AN ABSTRACT OF THE THESIS OF
Christina Cowger for the degree of Doctor of Philosophy in Botany and Plant Pathology,
presented on March 19, 2002. Title: Effects of Host Resistance on Mycosphaerella
graminicola Populations.
Abstract approved:
Redacted for Privacy
Christopher C. Mundt
Mycosphaerella graminicola (anamorpltSeptoria tritici) causes Septoria tritici blotch, a
globally important disease of winter wheat. Resistance and pathogenicity generally vary
quantitatively. The pathogen reprQducesbotksexually and asexually, and th pathogen
population is highly genetically variable. Several unresolved questions about the
epidemio logy ofihis pathosystentare addressed by thisresearch. Among them are
whether cultivar-isolate specificity exists, how partial host resistance affects pathogen
aggressiveness and sexualreproduction, and how host genotype mixtures influence
epidemic progression and pathogenicity.
Atits release in 1992, the cultivar Gene was highly resistant to M graminicola, but that
resistance had substantially dissolved by 1995. Six of seven isolates collected in 1997
ftm field plots of Gene were virulent to Gene seedlings in the greenhouse, while 14 of
15 isolates collected from two other cultivars were avirulent to Gene. Gene apparently
selected for strains of
M graminicola with specific virulence to it.
In a two-year experiment, isolates were collected early and late in the growing season
from field plots of three moderately resistant and three susceptible cultivars, and tested on
seedlings of the same cultivars in the greenhouse. Isolates were also collected from plots
of two susceptible cultivars sprayed with a fungicide to suppress epidemic development.
Isolate populations were more aggressive when derived from moderately resistant than
from susceptible cultivars, and more aggressive from fungicide-sprayed plots than from
unsprayed plots of the same cultivars.
Over 5,000 fruiting bodies were collected in three years from replicated field plots of
eight cultivars with different levels of resistance. The fruiting bodies were identified as
M graminicola ascocarps or pycnidia, or other. In all three years, the frequency of
ascocarps was positively correlated with cultivar susceptibility, as measured by area
under the disease progress curve, and was also positively associated with epidemic
intensity.
For three years, four 1:1 mixtures of a moderately resistant and a susceptible wheat
cultivar were planted in replicated field plots. Isolates from the plots were inoculated as
bulked populations on greenhouse-grown seedlings of the same four cultivars. Mixture
effects on disease progression varied among the years, and were moderately correlated
with mixture effects on pathogenicity.
Effects of Host Resistance on Mycosphaerella graminicola Populations
by
Christina Cowger
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctor of Philosophy
Presented March 19, 2002
Commencement June 2002
Doctor of Philosophy thesis of Christina Cowger presented on March 19. 2002.
APPROVED:
Redacted for Privacy
Major Professor, representing Botany and Plant Pathology
Redacted for Privacy
Chair of Department of Botany and Plant
Redacted for Privacy
Dean of tl&Graduate School
I understand that my thesis will become part of the permanent collection of Oregon State
University libraries. My signature below authorizes release of my thesis to any reader
upon request.
Redacted for Privacy
Christina Cowger, Author
ACKNOWLEDGEMENT
My heartfelt appreciation to Chris Mundt, my advisor, for excellence in guidance,
collaboration, teaching, values, and professional judgment. Without his consistent,
generous support and good management, the research would have been impossible and
maternity could not have accompanied my graduate education.
My sincere thanks to Molly Hoffer, Dan Coyle, and LaRae Wallace for their
skilled and cheerful participation in the technical aspects of the research.
Many thanks also to my committee members Carol Mallory-Smith, Jim Peterson,
Mary Powelson, and Dave Thomas, and to Lynda Ciuffetti, Ken Johnson, and Cliff
Pereira, for their patience, guidance, and contributions to the development of the thesis.
Appreciation is also extended for the grant that made these investigations
possible, award number 97-35303-453
7
of the NRI Competitive Grants Program/USDA.
Last but not least, I thank my family members and friends for their highly valued
love and support over the years of this work.
CONTRIBUTION OF AUTHORS
Molly Hoffer conducted greenhouse research with the isolates collected in 1992
that are described in Chapter II. Dr. Bruce McDonald developed the protocol used for
sampling fruiting bodies from wheat leaf tissue in Chapter IV, and assisted in writing and
editing that chapter.
TABLE OF CONTENTS
CHAPTERI: Introduction .................................................................................................. 1
CHAPTER II: Specific Adaptation by Mycosphaerella graminicola to a Vertically
ResistantWheat Cultivar ................................................................................................... 11
Abstract .................................................................................................................. 12
Introduction ............................................................................................................ 12
Materialsand Methods ........................................................................................... 14
Results .................................................................................................................... 20
Discussion .............................................................................................................. 23
Acknowledgements ................................................................................................ 29
References .............................................................................................................. 30
CHAPTER ifi: Aggressiveness of Mycosphaerella graminicola Isolates from
Susceptible and Partially Resistant Wheat Cultivars ......................................................... 33
Abstract .................................................................................................................. 34
Introduction ............................................................................................................ 35
Materials and Methods ........................................................................................... 37
Results .................................................................................................................... 45
Discussion .............................................................................................................. 50
Acknowledgements ................................................................................................ 56
LiteratureCited ...................................................................................................... 57
CHAPTER IV: Frequency of Sexual Reproduction by Mycosphaerella graminicola
on Partially Resistant Wheat Cultivars .............................................................................. 60
Abstract .................................................................................................................. 61
Table of Contents, Continued
Introduction............................................................................................................ 62
Materialsand Methods ........................................................................................... 64
Results.................................................................................................................... 72
Discussion .............................................................................................................. 79
Acknowledgements ................................................................................................ 84
LiteratureCited ...................................................................................................... 85
CHAPTER V: Effects of Wheat Cultivar Mixtures on Epidemic Progression of
Septoria Tntici Blotch and Pathogenicity of Mycosphaerella graminicola ...................... 89
Abstract .................................................................................................................. 90
Introduction............................................................................................................ 91
Materials and Methods ...........................................................................................94
Results.................................................................................................................... 99
Discussion ............................................................................................................ 108
Acknowledgements .............................................................................................. 112
LiteratureCited .................................................................................................... 113
CHAPTER VI: Summary ............................................................................................... 117
Cultivar-isolate specificity ................................................................................... 117
Selective effects on aggressiveness ..................................................................... 117
Pathogen sexual reproduction .............................................................................. 118
Host genotype mixture effects on epidemics and pathogenicity ......................... 119
Disease management implications ....................................................................... 120
BIBLIOGRAPHY ............................................................................................................ 121
LIST OF FIGURES
Figure
ig
11.1.
Mean percentage diseased leaf area caused by isolates of
Mycosphaerella graminicola collected from each of three cultivars in the
field and inoculated singly on each of the same cultivars as seedlings in
thegreenhouse ....................................................................................................... 21
11.2.
Percentage of soft white winter wheat area occupied by three cultivars in
the Willamette Valley of Oregon during the period 1990-97 ................................ 24
ifi. 1.
Regression of AL AE (late aggressiveness - early aggressiveness) of
Mycosphaerella graminicola as measured in the greenhouse on field
disease severity for two years ................................................................................ 48
N. 1.
Regression of Mycosphaerella graminicola ascocarps as a percentage of
all fruiting bodies sampled on area under the disease progress curve
(AUDPC) assessed in four replicated field plots of each host ............................... 74
P1.2.
Plot of Mycosphaerella graminicola ascocarps as a percentage of all
fruiting bodies sampled vs. disease severity assessed on 15 June 1998
and 17 June 1999 in four replicated field plots of seven and 12
treatments, respectively; 1998 = 0, 1999 = ......................................................... 75
P1.3.
Sexual and asexual Mycosphaerella graminicola fruiting bodies as
percentages of all fruiting bodies sampled at one-week intervals between
6 June and 27 June 2000 from cultivars Gene and Stephens (AUDPCs
788 and 2185, respectively)................................................................................... 76
V.1.
Disease progress curves for four 1:1 moderately resistant-susceptible
mixtures of winter wheat naturally inoculated with Mycosphaerella
graminicolain 1998 ........................................................................................... 101
V.2.
Disease progress curves for four 1:1 moderately resistant-susceptible
mixtures of winter wheat naturally inoculated with Mycosphaerella
graminicolain 1999 ........................................................................................... 102
V.3.
Disease progress curves for four 1:1 moderately resistant-susceptible
mixtures of winter wheat naturally inoculated with Mycosphaerella
graminicolain 2000 ........................................................................................... 103
List of Figures, Continued
Figure
V.4.
Relationship between effects of winter wheat mixtures on Septoria tntici
blotch disease severity in the field and their effects on pathogenicity of
Mycosphaerella graminicola populations derived from them ............................. 107
LIST OF TABLES
Table
11.1.
Percentage diseased leaf area of greenhouse-grown wheat seedlings
inoculated with Mycosphaerella graminicola isolates collected in 1997 .............. 18
11.2.
Analysis of variance using loge-transformed percentage disease severity data
from greenhouse inoculation of wheat seedlings with single isolates of
Mycosphaerella graminicola derived from the same cultivars in the field in
1997 ........................................................................................................................ 22
11.3.
Linear contrasts to dissect the cultivar-of-origin x tester cultivar interaction
for 1997 isolates of Mycosphaerella graminicola collected from three wheat
cultivars in the field and inoculated singly on the same three cultivars as
greenhouse-grown seedlings .................................................................................. 22
11.4.
Percent diseased leaf area of greenhouse-grown wheat seedlings inoculated
with Mycosphaerella graminicola isolates collected in 1992a
ifi. 1.
Field disease severity of winter bread wheat cultivars used in field and
greenhouse experiments to test selective effects of host resistance on
aggressiveness of Mycosphaerella graminicola populations ................................ 38
ffl.2.
Percent leaf area covered by lesions of Mycosphaerella graminicola for
pathogen populations collected from six winter wheat cultivars in the field
in 1998 and 1999 when subsequently tested on the same cultivars in the
greenhouse............................................................................................................. 46
ffl.3.
Analysis of variance of pathogenicity of Mycosphaerella graminicola
isolate populations derived from three moderately resistant and three
susceptible wheat cultivars in the field in 1998 and 1999 and tested on the
same cultivars as seedlings in the greenhouse ....................................................... 49
ffl.4.
Percent leaf area covered by lesions of Mycosphaerella graminicola for
pathogen populations collected in 1998 and 1999 from two winter wheat
cultivars treated with different levels of a protectant fungicide in the field
and tested on the same cultivars in the greenhouse ............................................... 51
IV. 1.
Percentages of Mycosphaerella graminicola, Phaeosphaeria nodorum, and
unidentified fruiting bodies sampled at the end of three growing seasons
from four replicated field plots of wheat cultivars with differing levels of
susceptibility to Septona diseases .......................................................................... 66
23
List of Tables, Continued
Table
P1.2.
Significance of explanatory variables from logistic regression analysis, with
response variable being the pseudothecial proportion of all identified
Mycosphaerella graminicola fruiting bodies in 1998, 1999 and 2000 .................. 73
P1.3.
Percentages of Mycosphaerella graminicola pycnidia sampled from field
plots of winter wheat leaves at the end of three growing seasons that
contained micropycnidiospores, macropycnidiospores, and both ......................... 78
V.1.
Conduciveness of three study years to epidemics of Septoria tritici blotch
(caused by Mycosphaerella graminicola) in winter wheat .................................. 100
V.2.
Disease severity caused by Mycosphaerella graminicola isolates from
mixtures vs. pure stands of wheat when inoculated on respective mixture
component cultivars in the greenhouse ................................................................ 105
Effects of Host Resistance on Mycosphaerella graminicola Populations
CHAPTER I: Introduction
Partial or quantitative resistance was defined by Parlevliet (3 3,34) as incomplete
resistance characterized by non-hypersensitive, susceptible reactions with large
differences in disease severity. Partial resistance is a relative measure, usually with
respect to a well-known susceptible cultivar. Parlevliet (34-36) held partial resistance to
be usually (although not always) race-nonspecific and substantially more durable than
resistance due to major, race-specific genes.
Numerous traits specify partial resistance, including longer incubation period
(16), lower rate of lesion expansion (3), reduced infection frequency, longer latent period,
and lower sporulation rate (33). These partially resistant phenotypes may result from the
action of genes with major or minor effects, in varying numbers and combinations. For
comparison, qualitative resistance based on major genes often leads to small necrotic
spots without sporulation, the visible result of a defensive hypersensitive response by the
host (35).
Many host plants have sources of partial resistance sensu Parlevliet. Among them
are biotrophs such as rusts, bunts, smuts, and powdery mildews; necrotrophs such as
helminthosporiums and altemarias; and hemibiotrophs such as Phytophthora infestans,
Rynchsporium secalis, and the septorias, including Phaeosphaeria nodorum and
Mycosphaerella graminicola (32,37).
2
Many quantitative host-pathogen interactions are relatively uninvestigated, and
the mechanisms of resistance in these systems are often unknown (35). For the most part,
relatively little is known about whether there is specificity at the cultivar level among
partially resistant hosts. Among biotrophs and hemibiotrophs, major-gene and racespecific resistance frequently occur together with partial resistance in the same host
genotypes. Knowing whether major genes are present bears upon the number of isolates
to be used in inoculating experimental material, and whether it is important to screen out
material bearing major genes in order to select for minor genes (35).
In recent years, there has been increased interest among breeders and pathologists
in using horizontal or partial resistance, for example, for the control of potato late blight
(9,13). However, there is little empirical evidence regarding how the deployment of
partial resistance affects pathogen evolution. Adaptation of pathogen populations to
quantitative resistance has been demonstrated in greenhouse and growth chamber
experiments (1,2,5,8,24-26), but is more difficult to investigate in the field. The
moderate resistance of a wheat cultivar to M. graminicola was observed to deteriorate
substantially over a 10-year period in the Willamette Valley (30). On the other hand,
Vanderplank (42) presented data suggesting that partial resistance in certain potato
cultivars to the late blight fungus, Phytophthora infestans, remained stable for more than
30 years.
The disease system chosen for our investigation of host-pathogen dynamics is that
involving wheat (Triticum aestivum) and the fungal foliar pathogen Mycosphaerella
graminicola (anamorph Septoria tritici). M. graminicola has received growing attention
3
during the last 15 years because the disease it incites, Septoria tritici blotch, has increased
in incidence and severity in certain regions (11), causing greater yield losses and
occasioning higher expenditures for monitoring and chemical protection.
M graminicola undergoes multiple cycles of asexual reproduction during the
growing season. Conidia are dispersed within a leaf layer under conditions of high
relative humidity, and to successively higher leaf layers by physical contact and high-
impact rainsplash. Until recently, it was assumed that the sexual stage of
M graminicola
initiated epidemics in the fall and early winter, but was otherwise unimportant in the life
cycle of the pathogen. However, in the UK, spore trapping revealed that ascospores were
present at most times during the year (15). Mating experiments indicated that a sexual
cycle could be completed in five weeks, and it was suggested that the fungus might
complete several sexual cycles in a single growing season (21).
Widespread interest in the interactions between
M graminicola and wheat has
turned this into something of a model system. Several areas have received particular
attention:
.
inheritance of partial resistance components (16,41) and their interaction with the
environment (39);
methodology for determining whether virulence, host-specificity, and gene-forgene interactions are present in a quantitative pathosystem (1,14,17-20,22);
4
development and use of molecular markers to study issues in fungal population
genetics such as population genetic structure, distribution of genetic diversity in
geographic space, and frequency of sexual recombination (4,6,7,27-29,31,38, 4447);
.
induced resistance and isolate-isolate interactions (12,43);
.
role and importance of the sexual stage in the life cycle of a facultatively sexual
plant pathogen (10,15,40);
functional genomics and analysis of gene expression via targeted gene disruption
(49), reporter genes (37), cDNAs (23), and gene cloning and characterization
(48).
Despite the research advances on several fronts with this pathosystem, several
issues have remained unresolved. Some have important consequences for breeding and
disease management. Data gathered on these problems will expand the general body of
knowledge about quantitative host-pathogen interactions.
1)
Is there cultivar-isolate specificity of the sort that can lead to a rapid breakdown
of resistance?
2) What is the selective effect of quantitative resistance on pathogen population
aggressiveness?
3) How do different levels of partial resistance affect the frequency of sexual
reproduction? How important is the sexual stage during the course of the
epidemic?
4) How do mixtures of partially resistant cultivars affect disease progression, and
what is their selective impact on the pathogen population? (Most of the research
on host multilines and cultivar mixtures has been conducted with mixtures of
qualitatively resistant/susceptible hosts.)
The following chapters report on experiments in these four areas.
LITERATURE CITED
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2. Aluned, H. U., Mundt, C. C., Hoffer, M. E., and Coakley, S. M. 1996. Selective
influence of wheat cultivars on pathogenicity of Mycosphaerella graminicola
(anamorph Septoria tritici). Phytopathology 86:454-458.
3. Berger, R. D., Bergamin Filho, A., and Amorim, L. 1997. Lesion expansion as
an epidemic component. Phytopathology 87:1005-1013.
4. Brown, J. K. M. 2000. Estimation of rates of recombination and migration in
populations of plant pathogens. Phytopathology 90:320-323.
5. Caten, C. E. 1974. Intra-racial variation in Phytophthora infestans and
adaptation to field resistance for potato late blight. Ann. Appi. Biol. 77:259-270.
6. Chen, R.-S., and McDonald, B. A. 1996. Sexual reproduction plays a major role
in the genetic structure of populations of the fungus Mycosphaerella graminicola.
Genetics 142:1119-1 127.
7. Chen, R.-S., Boeger, J. M., and McDonald, B. A. 1994. Genetic stability in a
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8.
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Coombs, J., Bisognin, D. A., and Felcher, K. J. 2001. Evaluation of wild
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and de Bree, J. 1996. Genetic variation for virulence and resistance in the wheatMycosphaerella graminicola pathosystem. I. Interactions between pathogen
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20. Kema., G. H. J., and van Silthout, C. H. 1997. Genetic variation for virulence and
resistance in the wheat-Mycosphaerella graminicola pathosystem. ifi.
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21. Kema, G. H. J., Verstappen, E. C. P., Todorova, M., and Waalwijk, C. 1996.
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22. Kema, G. H. J., Verstappen, E. C. P., and Waalwijk, C. 2000. Avirulence in the
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24. Kolmer, J. A., and Leonard, K. J. 1986. Genetic selection and adaptation of
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25. Lehman, J. S., and Shaner, G. 1997. Selection of populations of Puccinia
recondita f. sp. tritici for shortened latent period on a partially resistant wheat
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27. Linde, C., Zhan, J., and McDonald, B. A. 2002. Population structure of
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28. McDonald, B. A., and Martinez, J. P. 1990. DNA restriction fragment length
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10
45. Zhan, J., Mundt, C. C., and McDonald, B. A. 1998. Measuring immigration and
sexual reproduction in field populations of Mycosphaerella graminicola.
Phytopathology 88:1330-1337.
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length polymorphisms to assess temporal variation and estimate the number of
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Curr. Genet. 49:388-393.
11
CHAPTER II: Specific Adaptation by Mycosphaerella graminicola to a Vertically
Resistant Wheat Cultivar
C. Cowger, M. E. Hoffer, and C. C. Mundt
Department of Botany and Plant Pathology, 2082 Cordley Hall,
Oregon State University, Corvallis, OR, 97331-2902, USA
Corresponding author: Christina Cowger, cowgercbcc.orst.edu
Cowger, C., Hoffer, M. E., and Mundt, C. C. 2000. Specific adaptation by
Mycosphaerella graminicola to a vertically resistant wheat cultivar. Plant Pathol.
49:445-451.
12
ABSTRACT
Three cultivars of winter bread wheat (Gene, Madsen and Stephens) were each
inoculated as seedlings in the greenhouse with seven or eight individual isolates of
Mycosphaerella graminicola collected in 1997 from each of the same cultivars in the
field. Isolates collected from Gene were virulent to all three cultivars, while isolates
obtained from Madsen and Stephens were virulent to those two cultivars and, in all but
one case, avirulent to Gene. At its release in 1992, Gene was resistant to
M graminicola,
as indicated by both field observations and greenhouse tests, but by 1995 its resistance
had substantially deteriorated. This indicated that its resistance was vertical (sensu
Vanderplank) or race-specific, and that commercial cultivation of Gene rapidly selected
for strains in the local
M graminicola population that were specifically adapted to
overcome its resistance.
Keywords: Mycosphaerella graminicola, physiologic specialisation, septoria tritici
blotch, Triticum aestivum, winter bread wheat
INTRODUCTION
Septoria tritici blotch is a foliar disease of durum and bread wheat caused by
Mycosphaerella graminicola (anamorph Septoria tritici), which inflicts major losses on
wheat crops in areas of relatively high rainfall and moderate temperatures. Severity of
13
septoria tritici blotch appears to be increasing in many parts of the world (24), including
the Willamette Valley of Oregon, USA (20).
Host plant resistance is the method of choice for control of septoria tritici blotch.
Whether
M graminicola has specific interactions with its hosts is important in breeding
for resistance, and has been extensively debated. There is widespread agreement that it is
specialised at the level of the host species, on bread or durum wheat (5,6,8,11,23). Three
early reviews (14,21,25) suggested that
M graminicola probably does not possess
cultivar specificity. In these three reviews, the total evidence cited against physiological
races and specificity consists of two abstracts, one reference to unpublished data and two
published papers. However, numerous more recent studies have yielded evidence of
differential cultivar-isolate interactions including several that used only same-species
isolates and testers (i.e., bread-wheat isolates on bread wheats) (1,2,12,13). Kema et al.
(12,13) inferred gene-for-gene interaction in the M. graminicola-wheat pathosystem
because of significant interaction mean-square values in analysis of variance and
covariance, and cluster analyses indicating substantial genetic variance for virulence and
resistance among isolates and cultivars.
If isolate x cultivar and gene-for-gene interactions do occur, what is the practical
significance for the durability of resistance? Previous studies of
M graminicola-wheat
interaction have involved isolates and cultivars that were unrelated to each other; often
the choice of isolates was apparently arbitrary. Significant cultivar-isolate interactions in
these experiments tell only of the potential for specific adaptation, not whether such
14
adaptation has in fact occurred or will occur. Experiments in which isolates have been
selected on the cultivars studied can reveal actual patterns of adaptation to host.
This paper describes an experiment with M graminicola isolates collected in
1997 from three cultivars with different levels of resistance, and tested as single isolates
on those same cultivars in the greenhouse. This work provides evidence that the 1992
introduction of a cultivar with resistance to septoria tritici blotch was soon followed by
the evolution and retention in the local
M graminicola population of strains with specific
virulence to that cultivar, indicating that its resistance was vertical (sensu Vanderplank,
28) or race-specific.
MATERIALS AND METHODS
The experiment was conducted in 1998 with wheat seedlings grown in the
greenhouse and monopycnidial isolates of
M graminicola collected in July 1997. Pots of
wheat seedlings (testers) were inoculated with individual isolates, and the experimental
unit was the pot.
The experiment was a factorial of tester cultivar and isolate. The soft white
winter wheat cultivars Gene (P.!. 560129), Madsen (P.1. 511673), and Stephens (C.I.
017596) served both as sources of isolates in the field and also as tester cultivars in the
greenhouse. At its release in 1992, Gene was highly resistant to M graminicola, but that
resistance has declined markedly since then (20). Madsen has moderate resistance to
septoria tritici blotch, and Stephens is highly susceptible.
15
There were 22 monopycnidial isolates of M. graminicola (seven each derived
from Gene and Stephens, and eight from Madsen). The isolates had been collected at
random from 3 x 22 m experimental plots at the Hyslop Crop Science Field Research
Laboratory in Corvallis, Oregon. The Gene isolates came from five different Gene flag
or F-i leaves (in some cases, flag leaves were difficult to isolate from due to low levels of
infection), and the Madsen and Stephens isolates originated from four different flag
leaves of those cultivars, respectively. Thus, the probability that two isolates from a
particular cultivar were genetically identical was extremely low, as even pycnidia from
different lesions on the same leaf are highly likely to be genetically distinct (17). The
experiment was replicated three times.
In each replication, 10-cm pots were planted with 1 5 seeds of each cultivar. The
pots were fertilized with Osmocote 18-6-12 extended time-release fertilizer (Scott-Sierra
Horticultural Products Co., Marysville, Ohio) at a rate of about 1 g per pot at seeding, and
watered as needed. Soon after seedling emergence the pots were thinned to a density of
10 plants per pot.
M graminicola
isolates were maintained on refrigerated silica gel and yeast-malt
agar, and inoculum was prepared by growing the isolates for 5-7 days on yeast-malt agar
slants. The conidia were suspended in distilled water and the suspension was adjusted to
a concentration of approximately 1 x 106 spores per ml using a haemocytometer. For
each isolate, 50 ml of the suspension was used to inoculate seedlings (one pot of each of
the three cultivars) to the point of run-off. Pots were inoculated at 21 days after planting,
and placed in a mist chamber with high relative humidity for 96 hr. They were then
Ir
returned to the greenhouse bench, arranged in random order within each block and with
even spacing on the bench, and watered by drip irrigation to promote constant canopy
humidity. At 21 days after inoculation, symptoms of septoria tritici blotch were assessed.
The third leaf from the bottom of each plant was assayed for percentage of leaf area
covered by M graminicola lesions, irrespective of pycnidial density. A mean percentage
diseased leaf area (%DLA) was calculated for the approximately 10 plants in each pot.
Some researchers have assayed necrotic leaf area (6,7); others have measured
pycnidial coverage (5,11,13,22) or percentage of leaf area with necrotic lesions
containing pycnidia (26). In the present experiments percentage leaf area occupied by
Septoria tritici blotch lesions was recorded, rather than either necrotic leaf area or
pycnidial density, as a measure of disease. Some of the lesions observed contained few
or no pycnidia. It was possible to distinguish a lesion, even if it did not contain pycnidia,
from other necrotic leaf tissue, particularly if the necrosis on water controls was used as a
reference point and if trials were conducted during the period from November to March.
This choice was based on observations that diseased leaf area proportions accurately
reflected the susceptibility of cultivars in the field, and that naked-eye assessments of
lesion area seemed more accurate and reproducible than those of pycnidial coverage.
As a result of the size of this experiment, the assessment of symptoms in each
replication had to be conducted over two consecutive days. To account for disease
increase from the first to the second assessment day, between four and eight treatments in
each replication were assessed on both days, and the percentage change in diseased leaf
17
area was calculated. A linear regression model was used to adjust day 1 means to be
equivalent to day2 means Jbr each pot.
A previous greenhouse experiment using similar methods was conducted in 1994
with the same tester cultivars and isolates that had been collected in the field in 1992.
The only differences in method were that the experiment had thur replicates; 25 ml
instead of 50 ml of spore suspension wereapplied to each group of four pots in the
greenhouse (the three reported here plus a fourth); and the second rather than the third
leaf from the bottom was assessed.
Statistical Analyses: Data were subjected to analysis of variance. As disease severity
on the water controls was negligible (Table ILl), these values were excluded from the
analysis in order to avoid underestimating the mean square for error. To improve
homogeneity of variance, the data were
loge-xransfonned with the pianthy LO added to
alloriginalvalues). Fixed effects were origin, tester cukivar, and their interaction.
Replicate, isolate within origin, and interactions involving those two effects were treated
as random.. The origin-tester interaction was dissected with linear contrasts to determine
if isolates collected from Gene were specifically adapted to that cultivar. As an
additional test of specific adaptation, Pearson's chi-square test with Yates's continuity
correction was performed on a contingency table in which isolates were classified as
virulent or avirulent on Gene, and originating either from Gene or from
Madsen/Stephens. Origin and tester means were separated using Fisher's protected least
significant difference test.
19
Table 11.1., Continued
bMeans are the untransformed percent diseased leaf area over four replications.
Statistical analyses were conducted on lo&-transformed data.
To determine the importance of the isolate-tester interaction, the three 1997
replicates were analyzed separately because of a significant interaction between isolates
and replicates. For this assessment, the intraclass correlation (16), which measures the
similarity of observations within groups compared to observations between groups, was
calculated using untransformed data. In this case the intraclass correlation coefficients
indicate the correlation in values among testers inoculated with the same isolate. The
intraclass correlation coefficient is the ratio
aisolate(ongin)
aisolate(oi,gin)
+a
isolate(origin) x tester
Here the ratio can take on values from -V2 [-1 /(number of testers - 1)] to 1. A ratio close
to 1 indicates a strong correlation in disease readings among testers affected by the same
isolate; that is, variation between isolates is more important than that of isolate-tester
interaction. Conversely, a ratio toward the low end of the range indicates a lack of
correlation in disease levels among cultivars inoculated with the same isolate; that is, the
preponderance of variation can be attributed to the interaction of isolates and testers.
20
RESULTS
Isolates applied to tester Gene can be clearly separated into virulent (>10% DLA)
and avirulent (5% DLA) categories (Table 11.1). Of the seven 1997 isolates from Gene,
only one caused less than 5% DLA on Gene, and the mean was 2 1.3% (Fig. 11.1). The
Gene isolates were also strongly virulent to Madsen and Stephens, causing an average of
35.7% and 33.1% DLA, respectively. In fact, some isolates from Gene were more
virulent to Madsen and Stephens than were isolates from those cultivars (Fig. 11.lb).
However, nearly all isolates from Madsen and Stephens were avirulent to Gene, causing
mean % DLAs of 2.8% and 1.8%, respectively. Only one of those 15 isolates, number 11
derived from Madsen, was virulent to Gene (10.7% DLA).
The origin-tester interaction was significant (P = 0.03 16) (Table 11.2). Linear
contrasts designed to dissect the origin-tester interaction revealed no interaction between
origins and the testers Madsen and Stephens, but very strong interaction between the
origins and tester Gene versus testers Madsen and Stephens (Table 11.3). Chi-square
analysis confirmed that there was a significant difference between the proportion of
isolates from Gene that were virulent to Gene (six of seven) and the proportion of isolates
from Madsen/Stephens that were virulent to Gene (one of 15) (x2 = 10.3445, 1 d.f., P =
0.0013).
For the 1998 experiment, the point estimates of intraclass correlation were 0.62 1,
0.518, and 0.473 for replicates 1, 2, and 3, respectively. The 95% confidence intervals
for those estimates were (0.376, 0.811), (0.252, 0.748), and (0.200, 0.7 18), respectively.
21
0
10
0
0
0
Madsen
Gene
Stephens
Isolates from:
U Gene
phensaba
U Madsen
2 30
V0
0
'5
0
0
V
C
0
a
I-
b
0
Gene
Madsen
Stephens
Tester cultivars
Fig. 11.1. Mean percent diseased leaf area caused by isolates of Mycosphaerella
graminicola collected from each of three cultivars in the field and inoculated singly on
each of the same cultivars as seedlings in the greenhouse. Within a tester, bars topped by
the same letter are not significantly different at the 95% confidence level based on
Fisher's protected least significant difference. A. Two isolates were collected from each
cultivar in 1992 and the experiment was performed in 1994. Values are means of four
replications. B. Seven or eight isolates were collected from each cultivar in 1997 and the
experiment was performed in 1998. Values are means of three replications.
22
TABLE 11.2. Analysis of variance using lo&-transformed percent disease severity data
from greenhouse inoculation of wheat seedlings with single isolates of Mycosphaerella
graminicola derived from the same cultivars in the field in 1997
Source
DF
Rep
2
Origina
2
Isolate(origin)
19
Tester
2
Origin x tester
4
Isolate(origin) x tester 38
Mean sq.
F-value
Pr> F
11.465
13.438
3.069
53.715
5.466
4.965
2.755
21.214
4.099
1.420
0.1102
0.0268
0.0040
0.0051
4.240
0.5 18
0.03 16
0.0979
a()jgj = cultivar from which isolates originated.
TABLE 11.3. Linear contrasts to dissect the cultivar-of-origin x tester cultivar interaction
for 1997 isolates of Mycosphaerella graminicola collected from three wheat cultivars in
the field and inoculated singly on the same three cultivars as greenhouse-grown seedlings
Linear contrast
DF
Mean Sq. F-value
Madsen vs. Stephens x thigifla
Madsen & Stephens vs. Gene x Origiif
2
2
0.003825
0.197871
0.16
8.03
Pr> F
0.8580
0.0068
a()1.jfl = cultivar from which isolates originated.
None of the isolates collected in 1992 was virulent to Gene (Table 11.4), and the
origin-tester interaction was not significant (data not shown).
23
TABLE 11.4. Percent diseased leaf area of greenhouse-grown wheat seedlings
inoculated with Mycosphaerella graminicola isolates collected in 1992a
Cultivar
of Origin
Tester
Gene
Madsen
Stephens
Iso!. 1
Iso!. 2
0.33
0.48
8.15
4.03
5.78
6.10
4.75
3.54
Iso!. 3
Iso!. 4
0.43
0.38
4.60
5.75
6.35
16.28
3.79
7.47
Iso!. 5
Iso!. 6
0.43
0.53
3.45
6.25
3.98
7.45
2.62
4.74
Grand mean
0.43
5.37
7.66
4.49
Water contro!
0.30
0.20
0.20
0.23
Mean1'
Genec
Madsenc
Stephensc
aExperiment was conducted in 1994.
1'Means are the untransformed percent diseased leaf area over four replications.
Statistical analyses were conducted on logetransformed data.
cField reactions: Gene, highly resistant at the time isolates were collected; Madsen,
moderately resistant; Stephens, highly susceptible.
DISCUSSION
These results indicate that, following commercial introduction of Gene, strains
rapidly appeared in the local M graminicola population that were adapted to overcome
its vertical resistance. These strains persisted in the population even when Gene no
longer occupied a substantial share of commercial wheat area (Fig. 11.2).
24
I-
4-
Gene
60
DMadsen
o SteDher
40
>
0
4-
30
'I-
o 20
4-
C
0
10
I0)
0
0
1990
1991
1992
1993
1994
1995
1996
1997
Fig. 11.2. Percent of soft white winter wheat area occupied by three cultivars in the
Willamette Valley of Oregon during the period 1990-97. Data are compiled from
successive editions of Oregon Agriculture & Fisheries Statistics, published by the U.S.
Dept. of Agriculture and the Oregon Dept. of Agriculture.
At its release in 1992, Gene was almost completely resistant to M graminicola
(19,20). Gene is an F4-derived selection from a cross involving the resistant cultivar
Cleo, which is believed to carry the septoria tritici blotch resistance gene Sth4 (15,26). In
1994 and 1995, Gene quickly gained popularity among commercial growers in Oregon
(Fig. 11.2), and by 1995 it occupied over 15% of the Willamette Valley wheat area. The
data in Fig. 11.2 are valley-wide means; Gene's proportion of wheat area was substantially
higher in the south-central Willamette Valley, where the experiments were carried out.
By 1995 it was evident that M graminicola was rapidly overcoming the resistance of
Gene (20). Its susceptibility also to Leptosphaeria nodorum (anamorph =Septoria
nodorum) was an additional liability, and planting of Gene apparently increased the
25
prevalence of that pathogen in the Willamette Valley. Commercial production of Gene
declined and by 1997 the cultivar accounted for only 3.9% of Willamette Valley wheat
cultivation.
Consistent with this history, Ahmed et al. (1) detected avirulent reactions on Gene
for 14 monopycnidial isolates, including six from Oregon, that were all collected prior to
1992. Further, bulk populations of 20 isolates collected from each of four cultivars,
including Gene, in 1992 produced avirulent reactions on Gene (2). Its field performance
was consistently highly resistant prior to commercial production (19,20). The data in
Table 11.4 indicate avirulence to Gene in the six isolates collected in 1992; while these
data by themselves would be insufficient, they corroborate other evidence that M.
graminicola was avirulent on Gene before 1992.
The 1997 isolates obtained from Gene were virulent to all three cultivars with one
exception, while those from Madsen and Stephens were avirulent to Gene, again with one
exception. Inspection of Fig. 11.1 and the linear contrasts (Table 11.3) illustrate that most
of the origin-cultivar interaction resulted from differential interactions of isolates on
Gene. Similarly, Kema et al. (12) found considerable variation among subsets of host
cultivars and pathogen isolates as to whether they appeared to possess specific
resistance/virulence factors.
The rapid decline of resistance in Gene suggests that it is under the control of a
monogenic or oligogemc system. This report now provides a clear example of rapid
decline in resistance of a cultivar to M graminicola that was reported as missing from the
literature by Johnson (10) and Kema et al. (13). It should be noted that in the Willamette
26
Valley, M graminicola has near-optimal conditions for rapid evolution in response to
host characteristics. Epidemics vary from moderate to severe, the sexual stage is
abundantly present during more than one part of its life cycle (20; C.Cowger,
unpublished data), and genetic variability of the pathogen is extremely high (3, 4). In
contrast, environmental conditions are less favourable to septoria tritici blotch in the
Central Valley of California; even so, the resistance of cultivars planted extensively there
starting in the mid-1990s had already eroded significantly, although not completely, by
1998 (L. Jackson, University of California at Davis, personal communication). At least
one of those cultivars probably bears the Stb4 resistance gene.
While the sample of 22 isolates was limited in size, it was probably adequate
because of the high level of consistency in the response of Gene to isolates from each
cultivar. Isolates were either virulent or avirulent, and isolates from the two other
cultivars performed almost uniformly on Gene.
Average disease levels were considerably higher in the 1998 experiment than in
the small experiment performed in 1994. The difference could result from environmental
conditions in the greenhouse, a general increase in aggressiveness of the pathogen over
the intervening years, or both. Isolates from 1992 and 1997 were not compared in the
same test, and no claims are made about the relative aggressiveness of the two sets of
isolates. A simultaneous test of both sets of isolates, while interesting, would address a
different question from the main focus of this research, and would also introduce the
confounding factor of differential periods of maintenance of the pathogen in culture. The
27
data from the 1994 experiment simply help to establish the baseline of avirulence of
isolates to Gene, which is amply confirmed by field data and other greenhouse studies.
The relative disease levels on the 1998 greenhouse testers did not mirror those of
the same cultivars in the field (disease was, on average, most severe on Madsen, while
Stephens is the most susceptible of the three in the field). One possible explanation is
that the set of isolates we used was not perfectly reflective of the population as a whole.
Another explanation is that seedling and adult plant responses are not completely
correlated (13). However, previous greenhouse studies using bulked isolates (2) ranked
seedlings of these cultivars the same as adult plants in the field.
Kema et al. (13) showed that adult plants were more susceptible than seedlings in
a set of 22 cultivars each challenged with three individual M graminicola isolates. While
such developmental factors might explain the avirulence of Madsen and Stephens isolates
on our Gene seedlings, they would not account for the consistent and specific virulence
of Gene isolates on Gene that we observed.
It may be speculated why no isolates from Stephens and only one from Madsen
possessed virulence to Gene. A possible explanation is that virulence to Gene entails a
fitness cost under field competition that is not evident in single-isolate greenhouse
experiments. Alternatively, isolates adapted to Gene may comprise a small proportion of
the M. graminicola population, perhaps related to the relatively small area on which it
was cultivated. In either case, isolates virulent to Gene would be detected more easily
under the selective effect of that cultivar.
The data support the conclusion that specific cultivar-isolate interactions operate,
although not exclusively, in the M graminicola/Triticum aestivum pathosystem. The
isolate-tester interaction was significant at P = 0.0979 for the 1997 isolates. The
intraclass correlations estimated for the 1997 replicates were consistently about one-third
from the top of the range, and their 95% confidence intervals overlapped substantially.
This implies low to moderate specificity in our total set of isolates and testers, which is
consistent with the finding of a lack of specificity to Madsen and Stephens, and
specificity to Gene.
The specificity detected in an agricultural host-pathogen system will depend
greatly on the nature of resistance that is commercially deployed. Gene was the first
wheat cultivar with high resistance to septoria tritici leaf blotch to be grown
commercially in the Willamette Valley of Oregon, and it was possible to identify
cultivar-specific isolates within five years of its release.
Detection of clear specificity only after use of highly resistant cultivars has
occurred previously in other host-pathogen systems. For example, specificity in the rice
(Oryzae sativa)/Xanthomonas oryzae pv. oryzae system was debated into the I 970s.
However, the deployment and subsequent breakdown of resistant rice cultivars in Japan
and the Philippines in the 1 970s resulted in the clear identification of physiological races
(18). Subsequently, bacterial blight of rice has become one of the best molecularly
characterized gene-for-gene systems (9,27).
In agreement with Kema and van Silthout (13), it is suggested that the slow
evolution of virulence in M. graminicola that has often been observed results, in part,
29
from the incomplete nature of most resistances that have been used. However, it is
anticipated that qualitative resistance to M. graminicola will be no more durable than in
any other host-pathogen system, as illustrated by the performance of Gene in the
Willamette Valley of Oregon.
ACKNOWLEDGEMENTS
We thank Dave Thomas for statistical advice and Dan Coyle and Kris Kovar for
technical support. This research was supported by the U. S. Department of Agriculture
National Research Initiative grant 97-35303-4537.
30
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33
CHAPTER III: Aggressiveness of Mycosphaerella graminicola Isolates from
Susceptible and Partially Resistant Wheat Cultivars
Christina Cowger and Christopher C. Mundt
Department of Botany and Plant Pathology, 2082 Cordley Hall, Oregon State University,
Corvallis, OR 97331-2902.
Supported by award number 97-35303-4537 of the NRI Competitive Grants
Program/USDA
Accepted for publication 28 February 2002.
Corresponding author: C. Cowger; E-mail address: cowgercbcc.orst.edu
Cowger, C., and Mundt, C. C. 2002. Aggressiveness of Mycosphaerella graminicola
isolates from susceptible and partially resistant wheat cultivars. Phytopathology, in press.
34
ABSTRACT
The selective effect of quantitative host resistance on pathogen aggressiveness is
poorly understood. Because two previous experiments with a small number of bread
wheat cultivars and isolates of M graminicola had indicated that more susceptible hosts
selected for more aggressive isolates, we conducted a larger experiment to test that
hypothesis. In each of two years, six cultivars differing in their levels of partial
resistance were planted in field plots, and isolates were collected from each cultivar early
and late in the growing season. The isolates were inoculated as populations bulked by
cultivar of origin, field replicate, and collection date on seedlings of the same six
cultivars in the greenhouse. The selective impact of a cultivar on aggressiveness was
measured as the difference in aggressiveness between early and late isolates from that
cultivar. Regression of those differences on disease severity in the field yielded
significance values of 0.0531 and 0.0037 for the two years, with moderately resistant
cultivars selecting for more aggressive isolates. In a related experiment, the protectant
fungicide chlorothalonil was applied to plots of two susceptible cultivars to retard
epidemic development. When tested in the greenhouse, isolates of M. graminicola from
those plots were significantly more aggressive than isolates from the same cultivars
unprotected by fungicide.
Additional keywords: pathogenicity, resistance, Septoria tritici blotch, Triticum aestivum,
virulence.
35
INTRODUCTION
In this paper, we employ Vanderplank's (25) definitions of "pathogenicity" as the
sum of a pathogen's disease-causing abilities, including both "virulence" (specific
disease-causing abilities) and "aggressiveness" (non-specific disease-causing abilities).
Host selective effects on pathogenicity are an important aspect of host-pathogen
interactions. Research on a host's selective impact on virulence is relatively common,
with efforts more on qualitative than on quantitative interaction. Very few reports can be
found of host selective impacts on aggressiveness, despite the large number of
quantitatively varying pathosystems.
The data that are available tend to support the conclusion that resistant hosts
select for more aggressive pathogens than do susceptible hosts. In one example (24),
potato cyst nematodes (Globodera pallida) reared for several generations on resistant
potato cultivars exhibited an increased reproductive rate, while those raised on
susceptible potato cultivars did not. The authors of another report (21) suggested that
multiplication of Lettuce Mosaic Virus in lettuce (Lactuca sativa) cultivars with
relatively high levels of partial resistance may have contributed to the emergence of new,
more aggressive viral pathotypes.
A report from our laboratory (1), subsequently cited by others (2,3,5,6,1620,22,23), suggested that more susceptible cultivars of winter wheat (Triticum aestivum)
select for more aggressive isolates of Mycosphaerella graminicola. The evidence came
from two greenhouse experiments in which wheat seedlings were challenged with
isolates previously obtained from the same cultivars grown in field plots. Four cultivars
36
were used in the first study, and two cultivars in the second study. Similar results, though
smaller in magnitude, had been obtained with Xanthomonas
oryzae pv. oryzae on
rice
(Mundt, unpublished). When 80 isolates of the rice pathogen collected from each of two
cultivars in replicated field experiments at each of two locations were tested on the same
cultivars in the greenhouse, the isolates from the susceptible cultivar caused significantly
longer lesions than those from the moderately resistant cultivar.
Although evidence that susceptible cultivars select for greater aggressiveness was
surprising, it was hypothesized (1,19) that such an effect might be explained by a greater
variance in the pathogen's reproductive rate (lesions produced per lesion per day) on the
susceptible cultivars. Since gain due to selection is directly proportional to genetic
variance, one could thus expect a more rapid increase in aggressiveness on the
susceptible cultivars. Alternatively, more aggressive isolates could be selected directly
by some cultivar phenotype(s), perhaps pleiotropically by the characters conferring
susceptibility, and not as a result of the more intense epidemics occurring on those
cultivars.
M graminicola causes the foliar disease Septoria tritici blotch in wheat. This
pathosystem is useful for investigation of host selective effects on aggressiveness. In the
Willamette Valley of Oregon, the site of our research, the pathogen population possesses
high levels of genetic variability (26) and thus the basis for rapid adaptation. Disease
severity varies quantitatively, and most studies of resistance to M graminicola in bread
wheat have found that additive effects predominate (4,11,12). Moreover, host resistance
is the method of choice for controlling this economically increasingly important pathogen
37
(5,9), and it is therefore desirable to understand how the deployment of partial resistance
affects pathogenicity.
The present research tested two hypotheses. First, we tested the hypothesis that
host susceptibility was associated with greater aggressiveness among isolates. To do so,
we used a larger number of cultivars and isolates than did Ahmed et al. (1), and chose
cultivars that were not closely related. Our second hypothesis was that, on susceptible
cultivars, a higher pathogen reproductive rate was by itself sufficient to explain more
rapid selection for aggressiveness. This hypothesis was tested by applying lowered rates
of a protectant fungicide to susceptible cultivars to reduce pathogen reproduction.
MATERIALS AND METHODS
Overview
In each of two winter wheat seasons (1997-98 and 1998-99), field piots were
planted in a randomized, complete block design with four replicates. The treatments
were six soft white winter wheat cultivars (Table ffl.l) and four additional treatments in
which two of the susceptible cultivars were sprayed with a protectant fungicide to
artificially reduce the rate of epidemic development. Isolates collected each year were
used the subsequent winters in greenhouse experiments to measure aggressiveness.
38
TABLE 111.1. Field disease severity of winter bread wheat cultivars used in field and
greenhouse experiments to test selective effects of host resistance on aggressiveness of
M graminicola populations
Treatment
Accession
number
Lewjain
C.I. 17909
Cashup
PVP 8500178
Madsen
P.1. 511673
W-301
P.!. 559718
MacVicar P.1. 552427
Stephens
C.I.017596
Stephens 1.8 mi/L fung.z
Stephens 0.88 mi/L fung.
W301_1.8m1/Lfimg.z
W-301
0.88 mi/L fung.z
Disease severit?
1998
1999
43.3
61.0
71.3
77.5
73.5
79.0
25.8
30.0
43.3
51.8
57.8
66.0
67.0
41.8
43.8
41.4
44.0
31.3
--
'Mean percent diseased canopy area of four field replicates on 15 June 1998 and 17 June
1999.
zplOtS sprayed with the protectant fungicide chlorothalonil (ml Bravo per L water) to
reduce the rate of pathogen reproduction.
Field experiments
Plot establishment
Field plots were established at the Oregon State University Botany and Plant
Pathology Field Laboratory in Corvallis, Oregon, USA. Each of the four plots of each
treatment occupied two adjacent 1.5 x 6.1 m planting units. Equal-sized plots of wheat
39
and barley were alternated in a checkerboard pattern so that the barley provided a buffer
between the wheat plots.
Three of the cultivars (Stephens, Madsen, and MacVicar) were commonly grown
in western Oregon, where the experiment took place. The other three cultivars, not
commonly grown in this region, were chosen to provide a balance between moderately
resistant and susceptible cultivars. The moderately resistant cultivars had expressed
approximately one-half of the level of disease found on the susceptible cultivars in
western Oregon (DiLeone and Coakley, unpublished data). Heights of the six cultivars
were uncorrelated with susceptibility to M. graminicola.
Plots were planted on 20 October 1997 and 7 October 1998. Fertilization,
planting, and weed control were all conducted according to standard commercial
practices in the area.
Fungicide applications
Reduction in the reproductive rate of the pathogen was accomplished by applying
the non-systemic, protectant fungicide chlorothalonil (Bravo 720, 720 g chlorothalonil/L,
54% a.i.) to plots of two susceptible cultivars, Stephens and W-301. Chiorothalomi is a
broad-spectrum, multisite fungicide that was chosen because it was not expected to exert
specific selective influences on pathogen populations. The concentration was 1.8 ml
Bravo per L water, about 40% of manufacturer's recommended strength, with the
surfactant Triton B added at the rate of 0.176 milL. The rate of application was 0.84
L/plot (468 L/ha). In the first year, applications were made seven times at intervals of
2.5-5 wk, depending on the growth rate of the wheat, from December 10 to May 22. In
the second year of the experiment, in order to allow formation of more lesions from
which to obtain isolates, fungicide was applied only five times at approximately 3-wk
intervals between late January and late April. A lower dosage, 0.88 milL, was also
applied to plots of Stephens and W-301 in 1999.
Disease measurements
Visual assessments of percent diseased leaf area were conducted by two assessors
for each plot on a whole-canopy basis on 15 June 1998 and 17 June 1999.
Isolate sampling
Leaves infected with M graminicola were collected from the field plots on 19
February 1998 and 25 February 1999, before significant secondary infection had taken
place, and on 12 June 1998 and 24 June 1999, after there had been ample opportunity for
host treatments to exert selective influences on the pathogen populations. These
collection times will be referred to as "early" and "late." Leaves were collected as
follows: along a diagonal transect across each 12-row plot, the flag (1998) or F-i (1999)
leaf was collected from each of two adjacent plants chosen at random in each of the
innermost ten rows of the plot. F-i leaves were collected in 1999 because, due to the
lighter epidemic that year, flag leaves on the more resistant cultivars had too few lesions
to assure that isolates could be obtained. Leaves were bagged separately by row.
41
In the laboratoty, monopycnidial isolates of M graminicola were obtained from
the leaves separately by collection date, replicate, cultivar of origin, and row. Isolations
were made by placing leaf segments bearing lesions in petri dishes lined with moistened
filter paper overnight, and then transferring cirrhi individually with a sterile needle to
plates of yeast-malt agar amended with 50 mgfL streptomycin. The isolates were stored
at 4° C and transferred to fresh yeast-malt agar every 3-5 mo.
Greenhouse trials
In 1999 and 2000, pathogen populations collected in the field during the previous
growing season were evaluated in the greenhouse in two experiments addressing the first
and second hypotheses, respectively.
Tester plants
Tester cultivars used to address the first hypothesis, that susceptible cultivars
select for more aggressive isolates, were the same as the six cultivars from which the
isolates were obtained (Table ifi. 1). Testers for the second hypothesis, that a faster rate
of pathogen reproduction accounts for more aggressive isolates on susceptible cultivars,
were the two cultivars subjected to the rate-reducing fungicide treatment in the field
(Stephens and W-301) and the moderately resistant Madsen.
The experimental unit was a pot of seedlings. In the first year, plants were raised
in 10-cm plastic pots filled with a 1:1:1:2 mixture of peat:sand:loam:#8 pumice amended
42
with Osmocote 18-6-12 extended time-release fertilizer (Scott-Sierra Horticultural
Products Co., Marysville, OH) at a rate of about 1 g/pot at seeding. Approximately 13
seeds of a cultivar were sown in each pot, and seedlings were thinned to 10 plants per pot
at about 18 days after planting. The plants were grown under sodium halide lighting to
extend the daylength to 16 hr, and watered as needed. To control powdery mildew,
plants were treated as necessary with 50 ml of a soil drench of ethirimol (0.11 g a.i./L),
which has no influence on the development of M graminicola. The greenhouse
temperature was maintained at 20-25° C.
During the second greenhouse season, because of problems in the previous year
with emergence, growth, and non-disease-related necrosis, plants were raised in Black
Gold all-organic potting soil (Black Gold, Inc., Hubbard, OR) amended with about 2.5 g
Osmocote per pot. Pots were also fertilized weekly with 50 ml of Peters water-soluble
20-20-20 fertilizer (United Industries Corp., St. Louis, MO) at the rate of 3.8 g/L of
water. Watering, temperature, and lighting were as in the first year.
Inoculum preparation and inoculation
For greenhouse tests of 1998 isolates, each experiment had four replicates in time,
with each replicate testing a pathogen population from a different block in the field
experiment. Isolates were increased individually for four days on fresh yeast-malt agar.
Isolate populations were created by mixing together spores of each isolate from a given
field plot in an equiproportional suspension, which was adjusted to a concentration of
spores/ml just prior to inoculation.
i05
43
In the first experiment, which measured aggressiveness of early and late isolates,
the target number of isolates in a population was 10, corresponding to the 10 rows in the
plot from which isolates were sampled. In some cases, due to poor isolate growth or
contamination, the actual number of isolates in an equiproportional mixture was less than
10. However, in only five out of the 48 treatments comprising the four replicates did the
number of isolates in a population fall below eight (four, five, six, six, and seven
isolates), and all five involved different cultivars of origin.
When plants were 21 days old, a group of six pots comprising one pot of each
cultivar was inoculated with 50 ml of each isolate population plus one drop of surfactant,
which was sufficient to inoculate the plants to run-off. Inoculation was performed with
spray bottles and a turntable. After inoculation, the plants were kept in a greenhouse mist
chamber for 96 hours. Noninoculated control plants were sprayed with water and
surfactant only, but otherwise treated in the same manner as inoculated plants in order to
control for contamination among pots.
In the second experiment (effect of rate reduction), five isolates from each plot
were bulked, due to the shortage of isolates available from some of the sprayed plots. For
each isolate population, 25 ml was applied to the three pots of the tester cultivars.
In greenhouse tests of 1999 isolates, isolate production for the early/late
experiment was the same as before, except that one of the 48 bulked-isolate treatments
had to be omitted due to poor growth and contamination, and 11 other treatments
contained fewer than eight isolates (one had four, four had five, three had six, and three
had seven). For the rate-reduction experiment, five isolates were available from 16 of the
24 plots; four isolates from three; three from three; two from one; and one treatment
(W-301 high-spray replicate two) had to be omitted due to lack of isolates. As a result of
tests to optimize soil fertility and disease severity, inoculum concentrations were
increased to 106 spores/ml.
Disease assessment
In order to assess disease levels of all plants in a given experiment in the same
day, two workers conducted the assessments, each assessing half of the plants in each
pot. The percent of the second leaf from the base of the plant covered by Septoria tritici
blotch lesions and lesion-associated necrotic area was estimated visually for each plant in
each pot at 21 days after inoculation. The exception was the early/late experiment with
1999 isolates, in which the third leaf from the base was read because of high levels of
senescence on the second leaf.
Statistical methods
For each cultivar as an isolate source, the mean disease severity caused by early
isolates across all testers (AE) was subtracted from the mean disease severity caused by
late isolates across all testers (AL). These early-late differences in aggressiveness, AL
AE,
were regressed on disease severity as assessed in the field for each cultivar.
45
Data were subjected to analysis of variance, using the SAS PROC MIXED
procedure, which takes missing data into account (11 pots among the four replicates were
unusable the first year, and no early MacVicar isolates were available in replicate three of
the second year). As disease severity on the water controls in the greenhouse studies was
negligible, these values were excluded from the analysis in order to avoid
underestimating the mean square for error. Data were
loge-transformed to improve
homogeneity of variance. Fisher's protected least significant difference test was used to
separate means of testers and of isolate populations derived from moderately resistant and
susceptible cultivars by collection date. Linear contrasts were used to test the differences
in aggressiveness of populations collected at a particular date from cultivars with a
particular resistance level (MR or S).
RESULTS
Unlike Ahmed et al. (1), we found no evidence that susceptible cultivars selected
for more aggressive populations of M graminicola than moderately resistant cultivars
(Table ffl.2, Fig. ifi. 1). On the contrary, it is apparent from the regression of early-late
aggressiveness differences (AL
AE)
on disease severity (Fig. ifi. 1) that moderately
resistant cultivars selected for smaller declines or larger gains in aggressiveness than
susceptible cultivars, with P-values of 0.0531 and 0.0037 in 1998 and 1999, respectively.
There was no significant difference in aggressiveness between isolates from
moderately resistant and susceptible cultivars at either collection date in either year
(Table ffl.2). No significant interaction of cultivar as an isolate source and tester
46
TABLE 111.2. Percent leaf area covered by lesions of Mycosphaerella graminicola for
pathogen populations collected from six winter wheat cultivars in the field in 1998 and
1999 when subsequently tested on the same cultivars in the greenhouse
Teste?
Source of isolates''
Collection Resistance
time
level
Cultivar
1998
E
E
E
MacV
Steph
W-301
Cash Mads Steph W-301 MacV Lewj
22.2
35.0
20.0
25.7
27.6
14.6
24.6
28.0
22.6
25.8
25.5
23.5
23.4
20.9
28.6
26.3
20.2
15.2
16.1
21.5
25.0
4.3
8.3
8.9
7.4
11.5
6.7
6.6
8.3
15.4
25.0
28.6
11.1
15.8
17.7
26.3
Mean of all late
14.1
MacV
Steph
W-301
21.7
13.0
Cash
20.0
S
S
S
Mean of late S
L
MR
L
MR
L
MR
Mean of late MR
MacV
Steph
W-301
Cash
Lewj
Mads
Mean of 1998z
1999
E
E
E
S
S
S
Mean of early S
E
MR
E
MR
E
MR
Mean of early MR
17.4
20.1
12.0
22.0
17.3
L
L
L
Cash
Lewj
Mads
28.9
24.2
15.4
17.9
16.2
15.9
16.9
Mean of all early
Mean of early S
E
MR
E
MR
E
MR
Mean of early MR
29.0
21.8
28.7
23.8
23.5
25.0
23.8
24.5
33.2
27.2
25.3
30.0
23.0
24.3
20.4
22.6
S
S
20.0
22.3
24.8
22.3
31.7
23.9
29.0
28.8
26.7
30.3
29.9
29.0
28.8
16.6
16.8
13.6
15.7
19.5
18.5
18.9
19.0
13.7
16.5
8.4
12.9
18.4
13.1
14.7
15.4
S
18.8
17.8
21.4
19.0
18.8
15.8
17.9
24.1
20.5
16.4
16.5
14.6
16.0
15.5
12.7
15.6
14.2
20.7
21.5
21.6
18.1
16.2
19.8
17.4
15.2
28.6
19.1
20.2
18.5
15.7a 21.9bc 19.3ab 18.3ab 25.Ocd 25.6d
Lewj
Mads
21.1
18.3
15.0
14.6
16.5
13.1
25.6
18.3
16.1
16.3
17.2
16.5
16.5
23.2
24.4
13.3
7.9
15.2
18.8
23.7
18.5
20.3
24.1
14.9
19.8
20.3
25.8
27.3
23.9
25.7
Mean'
24.7
23.9
20.8
23.Oa
23.9
22.4
24.3
23.5a
19.2
20.0
15.1
18.lb
19.6
19.6
18.6
19.2b
23.3a
18.7b
21.7
15.5
16.6
20.4
17.7a
19.7
17.1
14.2
17.Oa
47
Table 111.2., Continued
L
L
L
S
S
S
Mean of late S
MR
MR
MR
Mean of late MR
L
L
L
Mean of all early
Mean of all late
Mean of all 1999z
MacV
Steph
W-301
6.3
12.7
25.1
14.7
5.1
Cash
25.6
24.3
10.8
6.3
6.2
7.8
21.4
22.8
7.7
17.3
16.7
Lewj
Mads
11.7
20.5
17.6
17.6
17.5b
7.0
7.2
6.5
7.1
7.5a
10.2
17.1
20.4
15.9
8.4
17.6
17.6
20.1
18.8
18.8
18.9
15.9
15.9
16.9
26.4
12.2
14.2
17.6
15.la
26.9
16.9
27.8
31.4
22.2
26.4
19.3a
16.2
18.1
20.4
19.3
19.6
17.lb 19.4b
24.0
22.9
35.1
12.9
18.8 22.8
19.9 22.0
19.5b 22.5b
14.1
14.2
16.9
22.1
13.6
17.3a
17.2a
17.3
wIsolates collected early (B) or late (L) in the growing season from susceptible (S)
cultivars W-301, MacVicar, and Stephens or from moderately resistant (MX) cultivars
Cashup, Madsen, and Lewjain.
xValues are least-squares means of three trials for 1998 and four trials for 1999, each
using isolates from a different field replicate.
'1n the final column, means not separated by a blank row and followed by the same letter
are not significantly different at P 0.05 according to Fisher's protected least significant
difference test.
ZWjfljn a row, means followed by the same letter are not significantly different at P
0.05 according to Fisher's protected least significant difference test.
occurred in either year (Tables ffl.2 and ffl.3), and the interaction of isolate source, tester,
and collection date was also nonsignificant in both years (Table ffl.3). Disease severity
varied significantly among testers in both years. There were significant differences in
aggressiveness of populations from different isolate sources across collection dates in
-2.5
L.
55
1998
1999
IIIiKII
-3.4
R2
0.5
237Md
0.65
R2 = 0.90
-2.0
w
..60
3.0
P =0.0037
W
Mc
-
_e$.5
35
45
55
65
Percent disease on June 15
75
85
25
35
45
55
65
75
Percent disease on June 17
Fig. 111.1. Regression of AL - AE (late aggressiveness - early aggressiveness) of Mycosphaerella graminicola as measured in
the greenhouse on field disease severity for two years. Moderately resistant winter wheat cultivars: Cashup (C), Lewjain (L),
arid Madsen (Md); susceptible cultivars: MacVicar (Mc), Stephens (S), and W-301 (W)
49
TABLE 111.3. Analysis of variance of pathogenicity of Mycosphaerella graminicola
isolate populations derived from three moderately resistant and three susceptible wheat
cultivars in the field in 1998 and 1999 and tested on the same cultivars as seedlings in the
greenhouse
1998
Source of variation
DF
F-value
P-value
Isolate source
Tester (T)x
4
0.6283
25
0.67
4.52
0.78
1.70
10.25
0.78
0.59
DF
F-value
P-value
3.73
10.45
0.58
0.30
0.69
0.09
0.38
0.0357
0.0002
0.9267
0.7627
(J)X
IxT
Adaptation to host
Collection date (C)z
IxC
IxTxC
1999
Source of variation
Isolate source (I)'
Tester (T)X
IxT
Adaptation to host
Collection date (C)z
IxC
IxTxC
5
20
1
1
5
4
5
20
1
1
5
25
0.0 103
0.7404
0.090 1
0.0493
0.5637
0.9413
0.47 18
0.9842
0.9967
xCUltjV
serving as isolate sources and testers were: Cashup, Lewjain and Madsen,
moderately resistant; and MacVicar, Stephens, and W-301, susceptible.
Linear contrast within I x T interaction to compare pathogenicity of late isolate
populations on their cultivars of origin to their pathogenicity on other tester cultivars.
zIsolates were collected either early (19 February 1998 and 25 February 1999) or late (12
June 1998 and 24 June 1999) in each growing season.
50
1999, but not in 1998 (Table ffl.3). Late isolate populations were significantly less
aggressive than early populations in 1998, but there was no difference in aggressiveness
between populations collected early and late in 1999. There was modest evidence that
fungal populations were selected for greater pathogenicity on their hosts of origin in 1998
(linear contrast, P = 0.090 1), but no such evidence in 1999.
Isolate populations from the fungicide-sprayed plots were significantly more
aggressive than populations from unsprayed plots of the same cultivars in both years
(Table ffl.4). In 1999, both fungicide dosages selected for more aggressive populations,
although aggressiveness was not statistically different between the two dosages. For
1998 populations, there was no significant interaction between the cultivar from which
populations originated (Stephens or W-301) and the spray level (P = 0.1872). However,
for 1999 populations there was some indication of such an interaction (P
0.0907), with
increasing fungicide dosages associated more strongly with increased pathogenicity of
isolates from Stephens than from W-30 1.
DISCUSSION
These experiments were undertaken to confirm unexpected evidence, obtained by
our research group, that susceptible cultivars selected for more aggressive isolates (1).
As it turned out, we obtained the opposite result with a larger set of cultivars and isolate
populations that were specifically chosen to test this hypothesis.
Our finding is consistent with the few other available reports on selective impact
of host resistance level on aggressiveness in quantitatively varying pathosystems (21,24).
51
TABLE 111.4. Percent leaf area covered by lesions of Mycosphaerella graminicola for
pathogen populations collected in 1998 and 1999 from two winter wheat cultivars treated
with different levels of a protectant fungicide in the field and tested on the same cultivars
in the greenhouse
Tester"
Isolates
from:x
W-301 Stephens Madsen
Mean
1998-0.0 milL
10.0
15.9
12.9a
11.1
17.6
14.3a
14.7
13.7
14.2a
11.9a
15.7b
13.8
1998-W-301
1998-Stephens
12.8
13.0
15.8
12.8
11.5
16.8
13.4a
14.2a
26.4
31.4
25.2a
18.2
6.8
14.9
13.0
11.7b
4.2
6.2
7.8
15.9b
17.4b
24.8
25.7
11.2
12.1
1998 - 1.8 mi/L
MEANZ
1999 - 0.0 mi/L
1999 - 0.88 mi/L
1999- 1.8m1/L
MEANZ
1999-W-301
1999-Stephens
6.lb
4.9
7.3
9.7a
14.3
13.6a
15.Oa
WValues are means of four replicates, each with inoculum from a different field replicate.
Xml/L are ml Bravo (a.i.: chiorothalonil) per L water, applied at rate of 0.84 L/plot (468
LJha)
seven times in 1998 and five times in 1999.
In the final column, means not separated by a blank row and followed by the same letter
are not significantly different at P = 0.05 according to Fisher's protected least significant
difference test.
zWithin a row, means followed by the same letter are not significantly different at P =
0.05 according to Fisher's protected least significant difference test.
52
It is not surprising that, in these systems, more resistant hosts should select for more
aggressive isolates. If partial resistance and aggressiveness are non-specific and are
conferred additively by several genes, fungal individuals selected by a resistant host
would likely possess one or more traits conferring an advantage on all host genotypes.
Indeed, the model of Gandon and Michalakis (8) predicts that increasing levels of
quantitative host resistance will select for increasing levels of damage caused by a
parasite to the host, while qualitative host resistance will not exercise a similar selective
effect.
Why did our results diverge from those of Ahmed et al. (1), even though the two
studies occurred in the same geographic region, utilized similar methodology, and shared
two cultivars (Madsen and Stephens) as sources of isolates and testers? For one thing,
the larger of two experiments conducted by Ahmed et al. was not designed to estimate the
selective effects of different levels of resistance on aggressiveness, since it included only
one highly resistant cultivar (Gene), one moderately resistant cultivar (Madsen), and two
susceptible cultivars (Stephens and Malcolm). The apparent association of cultivar
susceptibility and isolate aggressiveness was not borne out when the moderate and low
levels of resistance were replicated in the present study.
Moreover, Ahmed et al. chose cultivars that were relevant to commercial
production, and by chance the two susceptible cultivars were very closely related to each
other. The greater mean pathogenicity of isolate populations from Stephens and Malcolm
was almost entirely due to the greater disease severity incited by those isolates on the
same two cultivars. Because Stephens had been the dominant cultivar in the Willamette
53
Valley for many years, greater pathogenicity in isolate populations derived from it and
from its close relative Malcolm may merely have reflected the long period available for
specific adaptation to Stephens. In fact, the analysis of variance performed by Ahmed et
al. revealed a significant pathogen population x tester cultivar interaction, which is
indicative of specificity and clouds the interpretation of the significant main effect of
pathogen population.
The difference in outcomes of the two studies may also be partially attributable to
pathogen population shifts in the interval between them. Ahmed et al. used isolates
collected in 1992 and 1994, while the isolates for this work were collected in 1998 and
1999. In the intervening years, the dominant cultivar in the Willamette Valley shifted
from the susceptible Stephens to the moderately resistant Madsen, and epidemic seventy
generally appeared to increase (2,19). Additionally, Gene's resistance, which was nearly
complete at the time that isolates were collected from it for the earlier experiment (1992)
but had deteriorated substantially just three years later, appears to have been specific (2).
Examination of the data (1,2) reveals that Gene had a different selective impact on
aggressiveness before and after it selected for strains able to overcome its resistance.
Differences in aggressiveness were not significant in either year when comparing
MR-derived to S-derived populations as groups at the late collection date (Table ffl.2),
which was the criterion used by Ahmed et al. However, there was some variability in
aggressiveness among populations within those groups in the early collections (Table
ffl.2). There were also differences in cultivar resistance level within the MR and S
categories (Table ffl.1). Averaging across the variability within MR and S groups for
54
pathogen population aggressiveness and for degree of host resistance obscured the
selective effect of host resistance level. That selective effect only became significant
using linear regression of AL AE on disease severity, so that individual host effects
could be measured.
The interaction of isolate source with tester cultivar was nonsignificant in both
years of this study. Other studies have found both significant (1,2,7,14-16) and
nonsignificant (10) interactions of M graminicola isolates or isolate populations and
tester cultivars within a particular wheat species. As Kema Ct al. (15) showed, some
combinations of isolates and cultivars display high specificity, while other combinations
possess low specificity. We have detected evidence of both qualitative (2) and
quantitative (19) adaptation to hosts by M. graminicola. While what we observed in the
present study was probably selection for aggressiveness, the difficulty of distinguishing
aggressiveness from virulence in this pathosystem should be remembered (13).
For 1998 isolate populations, we observed both modest evidence of adaptation to
individual host genotypes and a mean decline in aggressiveness over the growing season.
Neither of these effects was detected for the 1999 populations. The epidemic of 1998
was considerably more intense than that of 1999, owing to the wet spring of the first year
and the unusually dry late spring of the second year. It is reasonable to suppose that
greater epidemic intensity might facilitate stronger selection. Why selection should have
favored decreased, rather than increased, aggressiveness in 1998 is unclear.
Given the findings of Ahmed et al. (1), we designed the rate-reduction experiment
to help expose the mechanism by which susceptible cultivars might select for more
55
aggressive isolates. That is, we expected that the lower rate of pathogen reproduction
induced by the fungicide would result in isolates that were less aggressive than isolates
from unsprayed plots of the same susceptible cultivars. In fact, we found the opposite,
that isolates from the sprayed treatments were significantly more aggressive than isolates
from the unsprayed treatments in both years of the experiment. This result is consistent
with our finding that more resistant cultivars select for more aggressive isolates. The
multisite fungicide and partial host resistance exercised qualitatively similar selective
pressures on the fungal populations exposed to them. The influence of the fungicide was
evidently more powerful than that of the resistance, as there were significant differences
in aggressiveness of isolates from sprayed and unsprayed plots in both years but not of
late isolates from MR and S plots in either year.
In both years, Lewjain was the most susceptible of the six tester cultivars (Table
ffl.2), although Lewjain had the lowest disease severity both years in our field
assessments (Table ffl.l). In the same vein, W-301 displayed high susceptibility in the
field, but low to intermediate susceptibility as a tester. One explanation for these
inconsistencies may be that genes governing resistance to M. graminicola differ during
juvenile and adult growth stages, as has been shown (16). Lewjain matures 7-9 days later
than most of the other cultivars in this study, so its adult resistance may be associated
with its different growth habit. One would expect the AL AE measure of within-season
selection for aggressiveness to be relatively robust to juvenile-adult plant resistance
differences. While the three susceptible testers and the moderately resistant tester
Cashup expressed roughly consistent disease ranking both within and between years,
56
Madsen's performance as a tester was inconsistent between years in both experiments
(Tables ffl.2 and ffl.4), and we are uncertain why. Theoretically, the presence ofracespecific resistance genes could account for such anomalies as those observed with
Lewjain, W-301 and Madsen. However, in both years the isolate-source x tester
interaction was nonsignificant and did not depend on collection date (Table ffl.3).
In summary, earlier suggestions (1; Mundt, unpublished) that host susceptibility
selects for increased pathogen aggressiveness may have been caused by artifacts in
experiments that were not originally designed to test this hypothesis. Results of the
present study suggest that disease management practices that suppress epidemic
development (e.g., quantitative resistance and protectant fungicides) may select for
increased pathogen aggressiveness, a result consistent with previous studies (21,24).
Conditions in the Willamette Valley of Oregon are highly favorable for adaptation of M.
graminicola (19). Thus, similar effects may not be detected within a single season for
pathogens that are less variable, in situations where epidemics are less frequent, or where
there is significant pathogen migration from areas with different selective influences.
ACKNOWLEGEMENTS
We thank Dan Coyle, Molly Hoffer, and Kellie Webb for expert technical
assistance, and Dave Thomas for statistical advice. We also appreciate the comments of
anonymous reviewers, which considerably strengthened the paper.
57
LITERATURE CITED
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influence of wheat cultivars on pathogenicity of Mycosphaerella graminicola
(anamorph Septoria tritici). Phytopathology 86:454-458.
2. Cowger, C., Hoffer, M. E., and Mundt, C. C. 2000. Specific adaptation by
Mycosphaerella graminicola to a resistant wheat cultivar. Plant Pathol. 49:445-451.
3. Cowger, C., Mundt, C. C., and Hoffer, M. B. 1999. A vertically resistant wheat
cultivar selects for specifically adapted Mycosphaerella graminicola strains. Pages
85-86 in: van Ginkel, M., McNab, A., and Krupinsky, J., eds. Septoria and
Stagonospora Diseases of Cereals: A Compilation of Global Research. Mexico, D.
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4. Dubin, J., and Rajaram, 5. 1996. Breeding disease-resistant wheats for tropical
highlands and lowlands. Annu. Rev. Phytopathol. 34:503-526.
5. Eyal, Z. 1999. Breeding for resistance to Septoria and Stagonospora diseases of
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on Cereals: A Study of Pathosystems. Wallingford, UK: CAB International.
6. Eyal, Z. 1999. The Septoria tritici and Stagonospora nodorum blotch diseases of
wheat. Eur. J. Plant Pathol. 7:629-641.
7. Eyal, Z., Amiri, Z., and Wahl, I. 1973. Physiologic specialization of Septoria tritici.
Phytopathology 63:1087-1091.
8. Gandon, S., and Michalakis, Y. 2000. Evolution of parasite virulence against
qualitative or quantitative host resistance. Proc. R. Soc. Lond. B 267:985-990.
9. van Ginkel, M., and Rajaram, S. 1999. Breeding for resistance to the
Septoria/Stagonospora blights of wheat. Pages 117-126 in: van Ginkel, M., A.
McNab, and Krupinsky, eds. Septoria and Stagonospora Diseases of Cereals: A
Compilation of Global Research. Mexico, D. F.: CIMMYT.
10. van Ginkel, M., and Scharen, A. L. 1988. Host-pathogen relationships of wheat and
Septoria tritici. Phytopathology 78:762-766.
11. Jlibene, M., and El Bouami, F. 1995. Inheritance of partial resistance to Septoria
tritici in hexaploid wheat (Triticum aestivum). Pages 117-124 in: Gilchrist, S. L.
van Ginkel, M., McNab, A., and Kema, G. M. J., eds. Proceedings of a Septoria
tritici workshop. Mexico, D. F.: CIIvIMYT.
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12. Jlibene, M., Gustafson, J. P., and Rajaram, S. 1994. Inheritance of resistance to
Mycosphaerella graminicola in hexaploid wheat. Plant Breed. 112:301-310.
13. Johnson, R. 1992. Past, present and future opportunities in breeding for disease
resistance, with examples from wheat. Euphytica 63:3-22.
14. Kema, G. H. J., Annone, J. G., Sayoud, R., van Silfhout, C H., Van Ginkel, M., and
de Bree, J. 1996. Genetic variation for virulence and resistance in the wheatMycosphaerella graminicola pathosystem. I. Interactions between pathogen isolates
and host cultivars. Phytopathology 86:200-2 12.
15. Kema, G. H. J., Sayoud, R., Annone, J. G., Van Silthout, C. H. 1996. Genetic
variation for virulence and resistance in the wheat-Mycosphaerella graminicola
pathosystem. II. Analysis of interactions between pathogen isolates and host
cultivars. Phytopathology 86:213-220.
16. Kema, G. H. J., and van Silthout, C. H. 1997. Genetic variation for virulence and
resistance in the wheat -Mycosphaerella graminicola patbosystem. ifi. Comparative
seedling and adult plant experiments. Phytopathology 87:266-272.
17. McDonald, B. A., Mundt, C. C., and Chen, R.-S. 1996. The role of selection on the
genetic structure of pathogen populations: Evidence from field experiments with
Mycosphaerella graminicola on wheat. Euphytica 92:73-80.
18. Milus, E. A., and Chalkley, D. B. 1997. Effect of previous crop, seedborne
inoculum, and fungicides on development of Stagonospora blotch. Plant Dis.
11:1279-1283.
19. Mundt, C. C., Hoffer, M. E., Ahmed, H. U., Coakley, S. M., DiLeone, J. A., and
Cowger, C. 1999. Population genetics and host resistance. Pages 115-130 in:
Lucas, J. A., Bowyer, P., Anderson, A. M., eds. Septoria on Cereals: A Study of
Pat hosystems. Wallingford, UK: CAB International.
20. Petersson, S., and Schnurer, J. 1999. Growth of Penicillium roqueforti, P. carneum,
and P. paneum during malfunctioning airtight storage of high-moisture grain
cultivars. Postharvest Biol. Tec. 1:47-54.
21. Pink, D. A. C., Lot, H., and Johnson, R. 1992. Novel pathotypes of lettuce mosaic
virus breakdown of a durable resistance? Euphytica 63:169-174.
22. Rubiales, D., Reader, S. M., and Martin, A. 2000. Chromosomal location of
resistance to Septoria tritici in Hordeum chilense determined by the study of
59
chromosomal addition and substitution lines in 'Chinese Spring' wheat. Euphytica
3:221-224.
23. Scharen, A. L. 1999. Biology of the Septoria/Stagonospora pathogens: an overview.
Pages 19-22 in: van Ginkel, M., McNab, A., and Krupinsky, J., eds. Septoria and
Stagonospora Diseases of Cereals: A Compilation of Global Research. Mexico, D.
F.: CIIvIMYT.
24. Schouten, H. J., and Beniers, J. E. 1997. Durability of resistance to Globodera
pal/ida. I. Changes in pathogenicity, virulence, and aggressiveness during
reproduction on partially resistant potato cultivars. Phytopathology 87:862-867.
25. Vanderplank, J. E. 1968. Disease Resistance in Plants. New York, USA: Academic
Press.
26. Zhan, J., Mundt, C. C., and McDonald, B. A. 2001. Using restriction fragment
length polymorphisms to assess temporal variation and estimate the number of
ascospores that initiate epidemics in field populations of Mycosphaerella
graminicola. Phytopathology 91:1011-1017.
CHAPTER IV: Frequency of SexuaL Reproduction by Mycosphaerella graminicola
on Partially Resistant Wheat Cultivars
Christina Cowger, Bruce A. McDonald, and Christopher C. Mundt
First and third authors: Department of Botany and Plant Pathology, 2082 Cordley Hall,
Oregon State University, Corvallis 9733 1-2902. Second author: Institute of Plant
Sciences, Phytopathology Group, ETH ZentrumlLFW, Universitätstrasse2, CH-8092
ZUrich, Switzerland.
Supported by award number 97-35303-4537 of the NRI Competitive Grants
Program/USDA
Accepted for publication
Corresponding author: C. Cowger; E-mail address: cowgercbcc.orst.edu
Cowger, C., McDonald, B. A., and Mundt, C. C. 200_. Frequency of sexual
reproduction by Mycosphaerella graminicola on partially resistant wheat cultivars.
Phytopathology, in review.
61
ABSTRACT
The frequency of sexual reproduction has a profound effect on the population
structure and the adaptive potential of a facultatively sexual parasite. Little is known
about the relationship of quantitative host resistance to the frequency of sex in pathogens.
We sampled over 5,000 fungal fruiting bodies from seven different wheat cultivars over a
three-year period. The cultivars possessed varying degrees of susceptibility to
Mycosphaerella graminicola, a facultatively sexual pathogen that is heterothallic and
bipolar. The fruiting bodies were classified as M. graminicola pycnidia or ascocarps,
other fungi, or unidentified. In all three years, area under the disease progress curve
(AIJDPC) explained a significant proportion of the variation in the percentage of M
graminicola ascocarps (P < 0.0005). The mean percentage of M graminicola ascocarps
from all cultivars was 63% in 1998, when the epidemic was intense, and 14% in 1999, a
year of low disease levels. In 2000, samples were taken at 7-day intervals from 6 June to
27 June from two cultivars with substantially different AUDPCs (788 and 2185
percentage-days). The less diseased cultivar yielded its first M. graminicola ascocarps
one week later than the more diseased cultivar, and respective mean ascocarp percentages
across sampling dates were 20.2% and 59.3%. The frequency of sexual reproduction by
M graminicola is likely to be strongly conditioned by infection density.
Additional keywords: microconidia, Phaeosphaeria nodorum, Red Queen hypothesis,
Septoria tritici blotch, Triticum aestivum
62
INTRODUCTION
As an evolutionaiy strategy, sexual reproduction can increase pathogenic fitness
in three ways. First, it can generate new combinations of alleles (new genotypes) through
independent assortment that have higher fitness as a result of interactions among alleles
at different loci (20,28,33). Second, through intragenic recombination it can create new
alleles (36), possibly at loci that affect pathogenicity (7). Third, sexual reproduction may
free beneficial alleles from disadvantageous alleles at other loci and prevent
accumulation of low-fitness alleles in a single genetic background (28,29,33,37). Thus,
sex can increase the effectiveness of selection and facilitate adaptation by the pathogen
population to host defenses or chemical pesticides (5,37).
Experimental evidence indicates that there is a trade-off between sexual and
asexual reproduction in many fungal species, and that the balance is influenced by several
genetic and environmental factors (9). Different ecological roles for sexual and asexual
propagules may help to maintain recombination in the life cycle of organisms that are
facultatively sexual, as are numerous plant-pathogenic fungi (8,34). The relative
contributions of sexual and asexual reproduction have a significant effect on pathogen
population structure (2,29), and have been investigated using direct and indirect
techniques for several plant pathosystems (6,31,35,40).
Whether facultative sexual reproduction by a pathogen is more frequent on
resistant or susceptible hosts is a matter with important epidemiological and disease-
management consequences (19). Little empirical evidence is available to shed light on
this issue. A study of potato cultivars with different levels of race-nonspecific resistance
63
to Phytophthora infestans indicated that oospore formation was highest in cultivars with
intermediate levels of resistance, perhaps because susceptible cultivars were destroyed
too quickly to allow oospore formation (21). Gemmill et al. (19) reported that facultative
sexual reproduction by parasitic nematodes was higher when challenged by a genotype-
specific immune response in rats than in the absence of the immune response. They
suggested that sex in pathogens might be an adaptation to combat facultative antiparasite
defenses. This would essentially be the converse of the Red Queen hypothesis, which
holds that sexual reproduction is maintained in hosts as a response to cyclical changes in
selection pressure caused by pathogens over time (5,11). A theory that accounts for
sexual reproduction in facultatively sexual organisms as a response to stress (3) might
also lead one to expect higher levels of pathogen sexual reproduction on more resistant
hosts.
Mycosphaerella graminicola, a fungus that is probably bipolar and heterothallic
(27), is a facultatively sexual pathogen of wheat (Triticum aestivum). This fungus causes
the increasingly damaging foliar disease Septoria tritici blotch. A good deal is already
known about the sexual stage of M graminicola in winter bread wheat. Ascospores are
not only the primary source of inoculum that initiates epidemics (38,39), but are also
produced under at least some conditions throughout the year (22,27). The sexual stage
has a major impact on the genetic structure of M. graminicola populations (10,32), which
are characterized by high levels of gene and genotypic diversity (31).
The role of sexual reproduction in disease progress, and the environmental and
host factors that influence the degree of sexual reproduction, are still uncertain. Kema et
64
al. (27) reported completion of a sexual cycle on a susceptible cultivar in five weeks, and
suggested that M. graminicola could complete several sexual cycles per season. Eriksen
et al. (17) predicted on the basis of mathematical modeling that, while ascospores should
have little impact on epidemic progress, the proportion of all pycnidia that were sexual
descendants could be as high as 20-30% by the end of the growing season. Similarly,
Zhan et al. (42,43) estimated 21% sexual recombinants in a sample of 600 isolates
gathered from field plots at the end of a growing season and analyzed using molecular
marker data.
To date, no published research about the role of sexual reproduction in this
disease system has specifically taken into account differences in host susceptibility. We
sampled fruiting bodies from wheat cultivars with varying levels of resistance to M.
graminicola. Our purpose was to better understand the relationship between degree of
host resistance and frequency of sexual reproduction.
MATERIALS AND METHODS
Field experiments
Cultivars and plot establishment
Three of the six cultivars selected in 1998 (Stephens, Madsen, and MacVicar)
were commonly grown in western Oregon, where the experiment took place. The other
three cultivars, not commonly grown in this region, were chosen to provide a balance
65
between moderately resistant and susceptible cultivars. The moderately resistant
cultivars had expressed approximately one-half of the level of disease found on the
susceptible cultivars in western Oregon (DiLeone and Coakley, unpublished data). Days
to heading, which is moderately correlated with resistance to M. graminicola (15), also
varied among cultivars (Table IV.!). Heights of the six cultivars were unassociated with
susceptibility to M graminicola.
In 1999, the cultivars Gene and Foote were added to the experiment in order to
enlarge the sample and because Foote was both moderately resistant and had a similar
maturity date to those of the susceptible cultivars Stephens, W-301, and MacVicar (Table
IV.1). At its release in 1992, Gene was highly resistant to M. graminicola, but that
resistance had largely disintegrated by 1995, and specific interactions between Gene and
the pathogen have been demonstrated (14). Gene matures approximately 10 days earlier
than the other susceptible cultivars in the experiment.
Field plots were established each year at the Oregon State University Botany and
Plant Pathology Field Laboratory in Corvallis, Oregon, USA. Each of the four plots of
each treatment occupied two adjacent 1.5 x 6.1 m. planting units. Plots of wheat and
barley were alternated in a checkerboard pattern so that the barley provided a buffer
between the wheat plots. Plots were planted on 20 October 1997, 7 October 1998, and 12
October 1999. Fertilization, planting, and weed control were all conducted according to
standard commercial practices in the area.
TABLE IV.1. Percentages of Mycosphaerella graminicola, Phaeosphaeria nodorum, and unidentified fruiting bodies
sampled at the end of three growing seasons from four replicated field plots of wheat cultivars with
differing levels of
susceptibility to Septoria diseases
M graminicola
Yea?
Cultivar
1998
Cashup
Lewjain
MacVicar
Madsen
Stephens
W-301
Days to
headingb
Sexual
(% of total)c
Asexual
(% of total)c
145.0
147.5
139.0
140.5
138.0
138.5
23.8
2.8
27.7
39.4
45.1
30.2
3.8
11.3
60.6
4.6
4.7
3.4
4.5
19.2
87.1
16.7
24.7
15.5
0.7
0.0
5.2
0.0
4.7
0.4
11.0
4.4
5.7
12.4
41.7
44.4
Steph-spray8
Mean
1999
Cashup
Foote
Gene
Lewjain
MacVicar
Madsen
Stephens
W-301
Steph-low8
Stephhig
145.0
138.5
128.0
147.5
139.0
140.5
138.0
138.5
15.1
70.5
23.0
31.6
23.9
24.5
31.6
37.3
Sexual
of
(% M. gram.)d
67.9
4.4
85.7
89.3
92.9
P. nodorum
(% of total)e
3.1
7.0
6.9
1.8
Unidentified
(% of total)1'
61.9
29.6
60.7
3.4
5.0
54.1
48.1
60.3
63.4
4.2
55.7
1.6
0.0
6.5
0.9
0.0
0.0
0.0
0.5
0.5
0.0
0.6
57.6
49.1
78.9
29.5
72.3
68.0
64.6
70.6
62.7
49.7
0.0
25.5
0.0
16.9
1.4
31.5
15.3
15.4
25.0
1.9
75.0
TABLE IV.1, Continued
Mean
2000
Mean
W301-low5
W301-high5
Gene
Stephens
128.0
138.0
1.8
10.9
3.3
25.9
13.5
0.0
0.0
0.7
45.9
4.8
52.4
31.4
35.6
5.2
23.4
10.9
8.7
32.1
72.9
56.3
16.8
27.6
14.3
9.8
52.5
36.6
39.4
57.7
58.8
51.1
aFmiting bodies sampled from leaves collected on 2 July 1998, 29 June 1999, and 27 June 2000.
bjuljan days in Corvallis, Oregon, averaged for 1994 and 1995,
except Foote for which the mean of Julian days to heading in
1997 and 1998 was adjusted by the average differences between the other cultivars' readings in the
two pairs of years
(23,24,25,26).
CM graminicola pycnidia or pseudothecia as a percentage of all fruiting bodies sampled.
dPseudothecia as a percentage of M. graminicola pseudothecia and pycnidia.
eObvious P. nodorum lesions were excluded when selecting leaf segments for fruiting body sampling in order to enrich
samples for M graminicola fruiting bodies.
1'Fruiting bodies of fungi recognizable as other than M graminicola and P. nodorum ranged from 0.0%
to 8.3%.
51n 1998, chiorothaloml was applied to each plot at a rate of 0.84 Liplot and a dosage of 3.7 ml chlorothalomllL water
seven
times during the growing season. In 1999, the dosages were 1.8 mi/L (high) and 0.88 mi/L (low) and five applications
were
made during the growing season.
Fungicide applications
Reduction of disease levels on susceptible cultivars was accomplished by
applying the non-systemic, protectant fungicide chlorothalonil (Bravo 720, 720 g
chlorothalonillL, 54% a.i.). In 1998, the concentration was 3.7 ml Bravo per L water,
about 80% of manufacturer's recommended strength, with the surfactant Triton B added
at the rate of 0.176 mlIL. Applications were made to plots of the cultivar Stephens at a
rate of 0.84 LIplot (468 Iiha) seven times at intervals of 2.5-5 wk, depending on the
growth rate of the wheat, from 10 December to 22 May.
In the second year of the experiment, lower fungicide dosages of 1.8 mi/L and
0.88 mi/L were utilized in order to effect more moderate disease reductions. The
fungicide was applied only five times at approximately 3-wk intervals between late
January and late April, and plots of a second susceptible cultivar, W-301, were also
sprayed.
Disease measurements
Visual assessments of percent diseased leaf area were conducted by two assessors
for each plot on a whole-canopy basis eight times at 10- to 27-day intervals between 6
February and 15 June 1998, 17 February and 17 June 1999, and 18 February and 6 June
2000.
Leafsampling
In 1998 and 1999, leaf sampling took place on 2 July and 29 June, respectively.
One F-4 leaf was randomly selected from each of five rows of each replicate plot of each
treatment, excluding the outermost rows to avoid edge effects, and the leaves were
bagged by replicate. In 1999, F-4 leaves were also collected in the same manner on 26
May from all replicates of Gene and Stephens in order to examine fruiting bodies present
prior to the end of the growing season.
In 2000, F-4 leaves were collected separately by replicate from plots of Gene and
Stephens four times at 7-day intervals starting 6 June and ending 27 June.
Fruiting body sampling and identification
Fruiting bodies were sampled from each field replicate as follows: approximately
7-8 cm of leaf tissue with a high density of fruiting bodies was frozen approximately 30
sec in liquid nitrogen, and then ground for 10-15 sec into a semi-fine powder with a
mortar and pestle. Working with a fine-pointed needle under a dissecting microscope,
3 0-70 fruiting bodies were selected at random from the powder, with the number selected
depending on availability. The exception in sample size was from the Stephens sprayed
treatment in 1998, from which only 52 fruiting bodies could be recovered from leaves
collected in all four field replicates combined, due to the small number of lesions on
those leaves.
70
A total of 5,454 fruiting bodies were examined for the three years: 1,154 from
1998, 2,841 from 1999, and 1,459 from 2000. Leaving aside the Stephens high-spray
treatment in 1998, a mean of 195 fruiting bodies were sampled from each host on each
sampling date in each year across all four field replicates, with sample sizes ranging from
137 to 290 fruiting bodies. The 26 May 1999 samples from the cultivars Gene and
Stephens contained no M graminicola pseudothecia, and those data are not included in
the presentation of results.
Fruiting bodies were sorted and examined by size because previous observations
had suggested that larger fruiting bodies tended to be M. graminicola pycnidia. Each
fruiting body was assigned by eye to one of three size categories (small, medium, and
large), such that there were roughly equal numbers of fruiting bodies in each category.
Members of a single size category were placed individually on a microscope slide in a
small drop of lactophenol blue stain. The fruiting bodies were squashed gently under a
cover slip, and allowed to absorb stain overnight.
Fruiting bodies were identified under a compound light microscope at 400X
magnification. Each fruiting body was classified as one of the following: M
graminicola ascocarp (pseudothecium), M. graminicola pycnidium, P. nodorum ascocarp
(pseudothecium), P. nodorum pycnidium, or unidentified. Fruiting bodies were only
determined to be ascocarps or pycnidia of M graminicola or P. nodorum if clearly
recognizable asci, ascospores, or pycnidiospores were present. Asci, ascospores, macroand micropycnidiospores of the two genera were distinguished according to the
descriptions of Eyal et al. (18).
71
Statistical methods
Logistic regression by the SAS GENMOD procedure (1) was used to assess the
effects of the explanatoiy variables of field replicate, AUDPC, and size on the
dichotomous response variable sexuallasexual for identified M. graminicola fruiting
bodies. Because goodness-of-fit criteria indicated variation other than binomial variation
in the data, a scaling option (d-scale) was used that multiplies the binomial variance by a
constant and performs F-tests rather than
x2 tests.
Thus, standard errors increased by a
factor of two, making it harder to achieve significance. In fitting the model, the inclusion
of the term AUDPC2 significantly improved the goodness of fit in 1998 and 1999 but not
in 2000.
Correlation analysis was performed in SAS to assess the strength of the linear
relationships between AUDPC and 1) pseudothecia as a percentage of M. graminicola
fruiting bodies, and 2) M graminicola pseudothecia as a percentage of all fruiting bodies
sampled. The correlation was also assessed between the percentage of unidentified
fruiting bodies and percentages in the total sample of M. graminicola pycnidia and
pseudothecia, respectively.
When comparing the years 1998 and 1999 for the effects of cultivar susceptibility
on pseudothecial percentages, mid-June disease severity was used rather than AUDPC as
the measure of susceptibility. This was because the shapes of the disease progress curves
from the two years differed, rendering the comparison of area under them misleading,
whereas for within-year comparisons AUDPC was preferable due to lower error.
72
RESULTS
In all three years, AUDPC was predictive of the frequency of M graminicola
ascocarps (Table IV.2, Fig. 1V.1), with P < 0.0005 by logistic regression. Correlation
analysis indicated a positive relationship between AUDPC and M. graminicola ascocarps
as a percentage of
M graminicola fruiting bodies (r= 0.90 and P = 0.0054 in 1998, r =
0.79 and P = 0.0022 in 1999). If the 1999 sprayed treatments were omitted from the
analysis, the association became stronger in that year (r
0.93, P = 0.0009). There was
also a positive relationship between AUDPC and M. graminicola ascocarps as a
percentage of all sampled fruiting bodies (r= 0.84 and P = 0.0173 in 1998, r = 0.60 and P
= 0.0396 in 1999). From logistic regression, a one-unit increase in AUDPC was
associated with increases in the odds of an identified M. graminicola fruiting body being
an ascocarp of 1.8%, 2.1%, and 1.4% for the three years, respectively.
Mean epidemic intensity and mean pseudothecial percentages varied among the
years. In 1998, the epidemic developed consistently and strongly throughout the season.
In 1999, there were relatively low levels of disease due to a dry late spring. In 2000,
disease increased slowly in the early season, but rapidly in May and June. Both mean
late-season disease seventies and mean pseudothecial percentages were greater in 1998
than in 1999 (Fig. IV.2). Means
of pseudothecia
as a percentage of M graminicola
fruiting bodies were 63.4% in 1998, 13.5% in 1999, and 52.5% in 2000 (Table IV.1).
In 1998, the application of fungicide reduced both disease severity and percent
pseudothecia on Stephens to near the levels on Lewjain, the most resistant cultivar
73
TABLE IV.2. Significance of explanatory variables from logistic regression analysis,
with response variable being the pseudothecial proportion of all identified M.
graminicola fruiting bodies sampled in 1998, 1999, and 2000
1998
Parameter
df
F-value
P-value
Rep
3
2
3.06
4.15
36.12
2
19.36
7.60
0.0269
0.0157
<0.0001
<0.0001
0.0224
Parameter
df
F-value
P-value
Rep
3
2
0.0081
0.1077
<0.0001
0.0005
0.7915
Sizea
AUDPCb
AUDPC2
Size*AUDPC
1999
1
1
AUDPC2
Size*AUDPC
2
3.93
2.23
16.17
12.04
0.23
2000
Parameter
df
F-value
P-value
Rep
3
2.52
10.66
3.19
12.65
0.72
0.19
0.0560
<0.0001
0.0410
0.0004
0.3948
0.8282
Sizea
AUDPCb
Datec
Sizea
AUDPCb
AUDPC2
Size*AUDPC
1
1
3
2
1
1
1
aBefore staining and identification, fruiting bodies were classified by eye as small,
medium, or large.
bMea under the disease progress curve was a continuous variable, calculated from visual
assessments of percent diseased leaf area conducted by two assessors for each plot on a
whole-canopy basis eight times at 10- to 27-day intervals between 6 February and 15
June 1998, 17 February and 17 June 1999, and 18 February and 6 June 2000.
Cj
2000, F-4 leaves were collected at 7-day intervals starting 6 June and ending 27 June.
100
s'.
Mi
1998
100
1999
80
r=0.79
P = 0.0022
60
40
40
G
W-high S-high
20
20
S-spray
0
1400
S
-,
1775
2150
AUDPC
2525
2900
1400
1750
2100
ALJPC
2450
2800
Fig. IV.1. Plot of pseudothecia as a percentage of all Mycosphaerella graminicola fruiting bodies on area under the disease
progress curve (AIJDPC) assessed in four replicated field plots of each host. Hosts: C = Cashup, F = Foote, G = Gene, L =
Lewjain, Mc = MacVicar, Md = Madsen, S Stephens, W = W-301. Low = low-spray, high = high-spray. To reduce
epidemic development, 0.84 L chiorothalonil was applied to each plot at a dosage of 3.7 mi/L water seven times during the
1998 growing season. During the 1999 growing season, five applications were made with dosages of 1.8 mi/L (high) and 0.88
mi/L (low).
100
1998+1999
e80
0
0
0
.
r=0.81
P <0 0001
60
0
a.
a.
4O
20
0
0
20
40
60
80
100
Mid-June dIsease severity
Fig. IV.2. Plot of pseudothecia as a percentage of all Mycosphaerella graminicola
fruiting bodies vs. disease severity assessed on 15 June 1998 and 17 June 1999 in four
replicated field plots of seven and 12 treatments, respectively; 1998 = 0, 1999 .
(Fig. IV. 1). In 1999, when the high-spray dosage was half that applied in 1998, high and
low dosages resulted in similar levels of disease reduction: 19.0%, 19.2%, 15.2%, and
17.3% in the Stephens low, Stephens high, W-301 low, and W-301 high spray plots,
respectively. Both fungicide dosages reduced pseudothecia as a percentage of M
graminicola fruiting bodies on Stephens, but only the low dosage did so on W-301 (Table
N.1).
In 2000, pseudothecia constituted a lower percentage of M graminicola fruiting
bodies on each sampling date and averaged over all sampling dates on Gene than on
Stephens (Fig. P/.3; means of 20.2% and 59.3%, respectively). In that year, AIJDPCs
were 788 and 2185 percentage-days for Gene and Stephens, respectively.
76
100%
80%
6-Jun
O Gene pseudothecia
i Steph pseudothecia
13-Jun
20-Jun
2000 sampling dates
27-Jun
Fig. IV.3. Pseudothecia as a percentage of Mycosphaerella graminicola fruiting bodies
sampled from leaves collected in field piots at one-week intervals between 6 June and 27
June 2000 from the winter wheat cultivars Gene and Stephens. Area under the disease
progress curve (percentage-days) in the field plots was 788 and 2185 for Gene and
Stephens, respectively.
While P. nodorum fruiting bodies were absent or at veiy low percentages on all
cultivars in 1998 and 1999, they comprised 56.3% and 15.9% of the fruiting bodies
sampled from Gene and Stephens, respectively, at an equivalent point in the 2000
growing season (Table IV. 1). In 2000, P. nodorum fruiting bodies increased from 7.4%
to 56.3% of all fruiting bodies sampled on Gene between 6 June and 27 June, and
increased from 10.5% to 33.7% of all fruiting bodies sampled from Stephens between 6
June and 20 June before dropping again to 16.9% of all fruiting bodies on 27 June.
Probably due to competition with P. nodorum, only 24.1% of sampled fruiting bodies
were identified as M. graminicola in 2000. In that year, 19.6% of fruiting bodies from
the two cultivars were M. graminicola pseudothecia., and 9.8% were pycnidia.
77
The mean frequency with which M graminicola pycnidia contained some or
exclusively micropycnidiospores was 27.7% in 1998 and 56.6% in 1999 (Table P1.3). In
2000, when only two hosts were sampled, the mean frequency of micropycnidiospores
was 30.4%.
Among M. graminicola fruiting bodies, smaller size was associated with a lower
frequency of pseudothecia (Table P1.2). In 1998, the relationship of size to proportion of
pseudothecia varied with AUDPC: higher pseudothecial proportions were found among
large fruiting bodies for higher AUDPCs and among medium-sized fruiting bodies for
lower AUDPCs. In 1999, higher pseudothecial proportions were found among larger
fruiting bodies; size was significant at P = 0.11. Across all sampling dates in 2000, size
was significant at P = 0.04 10 and higher pseudothecial proportions were found among
larger fruiting bodies. In general, fruiting bodies seemed to be smaller on moderately
resistant than on susceptible cultivars.
Correlation analysis indicated that in both 1998 and 1999, there was some degree
of negative association between the percentages of
M graminicola pycnidia and
unidentified fruiting bodies (r = -0.70 and P = 0.08 18 in 1998, r = -0.96 and P < 0.0001
in 1999). No association was found in either year between the percentages of M.
graminicola ascocarps and unidentified fruiting bodies (P
0 .8397 in 1998 and P =
0.4330 in 1999). In 2000, there was no significant association between the percentages
of M graminicola pycnidia or pseudothecia and that of unidentified fruiting bodies (P =
0.6421 and P = 0.4059, respectively). AUDPC was uncorrelated with percent of
78
TABLE IV.3. Mycosphaerella graminicola pycnidial types in samples from field plots
of winter wheat leaves at the ends of three growing seasons
Percenta
Treatment
1998
Cashup
Lewjain
MacVicar
Madsen
Stephens
W-301
Steph-spray
Micro
27.8
74.8
0.0
25.0
28.6
12.5
10.0
MEAN
25.5
1999
Cashup
Foote
Gene
Lewjain
MacVicar
Madsen
Stephens
W-301
Steph-high
Steph-low
W-high
W-Iow
73.6
56.3
51.1
84.2
41.7
44.3
59.6
70.7
46.2
46.8
2.8
46.3
MEAN
2000
Gene
Stephens
MEAN
52.0
10.5
43.8
27.2
Macro
72.2
22.1
100.0
62.5
71.4
87.5
90.0
72.3
18.5
40.8
46.1
13.2
46.4
52.1
32.5
26.2
51.4
50.2
93.7
50.8
43.5
89.5
50.0
69.8
Both
0.0
3.1
0.0
12.5
0.0
0.0
0.0
2.2
8.0
3.0
2.8
2.6
12.0
3.7
7.9
3.2
2.4
3.1
3.6
3.0
4.6
0.0
6.3
3.2
Total number
of pycnidia
18
131
8
8
7
8
10
27.1
121
75
35
146
61
73
50
50
63
66
43
89
72.7
19
16
17.5
apercenges of M graminicola pycnidia containing only micropycnidiospores, only
macropycnidiospores, and both.
summarized evidence that contact with mycelial extracts triggers sexual morphogenesis
and/or the switch from vegetative growth to reproduction in facultatively sexual fungi,
even across species lines.
It is also possible that certain genes conferring partial resistance in plant hosts
exercise a pleieotropic effect on the frequency of sexual reproduction in their pathogens,
e.g., by delaying the induction of sexual sporulation (9). However, the hypothesis that
lower rates of sexual reproduction are a function of lesser disease severity, and not of
resistance itself, is supported by the fact that, with one exception, fungicide applications
reduced pseudothecial percentages on susceptible cultivars relative to unsprayed plots of
the same cultivars. In the case of the W-301 high-spray treatment in 1999, the reason for
the higher pseudothecial percentage is unclear.
Given the moderate negative correlation between days to heading and
susceptibility to M graminicola, the possibility must be considered that more frequent
sexual reproduction is actually in some manner an outgrowth of more rapid progress to
maturity. However, the lower percentages of pseudothecia in sprayed plots of Stephens
in 1998 and on Foote in 1999, relative to other treatments with the same heading dates,
argue against maturity as a significant confounding factor, as do the lower pseudothecial
percentages on Gene than on Stephens in 2000.
Higher percentages of microconidia were found in 1999 than in the other two
years. In some fungal genera, spermatia (microconidia) are borne both in the same
conidioma as macroconidia and in separate spermatium-bearing structures resembling the
conidioma (12). However, it is unknown whether M. graminicola microconidia serve as
81
spermatia, and whether they are as infective as macroconidia. The higher percentages of
microconidja found in 1999 would be consistent with a sexual role for these spores, since
the epidemic that year was milder than those of 1998 and 2000, and fewer ascocarps had
formed by the sampling date. It is also possible that microconidia are formed by M.
graminicola in response to stress.
The negative correlation between the percentages of M. graminicola pycnidia and
unidentified fruiting bodies found in 1998 and 1999, coupled with the lack of a similar
correlation in the case of pseudothecia, suggests that the majority of the unidentified
fruiting bodies were spent pycnidia. The absence of a linear relationship between either
type of M graminicola fruiting body and the unidentified fruiting bodies in 2000,
together with the fact that the P. nodorum epidemic was severe that year, is consistent
with the hypothesis that a large fraction of the unidentified fruiting bodies in 2000 were
spent pycnidia of P. nodorum.
The higher frequency of unidentified fruiting bodies on more susceptible cultivars
in 1999 may reflect the higher rate of pseudothecial formation on those cultivars, and
could imply that immature pseudothecia account for a significant fraction of the
unidentified fruiting bodies in 1999, when sexual reproduction appears to have been
delayed by the mildness of the epidemic. The other possible explanations (that there is a
higher rate of pycnidial or pseudothecial exhaustion on susceptible cultivars, or a lower
rate of pycnidial formation) do not seem logical. There was also no evidence of a higher
frequency of other pycnidial pathogens in 1999.
Why was Gene susceptible to M graminicola in 1999, yet moderately resistant in
2000? In 1995, after isolates had appeared with specific virulence to Gene (14), the
cultivar occupied 16% of the Willamette Valley winter wheat area, and consequently
produced a significant share of total inoculum. Subsequently, the area planted to Gene
diminished by roughly 50% each year until it constituted 1.5% of the total wheat area in
1998 and 0.5% in 1999. Strains virulent to Gene presumably were at increasingly lower
frequencies during this period, and thus random events could at least partially account for
the difference between the two years. In addition, competition from P. nodoruin, to
which Gene is susceptible, may have suppressed overall M graminicola levels on Gene
in 2000. The unusually dry early spring of 2000 depressed Septoria tritici blotch levels
until mid-May and probably favored P. nodorum, which apparently releases its
ascospores later than M. graminicola (16) and is normally outcompeted in the Willamette
Valley.
While our data do not disprove the suggestion of Kema et al. (27) that M
graminicola is capable of multiple sexual cycles in a season, they do suggest one or a
very small number of sexual cycles per season in our environment. Relative to most
other wheat-growing regions, the Willamette Valley of Oregon is highly conducive to
severe M. graminicola epidemics and thus to the sexual stage of the fungus. In the 26
May 1999 samples from the susceptible cultivars Gene and Stephens, no pseudothecia
were found, while one month later, 5.2% of fruiting bodies from Gene and 11.0% from
Stephens were M. graminicola pseudothecia. In the more severe epidemic of 2000,
pseudothecial percentages for the first sampling date, 6 June, were 0.0% for Gene and
83
12.1% for Stephens, and increased on both cultivars on the second and third sampling
dates. It seems likely that the number of sexual cycles completed in a season is
dependent on cultivar susceptibility, and that large numbers of pseudothecia are only
produced toward the end of a growing season, in agreement with other findings
(13,17,42,43).
It should be noted that fruiting body counts do not give direct information about
the relative contributions of sexual and asexual reproduction to the genetic structure of
the M. graminicola population. Empirical data about the respective multiplication
efficiencies of pseudothecia and pycnidia (sensu Eriksen Ct al., 17) under different
environmental conditions are lacking. Molecular marker techniques are currently being
used to address the question of how differing levels of host susceptibility affect the
percentage of sexual descendants at the end of a growing season.
The effect of different levels of host resistance on the frequency of sexual
reproduction in facultatively sexual parasites will be modulated by particular factors in
each pathosystem. For example, the parasitic nematode Strongyloides ratti is
parthenogenetic (19), whereas M. graminicola and P. infestans are both bipolar
heterothallic fungi. For these plant pathogens, the frequency of sexual reproduction is
probably strongly conditioned by the density of infections. In the case of P. infestans, a
further limiting factor is the rapid tissue necrosis in susceptible cultivars, allowing
insufficient time for oospores to form.
Hanson and Shattock (21) conclude that potato cultivars with very high levels of
late blight resistance are likely to minimize formation of P. infestans oospores. Similarly,
84
moderately resistant wheat cultivars limit sexual reproduction in M. graminicola, thus
reducing the rate at which the pathogen can evolve to overcome host defenses or
chemical controls.
ACKNOWLEDGEMENTS
We thank Molly Hoffer and Dan Coyle for technical assistance, Jeffrey Stone for
mycological consulting, and Cliff Pereira for statistical advice.
85
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CHAPTER V: Effects of Wheat Cultivar Mixtures on Epidemic Progression of
Septoria Tritici Blotch and Pathogenicity of Mycosphuerella graminicola
Christina Cowger and Christopher C. Mundt
Department of Botany and Plant Pathology, Oregon State University, Cordley Hall 2082,
Corvallis, OR 97331-2902, USA
Accepted for publication 27 January 2002.
Corresponding author: Christina Cowger, cowgerc@bcc.orst.edu
Cowger, C., and Mundt, C. C. 200_. Effects of wheat cultivar mixtures on epidemic
progression of Septoria tritici blotch and pathogenicity of Mycosphaerella graminicola.
Phytopathology, accepted pending revisions.
ABSTRACT
The effects of host genotype mixtures on disease progression and pathogen
evolution are not well understood in pathosystems that vary quantitatively for resistance
and pathogenicity. We used four mixtures of moderately resistant and susceptible winter
wheat cultivars naturally inoculated with Mycosphaerella graminicola to investigate
impacts on disease progression in the field, and effects on pathogenicity as assayed by
testing isolate populations sampled from the field on greenhouse-grown seedlings. Over
three years, there was a correspondence between the mixtures' disease response and the
pathogenicity of isolates sampled from them. In 1998, with a severe epidemic, mixtures
were 9.4% less diseased than were their component pure stands (P = 0.0045), and
pathogen populations from mixtures caused 27% less disease (P = 0.085) in greenhouse
assays than did populations from component pure stands. In 1999, the epidemic was
mild, mixtures did not reduce disease severity (P
0.39), and pathogen populations from
mixtures and pure stands did not differ in pathogenicity (P = 0.42). In 2000, epidemic
intensity was intermediate, mixture plots were 15.2% more diseased than the mean of
component pure stands (P = 0.053), and populations from two of four mixtures were
152% and 156% more pathogenic than the mean of populations from component pure
stands (P = 0.043 and P = 0.059, respectively). Mixture yields were on average 2.4% and
6.2% higher than mean component pure-stand yields in 1999 and 2000, respectively, but
the differences were not statistically significant. The ability of mixtures challenged with
M. graminicola to suppress disease appears to be inconsistent. In this system, host
91
genotype mixtures evidently do not consistently confer either fitness benefits or liabilities
on pathogen populations.
Additional keywords: epidemiology, host genetic diversity, partial resistance,
quantitative pathosystems, Triticum aestivum
INTRODUCTION
While there is extensive literature about the use of multiline cultivars and cultivar
mixtures to manage disease (e.g., 6,13,36), the vast majority concerns pathosystems in
which pathogenicity (sensu Vanderplank, 35) and resistance vary qualitatively. In
particular, the study of pathogen evolution within mixtures has been almost exclusively
confined to such pathosystems. Little is known about the selective effects of cultivar
mixtures on pathogen evolution in disease systems where resistance is partial.
Mathematical modeling by Jeger et al. (14) indicated that cultivar mixtures may
either decrease, increase, or have no effect on the severity of disease caused by non-
specialized pathogens. In the case of Mycosphaerella graminicola, causal agent of the
foliar disease Septoria tritici blotch in wheat (Triticum aestivum), one might expect
mixtures to be relatively ineffective at disease suppression for two reasons. First, there is
considerable evidence that mixtures are less effective against splash-dispersed pathogens
than against pathogens whose secondary propagules are wind-borne (13). This is likely
due largely to the steeper dispersal gradient of splash-dispersed propagules, more of
which fall upon the hosts of origin (28), thus obviating the dilution advantage (16) of
92
mixing host genotypes. Second, mixtures are predicted to be less effective in suppressing
disease when initial inoculum is abundant and well-distributed (28). Indeed, Abbott et al.
(1) found little benefit from mixing barley cultivars to manage Rynchosporium secalis, a
splash-dispersed pathogen with numerous initial infection foci at their experimental site.
Data so far indicate some promise for controlling Septoria diseases with mixtures,
but most experiments have involved more than one pathogen. Mundt Ct al. (26) reported
that, in plots inoculated with Pseudocercosporella herpotrichoides, winter wheat
mixtures reduced Septona tritici blotch severity by 27%, 9%, and 15% compared to mean
seventies of pure components over three years. However, the greatest reductions in
disease severity were brought about by mixtures including the highly resistant cultivar
Gene, which has since been shown to select for isolates specifically adapted to it (9).
Mille and Jouan (25) found that winter wheat mixtures sustained an average of 8% less
disease caused by M. graminicola, Phaeosphaeria nodorum, and Puccinia recondita and
yielded an average of 7% better than the mean of component pure stands. Jeger Ct al.
(15) found that spring and winter wheat mixtures lightly infected with P. nodorum
(anamorph Stagonospora nodorum) were significantly less diseased and yielded
significantly better than the geometric mean of component pure stands. In an experiment
involving several foliar pathogens, Manthey and Febrmann (22) found that levels of P.
nodorum conidia on flag leaves of winter wheat cultivars in mixtures did not differ
statistically from similar counts for component pure stands.
Much of the theoretical and empirical investigation of pathogen evolution in
mixtures has concerned the potential for selecting complex races (those possessing
93
multiple virulence genes), the relative fitness of complex and simple races, and the extent
to which complex races, if favored, could erode the benefits of multiline cultivars and
cultivar mixtures (4,12,19,21,23,27,29,36). Although this discussion has focused on
qualitatively varying pathosystems, it may be relevant to quantitative systems as well.
Several researchers (8,10,12,20) have suggested that differential adaptation to host
genetic background could result in a higher reproductive rate for simple races as
compared to complex races. Differential adaptation could also operate in pathosystems
not characterized by gene-for-gene interactions or specificity, and thus reduce epidemic
progression in cultivar mixtures.
The M. graminicola-wheat system is useful for addressing these issues because
resistance in most cultivars is partial; also, the pathogen population is highly genetically
diverse (24) and thus there is strong potential for within-season adaptation. Further,
Septoria tritici blotch is a growing worldwide economic threat to wheat production (11),
and there has been insufficient investigation of the potential to manage this disease with
cultivar mixtures.
We conducted an experiment in which naturally inoculated populations of M.
graininicola
were subjected to host selection in plots of pure stands and mixtures of
winter wheat. The pathogenicity of isolates derived from pure stands was compared to
that of isolates from mixtures by testing the isolates as bulked populations on seedlings in
the greenhouse. We assessed disease severity and yield for field plots of mixtures and
pure stands in order to help evaluate the utility of cultivar mixtures for managing Septoria
diseases.
94
MATERIALS AND METHODS
Field experiments
Plot establishment and field disease assessment
Field plots of winter wheat were established during the 1997-98, 1998-99, and
1999-2000 growing seasons at the Oregon State University Botany and Plant Pathology
Field Laboratory in Corvallis, Oregon, USA. Four treatments consisted of the cultivars
Cashup and Madsen, moderately resistant (MR) to Septoria tritici blotch, and Stephens
and W-301, susceptible (S). In addition, the four possible 1:1 mixtures of MR and S
cultivars were planted. Stephens and Madsen were commonly grown in western Oregon,
where the experiment took place, while Cashup and W-301 were not. Mean Julian days
to heading, based on data from 1994 (17) and 1995 (18) for Corvallis, were: Cashup,
145; Madsen, 140.5; Stephens, 138; and W-301, 138.5.
Treatments were assigned randomly to plots in each of four replicate blocks. In
the first two years, each treatment occupied two adjacent 1.5 x 6.1 m planting units of six
rows each (20 cm between rows), while in the third year treatments were applied only to
single planting units in order to conserve space. Equal-sized plots of wheat and barley
were alternated in a checkerboard pattern so that the barley provided a buffer between the
wheat plots. Plots were planted on 20 October 1997, 7 October 1998, and 12 October
1999, using a plot drill and a rate of 2,740 seeds per planting unit. Plots received a
balanced starter fertilizer pre-planting, and 110 kg N/ha at late tillering. Weed control
was via appropriate herbicides, with hand weeding as necessary.
Visual assessments of percent diseased leaf area were conducted by two assessors
for each plot on a whole-canopy basis eight times each year between early-to-mid-
February and early-to-mid-June. Areas under the disease progress curve (AIJDPC) were
calculated for all plots using the mid-point method (7).
Isolate sampling
Isolates of M graminicola were collected from the field plots on 12 June 1998, 24
June 1999, and 27 June 2000 near the end of each growing season, after there had been
ample opportunity for host treatments to exert selective influences on the pathogen
population. In 1998 and 1999, leaves were collected as follows: along a diagonal
transect across each plot, the flag leaf (1998) or the leaf penultimate to the flag leaf (F- 1)
(1999) was collected from each of two adjacent plants chosen at random in each of the 10
innermost rows of the plot, the exterior rows being excluded to avoid edge effects. In
2000, when only four rows remained after excluding the outermost rows, F-i leaves were
sampled from each of two randomly chosen plants at 10 points along an S-shaped
transect, such that there were two or three sampling points in each row. F-i leaves were
collected in 1999 and 2000 because flag leaves on the more resistant cultivars had too
few lesions to assure that isolates could be obtained. Leaves were bagged separately by
row.
In the laboratory, one monopycnidial isolate of M graminicola was obtained for
each sampling point in each replicate of each cultivar of origin each year, such that there
were 10 isolates from each field plot, or 40 isolates per treatment. Isolations were made
by placing leaf segments bearing lesions in Petri dishes lined with moistened filter paper
overnight, and then transferring cirrhi individually with a sterile needle to plates of yeast-
malt agar amended with 50 mgIL streptomycin. The isolates were stored at 4° C and
transferred to fresh yeast-malt agar every 3-5 mo.
Greenhouse trials
Pathogen populations derived from each field plot were evaluated in the
greenhouse during the winter and spring following their collection in the field. Isolates
collected in 1999 were tested twice (in 2000 and 2001) to assess repeatability of the
methodology. In each year of greenhouse testing there were four replicates, conducted in
pairs, with each replicate testing a pathogen population from a different block in the field
experiment. Tester cultivars to which the populations were applied were the same as
those planted in pure stands in the field.
The experimental unit was a pot of seedlings. In 1999, plants were raised in 10cm plastic pots filled with a 1:1:1:2 mixture of peat:sand:loam:#8 pumice amended with
Osmocote 18-6-12 extended time-release fertilizer (Scott-Sierra Horticultural Products
Co., Marysville, OH) at a rate of about I g per pot at seeding. Approximately 13 seeds of
a cultivar were sown in each pot, and seedlings were thinned to ten plants per pot at about
18 days after planting. The plants were grown under sodium halide lighting to extend the
97
daylength to 16 hr, and watered as needed. To control powdery mildew, plants were
treated when necessary with 50 ml of a soil drench of ethirimol (0.11 g a.i.IL), which has
no influence on the development of M graminicola. The greenhouse temperature was
maintained at 20-25° C.
During the second greenhouse season, because of problems in the previous year
with emergence, growth, and non-disease-related necrosis, plants were raised in Black
Gold all-organic potting soil (Black Gold, Inc., Hubbard, OR) amended with about 2.5 g
Osmocote per pot. Pots were also fertilized weekly with 50 ml of Peters water-soluble
20-20-20 fertilizer (United Industries Corp., St. Louis, MO) at the rate of 3.8 g per L of
water. Watering, temperature, and lighting were as in 1999. Practices in the third
greenhouse season were the same as in the second.
Inoculum preparation and inoculation
Isolates of M graminicola were increased individually for four days on fresh
yeast-malt agar. Spores of each isolate were then mixed together by field plot in an
equiproportional suspension and adjusted to
i05
spores/mi for 1998 and 106 spores/ml for
1999 and 2000, the increased concentration having been selected as optimal in the course
of testing soil mix, fertilizers, and inoculum concentration.
When plants were 21 days old (2-3 fully expanded leaves), each spore population
was applied to a group of four pots, one pot of each tester cultivar, by means of a spray
bottle and turntable. For the 1998 isolates and the first trial of the 1999 isolates, 25 ml
were used, while 50 ml were used for the second trial of the 1999 isolates and the 2000
isolates. After inoculation, the plants were kept in a greenhouse mist chamber for 96
hours. Noninoculated control plants were sprayed with water and surfactant only, but
otherwise treated in the same manner as inoculated plants, in order to control for
contamination among pots.
Greenhouse disease assessment
In order to assess disease levels of all plants in a given experiment in the same
day, two workers conducted the assessments, each assessing half of the plants in each
pot. The percentage of the second leaf from the base of the plant covered by Septoria
tritici blotch lesions and lesion-associated necrotic area was estimated visually for each
plant in each pot at 21 days after inoculation.
Data analysis
Within a given year, AUDPC, yield, and greenhouse disease severity data were
analyzed using the SAS procedure PROC MIXED. Means were separated and
differences estimated using linear contrasts. The 2000 AUDPC and greenhouse data
were lo&-transformed to improve homogeneity of variances. Results of the two
greenhouse trials of 1999 isolates were similar, with nonsignificant by-trial interactions,
and so they were combined for statistical analysis.
A correlation analysis was performed across years to test the association between
1) the effect of mixtures on disease severity in the field, and 2) the effect of mixtures on
pathogenicity of isolate populations. The first variable was measured as the percent
difference between mixture AUDPCs and the mean of component pure stand AUDPCs.
The second variable was measured in the greenhouse as the percent difference between a)
the mean disease severity caused by a mixture population tested separately on the two
cultivars of origin (e.g., the mean of Cashup/Stephens tested on Cashup and
Cashup/Stephens tested on Stephens), and b) the mean disease severity caused by the two
component pure-stand populations tested separately on their respective cultivars of origin
(in this case, the mean of Cashup tested on Cashup and Stephens tested on Stephens).
RESULTS
The three study years varied in conduciveness to Septoria tritici blotch according
to measures developed by other researchers (Table V.1). In 1998, both temperature and
rainfall favored a severe epidemic. In 1999 and 2000, temperature was somewhat less
conducive than in 1998. In 1999, a wet early spring was followed by a dry late spring,
keeping ultimate disease seventies relatively low, while in 2000 those conditions were
reversed and, after a dry early season, disease developed strongly later.
As might be expected, disease progress varied considerably among the three study
years (Figs. V.1-3). It should be noted that the flat portions of the disease progress
curves in early season do not reflect an absence of disease increase; rather, pathogen
reproduction approximately kept pace with vegetative growth during those periods.
Overall, Septoria tritici blotch severity was greatest in 1998, followed closely by 2000
and then 1999.
100
TABLE V.!. Conduciveness of three study years to epidemics of Septona tritici blotch
(caused by Mycosphaerella graminicola) in winter wheat
Cumulative
generations
(Dec.June)a
RaindaysC
Number of 5-mm rain-days'
Year
MR S
Dec. Jan.
Feb.
Mar. April May June
1998
1999
6.70 8.17
6.32 7.59
6.39 7.66
6
12
14
18
9
10
2000
11
14
14
14
11
6
3
14
2
6
0
4
2
2
1
1 April14 June
11 May14 June
34
32
18
35
11
15
ausing temperature-based model of Shaw (32), with latent period as the time to the
maximum rate of change in sporulating lesion numbers.
b5
mm has been used as a threshold in identifying when pycmdiospores move between
leaf layers (33), and May rainfall has been found to be predictive of Septoria tritici blotch
severity (31,33,34).
eBased on Shaner and Finney (31); counts are of days with any amount of rain.
Performance of mixtures in suppressing disease varied among mixtures and
among years (Figs. V.1-3). In 1998, AUDPCs of mixtures were on average 9.4% lower
than the mean of their component pure stands (P = 0.0045), while in 1999, AUDPCs of
mixtures did not differ significantly from AUDPCs of component pure stands (P = 0.39).
In 2000, moderately resistant cultivars in pure stands sustained very low rates of disease
despite the later intensification of disease on susceptible cultivars (Fig. V.3), and
mixtures were on average 15.2% more diseased than the mean of their component pure
stands (P = 0.053).
replicates. field four of means are Data 0.0045). = P contrast, (linear stands pure component
for AUDPC mean the than lower 9.4% was mixtures for AUDPC mean The 1998.
in
graminicola
Mycosphaerella
with
inoculated naturally wheat winter of mixtures resistant-susceptible moderately 1:1 four for curves progress
Disease V.!. Fig.
N
-
Date
N
Madsen/W-301
w-301
Madsen(MR)
..c
j
80
I
N
-
N
-
-
A
.-.
N
.-O--.
U
i
W-30l
-
-
80
(
&ephens
(MR) Cashup
I
-
80
100
N
J114379IJ
Cashup/ephens
[
-
Cashup(MR)
(S)
N
N
.0--
&ephens
(
tte
4oJY
CashupfW-301
100
-
4334Q
Madsen/ephens
U
(MR) Madsen
I---0-.A
U
80
100
100
100
100
&ephens(
A
80
-J
1P0.47
40
t)
51.9
AUDPC,
.II
20
0
.
.
I
Ii
Maen (MR)
&ephens(
0-.. Madsen/&ephen
80
-O--- Cashup/&ephens
60
U
A
AUDPC1,
60
:
P -0.23
87.6
40
20
01
'
-
.
N
N
N
.
(1
>
i
N
'
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t
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100
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I JV
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U
A
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80.1-1
Cashup(MR)
W.301(
I
80
I
60
60
40
40
20
20
.5
0
0
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>s
I
I
4
d
N
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Date
>
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Q.,
-e
e
N
.
.
..6
.
.<
N
>..
>s
>1
II
.
0
Date
N
C
J
,
N
Fig. V.2. Disease progress curves for four 1:1 moderately resistant-susceptible mixtures of winter wheat naturally inoculated
with Mycosphaerella graminicola in 1999. The mean AUDPC for mixtures was 1.6% lower than the mean AUDPC for
component pure stands (linear contrast, P = 0.39). Data are means of four field replicates.
100
80
ri-i
£
0--1
60
V -0.99
100
&ephens (
80
thup/&ephens
/
117.9
I
&ephens
(
0
-. -0- -- Madsen/ephcns
0,.,,,,
-600.6
60
-1
inn_____________________________________
U
80
60
£
Cashup(MR)
W-301 (
I(IA
Madsen(MR)
80
--O-. Cashup/W-301
-336.9
/____
60
40
40
20
20
0
0
Date
£ _ W301(
---0--- MadsenlW-301
AUDPC
1P0.44
.IZ):
-175.1
p
O_ T1
Date
Fig. V.3. Disease progress curves for four 1:1 moderately resistant-susceptible mixtures of winter wheat naturally inoculated
with Mycosphaerella graminicola in 2000. The mean AIJDPC for mixtures was 15.2% higher than the mean AUDPC for
component pure stands (linear contrast, P 0.053). Data are means of four field replicates.
0
104
Yields were not recorded for 1998 due to poor stand density in many plots.
Yields of mixture plots exceeded the means of their component pure stands by an average
of 2.4% and 6.2% in 1999 and 2000, respectively, but no individual contrast or main-
effect contrast of mixtures and pure stands was statistically significant at P 0.13 (data
not shown).
The mean disease severity caused by an isolate population sampled from a
mixture and tested on its two component cultivars was compared to the mean disease
severity caused by the relevant two pure-stand isolate populations on their respective
cultivars of origin; e.g., the mean of Cashup/Stephens isolate populations tested on
Cashup and on Stephens vs. the mean of Cashup populations tested on Cashup and
Stephens populations tested on Stephens (Table V.2). In 1998, populations sampled from
mixtures in each case caused less disease than the mean of their counterpart pure-stand
populations, and mixture populations were on average less pathogenic than component
pure-stand populations (P = 0.085). In 1999, mixture populations caused approximately
the same amount of disease as the mean of their respective pure-stand populations. In
2000, populations from Cashup/Stephens and Madsen/Stephens caused greater disease
severity than their pure-stand counterparts (P = 0.043 and P
0.059). The mean severity
caused by mixture populations was not significantly different from that caused by
component pure-stand populations in either 1999 and 2000 (P = 0.42 and P = 0.29,
respectively). The higher levels of disease caused by the 1999 and 2000 populations
relative to the 1998 populations were likely due to the changes in greenhouse cultural
practices (see Materials and Methods).
105
TABLE V.2. Disease severity caused by Mycosphaerella graminicola isolates from
mixtures vs. pure stands of wheat when inoculated on respective mixture component
cultivars in the greenhouse
Disease severity
Trial"
Mixture
Mixture"' isolates'
1998
c/s
1999
2000
C/W
MIS
M/W
Mean
C/S
CIW
MIS
MJW
Mean
cis
CIW
MIS
M/W
Mean
Pure-stand
mean
Pr> (t)z
10.8
6.7
9.5
9.3
9.1
13.3
11.5
12.4
0.4303
0.4795
0.0513
0.5002
0.0850
29.8
44.5
29.3
39.8
23.0
33.6
31.4
0.9624
0.6363
0.1049
0.8387
0.4186
9.8
19.2
11.8
21.2
14.8
0.0430
0.8588
0.0592
0.1146
0.2940
39.1
31.6
36.2
24.7
27.6
30.2
13.9
24.1
9.0
15.9
vYe& of isolate collection; disease values reported for 1999 are means of two tests of
those isolates.
wlsolates were obtained from pure stands and 1:1 mixtures of moderately resistant and
susceptible cultivars. Moderately resistant: C = Cashup, M = Madsen. Susceptible: S =
Stephens, W = W-301.
xMean percent diseased leaf area on the two tester cultivars that comprised a mixture
(e.g., the mean for the Cashup/Stephens mixture population when tested separately on
Cashup and on Stephens). Values are means of four replicates.
106
TABLE V.2., Continued
Mean disease severity caused by isolate populations from pure stands of cultivars
comprising the mixture, when tested on seedlings of their cultivar of origin (e.g., the
mean severity when Cashup populations were tested on Cashup and Stephens populations
were tested on Stephens). Values are means of four replicates.
zSignificance based on linear contrasts of the difference in disease seventies caused by
mixture and pure-stand isolates.
Mixture effects on disease in the field were weakly correlated with their effects on
pathogenicity of populations derived from them (Fig. V.4; Pearson correlation coefficient
= 0.46, P = 0.13). A larger reduction in disease severity in mixture field plots was
associated with a larger reduction in pathogenicity of populations derived from those
plots.
In a further analysis, the performance of isolates from a given mixture on their
cultivars of origin was compared to their performance on the other two testers (data not
shown). Taken across mixtures, there was no evidence in any of the three years (linear
contrasts; P = 0.75, P = 0.86, P = 0.62, respectively) of a tendency for mixture isolates to
cause either more or less disease on their cultivars of origin. Similarly, there was no
evidence that pure-stand isolates were more pathogenic to their cultivars of origin than to
the other tester cultivars in any of the three years (data not shown; linear contrasts, P =
0.76, P = 0.99, and P = 0.30, respectively).
107
100
r0.46
50
-200
-40
-30
-20
-10
0
10
20
30
% change in Wsease
Fig. V.4. Relationship between effects of winter wheat mixtures on Septoria tritici blotch
disease severity in the field and their effects on pathogemcity of Mycosphaerella
graminicola populations derived from them. Along the X axis are plotted the percent
differences between areas under the disease progress curve (AUDPC5) in mixture plots
and mean AUDPCs of component pure stands. Along the Y axis are plotted the percent
differences between mean disease severities caused by mixture populations on the two
cultivars of origin and mean disease severity caused by populations from component pure
stands when tested on their respective cultivars of origin in the greenhouse. Data are
means of four replicates.
Isolates from the mixture of Madsen and Stephens, the two cultivars that together
accounted for >75% of the wheat area in the Willamette Valley during the period 1995-
2000, displayed inconsistency among the study years. In 1998, the Madsen/Stephens
population was least pathogenic relative to its pure-stand counterparts, while in 1999 and
2000 the pathogenicity of the Madsen/Stephens populations exceeded the mean of their
pure-stand counterparts to the greatest extent (Table V.2).
108
DISCUSSION
Mixture effects on epidemics
Cultivar mixtures influenced Septoria tritici blotch severity differently in each of
our three study years. In 1998, disease progress curves of three out of four mixtures
closely resembled those of the moderately resistant component throughout the season,
while in 2000, three out of four mixture progress curves closely resembled those of the
susceptible component. In 1999, disease progress curves for mixtures were intermediate
between the MR and S components. According to Wolfe (36), "With non-specialized
pathogens that infect mixtures, we can say generally that, if disease spread in the
susceptible component is hindered more than spread in the resistant component is
increased, then the overall infection tends to that of the resistant component grown
alone." Our results suggest that, in this pathosystem, environmental conditions may be
an important determinant of whether infection levels in a given mixture tend more toward
those of the resistant or the susceptible component.
A possible confounding factor is that our plot stands were generally sparser in
1998 than in the other two years; perhaps the denser stands in 2000 mixture plots
overcame the disease-suppressing mechanism of decreased spatial density of susceptible
plants (36). However, Jeger et al. (15) found no relationship between stand density and
severity of P. nodorum in mixed and pure-stand plots. In any case, one would expect that
increased stand density would merely neutralize the disease-suppressing benefit of
mixtures, instead of increasing disease in mixture plots relative to component pure stands,
as was actually observed.
Jeger et al. (14) hypothesized that, against nonspecific pathogens, cultivar
mixtures may either decrease, increase, or have no effect on epidemic progression,
depending on relative levels of sporulation and infection frequency of the mixture
components. This does not seem a likely explanation for our results, however, since the
variation in epidemiological effects that we observed was much greater among years than
within years among mixtures. Large interactions of resistance components with
environment would be required to alter the effects of host diversity on disease
progression qualitatively from year to year.
Mixture effects on pathogenicity
Our goal in measuring pathogenicity in the greenhouse was to investigate whether
disruptive selection reduced fitness of the pathogen population in mixtures, as was shown
by Chin and Wolfe (8) for barley powdeiy mildew. Selection is disruptive at the
population level if it operates on a given trait in different directions at different sites (5)
or, in this case, in interactions with different host phenotypes. In a mixture, such
disruptive selection requires adaptation to the genetic background of individual host
cultivars. Though quantitative adaptation of M. graminicola to host genetic background
has been demonstrated (2,3), we detected no such adaptation in our study. This was true
even in 1998, when there was some evidence for reduced fitness of pathogen populations
sampled from mixtures.
110
We used pathogen populations rather than single isolates in our greenhouse assays
of pathogenicity so as to better reflect field conditions. The population of M. graminicola
in the Willamette Valley of Oregon is very large and highly diverse, with different
pathogen genotypes commonly occurring even within the same lesion (24). Further,
lesion density on our greenhouse-inoculated plants was similar to that which we
commonly observe in the field. It has been demonstrated that mixtures of
M graminicola
isolates can cause more or less disease than the mean of individual isolates in greenhouse
inoculations (37), a potential complication for our results. For such interactions to
explain the differences in pathogenicity that we observed among years in this study,
however, would require substantially different pathogen populations among years, which
seems unlikely. Founder effects are highly unlikely for this pathogen in the Willamette
Valley of Oregon, where founding populations of at least 70 genetically distinct
individuals have been estimated per square meter in the field (38). Further, there were no
substantial changes in the number of cultivars or the proportion occupied by each cultivar
of total Willamette Valley wheat area during the period of 1998 to 2000.
Populations sampled from the Madsen/Stephens mixture in both
1999 and 2000
were significantly (P = 0.076 and P = 0.059) more pathogenic to Madsen and Stephens
than were the pure-stand populations (Table V.2). The
M graminicola population of
Oregon's Willamette Valley is likely to be adapted to Madsen and Stephens, the
dominant cultivars in commercial cultivation. From 1995 to 2000, the share of total soft
white winter wheat area in the valley that was planted to each of those two cultivars
ranged from 20% to over 60%. Why the
M graminicola populations sampled in 1998
111
from the Madsen/Stephens mixture were less pathogenic than those from the pure stands
is unclear.
Relationship of mixture effects on epidemics to those on pathogenicity
Effects of mixtures in suppressing epidemics were associated with selective
effects on pathogenicity. In 1998, AUDPCs of mixtures were lower than the pure stand
means and populations sampied from mixtures were less pathogenic than those sampied
from pure stands. In 1999, mixtures did not reduce disease severity and pathogenicity did
not differ significantly for populations sampled from mixtures and pure stands. In 2000,
there was on average significantly (P = 0.0528) more disease in mixtures relative to pure
stands in the field and two of the four populations sampled from mixtures were more
pathogenic as compared to those sampled from pure stands.
The question arises whether the differences in pathogemcity among years were
cause or effect; i.e., did mixtures exercise different selective influences among the years,
or did differences among the pathogen populations among years cause mixtures to vary in
epidemiological impact? While we have no definitive answer, it seems improbable that
the pathogen population differed greatly from year to year, as noted above.
A possible explanation for the different effects of mixtures on disease and
pathogenicity that were observed in different years is based on the interaction of leaf
emergence and rain-mediated spore dispersal. Our MR cultivars were later-maturing than
the S cultivars by between 2 and 7 days. Infection severity in a particular leaf layer is
highly dependent on the precise timing of leaf emergence in relation to high-impact rain
112
(30). Appropriately timed rain-splash could infect recently emerged leaves of an S
cultivar before the same leaves had fully emerged on an MR cultivar. After the latent
period, MR leaves would be infected by same-layer S leaves in mixture plots but not in
pure-stand MR plots. Under this scenario, more pathogen generations would occur on
MR cultivars in mixtures than in pure stands, allowing more opportunity for selection of
increased pathogenicity. Thus, the different effects of mixtures in 1998 and 2000, years
with similar average levels of ultimate disease severity, would be attributable to different
timings of splashy rainfall with respect to emergence of successive leaf layers.
Consistent with such an explanation is the fact that disease severity on the MR cultivars
in our study was considerably lower as a percentage of severity on the two S cultivars in
2000 than in the other two years.
In summary, host diversity effects on M. graminicola appear variable and
environmentally influenced. Overall, this set of mixtures did not reduce disease severity
or pathogemcity in the environments tested.
ACKNOWLEDGEMENTS
We thank Dan Coyle, Molly Hoffer, and Kellie Webb for invaluable technical
assistance; Cliff Pereira for essential statistical advice; and Kenneth B. Johnson and the
anonymous reviewers for thoughtful suggestions that improved the manuscript.
113
LITERATURE CITED
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114
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14. Jeger, M. J., Griffiths, E., and Jones, D. G. 1981. Disease progress of nonspecialised fungal pathogens in intraspecific mixed stands of cereal cultivars. I.
Models. Ann. Appi. Biol. 98:187-198.
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16. Jeger, M. J., Griffiths, E., and Jones, D. G. 1981. Effects of cereal cultivar
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116
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117
CHAPTER VI: Summary
TheLefictsofpartbl host resistmce on pathogen populations are not well
understood. From studies of the interactions of Mycosphaerella graminicola populations
andthekwintetwheathosts, usefuLinsights have beengained that may have relevance
for other disease systems.
CULTIVARISOLATE SPECIFICITY
At its release in 1992, the cultivar Gene was resistant to M graminicola, but by
1991 that resistance had beenniostly overcome.. Among monopycnidial isolates
collected in 1997 from Gene, Madsen (moderately resistant) and Stephens (susceptible),
and tested on seedlings of the same cultivars in the greenhouse, six of seven isolates from
Gene were virulent to Gene, while only one of 15 isolates collected from Madsen and
Stephens caused greater than lO% disease severity on Gene. It appears that Gene's
resistance was due to one or a very few genes, and rapidly selected for pathogen strains
with specific virulence to it. Specificity of pathogen strains tQ particular host cultivars
can exist in a pathosystem in which resistance and pathogenicity are mainly
quantitatively expressed.
SELECTIVE EFFECTS ON AGGRESSIVENESS
liteach of two years, cultivars witkdifferent levels of partial resistance to M
graminkola were planted in replicated field plots. Isolates were collected from the plots
118
early and late in each growing season, increased in the laboratory, and inoculated as
bulked populations by host cultivar, field replicate and collection date on greenhouse-
grown seedlings of the same cultivars. The selective impact of a cultivar on
aggressiveness was measured as the difference in aggressiveness between early and late
isolates from that cultivar. In both years, moderately resistant cultivars selected for more
aggressive isolates of M. graminicola than did susceptible cultivars. This result is
consistent with the few other available reports on selective impact of host resistance level
on aggressiveness in quantitatively varying pathosystems. It is reasonable to suppose
that, if partial resistance and aggressiveness are race-nonspecific, fungal individuals
selected by a resistant host would likely possess traits conferring an advantage on all host
genotypes.
PATHOGEN SEXUAL REPRODUCTION
The proportion of M graminicola fruiting bodies that were ascocarps
(pseudothecia) was studied for eight cultivars with varying levels of resistance over a
three-year period. The pseudothecial proportion of M graminicola fruiting bodies was
higher during years of intense epidemics and on susceptible cultivars. It is likely that the
greater density of infections present under those two circumstances contributes to a
higher frequency of sex. Possible mechanisms include more frequent encounters between
opposite mating types, and substances released by fungi that can stimulate sexual
morphogenesis andlor the switch from vegetative growth to reproduction.
119
HOST GENOTYPE MIXTURE EFFECTS ON EPIDEMICS AND
PATHOGENICITY
Four 1:1 mixtures of a moderately resistant and a susceptible cultivar were studied
during three years. Mixture effects on epidemics varied among the three years. There
was a correspondence between mixtures' disease response and the pathogenicity of
isolates sampled from them:
In 1998, disease progress curves of three of the four mixtures closely resembled
those of the moderately resistant component throughout the season. For each
individual mixture and on average for all mixtures, populations sampled from
mixtures caused less disease in greenhouse tests than the mean of their
counterpart pure-stand populations.
.
In 1999, disease progress curves for mixtures were intennediate between MR and
S components, and mixture populations caused approximately the same amount of
disease as the mean of their respective pure-stand populations.
In 2000, three out of four mixture progress curves closely resembled those of the
susceptible component. Populations from two mixtures caused greater disease
severity than their pure-stand counterparts.
A possible explanation for the differences among years in effects of mixture
effects on disease and pathogenicity was described based on the interaction of leaf
120
emergence and rain-splash-mediated spore dispersal. Effects of host genotype mixtures
on this pathogen appear to be variable and environmentally influenced.
DISEASE-MANAGEMENT IMPLICATIONS
Major-gene (qualitative) resistance to
M graminicola is likely present in winter
wheat and appears to be no more durable than in other pathosystems.
Partial resistance to
M graminicola is a valuable control strategy because it
reduces the need for fungicide applications. However, the evolutionary
consequences of deploying moderate resistance should be taken into account:
o as a disadvantage, selection for increased pathogen aggressiveness;
o as an advantage, reduction of the frequency of sexual reproduction.
Mixtures of moderately resistant and susceptible host genotypes may provide
modest yield benefits, but mixture effects on epidemic progression and on
pathogenicity are inconsistent and appear to depend strongly on environment.
121
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