data not shown).<br />
Azoxystrobin showed a very<br />
effective, long preventive effect<br />
against S. nodorum (Figure 2),<br />
lasting for three weeks or more. In<br />
comparison propiconazole +<br />
Propiconazole<br />
Azoxystrobin<br />
vs. 33-<br />
0<br />
100 75 50 25 12.5<br />
Percent <strong>of</strong> normal dose rate<br />
Figure 3. Percent attack <strong>of</strong> <strong>Septoria</strong> tritici at GS 75<br />
after application <strong>of</strong> different dosages<br />
<strong>of</strong> azoxystroin at different growth stages. 2 trials, 1998.<br />
% control<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
fenpropimorph had only a residual<br />
preventive effect for 10 days. In<br />
field trials the early season effect<br />
(GSZ 31) <strong>of</strong> azoxystrobin has been<br />
found to be inferior to the effect <strong>of</strong><br />
the co-formulation propiconazole +<br />
fenpropimorph, something which<br />
has not been observed following<br />
applications carried out around<br />
heading (Jørgensen <strong>and</strong> Nielsen,<br />
1998).<br />
Field trials using four different<br />
dosages <strong>of</strong> azoxystrobin, along<br />
with several other trial series<br />
(Jørgensen <strong>and</strong> Nielsen, 1998), have<br />
shown that azoxystrobin <strong>and</strong> other<br />
strobilurins may be used at a<br />
reduced dose (Figures 3 <strong>and</strong> 4).<br />
Different dosages, 12-100% <strong>of</strong> the<br />
normal azoxystrobin rate, have<br />
been tested in trials. When taking<br />
Propiconazole<br />
Azoxystrobin<br />
1 6 12 18 24<br />
Spraying day after inoculation<br />
Figure 2. Control <strong>of</strong> <strong>Stagonospora</strong> nodorum<br />
preventatively using 1 / 2 rate <strong>of</strong> two fungicides. 35%<br />
attack in untreated.<br />
t/ha<br />
1.6<br />
1.2<br />
0.8<br />
0.4<br />
0<br />
vs.39- vs.65- vs.33- vs.55- 100 75 50 25 12.5<br />
Percent <strong>of</strong> normal dose rate<br />
Figure 4. Net yield in winter wheat after one<br />
application af azoxystrobin for control <strong>of</strong> <strong>Septoria</strong><br />
tritici using different dosages <strong>and</strong> timing. 2 trials, 1998.
into account different timing, the<br />
most pr<strong>of</strong>itable dose under severe<br />
disease pressure in 1998 varied<br />
between 50 <strong>and</strong> 100% <strong>of</strong> the normal<br />
rate, depending on growth stage<br />
<strong>and</strong> disease pressure. Using<br />
optimal timing, 50% was sufficient.<br />
When summarizing results from all<br />
trials with azoxystrobin, the most<br />
pr<strong>of</strong>itable treatment is most likely<br />
applied between GSZ 39 <strong>and</strong> 55,<br />
using 25 to 50% <strong>of</strong> the normal dose.<br />
In three seasons, different<br />
thresholds have been investigated<br />
as background for recommending<br />
spraying azoxystrobin for septoria<br />
control. In 1998 the difference<br />
between using a threshold <strong>of</strong> 4 or 8<br />
days <strong>of</strong> precipitation above 1 mm<br />
was minor (Table 1). Similar results<br />
were found in two trials in 1997<br />
(data not shown). In two trials in<br />
1996, four days was the optimal<br />
threshold (Jørgensen et al., 1999).<br />
Mixing azoxystrobin with<br />
Adjusting Thresholds for <strong>Septoria</strong> Control in Winter Wheat Using Strobilurins 175<br />
Table 1. Results from trials using 4 <strong>and</strong> 8 days <strong>of</strong> precipitation as a threshold model for control <strong>of</strong> septoria diseases in winter wheat.<br />
Treatment 9 trials 1999 No. <strong>of</strong> appl. TFI a % septoria GS 69-71 Yield <strong>and</strong> yield inc. (t/ha) Net yield (t/ha)<br />
Untreated 0 0 21 6.67<br />
Tilt top 1.0 GSZ 45 1 1 8 1.05 0.47<br />
PC-P Tilt top 8 days 0.9 0.36 12 0.80 0.55<br />
PC-P Tilt top 4 days 1.4 0.65 7 0.91 0.47<br />
Amistar 1.0 GSZ 45 1 1 7 1.67 0.81<br />
PC-P Amistar 8 days 0.9 0.36 12 1.20 0.85<br />
PC-P Amistar 4 days 1.3 0.43 7 1.19 0.75<br />
PC-P Amistar+Folicur 8 days 0.9 0.44 12 1.26 0.90<br />
PC-P Amistar+Folicur 4 days 1.3 0.52 8 1.18 0.74<br />
LSD95 % septoria<br />
0.35<br />
Treatment 3 trials 1998 No. <strong>of</strong> appl. TFIa Flag leaf 2nd leaf Yield <strong>and</strong> yield inc. (t/ha) Net yield (t/ha)<br />
Untreated 0 0 33.2 80.6 7.40 -<br />
Folicur 1.0 GSZ 45 1 1 2.0 32.5 1.70 1.09<br />
PC-P Folicur 8 days 1 0.41 4.1 43.6 1.16 0.86<br />
PC-P Folicur 4 days 2 0.84 5.6 28.7 1.76 1.15<br />
Amistar 1.0 GSZ 45 1 1 2.8 42.3 2.00 1.14<br />
PC-P Amistar 8 days 1 0.41 5.5 55.7 1.79 1.39<br />
PC-P Amistar 4 days 1.3 0.42 9.2 44.7 1.89 1.46<br />
PC-P Amistar+Folicur 8 days 1 0.5 3.6 43.8 1.88 1.47<br />
PC-P Amistar+Folicur 4 days 1.3 0.5 10.9 35.8 1.91 1.45<br />
LSD95 6.8 10.9 5.3<br />
a TFI = Treatment frequency index.<br />
tebuconazole has given results<br />
similar to azoxystrobin applied alone<br />
at both 4 <strong>and</strong> 8 days. These results<br />
indicate that the curative effect <strong>of</strong><br />
azoxystrobin against S. tritici under<br />
Danish conditions are similar or<br />
better than the triazoles<br />
propiconazole <strong>and</strong> tebuconazole<br />
used on the Danish market.<br />
Based on collected information,<br />
so far the test model for strobilurins<br />
recommends spraying after four<br />
days <strong>of</strong> precipitation starting at GSZ<br />
32. If spraying is carried out before<br />
GSZ 51, 10 days’ protection can be<br />
expected before days <strong>of</strong> precipitation<br />
have to be counted again. If applying<br />
after GSZ 51, 20 days’ effect can be<br />
expected from azoxystrobin, which<br />
in practice means that no further<br />
application is needed. The<br />
recommended rates <strong>of</strong> azoxystrobin<br />
in the model vary between 35 <strong>and</strong><br />
45% <strong>of</strong> the normal rate.<br />
References<br />
Hansen, J.G., Secher, B.J., Jørgensen,<br />
L.N., <strong>and</strong> Welling, B. 1994.<br />
Threshold for control <strong>of</strong> <strong>Septoria</strong><br />
spp. in winter wheat based on<br />
precipitation <strong>and</strong> growth stage.<br />
Plant Pathology 43:183-189.<br />
Jørgensen, L.N., <strong>and</strong> Nielsen, G.C.<br />
1998. Reduced dosages <strong>of</strong><br />
strobilurins for disease<br />
management in winter wheat.<br />
Brighton Crop Protection<br />
Conference. Pest <strong>and</strong> <strong>Diseases</strong> 993-<br />
998.<br />
Jørgensen, L.N.; B.J.M. Secher, <strong>and</strong> H.<br />
Hossy. 1999. Decision support<br />
systems featuring <strong>Septoria</strong><br />
Management. In: <strong>Septoria</strong> on<br />
<strong>Cereals</strong>: a Study <strong>of</strong> Pathosystems.<br />
J.A. Lucas, P. Bowyer, <strong>and</strong> H. M.<br />
Anderson (eds.). CABI Publishing,<br />
Wallingford, UK. pp. 251-262.<br />
Secher, B.J., Jørgensen, L.N., Murali,<br />
N.S., <strong>and</strong> Boll, P.S. 1995. Field<br />
validation <strong>of</strong> a decision support<br />
system for control <strong>of</strong> pests <strong>and</strong><br />
diseases in cereals in Denmark.<br />
Pesticide Science 45:195-199.
176
Concluding Remarks<br />
The <strong>Septoria</strong>/<strong>Stagonospora</strong> Blotch <strong>Diseases</strong> <strong>of</strong> Wheat:<br />
Past, Present, <strong>and</strong> Future<br />
Z. Eyal (paper presented by A.L. Scharen)<br />
Department <strong>of</strong> Plant Sciences <strong>and</strong> the Institute for Cereal Crops Improvement (ICCI), Tel Aviv University, Tel Aviv,<br />
Israel<br />
Abstract<br />
<strong>Septoria</strong> tritici <strong>and</strong> stagonospora nodorum blotch <strong>of</strong> wheat are regarded as major diseases because <strong>of</strong> their impact on crop<br />
management <strong>and</strong> wheat production. Both pathogens mainly affect the crop’s grain filling processes. Early research dealt<br />
mostly with genetic, cultural, <strong>and</strong> chemical control measures. Chemical control remains one <strong>of</strong> the major means <strong>of</strong> protecting<br />
wheat production, mostly through the use <strong>of</strong> new families <strong>of</strong> systemic fungicides. Emphasis has been given to epidemiological<br />
studies, in many cases associated with chemical control; however, in recent years epidemiological studies have exp<strong>and</strong>ed to<br />
include the effect <strong>of</strong> primary inoculum initiated from dispersal <strong>of</strong> ascospores <strong>of</strong> both pathogens. That shift in focus has<br />
introduced issues such as disease cycle, mating types, <strong>and</strong> their effect—together with pathogenicity—on each pathogen’s<br />
population structure. Recent findings attributed population structure to the pathogens’ sexual rather than asexual stage. The<br />
association between virulence spectrum <strong>and</strong> pathogenicity <strong>and</strong> the contribution <strong>of</strong> the sexual stage remains to be investigated.<br />
The impact <strong>of</strong> such studies on population structure may dictate adapting the proper breeding strategies. A population that is<br />
ever changing due to recombination <strong>and</strong> gene flow may influence the use <strong>of</strong> sources for specific resistance. As for the<br />
introduction <strong>of</strong> DNA technology into <strong>Septoria</strong>/<strong>Stagonospora</strong>: wheat so far has been able to identify population genetic<br />
trends in both pathogens, but they have yet to be linked to virulence patterns. The use <strong>of</strong> genetic transformation in both<br />
pathogens makes it possible to follow post-penetration processes by following reporter genes. This may reveal that<br />
synchronous events associated with the disease cycle, such as the building <strong>of</strong> fungal biomes <strong>and</strong> the initiation <strong>of</strong> picnidia<br />
formation, are all under the control <strong>of</strong> host-parasite interaction. Mutations in virulence genes facilitate studying their<br />
function <strong>and</strong> genome mapping. Concurrently resistance genes can be identified, studied, <strong>and</strong> mapped. This is an exciting era<br />
for <strong>Septoria</strong> tritici/<strong>Stagonospora</strong> nodorum x wheat interaction.<br />
The septoria/stagonospora<br />
blotch diseases <strong>of</strong> wheat are incited<br />
by <strong>Septoria</strong> tritici Roberge in<br />
Desmaz. (teleomorph:<br />
Mycosphaerella graminicola (Fückel)<br />
J. Schrot. in Cohn) <strong>and</strong> by<br />
<strong>Stagonospora</strong> nodorum (Berk) E.<br />
Castellani <strong>and</strong> E. G. Germano<br />
(teleomorph: Phaeosphaeria nodorum<br />
(E. Müller) Hedjaroude),<br />
respectively. The two pathogens<br />
cause major foliar diseases <strong>of</strong><br />
wheat, inflicting considerable yield<br />
losses in many countries<br />
worldwide (King et al., 1983; Pnini-<br />
Cohen et al., 1998). It is <strong>of</strong> interest<br />
to note that these two pathogens<br />
are rare in many rice-wheat<br />
management systems in southeast<br />
Asia or others in Africa.<br />
The increased economic<br />
importance <strong>of</strong> these pathogens,<br />
especially <strong>of</strong> S. tritici, can be<br />
attributed to the cultivation <strong>of</strong><br />
susceptible cultivars with a<br />
concomitant enhancement <strong>of</strong><br />
resistance to other foliar pathogens<br />
(e.g., rusts, powdery mildew),<br />
predominance <strong>of</strong> wheat in crop<br />
management systems usually<br />
characterized by poor management<br />
<strong>of</strong> crop residues, increased nitrogen<br />
fertilization, high summer rainfall,<br />
earlier sowing, <strong>and</strong> resistance to<br />
MBC fungicides (Eyal, 1999).<br />
A surge in scientific research on<br />
these two pathogens occurred in<br />
the early 1980s, when a wide range<br />
<strong>of</strong> scientific data were published in<br />
177<br />
control-related disciplines (mostly<br />
chemical control <strong>and</strong><br />
epidemiology) <strong>and</strong> less on<br />
biological <strong>and</strong> genetic aspects<br />
associated with the pathogens<br />
(Eyal, 1999). Breeding for disease<br />
resistance lagged due to scarcity <strong>of</strong><br />
resistant germplasm <strong>and</strong> poor<br />
underst<strong>and</strong>ing <strong>of</strong> host-pathogen<br />
relationships <strong>and</strong> <strong>of</strong> how to<br />
manipulate resistance sources in<br />
breeding schemes.<br />
The shift in importance from<br />
stagonospora nodorum blotch to<br />
septoria tritici blotch (Pnini-Cohen<br />
et al., 1998) that occurred in certain<br />
European countries was explained<br />
by the biological differences in their<br />
disease cycles (shorter for S. tritici)
Concluding Remarks — Z. Eyal<br />
178<br />
<strong>and</strong> possibly due to differential<br />
sensitivity <strong>of</strong> the two pathogens to<br />
commercially used fungicides<br />
(Shaw, 1999). These explanations—<br />
which may reflect the situation in<br />
certain Western European<br />
countries—may not hold true in<br />
other wheat management systems.<br />
Still, the verification <strong>of</strong> the<br />
mechanism(s) affecting such shifts<br />
is extremely important, since it may<br />
dictate adoption <strong>of</strong> different control<br />
strategies.<br />
The sources <strong>of</strong> primary<br />
inoculum are rather variable:<br />
airborne ascospores,<br />
pycnidiospores from plant refuse,<br />
wild grasses, <strong>and</strong>, possibly, infested<br />
(infected?) seeds (Brokenshire,<br />
1975). Several wild grass species<br />
can serve as sources <strong>of</strong> inoculum to<br />
cultivated wheat, <strong>and</strong> some <strong>of</strong> them<br />
can contribute to speciation in<br />
pathogen populations. Artificial<br />
inoculation <strong>of</strong> wheat seedlings by<br />
isolates secured from grasses may<br />
not provide information on their<br />
contribution under natural<br />
infection. The possible contribution<br />
<strong>of</strong> infected seeds to the onset <strong>of</strong><br />
disease was confirmed for S.<br />
nodorum, but is not well established<br />
for S. tritici. The current world<br />
trade <strong>of</strong> grain <strong>and</strong>, for that matter,<br />
<strong>of</strong> infested/infected grain provides<br />
ample opportunities for the<br />
dissemination <strong>of</strong> inoculum<br />
worldwide if such a distribution<br />
system is operative.<br />
Global virulence surveys<br />
conducted by Eyal et al. (1985) <strong>and</strong><br />
Kema et al. (1996) have shown<br />
considerable diversity at the<br />
species level (Triticum spp.) <strong>and</strong><br />
differential response on the selected<br />
“differential” set <strong>of</strong> wheat cultivars.<br />
McDonald et al. (1999) stated that<br />
“since it has been shown that M.<br />
graminicola can infect seed<br />
(Brokenshire,1975), we consider it<br />
likely that this is also the<br />
mechanism for long distance gene<br />
flow in M. graminicola.” The<br />
authors quoted King et al. (1983)<br />
that in the case <strong>of</strong> S. nodorum, the<br />
most likely mechanism for<br />
intercontinental dispersal is<br />
infected seed. It should be noted<br />
that Brokenshire (1975) stated that<br />
“infection <strong>of</strong> seedlings from<br />
infected untreated seed samples<br />
has proved unsuccessful with<br />
Triticum dicoccum.” The report on<br />
seed infection <strong>of</strong> S. tritici by<br />
Brokenshire (1975) was not further<br />
verified <strong>and</strong> thus introduced<br />
ambiguity in the underst<strong>and</strong>ing <strong>of</strong><br />
the possible dissemination <strong>of</strong> this<br />
pathogen by seeds. The<br />
epidemiological implications <strong>of</strong><br />
naturally-infected grasses for shortscale<br />
dissemination <strong>and</strong> seeds<br />
(especially for S. tritici) for longdistance<br />
transport, warrant<br />
detailed investigations.<br />
The supposition that<br />
population genetics <strong>of</strong> M.<br />
graminicola using single-locus<br />
probes to measure gene diversity<br />
for individual RFLP loci ,<br />
population subdivision, <strong>and</strong><br />
genetic similarity among<br />
populations (McDonald et al., 1999)<br />
can be superimposed on virulence<br />
patterns was not supported in<br />
studies on populations from<br />
California <strong>and</strong> Oregon (Mundt et<br />
al., 1999). Isolates <strong>of</strong> S. tritici from<br />
these states differed significantly in<br />
their pathogenicity (Ahmed et al.,<br />
1996), yet they manifested<br />
similarity in allele frequencies as<br />
measured by RFLPs (Chen <strong>and</strong><br />
McDonald, 1996). The finding that<br />
the genetic diversity measured<br />
among 122 isolates collected in an<br />
S. tritici nursery at Patzcuaro,<br />
Mexico, was among the lowest,<br />
does not imply that the pathogen<br />
has a narrow virulence pattern. The<br />
Patzcuaro S. tritici population was<br />
reported to possess virulence to<br />
some <strong>of</strong> the most resistant wheat<br />
cultivars (e.g., Bobwhite”S” <strong>and</strong><br />
Kavkaz/K4500 L.6.A.4) (McDonald<br />
et al., 1999).<br />
Based on genetic population<br />
studies, McDonald et al. (1999)<br />
suggest that “if <strong>CIMMYT</strong> continues<br />
to use Patzcuaro as a field site to<br />
screen for resistance to M.<br />
graminicola <strong>and</strong> S. nodorum, it may<br />
want to consider introducing more<br />
diverse fungal populations from<br />
other parts <strong>of</strong> Mexico into this<br />
disease nursery.” It should be<br />
mentioned that some <strong>of</strong> the testing<br />
sites in the Patzcuaro area were<br />
artificially inoculated in the early<br />
1980s with a mixture <strong>of</strong> Mexican S.<br />
tritici isolates to ensure selection<br />
pressure on breeding materials<br />
(Santiago Fuentes, personal<br />
communication). The suggestion<br />
made by McDonald et al. (1999)<br />
implies that genetic diversity<br />
measured by r<strong>and</strong>om RFLP alleles<br />
can be used as an estimator <strong>of</strong><br />
virulence pattern. It is therefore<br />
proposed that unless genetic<br />
diversity measured by r<strong>and</strong>om<br />
alleles is not highly correlated with<br />
diversity in virulence patterns, the<br />
former should not be used as an<br />
estimator <strong>of</strong> virulence in disease<br />
resistance breeding schemes. The<br />
issue <strong>of</strong> selecting relevant fungal<br />
isolates in screening for disease<br />
resistance will be further discussed.<br />
The quantitative estimation <strong>of</strong><br />
host response percent (pycinidia<br />
<strong>and</strong>/or necrosis) for both S. tritici<br />
<strong>and</strong> S. nodorum <strong>and</strong> thereafter<br />
analyses <strong>of</strong> the interaction term <strong>of</strong><br />
cultivar x isolates by statistical<br />
means (ANOVA) have been used to<br />
estimate genetic variation in these<br />
pathosystems (Eyal <strong>and</strong> Levy,<br />
1987). Such a statistical measure
equires a predetermined set <strong>of</strong><br />
“differentiating cultivars,” which<br />
was not agreed to by <strong>Septoria</strong> tritici/<br />
<strong>Stagonospora</strong> nodorum workers,<br />
suitable methodology (sampling,<br />
culturing <strong>of</strong> pathogen, inoculum<br />
age <strong>and</strong> dosage, incubation<br />
categories, disease assessment,<br />
etc.), <strong>and</strong> controlled environmental<br />
conditions to ensure reliability <strong>of</strong><br />
results. The lack <strong>of</strong> a uniform<br />
methological approach <strong>and</strong>, in<br />
particular, <strong>of</strong> a common <strong>and</strong><br />
agreeable set <strong>of</strong> differentials<br />
introduces difficulties in comparing<br />
<strong>and</strong> drawing conclusions on a<br />
wider basis.<br />
The suggested speciation <strong>of</strong> M.<br />
graminicola on durum (Triticum<br />
durum) <strong>and</strong> common wheat (T.<br />
aestivum) may require the inclusion<br />
<strong>of</strong> differentiating cultivars <strong>of</strong> both<br />
species when they are grown<br />
together or in studies where both<br />
are tested (Kema et al., 1996;<br />
Saadaoui, 1987). Isolates from one<br />
species may not be pathogenic to<br />
the other Triticum species; still, they<br />
may show a significant interaction<br />
term when a proper set <strong>of</strong> cultivars<br />
<strong>of</strong> a species is being inoculated<br />
with isolates from the same species.<br />
Cross infection <strong>of</strong> the two species<br />
was reported for isolates secured<br />
from either Triticum species (Eyal,<br />
1999; Kema et al., 1996). The<br />
specific pathogenicity towards T.<br />
durum may have bearing on the<br />
structure <strong>of</strong> S. tritici populations<br />
(Saadaoui, 1987). Alternate<br />
cropping <strong>of</strong> bread wheat cultivars<br />
in a durum wheat management<br />
system may express a lower than<br />
expected infection level on the<br />
former than under uninterrupted<br />
bread wheat management.<br />
Today it is generally accepted<br />
that the specificity in virulence is<br />
low in S. nodorum, but can be<br />
The <strong>Septoria</strong>/<strong>Stagonospora</strong> Blotch <strong>Diseases</strong> <strong>of</strong> Wheat: Past, Present, <strong>and</strong> Future 179<br />
detected in S. tritici populations<br />
provided proper differentiating<br />
germplasm is used (Eyal, 1995).<br />
The magnitude <strong>of</strong> specificity in the<br />
S. tritici - wheat pathosystem <strong>and</strong><br />
its implications for breeding for<br />
disease resistance requires<br />
elaboration. Few dominant single<br />
genes conferring resistance were<br />
identified (e.g. Stb 1 - Bulgaria 88,<br />
Stb 2 - Veranopolis; Stb 4 - Tadinia)<br />
(Somasco et al., 1996). There are<br />
several reports that these resistant<br />
sources are not providing the<br />
claimed protection when moved<br />
across S. tritici populations with<br />
wide virulence patterns (Ballantyne<br />
<strong>and</strong> Thomson, 1995; Eyal, 1999).<br />
It is proposed that specificity<br />
<strong>and</strong> inheritance studies should be<br />
conducted with germplasm that is<br />
agronomically relevant to breeding,<br />
preferably exhibiting resistance to a<br />
wide virulence pattern (such as<br />
“Bobwhite“S”, IAS20-IASSUL, or<br />
other Frontana derivatives,<br />
Kavkaz/K4500 L.6.A.4, <strong>and</strong> other<br />
sources identified through<br />
multilocation testing) (Eyal et al.,<br />
1987). Special attention should be<br />
given to resistant wheat accessions<br />
developed in wide-cross programs.<br />
Some <strong>of</strong> these accessions may be<br />
susceptible to other foliar diseases<br />
that may need attention when<br />
incorporating <strong>Septoria</strong>/<strong>Stagonospora</strong><br />
resistance. Germplasm used in<br />
virulence studies is usually derived<br />
from disease nurseries naturally or<br />
artificially infected with the<br />
pathogen <strong>of</strong> interest. Only in a few<br />
cases has the virulence spectrum <strong>of</strong><br />
these populations been categorized.<br />
It is therefore likely that<br />
identified resistant germplasm may<br />
have only limited use either as a<br />
breeding source or as<br />
“differentials.” Resistant<br />
germplasm that has withstood<br />
prolonged testing to variable<br />
pathogen populations can be<br />
considered a potential source for<br />
breeding for resistance. Special<br />
attention should be given to avoid<br />
selecting tall stature <strong>and</strong> late<br />
maturing germplasm that may<br />
introduce such non-genetic factors<br />
into germplasm evaluation, or<br />
germplasm with poor agronomic<br />
characteristics.<br />
Isolates possessing virulence to<br />
important resistance sources can<br />
become the “core virulence<br />
spectrum” for screening<br />
germplasm. There is no criterion as<br />
yet for selecting “relevant” isolates<br />
in artificially inoculated breeding<br />
trials. The criteria dictating the<br />
choice <strong>of</strong> isolates for genetic studies<br />
<strong>of</strong> virulence (Kema et al., 1999) may<br />
use considerations other than<br />
breeding. The choice by Kema et al.<br />
(1999) <strong>of</strong> S. tritici isolates such as<br />
IPO323 (avirulent on Veranopolis,<br />
Kavkaz, <strong>and</strong> Shafir) <strong>and</strong> IP094269,<br />
which is virulent on these cultivars,<br />
in studying the genetics <strong>of</strong><br />
avirulence in this pathogen merits<br />
adoption by other <strong>Septoria</strong> tritici /<br />
<strong>Stagonospora</strong> nodorum investigators.<br />
The correlation between<br />
seedling <strong>and</strong> adult host response<br />
has been substantiated by several<br />
investigators (Eyal, 1999; Kema <strong>and</strong><br />
van Silfhout, 1997). Screening for<br />
resistance at the seedling stage<br />
does not provide an integral view<br />
<strong>of</strong> the tested germplasm. Seedling<br />
tests can serve as a supplementary<br />
measure <strong>and</strong> are an excellent tool<br />
for detailed, controlled studies on a<br />
multitude <strong>of</strong> biological issues<br />
associated with host-pathogen<br />
interactions. The seedling test as a<br />
screening measure is hampered by<br />
not knowing whether the used<br />
isolates are relevant to the<br />
virulence spectrum <strong>of</strong> the
Concluding Remarks — Z. Eyal<br />
180<br />
populations to which the test<br />
germplasm will be subjected under<br />
field conditions.<br />
Cross testing <strong>of</strong> germplasm to<br />
both seedling <strong>and</strong> field conditions<br />
is usually performed with<br />
predetermined isolate(s) that can<br />
serve as a “basic virulence set”<br />
(Eyal, 1999). Its composition may<br />
change by introducing isolates<br />
found to be virulent on specific<br />
resistant germplasm, or omitting<br />
others. The set should include<br />
isolate(s) whose virulence on<br />
specific accessions has been<br />
repeatedly confirmed. The<br />
multilocation <strong>Septoria</strong> Monitoring<br />
Nursery initiated by <strong>CIMMYT</strong> can<br />
provide information as to the<br />
magnitude <strong>of</strong> protection <strong>of</strong> some <strong>of</strong><br />
the identified sources <strong>and</strong><br />
contribute information on<br />
worldwide virulence patterns.<br />
The contribution <strong>of</strong> the sexual<br />
stage to the virulence spectrum<br />
needs special attention. Evidence<br />
from studies on population<br />
genetics emphasizes the major<br />
influence <strong>of</strong> the sexual stage on the<br />
genetic diversity <strong>of</strong> the population<br />
(Chen <strong>and</strong> McDonald, 1986; Eyal<br />
<strong>and</strong> Levy, 1987; Eyal et al., 1985;<br />
Jlibene et al., 1994; McDonald et al.,<br />
1999; Zhan et al., 1998). McDonald<br />
et al. (1999) stated that asexual<br />
reproduction may have an<br />
important impact over a limited<br />
area (e.g. few square meters),<br />
whereas sexual reproduction has<br />
much greater consequences for<br />
population genetics.<br />
The limited scope <strong>of</strong> clonality in<br />
M. graminicola <strong>and</strong> P. nodorum<br />
populations strengthens the need<br />
for comparative studies on<br />
virulence. The limitation <strong>of</strong> such<br />
studies resides with selected<br />
“wheat differentials.” This<br />
difficulty can be partly overcome<br />
by tracing specific virulences<br />
within the pathogen population on<br />
certain resistance accessions (e.g.<br />
Kavkaz/K4500 L.6.A.4). Low<br />
frequency <strong>of</strong> virulence to Kavkaz/<br />
K4500 L.6.A.4 was reported for the<br />
first time in Israel (Ezrati et al.,<br />
1998), interestingly, in Nahal Oz,<br />
where McDonald et al. (1999)<br />
detected the greatest genetic<br />
diversity using r<strong>and</strong>om RFLP<br />
alleles. Low frequency <strong>of</strong> virulence<br />
on Kavkaz/K4500 L.6.A.4 was<br />
reported in the global virulence<br />
surveys conducted by Eyal et al.<br />
(1995) <strong>and</strong> Kema et al. (1996). This<br />
germplasm was not used in<br />
international <strong>and</strong> Israeli breeding<br />
programs in the past <strong>and</strong> therefore<br />
no selective advantage can explain<br />
its detection. It is expected that the<br />
presence <strong>of</strong> virulence on this<br />
accession at Nahal Oz can be<br />
accounted for by the fact that the<br />
sexual stage is operative there, as<br />
implied by McDonald et al. (1999).<br />
Chen <strong>and</strong> McDonald (1996)<br />
hypothesized that M. graminicola<br />
isolates can be produced via<br />
genetic recombination with the<br />
same combination <strong>of</strong> virulence<br />
genes that may not have the same<br />
recent ancestors, thus contributing<br />
to multiple clonal lineages in the<br />
population. The distribution <strong>of</strong> this<br />
virulence in space (locations) <strong>and</strong><br />
time (years) is under study.<br />
The expression <strong>of</strong> virulence on<br />
certain wheat germplasm was<br />
reported to be altered upon<br />
inoculation with mixtures <strong>of</strong> S.<br />
tritici isolates (Ezrati et al., 1998;<br />
Zelikovitch <strong>and</strong> Eyal, 1991). When<br />
a resistant cultivar (e.g., Seri 82) is<br />
inoculated with a mixture <strong>of</strong><br />
avirulent (ISR398) <strong>and</strong> virulent<br />
(ISR8036) isolates, the level <strong>of</strong><br />
pycnidia produced on the seedlings<br />
<strong>and</strong> adult plants in the field<br />
resembles that produced by the<br />
avirulent isolate inoculated singly<br />
(Ezrati et al., 1998). The virulent<br />
isolate ISR8036 predominated in<br />
the population <strong>of</strong> pycnidia on both<br />
seedlings <strong>and</strong> adult plants <strong>of</strong> the<br />
susceptible cultivar Shafir<br />
inoculated with a 1:1 mixture <strong>of</strong> the<br />
two isolates. It was suggested that<br />
isolate ISR8036, which induced the<br />
same level <strong>of</strong> pycnidia on Shafir as<br />
ISR398, was more aggressive than<br />
the latter, giving it a competitive<br />
advantage in the population.<br />
The suppression phenomenon<br />
was also operative when an array<br />
<strong>of</strong> resistant cultivars (e.g.<br />
Bobwhite”S”, IAS 20-IASSUL, <strong>and</strong><br />
Kavkaz/K4500 L.6.A.4) were<br />
inoculated with appropriate<br />
mixtures <strong>of</strong> avirulent <strong>and</strong> virulent<br />
isolates (Ezrati et al., 1998). These<br />
findings are indicative <strong>of</strong> the<br />
commonality <strong>of</strong> the phenomenon<br />
where resistance induced by an<br />
avirulent S. tritici isolate can<br />
provide tissue protection against<br />
the virulent isolate. The level <strong>of</strong><br />
protection, the conditions under<br />
which it is expressed, <strong>and</strong> the<br />
mechanism(s) associated with the<br />
induction needs further<br />
investigation.<br />
It is possible that under field<br />
conditions, induced resistance may<br />
be operative under low to<br />
moderate S. tritici epidemics,<br />
provided a certain level <strong>of</strong> genetic<br />
resistance is present in the infected<br />
wheat cultivar. The reported<br />
differences in aggressiveness<br />
among isolates (Ahmed et al., 1996;<br />
Ezrati et al., 1998; Mundt et al.,<br />
1999) suggest that the structure <strong>of</strong><br />
an asexual population<br />
progressively developed on wheat<br />
plants can be strongly affected by<br />
both virulence <strong>and</strong> aggressiveness.<br />
If the sexual stage is the sole
provider <strong>of</strong> primary inoculum from<br />
distant sources (Shaw <strong>and</strong> Royle,<br />
1989) or from within the crop<br />
(Gilchrist <strong>and</strong> Velazquez, 1994), the<br />
structure <strong>of</strong> the population will<br />
probably change annually.<br />
The vertical progression within<br />
the crop <strong>of</strong> pseudothecia from<br />
lower to upper leaves (Gilchrist<br />
<strong>and</strong> Velazquez, 1994) may give an<br />
advantage to “local” populations,<br />
though it does not exclude the<br />
possibility that the primary<br />
inoculum may come from a<br />
distance <strong>and</strong> then perpetuate itself<br />
within the crop. Hunter et al. (1999)<br />
stressed that the continuous<br />
development <strong>of</strong> a functional sexual<br />
stage suggests that there is genetic<br />
exchange throughout the growing<br />
season that can respond over time<br />
to selection pressure exerted by<br />
resistant germplasm. The role <strong>of</strong><br />
the asexual stage in epidemics <strong>and</strong><br />
in formulating the virulence<br />
structure <strong>of</strong> the population under<br />
such conditions is not clear.<br />
The genetic variation for<br />
virulence <strong>and</strong> resistance in the<br />
wheat-S. tritici pathosystem, in<br />
addition to having implications for<br />
breeding for disease resistance,<br />
provides an opportunity to exp<strong>and</strong><br />
our knowledge on the biology <strong>and</strong><br />
genetics <strong>of</strong> the interaction. The<br />
recognized specificity in the<br />
relationship linked with the<br />
implementation <strong>of</strong> new DNA<br />
technology allows for a more<br />
thorough underst<strong>and</strong>ing <strong>of</strong> the<br />
pathogen, the host, <strong>and</strong> the<br />
interaction. This makes it possible<br />
to adopt methodologies from other<br />
host-pathogen systems <strong>and</strong> apply<br />
them to the economically important<br />
wheat-S. tritici/S. nodorum<br />
pathosystems.<br />
The <strong>Septoria</strong>/<strong>Stagonospora</strong> Blotch <strong>Diseases</strong> <strong>of</strong> Wheat: Past, Present, <strong>and</strong> Future 181<br />
Issues associated with the<br />
ability to infect wheat <strong>and</strong><br />
processes related to the infectioncycle<br />
(biochemical, involvement <strong>of</strong><br />
toxins, tissue necrosis, induction <strong>of</strong><br />
pycnidia formation, <strong>and</strong><br />
suppression <strong>of</strong> production),<br />
virulence (genotypic diversity <strong>and</strong><br />
population structure), pathogen<br />
migration, the potential <strong>of</strong> genetic<br />
recombination on pathogen<br />
structure <strong>and</strong> then on disease<br />
management, <strong>and</strong> genetic host<br />
resistance (specific, non-specific)<br />
are currently being investigated<br />
with the aid <strong>of</strong> molecular tools<br />
(Caten, 1999; Eyal, 1999; Kema et<br />
al., 1999; Baker et al., 1997; Eyal et<br />
al., 1985). Genetic diversity <strong>and</strong><br />
population structure are being<br />
revealed with the aid <strong>of</strong> RFLP,<br />
RAPD probes, <strong>and</strong> DNA<br />
fingerprinting (McDonald et al.,<br />
1999).<br />
The issue <strong>of</strong> mating types <strong>and</strong><br />
possible sequencing <strong>of</strong> genes<br />
associated with these types, <strong>and</strong><br />
mapping <strong>of</strong> the M. graminicola <strong>and</strong><br />
P. nodorum genomes are being<br />
investigated by Caten (1999) <strong>and</strong><br />
Kema et al. (1999). Genetically<br />
transformed S. tritici isolates <strong>and</strong><br />
tagged mutants with altered<br />
virulence are being investigated by<br />
Pnini-Cohen et al. (1998). The use<br />
<strong>of</strong> reporter gene(s) (e.g. GUS, GFP)<br />
in genetically transformed S. tritici<br />
<strong>and</strong> S. nodorum isolates <strong>and</strong><br />
immunological assays can greatly<br />
contribute to the underst<strong>and</strong>ing <strong>of</strong><br />
in planta qualitative <strong>and</strong><br />
quantitative post-inoculation<br />
events. The interrelations between<br />
isolates in a population <strong>and</strong> the<br />
effect <strong>of</strong> induced resistance <strong>and</strong><br />
competition on a pathogen<br />
population are elucidated with the<br />
aid <strong>of</strong> isolate-specific PCR primers<br />
(Ezrati et al., 1998). The elucidation<br />
<strong>of</strong> loci associated with host<br />
resistance employ QTL analyses,<br />
<strong>and</strong> genomic analysis may some<br />
day contribute to the identification<br />
<strong>of</strong> sequences linked to resistance.<br />
It is likely that genes associated<br />
with resistance to S. tritici <strong>and</strong> S.<br />
nodorum share products with<br />
structural similarities to other plant<br />
defense systems (Baker et al., 1997).<br />
The structural features shared by<br />
several R (resistance) gene products<br />
are a leucine-rich repeat (LRR)<br />
motif or a serine-threonine kinase<br />
domain. Some <strong>of</strong> these genes<br />
encode cytoplasmic receptor-like<br />
proteins that contain an LRR<br />
domain <strong>and</strong> a nucleotide binding<br />
site (NBS). It is therefore possible<br />
that these gene products are<br />
operative in wheat <strong>and</strong> can be<br />
tagged. The identification <strong>of</strong><br />
specific interactions between<br />
avirulence genes in S. tritici<br />
(AVRmg) <strong>and</strong> genes for resistance<br />
in the wheat plant may pave the<br />
way for the “genetic dissection <strong>of</strong> R<br />
gene-mediated induction <strong>of</strong><br />
hypersensitive (?)” (or suppression<br />
<strong>of</strong> symptoms in isolate mixtures ?)<br />
host defense. This insight can be<br />
used to genetically engineer wheat<br />
cultivars resistant to a broad<br />
spectrum <strong>of</strong> pathogens. It will<br />
require a better underst<strong>and</strong>ing <strong>of</strong><br />
avirulence, specific resistance, <strong>and</strong><br />
host-pathogen interactions <strong>and</strong><br />
their products in those<br />
pathosystems, prior to the<br />
formulation <strong>of</strong> a protection<br />
strategy. As a consequence, the<br />
economic <strong>and</strong> ecological (chemical<br />
protection) impact on wheat<br />
production will be reduced.
Concluding Remarks — Z. Eyal<br />
182<br />
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varying in virulence.<br />
Phytopathology 88<br />
(Supplement):S27.<br />
Gilchrist, L., <strong>and</strong> Velazquez, C. 1994.<br />
Interaction to <strong>Septoria</strong> tritici isolate<br />
<strong>of</strong> wheat on adult plant under field<br />
conditions. Pages 111-114 in:<br />
Proceedings <strong>of</strong> the 4 th International<br />
<strong>Septoria</strong> <strong>of</strong> <strong>Cereals</strong> Workshop. E.<br />
Arseniuk, T. Goral, <strong>and</strong> P. Czembor,<br />
eds.IHAR, Radzikow, Pol<strong>and</strong>.<br />
Hunter, T., Coker, R.R., <strong>and</strong> Royle, D.J.<br />
1999. The teleomorph stage,<br />
Mycosphaerella graminicola, in<br />
epidemics <strong>of</strong> septoria tritici blotch<br />
on winter wheat in the UK. Plant<br />
Pathology 48:51-57.<br />
Jlibene, M., Gustafson, J.P., <strong>and</strong><br />
Rajaram, S. 1994. Inheritance <strong>of</strong><br />
resistance to Mycosphaerella<br />
graminicola in hexaploid wheat.<br />
Plant Breeding 112:301-310.<br />
Kema, G.H.J., <strong>and</strong> van Silfhout, C.H.<br />
1997. Genetic variation for virulence<br />
<strong>and</strong> resistance in the wheat-<br />
Mycosphaerella graminicola<br />
pathosystem III. Comparative<br />
seedling <strong>and</strong> adult plant<br />
experiments. Phytopathology<br />
87:266-272.<br />
Kema, G.H.J., Sayoud, R., Annone, J.G.,<br />
<strong>and</strong> van Silfhout, C.H. 1996. Genetic<br />
variation for virulence <strong>and</strong><br />
resistance in the wheat-<br />
Mycosphaerella graminicola<br />
pathosystem II. Analysis <strong>of</strong><br />
interactions between pathogen<br />
isolates <strong>and</strong> host cultivars.<br />
Phytopathology 86:213-220.<br />
Kema, G.H.J., Verstappen, E.C.P.,<br />
Waalwijk, C., Bonants, P.J.M., de<br />
Koning, J.R.A., Hagenaar-de Weerdt,<br />
M., Hamza, S., Koeken, J.G.P., <strong>and</strong><br />
van der Lee, T.A.J. 1999. Genetics <strong>of</strong><br />
biological <strong>and</strong> molecular markers in<br />
Mycosphaerella graminicola, the cause<br />
<strong>of</strong> septoria tritici leaf blotch <strong>of</strong><br />
wheat. Pages 161-180 in: <strong>Septoria</strong> on<br />
<strong>Cereals</strong>: A Study <strong>of</strong> Pathosystems.<br />
J.A. Lucas, P. Bowyer, <strong>and</strong> H.M.<br />
Anderson, eds. CAB International,<br />
Wallingford, UK.<br />
King, J.E., Cook, R.J., <strong>and</strong> Melville, S.C.<br />
1983. A review <strong>of</strong> septoria diseases<br />
<strong>of</strong> wheat <strong>and</strong> barley. Annals <strong>of</strong><br />
Applied Biology 103:345-373.<br />
McDonald, B.A., Zhan, J., Yarden, O.,<br />
Hogan, K., Garton, J., <strong>and</strong> Pettway,<br />
R.E. 1999. The population genetics<br />
<strong>of</strong> Mycosphaerella graminicola <strong>and</strong><br />
<strong>Stagonospora</strong> nodorum. Pages 44-69<br />
in: <strong>Septoria</strong> on <strong>Cereals</strong>: A Study <strong>of</strong><br />
Pathosystems. J.A. Lucas, P. Bowyer,<br />
<strong>and</strong> H.M. Anderson, eds. CAB<br />
International, Wallingford, UK.<br />
Mundt, C.C., H<strong>of</strong>fer, M.E., Ahmed,<br />
H.U., Coakley, S.M., DiLeone, J.A.,<br />
<strong>and</strong> Cowger, C. 1999. Pages 115-130<br />
in: <strong>Septoria</strong> on <strong>Cereals</strong>: A Study <strong>of</strong><br />
Pathosystems. J.A. Lucas, P. Bowyer,<br />
<strong>and</strong> H.M. Anderson, eds. CAB<br />
International, Wallingford, UK.<br />
Pnini-Cohen, S., Zilberstein, A., <strong>and</strong><br />
Eyal, Z. 1998. Molecular tools for<br />
studying <strong>Septoria</strong> tritici virulence. In:<br />
Proceedings 7 th International<br />
Congress Plant Pathology. 9-16<br />
August, 1998, Edinburgh, Scotl<strong>and</strong>,<br />
UK.<br />
Polley, R.W., <strong>and</strong> Thomas, M.R. 1991.<br />
Surveys <strong>of</strong> diseases <strong>of</strong> winter wheat<br />
in Engl<strong>and</strong> <strong>and</strong> Wales, 1976-1988.<br />
Annals <strong>of</strong> Applied Biology 119:1-20.<br />
Saadaoui, E.M. 1987. Physiologic<br />
specialization <strong>of</strong> <strong>Septoria</strong> tritici in<br />
Morocco. Plant Disease 71:153-155.<br />
Shaw, M.W. 1999. Population dynamics<br />
<strong>of</strong> <strong>Septoria</strong> in the crop ecosystem.<br />
Pages 82-95 in: <strong>Septoria</strong> on <strong>Cereals</strong>:<br />
A Study <strong>of</strong> Pathosystems. J.A.<br />
Lucas, P. Bowyer, <strong>and</strong> H.M.<br />
Anderson, eds. CAB International,<br />
Wallingford, UK.<br />
Shaw, M.W., <strong>and</strong> Royle, D.J. 1989.<br />
Airborne inoculum as a major<br />
source <strong>of</strong> <strong>Septoria</strong> tritici<br />
(Mycosphaerella graminicola)<br />
infections in winter wheat crops in<br />
the UK. Plant Pathology 38:35-43.<br />
Somasco, O.A., Qualset, C.O., <strong>and</strong><br />
Gilchrist, D.G. 1996. Single-gene<br />
resistance to septoria tritici blotch in<br />
the spring wheat cultivar Tadinia.<br />
Plant Breeding 115:261-267.<br />
Zelikovitch, N., <strong>and</strong> Eyal, Z. 1991.<br />
Reduction in pycnidial coverage<br />
after inoculation <strong>of</strong> wheat with<br />
mixtures <strong>of</strong> isolates <strong>of</strong> <strong>Septoria</strong> tritici.<br />
Plant Disease 75:907-910.<br />
Zhan, J., Mundt, C.C., <strong>and</strong> McDonald,<br />
B.A. 1998. Measuring immigration<br />
<strong>and</strong> sexual reproduction in field<br />
populations <strong>of</strong> Mycosphaerella<br />
graminicola. Phytopathology<br />
88:1330-1337.
List <strong>of</strong> Participants<br />
Mr. Shaukat Ali<br />
Graduate Student<br />
North Dakota State University<br />
320 Walster Hall<br />
Fargo ND 58105<br />
Phone: (701) 231-7855<br />
Email: sali@prairie.NoDak.edu<br />
Ms. Lia Arraiano<br />
Phd Student<br />
John Innes Centre<br />
Colney Lane<br />
Norwich Research Park<br />
Norwich Norfolk NR4 7UH<br />
United Kingdom<br />
Phone: +44 (1603) 452571 ext. 2618<br />
Fax: +44 (1603) 502241<br />
Email: lia.arraiano-e-castroalves@bbsrc.ac.uk<br />
Pr<strong>of</strong>essor Edward Arseniuk<br />
Plant Breeding <strong>and</strong> Acclimatization<br />
Institute<br />
Radzikow<br />
05-870 Blonie<br />
Pol<strong>and</strong><br />
Phone: (48-22) 725 4536; (48-22) 349 470<br />
[home]<br />
Fax: (48-22) 725 4714<br />
Email: e.arseniuk@ihar.edu.pl<br />
Dr. Gary C. Bergstrom<br />
Pr<strong>of</strong>essor<br />
Department <strong>of</strong> Plant Pathology<br />
Cornell University<br />
334 Plant Science Building<br />
Ithaca NY 14853-4203<br />
Phone: +1(607) 255-7849<br />
Fax: +1(607) 255-4471<br />
Email: gcb3@cornell.edu<br />
Dr. Penny Brading<br />
Post-Doctoral Scientist<br />
<strong>Cereals</strong> Research Dept.<br />
John Innes Centre<br />
Colney Lane<br />
Norwich Research Park<br />
Norwich Norfolk NR4 7UH<br />
United Kingdom<br />
Phone: +44 (1603) 452571 ext. 2618<br />
Fax: +44 (1603) 502241<br />
Email: penelope.brading@bbsrc.ac.uk<br />
Dr. James K.M. Brown<br />
Pathologist & Geneticist<br />
<strong>Cereals</strong> Research Department<br />
John Innes Centre<br />
Colney Lane<br />
Norwich Norfolk NR4 7UK<br />
United Kingdom<br />
Phone: +44 (1603) 452571<br />
Fax: +44 (1603) 456844<br />
Email: james.brown@bbsrc.ac.uk<br />
Dr. Cristina C. A. Cordo<br />
Facultad de Agronomia<br />
Catedra de Fitopatologia La Plata<br />
Universidad Nacional de la Plata<br />
Calle 60 y 119; c.c. 31<br />
La Plata 1900<br />
Argentina<br />
Phone: +54 0221 4 83 18 31<br />
Fax: +54 0221 4 25 23 46<br />
Email: criscordo@infovia.com.ar<br />
Ms. Christina Cowger<br />
Graduate Research Assistant<br />
Botany Dept.<br />
Oregon State University<br />
2082 Cordley Hall<br />
Corvallis OR 97331-2902<br />
Phone: +1 (541) 737-4408<br />
Fax: +1 (541) 737-3573<br />
Email: cowgerc@bcc.orst.edu<br />
Dr. Barry M. Cunfer<br />
Pr<strong>of</strong>essor<br />
Dept. Plant Pathology<br />
University <strong>of</strong> Georgia<br />
1109 Experiment St.<br />
Griffin GA 30223-1797<br />
United States<br />
Phone: +1(770) 412 4012<br />
Fax: +1(770) 228 7305<br />
Email: bcunfer@gaes.griffin.peachnet.edu<br />
Dr. Pawel Czembor<br />
Research Assistant<br />
Plant Breeding & Acclimatization Inst.<br />
Radzikow<br />
05-870 Blonie<br />
Pol<strong>and</strong><br />
Phone: +48 (22) 7253611<br />
Fax: +48 (22) 7254714<br />
Email: p.czembor@ihar.edu.pl<br />
Pr<strong>of</strong>. Amos Dinoor<br />
Pr<strong>of</strong>essor <strong>of</strong> Plant Pathology<br />
Faculty <strong>of</strong> Agriculture, Food <strong>and</strong><br />
Environmental Quality Sciences<br />
The Hebrew University <strong>of</strong> Jerusalem<br />
P.O. Box 12<br />
Rehovot 76100<br />
Israel<br />
Phone: + 972 (8) 9481358<br />
Fax: +972 (8) 9466794<br />
Email: dinoor@agri.huji.ac.il<br />
Dr. Annika Djurle<br />
Plant Pathology 1<br />
Department <strong>of</strong> Ecology <strong>and</strong> Crop<br />
Production Sciences<br />
SLU<br />
P.O. Box 7043<br />
SE-75007 UPPSALA<br />
Sweden<br />
Phone: +46 (18) 67 16 02<br />
Fax: +46 (18) 67 28 90<br />
Email: annikad@pinus.slu.se<br />
Mr. Keith E. Duncan<br />
Biologist<br />
DuPont Agricultural Biotechnology<br />
E. I. DuPont de Nemours Co., Inc.<br />
E402 Experimental Station<br />
Wilmington , DE 19880-0402<br />
United States<br />
Phone: 302 695 4298; 302 695<br />
3891(laboratory)<br />
Fax: 302 695 4509<br />
Email: keith.e.duncan@usa.dupont.com<br />
Ms. Clare Marie Ellerbrook<br />
Research Scientist<br />
John Innes Centre<br />
Colney Lane<br />
Norwich UK NR4 7UH<br />
United Kingdom<br />
Phone: +44 (1603) 452571<br />
Fax: +44 (1603) 456844<br />
Email: clare.ellerbrook@bbsrc.ac.uk<br />
Ms. Smadar Ezrati<br />
Ph. D. Student<br />
Department <strong>of</strong> Botany<br />
Tel-Aviv University<br />
Tel Aviv 69978<br />
Israel<br />
Phone: +972 (3) 640 9766<br />
Fax: +972 (3) 640 9380<br />
Email: ezrati@post.tau.ac.il<br />
Dr. Lucy Gilchrist<br />
Pathologist<br />
Wheat Program<br />
<strong>CIMMYT</strong><br />
Lisboa 27, Col. Juárez<br />
Delegación Cuauhtemoc<br />
Apdo. Postal 6-641<br />
06600 Mexico D.F.<br />
Mexico<br />
Phone: +52 5804 2004<br />
Fax: +52 5804 7558/9<br />
Email: l.gilchrist@cgiar.org<br />
183<br />
Ing. Rebeca Margarita Gonzalez Iñiguez<br />
Investigador de Trigo y Triticale<br />
Instituto Nacional de Investigaciones<br />
Forestales y Agropecuarias<br />
Silvestre Guerrero 449, Colonia 5 de<br />
Diciembre<br />
58280 Morelia Mich.<br />
Mexico<br />
Phone: (43) 15 9489 y 15 9021<br />
Fax: (43) 151091<br />
Dr. Stephen B. Goodwin<br />
Plant Pathologist<br />
Department <strong>of</strong> Botany <strong>and</strong> Plant Pathology<br />
USDA/ARS Purdue University<br />
1155 Lily Hall<br />
West Lafayette IN 47907-1155<br />
United States<br />
Phone: +1(765) 494-4635<br />
Fax: +1(765) 494-0363<br />
Email: goodwin@btny.purdue.edu
184<br />
Dr. Patrice Halama<br />
Pr<strong>of</strong>essor<br />
Institut Superieur d’Agriculture<br />
41 rue du port<br />
59046 Lille cedex<br />
France<br />
Phone: +33 (03) 28 38 48 48<br />
Fax: +33 (03) 28 38 48 47<br />
Email: p.halama@isa.fupl.asso.fr<br />
Dr. Sonia Hamza<br />
Associate Pr<strong>of</strong>essor<br />
Laboratoire de Genetique<br />
INAT<br />
43 Av. Chrales Nicolle<br />
1082 El Mahrajene Tunis<br />
Tunisia<br />
Phone: +216 (1) 840 270<br />
Fax: +216 (1) 799 391<br />
Email: hamza.sonia@inat.agrinet.tn<br />
Dr. Karen K. Hanson<br />
Cereal Pathology Specialist<br />
Plant Pathology<br />
Zeneca Agrochemicals<br />
Jealott’s Hill Research Station<br />
RG42 6ET Bracknell Berishire<br />
Ukraine<br />
Phone: +44 (01344) 414488<br />
Fax: +44 (01344) 414502<br />
Email:<br />
Karen.Hanson@AGUK.ZECECA.com<br />
Pr<strong>of</strong>. Mouncef Harrabi<br />
Pr<strong>of</strong>essor <strong>and</strong> Director General<br />
Crop Science Department<br />
INAT<br />
43 Ave. Charles Nicolle<br />
1082 Tunis<br />
Tunisia<br />
Phone: +216 (1) 840270<br />
Fax: +216 (1) 799391<br />
Email: harrabi.moncef@inat.agrinet.tn<br />
Dr. Pavel Horcicka<br />
Head<br />
Wheat Breeding Department<br />
SELGEN<br />
Plant Breeding St. Stupice<br />
25084 Sibrina<br />
Czech Republic<br />
Phone: +42 (2) 81972462<br />
Fax: +42 (2) 81970465<br />
Email: horcicka@zero.cz<br />
Dr. Jerry W. Johnson<br />
Wheat Breeder<br />
Crop <strong>and</strong> Soil Sciences Division<br />
University <strong>of</strong> Georgia<br />
Georgia Station<br />
Griffin GA 30223<br />
United States<br />
Phone: +1 (770) 228 7321<br />
Fax: +1 (770) 229 3215<br />
Email: jjohnso@gaes.griffin.peachnet.edu<br />
Ms. Lise Nistrup Jorgensen<br />
Senior Scientist<br />
Research Centre Flakkebjerg<br />
Danish Institute <strong>of</strong> Agricultural Sciences<br />
Slagelse<br />
DK-4200 Flakkebjerg<br />
Denmark<br />
Phone: +45 (58) 113300<br />
Fax: +45 (58) 113301<br />
Email: LiseN.Jorgensen@agrsci.dk<br />
Dr. Ute Kastirr<br />
Scientific Collatorator<br />
Federal Centre for Breeding Research on<br />
Cultivated Plants<br />
Institute for Resistance Research <strong>and</strong><br />
Pathogendiagnostics<br />
Theodor-Roemer-Weg 4<br />
D-06449 Aschersleben<br />
Germany<br />
Phone: +49 (0) 3473 879197<br />
Fax: +49 (0) 3473 879200<br />
Email: u.kastirr@bromo.qlb.bafz.de<br />
Dr. Gert H.J. Kema<br />
Senior Scientist<br />
IPO-DLO<br />
P.O. Box 9060<br />
6700 GW Wageningen<br />
Netherl<strong>and</strong>s<br />
Phone: +31 317 476149<br />
Fax: +31 317 410113<br />
Email: G.H.J.Kema@IPO.DLO.NL<br />
Mr. Awgechew Kidane<br />
Quarantine Pathologist<br />
Ethiopian Agricultural Research<br />
Organization,<br />
Holetta ARC<br />
P.O. Box 2003<br />
Addis Ababa<br />
Ethiopia<br />
Phone: +251<br />
Fax: +251<br />
Email: <strong>CIMMYT</strong>-Ethiopia@cgiar.org<br />
Dr. Theodore J. Kisha<br />
Research Agronomist<br />
Agronomy Department<br />
Purdue University<br />
1150 Lilly Hall<br />
West Lafayette IN 47907<br />
United States<br />
Phone: +1 (765) 496 1917<br />
Fax: +1 (765) 496 2926<br />
Email: tkisha@purdue.edu<br />
Dr. Holger Klink<br />
Plant Pathologist at the University <strong>of</strong> Kiel<br />
Inst. for Phytopathlogie<br />
University <strong>of</strong> Kiel<br />
Hermann-Rodewald-Str.9<br />
D-24118 KIEL<br />
Germany<br />
Phone: +49 431 880 2994<br />
Fax: +49 461 880 1583<br />
Email:<br />
Barbara.Muth@ageurope.zeneca.com;<br />
Evelyn.Badeck@ageurope.zeneca.com<br />
Notes: HK@phytomet.uni.kiel.de<br />
Dr. Manfred Konradt<br />
Technical Manager<br />
ZENECA Agrochemicals<br />
Cmil-von-Behringstr.2<br />
D-60439 Frankfurt/Main<br />
Germany<br />
Phone: +49 069 58 01 414<br />
Fax: +49 069 5801 672<br />
Email: Barbara.Muth@ageurope.zeneca.com;<br />
Evelyn.Badeck@ageurope.zeneca.com<br />
Notes:<br />
Manfred.Konradt@geurope.zeneca.com<br />
Dr. Joseph M. Krupinsky<br />
Research Plant Pathologist<br />
Northern Great Plains Research Lab<br />
USDA-ARS<br />
P.O. Box 459<br />
M<strong>and</strong>an ND 58554-0459<br />
United States<br />
Phone: +1(701) 667-3011<br />
Fax: +1(701) 667-3054<br />
Email: Dvorakl@m<strong>and</strong>an.ars.usda.gov;<br />
krupinsj@m<strong>and</strong>an.ars.usda.gov<br />
Dr. Steven Leath<br />
Research Plant Pathologist<br />
Department <strong>of</strong> Plant Pathology<br />
USDA-ARS<br />
Box 7616, NCSU<br />
Raleigh NC 27695<br />
United States<br />
Phone: 919-515 6819<br />
Fax: 919-515 7716<br />
Email: Judith_Sulentic@ncsu.edu<br />
Notes: steven_leath@ncsu.edu<br />
Dr. Robert Loughman<br />
Senior Plant Pathologist<br />
Plant Protection Branch<br />
Agriculture Western Australia<br />
Plant Research <strong>and</strong> Development Services<br />
Locked Bag No. 4<br />
Bentley Delivery Centre W.A. 6983<br />
Australia<br />
Phone: +61 (618) 93683691<br />
Fax: +61 (618) 93672625<br />
Email: jtoms@agric.wa.gov.au;<br />
rloughman@agric.wa.gov.au<br />
Pr<strong>of</strong>. Dr. Bruce McDonald<br />
Group Leader<br />
Institute <strong>of</strong> Plant Sciences/Phytopathology<br />
Federal Insitute <strong>of</strong> Technology<br />
ETH-Zentrum, LFW<br />
Universitaetstr 2/LFW-B16<br />
CH-8092 Zuerich<br />
Switzerl<strong>and</strong><br />
Phone: +41 (1) 632 3847<br />
Fax: +41 (1) 632 15 72<br />
Email: Bruce.McDonald@ipw.agrl.ethz.ch
Dr. Ehud Meidan<br />
Wheat Breeder<br />
HAZERA Quality Seeds<br />
Mivhor<br />
M.P. Lachish-Dargm 75354<br />
Israel<br />
Phone: +972 (7) 6878155<br />
Fax: +972 (7) 6814057<br />
Email: Udi_Meidan@hazera.com<br />
Dr. Eugene Milus<br />
Associated Pr<strong>of</strong>essor<br />
Department <strong>of</strong> Plant Pathology<br />
University <strong>of</strong> Arkansas<br />
217 Plant Science Bldg.<br />
Fayetteville AR 72701<br />
United States<br />
Phone: +1(501) 575-2676<br />
Fax: +1(501) 575-7601<br />
Email: gmilus@comp.uark.edu<br />
Miss Mihaela V. Mincu<br />
Junior Research worker<br />
Research Institute for <strong>Cereals</strong> <strong>and</strong><br />
Industrial Crops<br />
FUNDULEA<br />
CP 22-171<br />
Bucuresti<br />
Romania<br />
Phone: +40 3154040<br />
Fax: +40 3110722<br />
Email: fundulea@cons.incerc.ro<br />
Mrs. Rose John Mongi<br />
Wheat Breeder<br />
MARTI-Uyole<br />
<strong>CIMMYT</strong><br />
P.O. Box 400<br />
Mbeya<br />
Tanzania<br />
Phone: + 255 (1) 614-645<br />
Fax: +255<br />
Email: c/o <strong>CIMMYT</strong>-Ethiopia@cgiar.org<br />
Dr. Chris C. Mundt<br />
Pr<strong>of</strong>essor<br />
Dept. <strong>of</strong> Botany <strong>and</strong> Plant Pathology<br />
Oregon State University<br />
2082 Cordley Hall<br />
Corvallis OR 97331-2902<br />
United States<br />
Phone: +1 541 737 5256<br />
Fax: +1 541 737 3573<br />
Email: mundtc@bcc.orst.edu<br />
Dr. Noel E.A. Murphy<br />
Research Fellow<br />
Murdoch University<br />
DSE South St<br />
Murdoch 6150<br />
Australia<br />
Phone: +61 (8) 9360 6097<br />
Fax: +61 (8) 9360 6303<br />
Email: nmurphy@central.murdoch.edu.au<br />
Dr. Lloyd R. Nelson<br />
Wheat Breeder <strong>and</strong> Pr<strong>of</strong>essor<br />
Texas A&M Research <strong>and</strong> Extension Center<br />
P.O. Box 200<br />
Overton TX 75684-0200<br />
United States<br />
Phone: +1(903) 834 6191<br />
Fax: +1(903)-834 7140<br />
Email: Ir-nelson@tamu.edu<br />
Ms. Guita Cordsen Nielsen<br />
Senior Adviser<br />
The National Department <strong>of</strong> Plant<br />
Production<br />
The Danish Agricultural Advisory Centre<br />
Udkaersvej 15<br />
DK-8200 Skejby Aarhus<br />
Denmark<br />
Phone: +45 (87) 405439; +45 (87) 405000;<br />
+45 20282695 mobile<br />
Fax: +45 (87) 405010<br />
Email: gcn@lr.dk<br />
Notes: Damgaard, Vroldvej 168; DK-8660<br />
Skaderborg; Phone: +45 (86) 57 98 00<br />
Dr. Thomas S. Payne<br />
Wheat Breeder/Pathologist<br />
Wheat Program<br />
<strong>CIMMYT</strong>-East Africa<br />
P.O. Box 5689<br />
Addis Ababa<br />
Ethiopia<br />
Phone: +251 (1) 615-127<br />
Fax: +251 (1) 614-645<br />
Email: t.payne@cgiar.org<br />
Telex: 21207 ILCA ET<br />
Dr. Sanjaya Rajaram<br />
Director<br />
Wheat Program<br />
<strong>CIMMYT</strong><br />
Lisboa 27, Col. Juárez<br />
Delegación Cuauhtemoc<br />
Apdo. Postal 6-641<br />
06600 Mexico D.F.<br />
Mexico<br />
Phone: +52 5804 2004<br />
Fax: +52 5804 7558/9<br />
Email: s.rajaram@cgiar.org<br />
Dr. Albert L. Scharen<br />
Pr<strong>of</strong>essor Emeritus<br />
Department <strong>of</strong> Plant Sciences<br />
Montana State University<br />
Bozeman MT 59717<br />
United States<br />
Phone: +1(406) 994-5162<br />
Fax: +1 (406) 994-1848<br />
Email: uplas@montana.campuscwix.net<br />
Mrs. Silvia Schuster<br />
Research Assitant<br />
Plant Sciences Department<br />
Tel-Aviv University<br />
Tel Aviv<br />
Israel<br />
Phone: +972 (3) 640 9766<br />
Fax: +972 (3) 640 9380<br />
Email: schus@post.tau.ac.il<br />
Dr. Gregory Shaner<br />
Pr<strong>of</strong>essor<br />
Botany <strong>and</strong> Plant Pathology<br />
Purdue University<br />
1155 Lilly Hall <strong>of</strong> Life Sciences<br />
West Lafayette IN 47907-1155<br />
United States<br />
Phone: +1(765) 494 4651<br />
Fax: +1 (765) 494 0363<br />
Email: shaner@btny.purdue.edu<br />
Dr. Michael W. Shaw<br />
Reader, Sch. <strong>of</strong> Plant Sciences<br />
The University <strong>of</strong> Reading<br />
Whiteknights<br />
Reading, RG6 6AS UK<br />
United Kingdom<br />
Phone: +44 118 931 8091<br />
Fax: +44 118 931 6577<br />
Email: M.W.Shaw@reading.ac.uk<br />
Dr. Ravi P. Singh<br />
Rust Genetist<br />
Wheat Program<br />
<strong>CIMMYT</strong><br />
Lisboa 27, Col. Juárez<br />
Delegación Cuauhtemoc<br />
Apdo. Postal 6-641<br />
06600 Mexico D.F.<br />
Mexico<br />
Phone: +52 5804 2004<br />
Fax: +52 5804 7558/9<br />
Email: r.singh@cgiar.org<br />
Dr. Bent Skovm<strong>and</strong><br />
Head, Wheat Genetic Research<br />
Wheat Program<br />
International Maize <strong>and</strong> Wheat<br />
Improvement Center<br />
Lisboa 27, Col. Juárez<br />
Delegación Cuauhtemoc<br />
Apdo. Postal 6-641<br />
06600 México D.F.<br />
Mexico<br />
Phone: +52 5804 2004 ext. 2226<br />
Fax: +52 5804 7558/9<br />
Email: b.skovm<strong>and</strong>@cgiar.org<br />
Dr. Brian J. Steffenson<br />
Associate Pr<strong>of</strong>essor<br />
Department <strong>of</strong> Plant Pathology<br />
North Dakota State University<br />
P.O. Box 5012<br />
Fargo ND 58105-5012<br />
United States<br />
Phone: +1(701)231-7078<br />
Fax: +1(701)231-7851<br />
Email: bsteffen@badl<strong>and</strong>s.nodak.edu<br />
Dr. Enrique Torres<br />
Calle 72 A No. 16-15 (301)<br />
Bogota<br />
Colombia<br />
Phone: +57-1-2359861, dentro Colombia:<br />
91-2359861<br />
Fax: +57-1-2359861<br />
Email: etorres@hotmail.com<br />
185
186<br />
Dr. Hala Toubia-Rahme<br />
Research Associate<br />
Dept. <strong>of</strong> Plant Pathology<br />
North Dakota State University<br />
P.O. Box 5012<br />
Fargo ND 58105-5012<br />
United States<br />
Phone: +1 (701) 231 7018<br />
Fax: +1 (701) 231 7851<br />
Email:<br />
Hala_Toubia_rahme@ndsu.nodak.edu<br />
Dr. Ludvik Tvaruzek<br />
Research Worker<br />
Agricultural Research Insitute Kromeriz<br />
Havlickova 2787<br />
767 01 Kromeriz<br />
Czech Republic<br />
Phone: +42 634317138<br />
Fax: +42 63422725<br />
Email: tvaruzek@vukrom.cz<br />
Dr. Peter Ueng<br />
Scientist<br />
Plant Molecular Biology Laboratory<br />
USDA-ARS<br />
MPPL, BARC-West, Bldg. 011A<br />
Beltsville MD 20705<br />
United States<br />
Phone: +1 (301) 504 6308<br />
Fax: +1 (301) 504 5449<br />
Email: pueng@asrr.arsusda.gov<br />
Dr. Maarten van Ginkel<br />
Head, Bread Wheat Program<br />
Wheat Program<br />
<strong>CIMMYT</strong><br />
Lisboa 27, Col. Juárez<br />
Delegación Cuauhtemoc<br />
Apdo. Postal 6-641<br />
06600 México D.F.<br />
Mexico<br />
Phone: +52 5804 2004<br />
Fax: +52 5804 7558/9<br />
Email: m.van-ginkel@cgiar.org; http://<br />
www.cimmyt.mx<br />
Ms. Carmen Velazquez<br />
Wheat Program<br />
<strong>CIMMYT</strong><br />
Lisboa 27, Col. Juarez<br />
Delegacion Cuauhtemoc<br />
Apdo. Postal 6-641<br />
Mexico D.F.<br />
Mexico<br />
Phone: +52 5804 2004<br />
Fax: +52 5804 7558/9<br />
Email: c.velazquez@cgiar.org<br />
Pr<strong>of</strong>. Dr. Joseph Alex<strong>and</strong>er Verreet<br />
Pr<strong>of</strong>essor<br />
Inst. for Phytopathlogie<br />
University <strong>of</strong> Kiel<br />
Hermann-Rodewald-Str.9<br />
D-24118 KIEL<br />
Germany<br />
Phone: +49 431 880 2996<br />
Fax: +49 461 880 1583<br />
Email:<br />
Barbara.Muth@ageurope.zeneca.com;<br />
Evelyn.Badeck@ageurope.zeneca.com<br />
Notes: JAV@phytomet.uni.kiel.de<br />
Mr. Robin Wilson<br />
Senior Wheat Breeder<br />
Crop Industries<br />
Agriculture Western Australia<br />
Locked Bag No. 4<br />
Bentley Delivery Centre W.A. 6983<br />
Australia<br />
Phone: +61 (618) 9368 3691<br />
Fax: +61 (618) 9367 2625<br />
Email: rwilson@agric.wa.gov.au<br />
Dr. Bruno Zwatz<br />
Head <strong>of</strong> the Institute<br />
Federal Office <strong>and</strong> Research Centre for<br />
Agriculture<br />
Institute <strong>of</strong> Phytomedicine<br />
Spargelfeldstrasse 191<br />
Vienna Wien A-1226<br />
Austria<br />
Phone: +43(1)73216-5500<br />
Fax: +43(1)73216-5194<br />
Email: bzwatz@bfl.gv.at;<br />
margarethe.zaufal@relay.bfl.at
ISBN: 970-648-035-8<br />
International Maize <strong>and</strong> Wheat Improvement Center<br />
Centro Internacional de Mejoramiento de Maíz y Trigo<br />
Lisboa 27, Apartado postal 6-641 México, D.F., México