PATHOGENIC CHARACTERIZATION, DISTRIBUTION IN OHIO AND WHEAT GENOTYPE REACTIONS TO STAGONOSPORA NODORUM AND PYRENOPHORA TRITICI-REPENTIS
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University
By Jessica S. Engle, M. S.
The Ohio State University 2005
Dissertation Committee: Approved by Professor Patrick E. Lipps, Adviser Professor Laurence V. Madden ______Professor Clay Sneller Professor Margaret Redinbaugh Advisor Professor Sophien Kamoun Plant Pathology Graduate Program ABSTRACT
Two fungal pathogens, Stagonospora nodorum and Pyrenophora tritici- repentis infect wheat leaves, the spike and impact seed quality. Both fungi produce host specific phytotoxins that elicit distinct lesion types. The distribution and pathogenic characterization of these pathogens was determined in seven wheat growing regions of Ohio. Both pathogens were recovered from wheat flag leaves from all seven regions, but S. nodorum was most prevalent. There was an association between S. nodorum isolate aggressiveness and region of the state. Isolates from North Eastern Ohio were less aggressive than those from the remaining 6 regions, while isolates from Central North West Ohio were more aggressive. P. tritici-repentis exists in the U.S. as a population of races, with the most common being races 1 - 4. These races are characterized by the toxin(s) produced causing specific reactions on a differential set of wheat cultivars and lines. Isolates of P. tritici-repentis in Ohio were determined to be races 1, 2 and rarely race 3, representing 60%, 35% and 5% of the population, respectively.
Seedling reactions of 15 wheat cultivars and one breeding line to races 1 and 2
of P. tritici-repentis indicated that some cultivars were more susceptible to race 2
ii than race 1. This same set of cultivars was inoculated with isolates of S. nodorum producing either predominately phytotoxins SnTox1 or SnTox2. Most genotypes were more sensitive to S. nodorum isolate producing predominately
SnTox2. Spike and flag leaf reactions to S.nodorum of 13 wheat cultivars were examined in the greenhouse and field. Cultivar Coker 9663 was identified as having moderately high resistance to both leaf blotch and glume blotch. Seed harvested from field plots were examined for pathogen infestation. S. nodorum was the predominant pathogen on seed obtained from both inoculated and non- inoculated field plots. P. tritici-repentis and Bipolaris sorikiniana were also present on seed from non-inoculated plots. This same set of cultivars was examined for P. tritici-repentis resistance in the greenhouse and field. The resistance levels to P. tritici-repentis of the 13 cultivars varied in the field with no significant differences detected among cultivar reactions. Two greenhouse experiments examined the response of wheat cultivars to P. tritici-repentis on adult flag leaves. One experiment used 10 isolates of P. tritici-repentis obtained from Ohio and the other experiment used either a race 1 or race 2 isolate from
North Dakota. Cultivar reactions were similar for these two greenhouse experiments regardless of origin of the isolate or isolate race. Cultivar reactions in the field and greenhouse experiments were correlated, and disease severities among cultivars were significantly different. Coker 9663 was more susceptible than the other cultivars tested to P. tritici-repentis. All cultivars tested were sensitive to P. tritici-repentis and S. nodorum toxins. These results indicated that
S. nodorum is the predominate leaf blotching pathogen in Ohio, but P. tritici-
iii repentis may be impacting leaf disease levels in the state. Moderately high resistance to S. nodorum and P. tritici-repentis was detected in several wheat cultivars indicating that breeding for resistance to both pathogens is possible.
iv
Dedicated to my parents.
v ACKNOWLEDGMENTS
Dr. Patrick Lipps not only provided the financial support for this dissertation, but
scholarly guidance and support.
Dr. Larry Madden gave me all of the necessary statistical analysis advice. He
also lent an ear when needed.
Drs. Redinbaugh, Kamoun and Sneller provided invaluable advice throughout my project. The editorial comments on my dissertation were very much appreciated.
Mr. Richard J. Minyo, Jr. provided the opportunity for natural inoculum plots
across Ohio.
Mr. Larry Herald taught me not only how to cross wheat, but also how to deal
with problems on the job.
Mrs. Lynn West was helpful during work and in life.
Ms. Audrey L. Johnston helped with multiple technical details.
vi VITA
Born: June 17, 1978 in Warren, Ohio
EDUCATIONAL BACKGROUND
M. S. (Plant Pathology)- The Ohio State University, June 2002 Advisor – Dr. Patrick E. Lipps Thesis Title – Evaluation and Characterization of Resistance to Fusarium graminearum in Winter Wheat
B. S. (Plant Pathology)- The Ohio State University, June 1999 Minor Plant Biology
PROFESSIONAL EXPERIENCE
2004-2005………....Collaborative work with Dr. Tim Freisen’s laboratory on race determination of Pyrenophora tritici-repentis and Stagonospora nodorum toxin screening.
2004-2005…………Collaborative work on a regional Stagonospora nodorum leaf blotch and glume blotch screening nursery and field work associated with that nursery.
2002 – 2005……….Graduate Research Assistant in the laboratory of Dr. Patrick E. Lipps at The Ohio State University. Duties included vii technical assistance with on-going research projects in the greenhouse and field; collection of field samples from across the state for species identification; assembly of a field disease screening nursery for breeding purposes, creating a breeding population for QTL analysis for Stagonospora nodorum resistance; hand spraying of fungicides; assistance with biological agent application; purchasing lab equipment; use of common lab equipment; attending several extension meetings dealing with field crops; writing extension bulletins; assisting in managing summer help.
2004………………..Participation in the graduate student seminar exchange program. Presented seminar at Cornell University.
1999 – 2002……….Graduate Research Assistant in the laboratory of Dr. Patrick E. Lipps at The Ohio State University. Duties included technical assistance with on-going research projects in the greenhouse and field; assembly of a field nursery for disease screening; hand spraying of fungicides; assistance with biological agent applications; purchasing lab equipment; use of common lab equipment such as balances, pH meter, microscopes, autoclaves and flow hoods; incorporating serial dilutions of endogenous wheat spike compounds into media; attending several extension meetings dealing with field crops; writing two extension bulletins; assisting in managing summer help.
2000…………………Teaching Assistant at The Ohio State University. Taught a laboratory section of PP401 Introductory Plant Pathology.
viii
1998 – 1999……….Student worker in Dr. Terrance Graham’s laboratory in the department of plant pathology at The Ohio State University. Work involved maintaining Arabidopsis thaliana plants and visual evaluation of populations for mutations that allowed growth in the presence of Rose Bengal. Mutants were then analyzed using high pressure liquid chromatography (HPLC).
1997 – 1998……….Student worker in Dr. Fred Sack’s laboratory in the department of plant biology at The Ohio State University. Work involved maintaining Arabidopsis thaliana plants for genetic experiments and visual evaluation of populations for stomotal mutations.
PUBLICATIONS REFERED JOURNALS
1. Engle, J. S., Lipps, P. E., Graham, T. L. and Boehm, M. J. 2003. Effects of choline, betaine and wheat floral extracts on growth of Fusarium graminearum. Plant Dis. 88:175-180.
2. Engle, J. S., Madden, L. V. and Lipps, P. E. 2003. Evaluation of Inoculation Methods to Determine Resistance Reactions of Wheat to Fusarium graminearum. Plant Dis. 87:1530-1535.
ix EXTENSION PAPERS
1. Engle, J. S., De Wolf, E. D. and Lipps, P. E. 2001. A visual scale for estimating damage to soft red winter wheat kernels by Fusarium Head Blight. The Ohio State University. Ohio State University Extension.
2. Engle, J. S., Lipps, P. E. and Mills, D. 2003. Fusarium Head Blight severity visual scale of non-awned wheat. The Ohio State University. Ohio State University Extension. Fact Sheet AC-49-03.
3. Engle, J. S., Lipps, P. E. and Mills, D. 2004. Tan spot: yellow leaf spot or blotch. The Ohio State University. Ohio State University Extension. Fact Sheet:AC-50-04.
4. Engle, J. S., Lipps, P. E. and Mills, D. 2005. Spot blotch and common root rot. The Ohio State University. Ohio State University Extension. Fact Sheet:AC-51-05.
FIELDS OF STUDY
Major Field: Plant Pathology
x TABLE OF CONTENTS
Page
Abstract………………………………………………………………………………...…ii
Dedication………………………………………………………………………………..v
Acknowledgments………………………………………………………………………vi
Vita……………………………………………………………………………………….vii
List of Tables………………………………………………………………………..…xvii
List of Figures………………………………………………………………………….xxi
Chapters:
1. Distribution and Pathogenic Characterization of Pyrenophora tritici- repentis and Stagonospora nodorum in Ohio………………………..………1
1.1 Abstract………………………………………………………………..……1
1.2 Introduction……………………………………………………………...….2
1.3 Objectives……………………………………………………………..……6
1.4 Materials and Methods…………………………………………..………..7
1.4.1 Collection of samples………………………………………...….7
1.4.2 Isolation of pathogens……………………..…………………….8
1.4.3 Single spore isolation and storage……………………….…..10
1.4.4 P. tritici-repentis race determination……………………….…11
xi
1.4.5 P. tritici-repentis inoculum and inoculation…..……….……..12
1.4.6 S. nodorum aggressiveness…………………………….…….14
1.4.7 Statistical analysis-survey of fields………………..………....15
1.4.8 Statistical analysis-S.nodorum aggressiveness…………….17
1.5 Results……………………………………………………………….…….18
1.5.1 Distribution of leaf blotching pathogens………………..…….18
1.5.2 Occurrence of S. nodorum…………………………….………18
1.5.3 Occurrence of P. tritici-repentis...…………………………….19
1.5.4 P. tritici-repentis race differentiation……………………….…20
1.5.5 S. nodorum aggressiveness……………………………….….20
1.6 Discussion………………………………………………………………...21
1.7 Literature cited…………………………………………………….………27
2. Seedling Reaction of Winter Wheat to Stagonospora nodorum Isolates
Producing SnTox1 or SnTox2 and Pyrenophora tritici-repentis Race 1 or
Race 2…...……………………………………………………………….……..45
2.1 Abstract……………………………………………………………………45
2.2 Introduction………………………………………………………….…….46
2.3 Objectives…………………………………………………………………50
2.4 Materials and Methods…………………………………………………..51
2.4.1 Genotypes and seedling production……………...………….51
2.4.2 Inoculum and inoculation……………………..………….……52
xii 2.4.3 Cultivar reaction assessments………………………….…….54
2.4.4 Experimental design and statistical analysis………….…….54
2.5 Results……………………………………………………………….…….55
2.5.1 S. nodorum reactions…………………………………….…….55
2.5.2 P. tritici-repentis reactions………………………….…….……56
2.6 Discussion………………………………………………………………...56
2.7 Literature cited……………………………………………………….……60
3. Reaction of Thirteen Soft Red Winter Wheat Cultivars to Pyrenophora tritici-repentis in the Field and Greenhouse…………………..……….……75
3.1 Abstract……………………………………………………………………75
3.2 Introduction………………………………………………………….…….76
3.3 Objectives…………………………………………………………………80
3.4 Materials and Methods…………………………………………………..81
3.4.1 Greenhouse experiments………………………………..…….81
3.4.2 Plant growth……………………………………………….……82
3.4.3 Greenhouse multiple isolate inoculation……………….…….83
3.4.4 Greenhouse inoculations with race 1 and race 2…….……..85
3.4.5 Race 1 and race inoculum and inoculations………………...85
3.4.6 Field experiments…………………………………………....…86
3.4.7 Statistical analysis………………………………………….…..88
3.5 Results……………………………………………….…………………….89
3.5.1 Greenhouse experiments…………………..………………….89
xiii 3.5.2 Multiple isolate inoculation…………………………….………89
3.5.3 Race 1 and race 2 inoculations..………………………….….90
3.5.4 Field reactions…………………………………………….…….91
3.5.5 Correlations…………………………………………….……….91
3.6 Discussion………………………………………………………………...92
3.7 Literature cited…………………………………………………….………94
4. Reaction of Commercial Soft Red Winter Wheat Cultivars to Stagonospora
nodorum in the Greenhouse and Field ….………………………………...105
4.1 Abstract………………………………………………………………..…105
4.2 Introduction…………………………………………………………..….106
4.3 Objectives………………………………………………………………..110
4.4 Materials and Methods………………………………………………....110
4.4.1 Cultivar information…………………………………………...110
4.4.2 Greenhouse experiments………………………………….…111
4.4.3 Plant production………………………………………….……111
4.4.4 Inoculum production and inoculation……………………….112
4.4.5 Field experiments………………………………………….….115
4.4.6 Inoculated plots……………………………………………….115
4.4.7 Field inoculum and inoculations……………………………..115
4.4.8 Naturally infested plots……………………………………….116
4.4.9 Seed infestation…………………………………………….…118
4.4.10 Statistical analysis…………………………………………..119
xiv 4.5 Results…………………………………………………………………...120
4.5.1 Greenhouse inoculations…………………………………….120
4.5.2 Field experiments: inoculated plots…………………………121
4.5.3 Naturally infested plots…….………………………………....121
4.5.4 Corrrelations…………………………………………………..123
4.6 Discussion…………………………………………………………….....124
4.7 Literature cited…………………………………………………………..129
5. Reactions of Wheat Genotypes to Isolates of Stagonospora nodorum
from Ohio……………………………………………………………..……….145
5.1 Abstract………………………………………………………………..…145
5.2 Introduction………………………………………………………………146
5.3 Objective…………………………………………………………………150
5.4 Materials and Methods………………………………………………....150
5.4.1 Greenhouse inoculations-Plant production.………………..151
5.4.2 Inoculum production and inoculation…………………….....152
5.4.3 Experimental design and statistical analysis…………..…..153
5.4.4 Field plots……………………………………………………...154
5.4.5 Field inoculations……………………………………….....….155
5.4.6 Field experimental design and statistical analysis………...155
5.5 Results………………………………………………………………..….156
5.5.1 Greenhouse reactions……………..……………………...….156
5.5.2 Field nursery………………………………..…………………157
xv 5.6 Discussion……………………………………………………………...158
5.7 Literature cited…………………………………………………………160
APPENDICES
Appendix A. Number of leaf blotching pathogens in Ohio Counties
sampled in 2002…...... 171
Appendix B. Number of leaf blotching pathogens in Ohio Counties
sampled in 2003…...... 173
Appendix C. Designation of the populations obtained from a partial
diallele cross of thirteen commercial cultivars screened for Stagonospora
nodorum and Pyrenophora tritici-repentis resistance…………………....175
Appendix D. General Conclusions…………………………………………………177
Literature cited…………………………………………………………………..……184
xvi LIST OF TABLES
Table Page
1.1 Distribution of leaf blotching pathogens collected from seven regions in
Ohio in 2002 and 2003……………………………………….………...……..37
1.2 The number of fields in which Stagonospora nodorum and Pyrenophora
tritici-repentis were isolated in Ohio in 2002 and 2003…………………....38
1.3 Estimated probability of obtaining either Stagonospora nodorum or
Pyrenophora tritici-repentis from diseased flag leaves in seven regions of
Ohio in 2002 and 2003……………………………………………………..…39
1.4 Chi-square values for comparing the probability of S. nodorum and P.
tritici-repentis occurrence within fields in seven regions of Ohio during
2002 and 2003…………………………………………………...………….…40
1.5 Occurrence of Pyrenophora tritici-repentis in seven regions of Ohio in
2002 and 2003………………………………...……………………………….41
xvii 1.6 Median ratings and estimated relative marginal effects of two susceptible
wheat genotypes inoculated with Stagonospora nodorum isolates obtained
in Ohio in 2002 and 2003…..…………………………………………………42
1.7 F-values for comparing estimated marginal effects of Stagonospora
nodorum aggressiveness for isolates obtained in Ohio in 2002 and
2003……………………………………………………………………………..43
1.8 Region 6 average weather conditions in 2002 and 2003 for April 1 to July
15 calculated from hourly data…………………………………………….....44
2.1 Reaction of soft red winter wheat cultivars to seedling inoculation with
Stagonospora nodorum isolates SN2000 (higher SnTox1 producer) and
SN69-1 (higher SnTox2 producer)……………………………….………….72
2.2 Reaction of soft red winter wheat cultivars to seedling inoculation with race
1 and 2 isolates of Pyrenophora tritici-repentis……………...……………..73
3.1 Stagonospora nodorum field reactions of selected soft red winter wheat
cultivars during 2000 and 2001 in Ohio and 2002 in Pennsylvania…….100
3.2 Mean disease severity of 13 wheat cultivars after inoculation of flag leaf
with Pyrenophora tritici-repentis isolates from Ohio in the greenhouse..101
xviii
3.3 Mean disease severity, median lesion rating, and relative marginal effects
of adult plants 28 days after inoculation with race 1 or race 2 of Pyrenophora tritici-repentis isolates………………………………………………………………..103
3.4 Mean tan spot severity and percentage seed infestation of 13 winter wheat
cultivars evaluated in two inoculated field plots in Ohio during 2004…..104
4.1 Field reaction of 13 soft red winter wheat cultivars to Stagonospora
nodorum during 2000 and 2001 in Ohio and 2002 in Pennsylvania...….137
4.2 Leaf and glume blotch reactions of commercial soft red winter wheat
cultivars inoculated with a composite of ten Stagonospora nodorum
isolates in the greenhouse……………..…………………………………...138
4.3 Reaction of fifteen cultivars in inoculated plots in Wayne County 2003
and Wayne and Wood Counties in 2004……………………………….….140
4.4 Mean Stagonospora leaf and glume blotch severities of the thirteen
cultivars planted in naturally infested plots in Wayne and Pickaway
Counties in 2003 and 2004……………………………………………..…..142
xix 4.5 Percentage of seed infested with Pyrenophora tritici-repentis and Bipolaris
sorokiniana of the thirteen cultivars harvested from naturally infested field plots in
Pickaway and Wayne Counties in 2004…………..……...………………..………143
4.6 Pearson’s correlation coefficients between mean cultivar assessments in the
greenhouse and field plots………………………...………………………………..144
5.1 Characteristics of the wheat genotypes used in this study………………………167
5.2 Least squares means of Stagonospora leaf blotch severity 21 days after
inoculating flag leaves of wheat genotypes in the greenhouse………...…….....168
5.3 Least squares mean estimate of leaf blotch and glume blotch severity for
genotypes inoculated in a field plot at Wooster, OH in 2004……………………169
A.1 Number of fields with leaf blotching pathogens in Ohio Counties sampled in
2002…………………………………………………………………………………...171
B.1 Number of fields with leaf blotching pathogens in Ohio Counties sampled in
2003…………………………………………………………………………………...173
C.1 Designation of the populations obtained from a partial diallel cross of thirteen
commercial cultivars screened for Stagonospora nodorum and Pyrenophora
tritici-repentis resistance………………………………………………………….…175
xx LIST OF FIGURES
Table Page
1.1 Map of Ohio with sampled regions outlined………………………….……..36
xxi CHAPTER 1
DISTRIBUTION AND PATHOGENIC CHARACTERIZATION OF
PYRENOPHORA TRITICI-REPENTIS AND STAGONOSPORA NODORUM IN
OHIO
Abstract
Stagonospora nodorum and Pyrenophora tritici-repentis have world wide distribution and affect all classes of wheat. In Ohio, S. nodorum is the major leaf blotching pathogen and it frequently occurs on the same plants with other leaf blotching pathogens. To determine the distribution of these pathogens on wheat in Ohio, flag leaves with lesions were collected and examined from 37 and 35 wheat producing counties in 2002 and 2003, respectively. Counties were grouped into seven regions based on soil type and annual precipitation. Log- linear analysis of the occurrence of leaf blotching pathogens within regions indicated that the occurrence of S. nodorum was independent of the occurrence of P. tritici-repentis. A logistic analysis revealed that the occurrence of both pathogens varied by regions in one or both years, with aggressiveness of S. nodorum isolates was determined by inoculating two susceptible genotypes with a sub-sample of isolates from each region from both years. S. nodorum isolates
1 obtained from North Eastern Ohio (an isolated region with little wheat) were less
aggressive than those from other regions. Isolates obtained from the Western
Central area of the state were significantly more aggressive than those obtained
from fields in the remaining regions. Isolates from the five other regions did not
differ significantly (P > 0.05) in aggressiveness from each other. These results
indicate that the aggressiveness of S. nodorum isolates was not, in general,
contributing to differences in cultivar reactions across most of the state. Races 1,
2, and a few race 3 isolates of P. tritici-repentis were detected in the state. The
distribution of P. tritici-repentis races 1 and 2 were not associated with any region
of Ohio, although the prevalence of race 1 was three times greater than race 2.
The rarer race 3 was associated with dispersed regions 5 (South), 6 (East North
Central) and 7 (Western Central).
Introduction
The wheat (Triticum aestivum L.) foliar pathogens Stagonospora nodorum
(Berk.) (Castellani and E. G. Germano) (teliomorph Phaeosphaeria nodorum (E.
Muller (Hedjaroude))), and Drechslera tritici-repentis (Died.) (Shoemarker)
(teliomorph Pyrenophora tritici-repentis(Died.) (Drechs)) are common throughout wheat growing regions of the world (73). S. nodorum causes Stagonospora leaf and glume blotch on wheat and other cereals. P. tritici-repentis causes tan (or yellow) spot of wheat. Although both of these pathogens have a world wide distribution, the prevalence and intensity of disease depends on environmental factors.
2 S. nodorum became one of the major wheat leaf spotting pathogens in the mid-west by the late 1980’s, coinciding with the increase of conservation tillage
(67). Stagonospora leaf and glume blotch affects both grain quality and yield.
Yield may be reduced as the result of lower test weights (29, 47) due to reduced photosynthetic area of the upper leaves, resulting in decreases in the level of carbohydrates available for seed fill (22, 72). The milling quality of grain may be reduced by infection of the developing seed, causing shriveling and reduced flour yield (22). Infection of wheat plants may occur at any stage of development (51,
71).
Management of this disease is achieved through cultural practices, fungicide application and the use of resistant cultivars (28, 36-37, 43, 70).
Primary cultural practices include crop rotation combined with burying infested residue by tillage to reduce the survival of the fungus from one crop to the next
(2, 28, 36-37, 43, 70). Control of seed-borne inoculum is achieved through application of fungicide seed treatment to reduce seed infection (2, 27, 36-38, 43,
64-65). Application of foliar fungicides can be effective in managing
Stagonospora leaf blotch in epidemic years, but cost can be economically prohibitive when the price of wheat is low (35-37, 43, 71). Moderately resistant cultivars are available, but there are currently no soft red winter wheat cultivars with high levels of resistance to this pathogen (35). Resistance to S. nodorum has been shown to be quantitatively inherited (8, 11, 16, 34, 44-46, 56-60, 62,
70).
3 In addition to quantitatively inherited resistance genes, loci for sensitivity
to toxins produced by S. nodorum have also been identified. A single nuclear
gene on chromosome 1BS has been identified as the sensitivity locus for the
proteinaceous toxin, SnTox1, that conditions the necrotic response (33). This
toxin appears to be a major virulence factor (33), although it is not required for disease development (34). This locus has been named Snn1 and has been
shown to be dominantly inherited (34). Other toxins have been reported, but not
fully characterized (25).
S. nodorum has been the subject of numerous population studies. These studies were focused on determining the spatial variation in aggressiveness among both large scale geographical areas and within lesions (59). Since S. nodorum reproduces sexually and asexually, could lead to determination of important inoculum sources by examining the allelic variation in the genome.
Molecular marker technology has been used to examine the genetic structure of
S. nodorum populations within a field and substantiated the hypothesis that the
initiation of epidemics were not influenced by seed source and/or the cultivars
planted (27). In an examination of aggressiveness differences among
geographically separated populations of S. nodorum, Scharen et al. (1985) found that isolates from South America and Canada had lower aggressiveness than isolates from the United States, and isolates from the U.S. were significantly more aggressive than isolates obtained in Europe (59). Aggressiveness of S. nodorum isolates is thought to be the main determinate in cultivar reactions,
4 with less aggressive isolates not discriminating resistant and susceptible reactions (56).
Environmental conditions in Ohio favor infection and spread of S. nodorum in wheat fields making leaf blotch resistance a preferred management tool.
Some years frequent precipitation events after head emergence favor leaf blotch development and subsequent development of glume blotch. Monitoring
Stagonospora leaf blotch progression through the season has proven difficult in
Ohio and other parts of the world due to the occurrence of P. tritici-repentis that causes tan spot with similar appearing lesions (5).
In the Northern Great Plains of North America, tan spot is considered one of the major foliar diseases of wheat, with increased occurrence in recent decades after the adoption of conservation tillage (1, 49-50, 63, 74-75). Severe tan spot causes losses in yield, lower thousand-kernel weight and reduced number of grains per head (5-6). Harvested grain may be shriveled and discolored (5-6). P. tritici-repentis exists as eight different races and is capable of producing at least three phytotoxins (1, 9, 19, 31). The most abundant races in the United States have been race 1 and race 2, with some occurrences of races 3, 4, and 5 (1, 18). Races 6-8 have been found in other areas of the world
(1, 31). Tan spot is currently managed by crop rotation, mold-board plowing to bury wheat stubble and foliar fungicide application (6, 52-53). Genetic resistance in commercial cultivars is considered the most environmentally safe and economical means of disease management (15, 17, 50, 52). The specific genes controlling resistance and the level of resistance expression for the seedling and
5 leaf phases of this disease have been investigated (14). Most studies concur
that resistance to P. tritici-repentis is controlled by a few major genes for toxin
sensitivity and these and other minor genes condition expression of partial
resistance to other virulence factors (20).
Two distinct symptoms develop on susceptible cultivars subsequent to
infection, tan necrosis and/or chlorosis as a response to toxins produced by P.
tritici-repentis (31, 75). The necrosis toxin, Ptr ToxA, is secreted by the fungus in
an inactive form that the plant enzymatically cleaves into an active form (31, 39,
42). Ptr ToxA has been shown to be localized to the chloroplast membrane in
plant cells (39). Ptr ToxB causes chlorosis (18, 31, 40) and the response has
been shown to be associated with the wheat A and D genomes (75). The third
toxin, Ptr ToxC, also a chlorosis toxin, has been isolated from cultures of P. tritici-
repentis obtained from the Turkish-Syrian border (31). Wheat nuclear genes
control reactions to all races and toxins produced (20). Additional virulence
factors apparently influence disease severity, but these have not been well
characterized (17-18, 31).
The occurrence and distribution of P. tritici-repentis races in Ohio has not been studied. No information is available on the occurrence of specific P. tritici- repentis races in most soft red winter wheat growing regions of the U. S.
Objectives
The objectives of this study were to: 1) determine the distribution of the predominant wheat leaf blotching pathogens in Ohio; 2) examine the variation in
6 aggressiveness of S. nodorum populations; and 3) determine the race structure of P. tritici-repentis within the state.
Materials and Methods
Collection of samples
A form of stratified random sampling (68) was utilized in 2002 and 2003 to obtain flag leaf samples form wheat fields in Ohio. Strata consisted of the seven wheat growing regions in the state, which were designated based on soil type and annual precipitation (61) with borders set arbitrarily by county borders
(Figure 1). The seven regions were: Region 1 (North East, acid soils, poorly drained and 0.48-0.58 m of rain/yr) Ashtabula and Trumbull Counties; Region 2
(North Central, limestone soils, moderately well drained and 0.48-0.58 m of rain/yr) Crawford, Huron, Morrow, and Richland Counties; Region 3 (North West, high organic matter soils, very poorly drained and 0.48-0.53 m of rain/yr)
Defiance, Henry, Paulding, Putnam, Sandusky, Williams and Wood Counties;
Region 4 (South West, high organic matter soils, poorly drained and 0.48-0.58 m of rain/yr) Darke, Logan, Preble, Shelby and Union Counties; Region 5 (South, low organic matter soils, poorly to well drained 0.53-0.64 m of rain/yr) Clark,
Clinton, Fayette, Highland, Madison, Pickaway and Ross Counties; Region 6
(North Central East, limestone soils, well drained and 0.48-0.53 m of rain/yr)
Ashland, Lorain and Wayne Counties; Region 7 (West Central, medium textured soils, somewhat poorly drained and 0.48-0.58 m of rain/yr) Allen, Auglaize,
Hancock, Hardin, Marion, Mercer, Seneca, Van Wert and Wyandot Counties.
7 The number of fields sampled per region was roughly proportional to the area of wheat grown in each county (using 2001 production-year statistics). Fields in a total of 37 and 35 counties were sampled in 2002 and 2003, respectively. As many as 15 fields per county were sampled during the period from flowering through senescence (Feekes growth stages 10.5.4 to 11.1 (32)).
Three flag leaves with leaf lesions were collected from each field. Flag leaves were collected at least 1 meter apart within a 3.3-m radius from the edge of the field. Leaves heavily infected with rust (Puccinia triticina ((DC.) E. O.
Speer)) and powdery mildew (Blumeria graminis f. sp. tritici (Ericks)) were discarded. The flag leaves were placed in numbered coin envelopes, with the approximate location of the field indicated by the nearest road intersection. Field samples were placed in an air flow hood each evening and left to dry for approximately 2 days after collection. After drying, the samples were stored at room temperature in a plastic container until isolation of leaf pathogens was attempted.
Isolation of pathogens
Dried flag leaf samples were placed in moisture chambers to enhance sporulation of leaf blotching pathogens. The moisture chambers consisted of 9- cm diameter Pyrex Petri dishes containing 9-cm diameter filter paper (Fisher
Scientific) moistened with 5 ml of water underneath a 6- x 6-cm wire mesh screen that supported a glass slide above the moistened filter paper. The glass slide had two pieces of double sided sticky tape placed evenly across the top for
8 adherence to leaves. This tape prevented the flag leaf from rolling during incubation in the moisture chamber.
Flag leaves were disinfested in a solution of 10% sodium hydrochloride
(5.25% Snow-EE, Mars, PA) solution for 2 minutes and rinsed in water for 30 seconds before being pressed onto the glass slides. The moisture chambers were placed under a bank of fluorescent and ultraviolet lights for 48 hours before the first isolation attempt and 72 hours before the second isolation attempt was made. For each field sample, a 5-cm section of one flag leaf was placed in each moisture chamber. Leaf sections were chosen based on the presence of discrete leaf lesions and on leaves with the least amount of senescence. A second set of flag leaf sections were prepared similarly after isolations were made from the first section. The two different isolation attempts per field sample were separately recorded.
Each glass slide with a flag leaf section was removed from the moisture chamber to a dissecting scope in a laminar air flow bench. A sterile sewing needle was employed to lift the spore mass from a single conidiophore and place the spore mass on V8 agar amended with 50 mg/liter of streptomycin sulfate
(Sigma-Aldrich, St. Louis, MO) to control bacterial growth. Four isolation attempts of each leaf blotching pathogen were made from each flag leaf.
Isolates were designated with letters following the field sample number. The glass slides were placed back into the moisture chambers and returned to under the light banks until the next day. Another isolation attempt was made the next day after placing the samples in the moisture chambers. This second isolation
9 attempt was done because the first isolation did not consistently provide
uncontaminated, mature spores. After all isolation attempts had been made, the
flag leaf samples were again air dried and placed at 2.2°C for storage.
Isolation plates were placed under the bank of lights previously described
until fungal species could be determined. Contaminated colonies were further
transferred into fresh plates of streptomycin amended V8 agar to separate the
leaf blotching pathogen from contaminants. Once a fungal isolate was
considered free of contaminants, a single spore isolation was made. The species was determined by pycnidium, conidia and colony morphology (13, 76), then placed in storage.
Single spore isolation and storage
Isolates of each species were single spored by removing a spore mass from individual colonies using a sterile sewing needle. This spore mass was placed in a sterile eppendorf tube containing 1 ml of sterile distilled water. The tube was mixed using a vortex, then 100 µL-volume of the spore suspension was pippetted from the eppendorf tube and placed into another sterile eppendorf tube containing 0.5 ml of sterile distilled water. This second tube was again mixed, then the spore suspension was decanted and spread onto 1.5% water agar using a sterile bent glass rod. After approximately 24 hours, individual germinated conidia were transferred from the water agar onto V8 agar plates. The isolates were then placed under the light bank previously described to enhance colony growth and conidiophore development.
10 Once each isolate had produced mature pycnidia or conidiophores,
isolates were prepared for storage. All species were stored in the same manner.
Sterile eppendorf tubes and cryogenic vials were labeled with the isolate number.
Isolates were numbered sequentially based on field collection information. Agar plugs with condiophores or pycnidia were sterily removed from the V8 agar plates under an air flow bench. One agar plug was placed on a fresh V8 agar plate, three agar plugs were placed in each cryogenic vial, and the remaining agar plugs were placed in an eppendorf tube.
The V8 agar plate with the colonized agar plug was placed under the light bank for colony production. When the isolate had grown sufficiently, this plate was placed in a 2.2°C refrigerator for storage. Vegetable oil-nitrate agar was added to each cryogenic vial and the vials were stored in a -10ºC freezer.
Vegetable oil-nitrate agar consisted of 2 ml Crisco Pure Vegetable Oil (canola and soybean oil, Procter and Gamble Inc., Cincinnati, OH), 1.5 g sodium nitrate,
1.0 g potassium hydrophosphate, 0.5 g magnesium sulfate*7 water, 2 ml of 10% thiamine hydrochloride and 13 g of agar per liter (24). The eppendorf tubes containing colonized agar plugs were left in the laminar air flow bench for 2 days to desiccate the plugs. The tubes were sealed and then placed in a 0ºC freezer for storage.
P. tritici-repentis race determination
A subset of 37 P. tritici-repentis isolates collected in 2002 were sent to Dr.
Tim Friesen at the USDA-ARS Red River Valley Agriculture Research Center
11 Fargo, ND for race determination. A single isolate from each county collected in
2002 was placed on a set of differential wheat cultivars to determine race. Dried
plugs of each isolate in eppendorf tubes were shipped (USDA-APHIS permit No.
61766) in a sealed container. Race determination was repeated in Ohio as
described below with a subset of the 2002 collection (18 isolates) and the 2003
collection (46 isolates).
Seed of the six differential cultivars and lines were obtained from Dr.
Friesen. This seed was maintained at 3.5°C until use. Two seeds of each
cultivar or line were placed into 20.5- x 4-cm conetainers (Stuewe and Sons, Inc.,
Corvallis, OR) of 3M Metro Mix (Conrad Fafard, Inc., Agawam, MA). The seeds were germinated in a 44.5 m2 greenhouse maintained with an average 20.8°C day and 19.2°C night temperature, with a 12-hour light period provided by one
1000-watt metal halide lamp and four 1000-watt high pressure sodium lights
(Rudd lighting Co., Solon, OH). During the course of the experiment, pots were fertilized weekly with 100 ml of a triple 20 Peter’s® fertilizer solution (N 20%, P
20%, K 20%; 4.8 g/liter water) (Scotts, Inc., Marysville, OH). For insect control, plants were sprayed weekly with S-kinoprene (Enstar®, Wellmark, Bensenville,
IL) (0.01 g ai/liter water) or abamectin (Avid®, Syngenta Crop Protection, Inc.,
Greensboro, NC) (0.01 g ai/liter water).
P. tritici-repentis inoculum and inoculation
Seedlings were inoculated when they were 2 weeks old, at approximately the two-leaf stage. Inoculum consisted of a 3x103 conidia/ml suspension from
12 individual isolates of P. tritici-repentis. Plates were washed with tap water and
the mycelial mat with conidia were scraped from the surface of cultures grown for
2 weeks on V8 agar modified as described above. The mycelial mats were
strained through 2 layers of cheese cloth to separate mycelia from conidia.
Spore numbers were determined using a Spotlite Hemacytometer® (American
Scientific Products, McGaw Park, IL) according to the protocol for counting large spores (69). The inoculum was atomized with a hand mister onto the surface of the leaves to the point of runoff. Control inoculations consisted of a set of the differentials inoculated with a confirmed race 1 isolate and another set inoculated with a race 2 isolate for each repetition of the experiment.
After inoculation, plants were placed into a mist chamber. The mist
chamber consisted of a 2.3- x 1.4- x 1.0-m PVC skeleton enclosed by
polyethylene sheeting with adjustable sides. An air atomizer with a fluid nozzle
(#2850 SS, Spraying Systems, Co., Spokane, WA) (0.56 kg/cm3 water) and an
air nozzle(#70) (4.2 kg/cm3 air) misted 30-ml of water for 30 seconds every 3 minutes above a polyethylene canopy over the plants. The temperature in the mist chamber was maintained at approximately 22°C and the relative humidity was approximately 100%. Inoculated plants were incubated in the mist chamber for 24 hours.
After the incubation period in the mist chamber, conetainers were returned to the greenhouse. Seedlings were visually assessed daily after inoculation for
symptom development and examined for disease symptoms 7 days after
13 inoculation. The lesion rating system of Lamari and Bernier was used for
determining the reactions which accounts for chlorosis and necrosis symptoms
(30).
S. nodorum aggressiveness
A subset of S. nodorum isolates from the 2002 (34 isolates) and 2003 (46
isolates) collections were evaluated for aggressiveness. A spore mass was
taken from the stored culture plates and placed into an eppendorf tube containing
1 ml autoclaved distilled water. The eppendorf tube was mixed using a vortex and spread on a fresh V8 plate with a sterile bent glass rod to enhance pycnidiospore production. Plates were placed under the light bank previously described for conidial production. After 1 week under the light bank, pycnidiospores were harvested from plates. The 8-cm-diameter agar discs from petri dishes of each isolate were inverted onto a 15-cm square of cheese cloth
(four layers) placed into glass beakers of tap water (100 ml) for several minutes, dipped several times and then squeezed to release pycnidiospores. Conidial suspensions were adjusted to 1x106 spores/ml using a hemacytometer according to the protocol for counting small spores (69).
The hard red spring wheat line ND2375 and the soft red winter wheat cultivar AGRA GR863 were used for evaluating isolate aggressiveness. Seed of
ND2375 was obtained from the North Dakota State University and maintained at
3.5°C under low humidity. Two seeds were sown in each conetainer of 3M Metro
Mix. Conetainers were placed on benches in a greenhouse under the conditions
14 previously described. Two weeks after planting, at approximately the two-leaf stage, seedlings were inoculated. For each isolate, 10 plants each of ND2375 and of AGRA GR863 were inoculated. Each repetition had control inoculations of two isolates, SN8 and SN39, which were isolated during 2002.
The spore suspensions from individual S. nodorum cultures were sprayed onto the seedlings to the point of run-off with an atomizer. After inoculation, the seedlings were allowed to dry for 1 hour before being placed in the previously described misting chamber for 48 hours.
After incubation in the mist chamber, seedlings were returned to the greenhouse. Conetainers were randomly placed on greenhouse benches.
Seedlings were examined daily for symptom development and visually rated for disease severity 7 days after inoculation. The lesion rating scale of Liu et al. (34) was used to determine reaction type.
Statistical analysis-survey of fields
Each isolate of both species was coded for geographic region to determine if prevalence of leaf blotching pathogens from diseased leaves was related to region and if there was an association of the two pathogens on diseased leaves. Analogous to the concept of disease prevalence in Nutter (48), we consider pathogen prevalence and pathogen occurrence to represent the proportion of fields in which the pathogen of interest was isolated from a diseased leaf. Binary variables were defined for the presence/absence of S. nodorum and P. tritici-repentis in each field.
15 A logisitic model was fitted to the occurrence (presence/absence) data for
each pathogen species using maximum likelihood, with region as a factor (i.e., class) variable (10). The CATMOD procedure of SAS (SAS Institute Inc., Cary,
NC version 9.1) was used for the analysis. It was assumed that the binary variable had a binomial distribution. A likelihood ratio test was used to determine if region significantly affected the occurrence of each pathogen in diseased wheat leaves. The likelihood ratio is compared to a chi-square value to determine significance. Maximum likelihood estimates of model parameters were used (after transformation) to determine the estimated probability of pathogen occurrence in each region, and contrasts of the estimated logits for each region were used to determine significant differences in the probability of occurrence between regions. A separate analysis was done for each year.
A log-linear model was fitted to the joint occurrence (presence/absence) data for both species using maximum likelihood (41). The CATMOD procedure
of SAS was used for the analysis. In the log-linear model, the response variable
was the count of number of fields with a given combined pathogen occurrence:
neither pathogen found, both pathogens found, S. nodorum only found, and P.
tritici-repentis only found. It was assumed that the count had a Poisson
distribution. Predictor variables were: region; indicator variables for S. nodorum
and P. tritici-repentis occurrence; and their interactions. Likelihood ration tests
were used to determine significant effects. A significant interaction of the
pathogen indicator variable and region in the log-linear model is the same as a
significant main effect of region in the logistic model above. Of particular
16 relevance here is the interaction of the indicator variables for the two species,
which would indicate if there was a significant association of the occurrence of
the two species in the sampled wheat fields. If the occurrence of the two species
was independent (i.e., the presence of one pathogen species neither increases
nor decreases the probability that the other species is present), then the
interaction term would not be significant. An additional likelihood ratio test was
performed for goodness-of-fit of the model to the data. Likelihood ratios are
compared to chi-square values to determine significance. A separate analysis
was done for each year.
Statistical analysis-S. nodorum aggressiveness
To determine if aggressiveness of S. nodorum isolates varied by region
and year of isolation, a nonparametric marginal effects analysis was performed
on the lesion rating measurements (66, 7). This nonparametric procedure is
appropriate for ordinal measurements (such as the 0-5 rating scale used here)
and continuous measurements scale. Region, cultivar and year were considered fixed effects. A so-called ANOVA-type statistic (ATS) was used for testing for fixed effects (7). Pathogen aggressiveness for each region was represented by the estimated relative marginal effect, p, which is a statistic (varying between 0 and 1) that summarizes the entire distribution of rating values for each region relative to the distribution of ratings for all regions combined (66). Contrasts were used to compare the p values between regions. The MIXED procedure of
SAS, with options explained by Shah and Madden (66), was used for this
17 analysis. For presentation purposes, the median disease ratings were also
calculated.
Results
Distribution of leaf blotching pathogens
In 2002, 359 fields in seven designated wheat growing regions of Ohio
were sampled, and in 2003, 368 fields were sampled (Figure 1.1; Table 1.1).
One hundred twenty-five fields in 2002 had neither S. nodorum nor P. tritici- repentis, while only 50 fields in 2003 had neither pathogen (Table 1.2). Both pathogens were recovered from flag leaves from 54 fields in 2002 and 50 fields in 2003.
A likelihood ratio test indicated that the log-linear model provided a good fit to the presence/absence data for the two pathogens for both 2002 and 2003.
There was no significant interaction of the indicator variables for the occurrence of the two pathogen species on the response counts (chi-square statistic = 0.03,
P = 0.854 in 2002; chi-square statistic = 0.72, P = 0.38 in 2003). Thus, the occurrence of one pathogen in sampled diseased leaves in a field was independent of the occurrence of the other.
Occurrence of S. nodorum
There was a significant effect of region on the occurrence of S. nodorum for both years (chi-square = 35.8, P < 0.001 for 2002, chi-square = 31.2, P <
0.001 for 2003) based on the logistic analysis. The regions varied substantially
18 in the estimated probability of occurrence of S. nodorum (Table 1.3). Contrasts
for pairs of regions revealed several significant differences (Table 1.4). In 2002,
the estimated probability of occurrence was lowest for Regions 1 and 3 (~0.23),
and highest for Regions 2, 4 and 5 (0.85 - 0.90). The Region 1 estimated
probability was less precise than for Region 3 (standard error of 0.10 versus
0.05) because of the smaller number of fields in Region 1. Thus, there were
fewer significant differences in estimated probabilities between Region 1 and the
rest, compared with Region 3 and the rest.
In 2003, occurrence of S. nodorum was higher than in 2002 (Table 1.1).
The smallest probability of S. nodorum occurrence was in Region 3 (0.58), which was the same region with the lowest probability in 2002. However, Region 1 and
all the other regions had estimated probabilities of occurrence of at least 0.73,
and there were few significant differences among regions (Table 1.3).
Occurrence of P. tritici-repentis
Region did not have a significant effect on the occurrence of P. tritici-
repentis in 2002 (chi-square = 3.2, P = 0.78), but it did have a significant effect in
2003 (chi-square = 31.0, P < 0.001). The estimated probability of occurrence of
P. tritici-repentis varied only between 0.27 and 0.40 in 2002. In 2003, however, there were large differences in occurrence probabilities, which ranged from 0
(Region 1) to 0.38 (Region 3). In general, the prevalence of this pathogen was lower in 2003 than in 2002, which was the opposite trend found for S. nodorum
(Table 1.3). Contrasts for pairs of regions in 2003 revealed several significant
19 differences in the occurrence of P. tritici-repentis (Table 1.4) in 2003. In general,
regions with low probability of occurrence (Regions 4 and 7) were significantly
different from regions with higher probabilities (Regions 3 and 5). Because of the
low precision of the estimated probability for Region 6 (standard error = 0.11),
this region was generally not different from the others in terms of pathogen
occurrence.
P. tritici-repentis race differentiation
The majority of P. tritici-repentis isolates recovered from leaf samples
were races 1 and 2, with rare occasions of race 3 recovery. Race 1 was the
predominant race obtained in 2002 (39 isolates or 71%) and 2003 (32 isolates or
68%) (Table 1.5). Regions 2, 4 and 6 had lower occurrence of race 1 than the
other regions sampled. Race 2 was obtained from all seven regions, but less
frequently in 2002 (13 isolates or 24%) and 2003 (12 isolates or 26%). Regions
1, 4, 5 and 6 had lower frequency of race 2 isolates than other regions. Race 3
was rarely found in Ohio in 2002 (3 isolates or 5%) and 2003 (2 isolates or 4%), occurring predominately in Regions 5, 6, and 7.
S. nodorum aggressiveness
The nonparametric analysis (66) of disease ratings indicated that region
and cultivar had significant effects (P < 0.02) on pathogen aggressiveness.
However, year did not have an effect (P = 0.36). The interactions were not
significant (P > 0.02). Isolates recovered from Region 1, representing North
20 Eastern Ohio, were lower in aggressiveness (i.e., a lower marginal effect) than isolates from the other six regions (Tables 1.6 and 1.7). Median ratings were between 2.3 and 2.8 for Region 1. The relative marginal effect of Region 7, the
West Central area, was greater than for three other regions (see Table 1.7) and had isolates that were generally more aggressive (with a median value of 3 - 4).
The remaining wheat growing regions (Regions 2-6) had isolates with similar aggressiveness (similar relative marginal effects).
Discussion
The distribution of two foliar pathogens of wheat was determined in Ohio in 2002 and 2003. Wheat growing areas of the state were separated into the seven individual regions based on the environmental factors of soil type and annual precipitation, as well as arbitrary county boundaries. There were significant (P = 0.05) differences in the estimated probability of obtaining an isolate of S. nodorum from a diseased leaf among the different regions, and the differences varied somewhat between years. S. nodorum had a higher probability of being obtained from fields in 2003 than in 2002 regardless of region
(Tables 1.1 and 1.3). These results indicate that 2003 probably had more favorable environmental conditions for development of this disease, at least relative to other diseases. In 2002, the highest occurrence of S. nodorum in diseased leaves was in Regions 2, 4, and 5. In 2003, Regions 1, 2, 4, 5, and 7 had the highest probability of obtaining S. nodorum isolates. Greatest difference between years was for Region 1, where occurrence went from low to high. In
21 both 2002 and 2003, Region 3 had the lowest probability of obtaining isolates of
S. nodorum. Regardless of variation in the probability of obtaining S. nodorum isolates in the seven regions, S. nodorum was the predominant leaf blotching pathogen obtained in both years.
P. tritici-repentis was obtained from the flag leaf samples in both years, but with significant (P < 0.05) differences among regions in the probability of obtaining an isolate from diseased leaves only in 2003. Most notably, in the geographically separated North Eastern region of Ohio (Region 1), no isolates of
P. tritici-repentis were obtained in 2003 (Table 1.1). In this region, S. nodorum was isolated from all fields. The probability of obtaining a P. tritici-repentis isolate was high in both years for Region 3, North West Ohio (Table 1.3). These results indicate that tan spot has the potential to occur annually, with differences in prevalence within regions most probably due to disease response to environmental conditions. For example, in Region 6 in 2002, six isolates of P. tritici-repentis were obtained in three counties (20 fields) while in 2003 four isolates were obtained in two counties sampled (15 fields). In April, Region 6 had 3.3 cm less precipitation in 2002 than in 2003 (Table 1.8), which may have assisted in conidial dispersal early in the season. P. tritici-repentis has wind- blown conidia (73), which may be washed out of the air by the precipitation events, such as those that were frequent in later months of 2003, especially July.
These results indicate that in drier springs (49), tan spot may be more important
relative to Stagonospora leaf blotch, which requires more frequent precipitation
events.
22 The greater prevalence of S. nodorum in 2003 than in 2002 was also
probably due to the different environmental conditions that occurred each year.
For example, in 2002, nine isolates of S. nodorum were obtained in three
counties of Region 6 while eleven isolates were obtained in 2003 in two counties
(Table 1.1). This increase in frequency of S. nodorum isolation occurred despite
the fewer fields sampled in 2003. In Region 6, hourly weather conditions were
recorded during both years from 1 April through 15 July allowing for some
comparisons (Table 1.8). The number of precipitation events (i.e., days with
>0.28 cm) were more frequent in 2002 (28 days) than in 2003 (26 days), but
there was more precipitation in 2003 (41.2 cm) than in 2002 (30.7 cm). The
temperatures in June 2003 were slightly higher than in 2002 (21.6ºC in 2003,
18.8ºC in 2002). The greater amounts of precipitation in July of 2003 in combination with the frequent rain events allowed for more days with a relative humidity ≥ 75% (2 days in 2002 and 15 days in 2003). Since 2002 was drier in
April as the wheat canopy was developing, the pathogen may not have become established on the lower leaves as early as in 2003, causing disease development to be delayed. Adequate precipitation early in the season is necessary to favor pycnidium development and pycnidiospore liberation and dissemination (3, 26). Pycnidiospores of S. nodorum are splash dispersed from the surface to seedling leaves (3-4, 23, 44, 55). Once established on seedlings, pycnidiospores are spread throughout the canopy by rain-splash (3-4, 23, 44,
55). The greater number of days and more frequent and higher amounts of precipitation in July 2003 apparently allowed for greater dispersal of
23 pycnidiospores in the upper canopy of the wheat fields and favored spread of
disease.
Although there were differences in the occurrence of the two pathogens
among regions or years, based on the log-linear model analysis, occurrence of
one pathogen in sampled diseased leaves in a field was independent of the
occurrence of the other pathogens. Thus, any differential effect of environment on occurrence of the pathogens was not large enough to result in a negative association at the field scale. Rather, differential environmental effects were manifested at the state-wide or regional scales.
Recovery of P. tritici-repentis isolates from flag leaves collected in 2002 and 2003 indicated race 1 predominated both years in all regions of Ohio, but race 2 was also collected frequently (Table 1.5). Race 3 was rarer both years, being recovered from only a few regions (Regions 5, 6 and 7). Due to the relatively small sample size and limitations of log-linear or logistic models, statistical analysis could not be conducted on the small number of isolates that were race identified (101 isolates total from seven regions). Although, race 1 was detected in the North Eastern Ohio, only two isolates of P. tritici-repentis were race identified from this region in 2002 and no isolates were obtained in
2003. Leaves from fields in Region 7 yielded more isolations that included races
1, 2 and 3 during both years of sampling. In 2002, Region 7 had the most occurrences of race 2, but in 2003 Region 3 had more race 2 isolates identified than the other regions. Race 1 and 2 are well distributed throughout Ohio wheat
24 growing regions indicating a need for P. tritici-repentis race 1 and 2 resistance in wheat cultivars.
Aggressiveness differences among S. nodorum isolates were evident from
region to region, but not year to year. The relationship between aggressiveness
of isolate and region of Ohio was examined by determining the relative marginal
region effects. The geographically separated North Eastern region of Ohio was
characterized by significantly (P = 0.007) less aggressive isolates than other regions (e.g., median lesion rating = 2.75 on AGRA GR863). Although this region has more snowfall with longer winters and more acid soils than other regions, the impact of these environmental factors on aggressiveness is uncertain. Probably more importantly, North Eastern Ohio is also geographically
separated from the other wheat producing areas of the state (Figure 1.1) and has fewer fields planted to wheat annually. The low number of wheat fields planted annually would limit the amount of initial inoculum and limit the genetic variability in the geographically isolated population. The direct impact of these factors on aggressiveness is unknown, but isolates from this region of the state could be used to assess genetic variation in the S. nodorum population in Ohio.
Interestingly, this region had the highest variability in pathogen occurrence between the 2 years (Table 1.3).
In contrast, isolates from Region 7 were statistically (P = 0.01) more aggressive than isolates from other regions (e.g., median lesion rating = 4.0 on
AGRA GR863). This region is characterized by winters with less snow cover, loamy, fertile soils and a high number of fields planted annually. The producers
25 in this area tend to use more reduced tillage practices than other regions that
have soils with higher clay contents. The reduced tillage practice has the
potential to enhance pathogen over-wintering, sexual reproduction and
production of higher levels of initial inoculum. S. nodorum ascospores are wind- blown in the fall and spring (3-4), hence crop rotation will not exclude infection events when neighboring fields with wheat residue are managed using reduced tillage. Therefore, Region 7 has conditions that may increase the likelihood of genetic recombination possibly leading to more aggressive isolates than some other regions sampled. The aggressiveness studies indicate that the majority of
Ohio wheat growing regions (2-6) have isolates of S. nodorum with similar aggressiveness levels and differences in cultivar reactions from one location to another in these regions are probably not due to difference in aggressiveness.
In conclusion, the results of this study indicate that S. nodorum is the most prevalent leaf blotching pathogen in Ohio. Studies to determine differences in aggressiveness of S. nodorum isolates obtained in the seven regions indicated that across the majority of the regions there were no major differences in aggressiveness. P. tritici-repentis was also isolated in all seven regions in at least one year, indicating that this pathogen is also wide spread in Ohio. Race differentiation of P. tritici-repentis isolates obtained in both years indicated that over 60% of isolates were race 1, but race 2 was also common (approximately
35% of the sampled population). There were only a few occurrences of race 3.
There was no positive or negative association between S. nodorum and P. tritici- repentis occurrence on flag leaves during the 2 years of this study, at the field
26 scale sometimes both pathogens occurred on the same leaf. Positive
identification of leaf blotching pathogen was not able to be done in the field due
to the similarity of lesion types caused by S. nodorum and P. tritici-repentis (13).
Differences in recorded cultivar reactions for Stagonospora leaf blotch at different sites and in different years were probably a result of mixed infections of both pathogens, not differences in aggressiveness of S. nodorum isolates from one location to the other.
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35 1
3 2 7 6
4
5
Figure 1.1: Map of Ohio with sampled regions outlined.
36
each county sampled. thogens in a was sampled with a maximum of 15 Ohio in 2002 and 2003. from diseased flag leaves. re 1.1. per county u blotching p f were obtained from diseased flag leaves. t one field s s from seven regions in rrence of lea ate d l cte iso isolates were obtained entis gion. At lea 03) for occu ens colle d in each re see Materials and Methods Fig ch region. Stagonospora nodorum Pyrenophora tritici-rep e e r r each region, 02) and Appendix B (20 of leaf blotching pathog
f of fields whe of fields whe Distribution
Table 1.1: a = For description o b = The number of counties in ea c = The total number of fields sample fields sampled in each county. d = Number e = Number f = See Appendix A (20 37 2002 2003 Pyrenophora tritici-repentis S. nodorum --a + -- + -- 125 67 50 20 + 113 54 248 50
Table 1.2: The number of fields in which Stagonospora nodorum and
Pyrenophora tritici-repentis were isolated in Ohio in 2002 and 2003. a = -- indicates neither pathogen was isolated; + indicates the designated pathogen was obtained.
38
Stagonospora nodorumb Pyrenophora tritici-repentisb Regiona 2002 2003 2002 2003 1 0.24c (0.10) 1.0 (--)d 0.29 (0.11) 0.0 (--) 2 0.64 (0.07) 0.87 (0.05) 0.27 (0.07) 0.19 (0.05) 3 0.23 (0.05) 0.58 (0.06) 0.40 (0.06) 0.38 (0.06) 4 0.67 (0.07) 0.85 (0.05) 0.31 (0.07) 0.04 (0.03) 5 0.60 (0.09) 0.90 (0.04) 0.38 (0.07) 0.29 (0.07) 6 0.45 (0.11) 0.73 (0.11) 0.30 (0.10) 0.27 (0.11) 7 0.45 (0.05) 0.87 (0.03) 0.33 (0.05) 0.09 (0.03)
Table 1.3: Estimated probability of obtaining either Stagonospora nodorum or
Pyrenophora tritici-repentis from diseased flag leaves in seven regions of Ohio in
2002 and 2003. a = For a description of each region, see Materials and Methods and Figure 1.1. b = The species occurrence (i.e. presence/absence) data were analyzed separately using a logistic model in the CATMOD procedure of SAS. Separate analysis was run for each year. c = Maximum likelihood predicted (i.e. estimated) values for probability of obtaining the species in that region from a diseased flag leaf. Standard error in parenthesis. d = When probability is 0 or 1, standard error is undefined.
39
Stagonospora nodorumb Pyrenophora tritici-repentisb 2002 2003 2003 Regiona Chi- Chi- contrast square P value square P value Chi-square P value 1 vs 2 7.42 c 0.0065 0.72 0.3958 1.07 0.3001 1 vs 3 0.00 0.9680 2.37 0.1234 2.15 0.1421 1 vs 4 8.62 0.0033 0.80 0.3720 0.08 0.7737 1 vs 5 5.91 0.0151 0.52 0.4705 1.65 0.1991 1 vs 6 1.81 0.1787 1.45 0.2292 1.45 0.2292 1 vs 7 2.54 0.1111 0.68 0.4085 0.45 0.5015 2 vs 3 19.12 <0.0001 11.10 0.0009 5.24 0.0221 2 vs 4 0.09 0.7768 0.03 0.8606 4.42 0.0356 2 vs 5 0.23 0.6307 0.21 0.6484 1.41 0.2346 2 vs 6 2.12 0.1458 1.46 0.2272 0.41 0.5207 2 vs 7 4.80 0.0284 0.02 0.8897 3.14 0.0764 3 vs 4 22.52 <0.0001 9.49 0.0021 12.18 0.0005 3 vs 5 15.78 <0.0001 12.12 0.0005 1.04 0.3068 3 vs 6 3.67 0.0554 1.22 0.2700 0.70 0.4012 3 vs 7 8.78 0.0030 20.09 <0.0001 20.81 <0.0001 4 vs 5 0.62 0.4296 0.38 0.5388 8.12 0.0044 4 vs 6 2.90 0.0886 1.12 0.2889 5.23 0.0222 4 vs 7 6.67 0.0098 0.12 0.7325 1.17 0.2804 5 vs 6 1.19 0.2748 2.30 0.1290 0.03 0.8516 5 vs7 2.84 0.0919 0.15 0.6939 9.71 0.0018 6 vs 7 0.00 0.9777 2.05 0.1522 3.67 0.0553
Table 1.4: Chi-square values for comparing the probability of S. nodorum and P. tritici- repentis occurrence within fields in seven regions of Ohio during 2002 and 2003. a = For a description of each region, see Materials and Methods and Figure 1.1. b = The species were analyzed separately using a logistic model in the CATMOD procedure of SAS. Separate analysis for each years. c = Chi-square and P values were obtained using contrasts of maximum likelihood estimates of logits for pairs of regions.
40
Total no. Race 1 Race 2 Race 3
Regiona isolatesb 2002 2003 2002 2003 2002 2003
1 2 2c 0 0 0 0 0
2 7 3 1 1 2 0 0
3 32 10 13 3 6 0 0
4 8 4 2 2 0 0 0
5 18 6 8 0 2 1 1
6 6 2 1 2 0 1 0
7 28 12 7 5 2 1 1
Total 101 39 32 13 12 3 2
Table 1.5: Occurrence of Pyrenophora tritici-repentis races in seven regions of
Ohio in 2002 and 2003. a = For a description of each region, see Materials and Methods and Figure 1.1. b = The total number of isolates recovered in each region in both years. c = Number of isolates that were identified as each specific race on the set of P. tritici-repentis differential cultivars and lines.
41
Median ratingsb Mean estimate of pc
AGRA AGRA
Regiona GR863 ND2375 GR863 ND2375 Mean
1 2.8d 2.3 0.35e 0.33 0.34
2 3.5 3.0 0.54 0.47 0.50
3 4.5 3.0 0.55 0.39 0.47
4 3.5 3.0 0.54 0.40 0.47
5 3.3 3.0 0.54 0.44 0.49
6 3.3 3.0 0.52 0.45 0.48
7 4.0 3.0 0.62 0.51 0.56
Table 1.6: Median ratings and estimated relative marginal effects of two susceptible wheat genotypes inoculated with Stagonospora nodorum isolates obtained in Ohio in 2002 and 2003. a = For a description of each region, see Materials and Methods and Figure 1.1. b = Ratings were obtained 7 days after inoculating each isolate onto 2-week-old seedlings of AGRA GR863 and ND2375. The lesion rating scale was 1 (resistant) to 5 (susceptible) (30). Median ratings were based on 10 seedlings of each cultivar. c = The estimate of marginal region effects was based on mid-ranks of data (61).
42
Region contrasta F-value P value
1 vs 2-7b 12.67 0.007
2 vs. 3 0.54 0.51
2 vs 4 1.06 0.31
2 vs 5 0.06 0.81
2 vs 6 0.12 0.73
3 vs 4 0.00 0.99
3 vs 5 0.15 0.71
3 vs 6 0.06 0.81
4 vs 5 0.23 0.63
4 vs 6 0.09 0.77
5 vs 6 0.01 0.91
7 vs 2-6b 6.94 0.01
Table 1.7: F-values for comparing estimated marginal effects of Stagonospora nodorum aggressiveness for isolates obtained in Ohio in 2002 and 2003. a = For a description of each region, see Materials and Methods and Figure 1.1. b = Regions 1 and 7 were significantly different from other regions, therefore a single contrast was used for both regions with the remaining regions of the state.
43
2002 2003 April Days with >0.28cm precipitation 11 4 Days with ≥75% relative humidity 15 10 Total precipitation 7.1 10.4 Average air temperature 11.0a 10.9 May Days with >0.28cm precipitation 10 9 Days with ≥75% relative humidity 18 25 Total precipitation 13.7 10.2 Average air temperature 14.7 13.3 June Days with >0.28cm precipitation 7 6 Days with ≥75% relative humidity 20 20 Total precipitation 9.9 8.4 Average air temperature 18.8 21.6 July 1 - 15 Days with >0.28cm precipitation 0 7 Days with ≥75% relative humidity 2 15 Total precipitation 0.03 12.2 Average air temperature 23.3 22.3
Table 1.8: Region 6 average weather conditions in 2002 and 2003 for April 1 to
July 15 calculated from hourly data. a = Temperatures in ºC.
44 CHAPTER 2
SEEDLING REACTION OF WINTER WHEAT TO STAGONOSPORA
NODORUM ISOLATES PRODUCING SNTOX1 OR SNTOX2 AND
PYRENOPHORA TRITICI-REPENTIS RACE 1 OR RACE 2
Abstract
The wheat pathogens Stagonospora nodorum and Pyrenophora tritici-
repentis produce phytotoxins that promote the infection process and disease development. Seedlings of 15 soft red winter and one hard red spring wheat cultivars were tested for reaction to isolates of S. nodorum producing high levels
of either SnTox1 or SnTox2. This same set of wheat cultivars was also tested for
reaction to P. tritici-repentis races 1 or 2. The isolate by cultivar interaction was
found to be significant (P < 0.0001) after inoculation with either pathogen.
Seedlings of Bouillon, Coker 9025 and Coker 9474 had significantly (P < 0.05) greater sensitivity to SnTox1, rather than SnTox2, than the other cultivars tested.
A moderately resistant reaction was observed on seedlings of cultivars Coker
9025, Coker 9663, OH708, Patterson and Sisson in response to inoculation of
the SnTox1 producing isolate. In contrast, seedlings of Bravo, Patterson and
OH708 had significantly (P < 0.05) greater sensitivity to SnTox2. Cultivars
45 Bouillon, Coker 9025, Coker 9663, and Roane had moderately resistant
reactions to SnTox2. Results of relative marginal effects analysis indicated a
significant (P < 0.05) difference among cultivar reactions after inoculation with P. tritici-repentis races 1 and 2. All cultivars tested, except for Coker 9474 and
Hopewell, had moderately resistant reactions to race 1. All cultivars tested, except for Bouillon, Coker 9474 and Hopewell, had moderately resistant reaction to race 2. Relative marginal effects analysis indicated 9 of the 16 cultivars were more sensitive to race 2 These results indicated that the wheat cultivars tested generally had a greater sensitivity to P. tritici-repentis race 2 than race 1.
Introduction
The wheat (Triticum aestivum L.) foliar pathogens Stagonospora nodorum
(Berk.) (Castellani and E. G. Germano) (teliomorph Phaeosphaeria nodorum (E.
Muller (Hedjaroude))) and Drechslera tritici-repentis (Died.) (Shoemarker)
(teliomorph Pyrenophora tritici-repentis (Died.) (Drechs)) occur world-wide (86).
Stagonospora nodorum causes Stagonospora leaf and glume blotch on wheat and other cereals. Pyrenophora tritici-repentis causes tan or yellow spot of wheat. The prevalence and intensity of these diseases depends on inoculum levels in the field and environmental factors and host.
S. nodorum became one of the major wheat leaf spotting pathogens in
Indiana by the late 1980’s, coinciding with the increase of conservation tillage
(78). Stagonospora leaf and glume blotch affects both grain quality and yield.
Yield may be reduced as the result of shriveling of the grain and lower test
46 weights (38, 57) due to reduced photosynthetic area of upper leaves resulting in decreased carbohydrate level available for seed fill (26, 83). Infection of wheat plants may occur at any stage of development (61, 82). Current management of this disease is achieved through cultural practices, fungicide application and use of resistant cultivars (34, 47-48, 53, 81). Control of seed-borne inoculum is achieved through fungicide seed treatment to reduce seedling infection and reduce subsequent epidemic development (3, 31, 47-49, 53, 75-76). Moderately resistant cultivars are available, but currently there are no soft red winter wheat cultivars with high levels of resistance to this pathogen (46).
Resistance to S. nodorum has been shown to be quantitatively inherited
(9, 12, 21, 45, 54-56, 65, 67, 68-71, 73, 81). Resistance reactions at different stages of wheat development appear to be independent and not highly correlated
(2, 7, 11, 21, 29, 33, 65, 85). Quantitative trait loci (QTL) for S. nodorum resistance for the seedling, leaf blotch and glume blotch phases have been examined in resistant germplasm sources (81). QTL for leaf blotch resistance of the flag leaf were found on chromosomes 3A, 4A and 3BS, and glume blotch resistance was located at those same loci as well as chromosomes 7A and 4BL
(29, 72). Identification of these QTL were based on partial resistance components, such as disease severity. Results of most studies indicate that there are a few major QTL controlling partial resistance as well as qualitative resistance for toxin sensitivity (11).
Loci conditioning sensitivity to toxins produced by S. nodorum have been identified. A single nuclear gene on chromosome 1BS contains the sensitivity
47 locus for the proteinaceous toxin, SnTox1, that conditions the necrotic response
(44). This toxin appears to be a major virulence factor (44), although it is not required for disease development (45). This locus has been named Snn1 and shown to be dominantly inherited (45). Other toxins have been previously reported, but not fully characterized (30).
S. nodorum is the major leaf blotching pathogen in Ohio wheat fields making leaf blotch resistance an important disease management tool. Some years frequent precipitation events after head emergence favor continued leaf blotch development and infection of the wheat spikes for development of glume blotch. Monitoring Stagonospora leaf blotch progression through the season in
Ohio and other parts of the world has been complicated by the occurrence of tan spot with similar appearing lesions (5).
In the Northern Great Plains of North America, tan spot is considered one of the major foliar diseases of wheat, with increased occurrence in recent decades after the adoption of conservation tillage (1, 59-60, 74, 87, 89).
Significant losses occur in yield, due to reduced on thousand-kernel weight and number of grains produced per spike (5, 6).
Harvested grain may be shriveled and discolored (5, 6). There are numerous graminaceous host species of P. tritici-repentis that permit over winter survival of the fungus (1, 27, 35-37). Wheat residue left on the soil surface appears to be a major reservoir for P. tritici-repentis (89). This pathogen exists as eight races and is capable of producing at least three phytotoxins (1, 10, 24,
43). The most abundant races found in the United States have been race 1 and
48 race 2, with some occurrences of race 3, 4, and 5 (1, 23). P. tritici-repentis races
1, 2 and 3 have been found in Ohio (16). Races 6-8 are found in other areas of
the world (1). Tan spot is currently managed by crop rotation, mold-board
plowing to bury wheat residue and fungicide application (6, 62, 64).
Genetic resistance in commercial cultivars is considered the most
environmentally safe and economical means of tan spot management (18, 22,
60, 62). A study of fourteen hundred bread wheat cultivars for race-specific
resistance indicated no genotypes express complete resistance (60).
Quantitative trait loci (QTL) for resistance to P. tritici-repentis race 1 (Ptr ToxA
and C) occur on chromosomes 1A, 3B, 5A and 5B, with most of the genetic
variation being explained by the 1AS marker (15).
P. tritici-repentis produces phytotoxins that elicit two distinct symptoms on
susceptible cultivars, tan necrosis and/or chlorosis (43, 89). These toxins disrupt
mesophyll membranes resulting in electrolyte leakage during the infection
response (4, 14, 28, 39). The necrosis toxin, Ptr ToxA, is secreted by the fungus
in an inactive form that the plant enzymatically cleaves into an active form (43,
50, 52). Ptr ToxA has been shown to be localized to the chloroplast membrane
in plant cells (50). Ptr ToxB is a chlorosis toxin (23, 43, 51). There are multiple
copies of the gene encoding Ptr ToxB in the P. tritici-repentis genome and non-
functional copies have been detected in the non-pathogenic race 4 (51). The
chlorosis response to Ptr Tox B has been shown to be associated with the wheat
A and D genomes (89). Genes encoding both Ptr ToxA and B have been cloned and characterized (43, 51). A third partially characterized toxin, Ptr ToxC also a
49 chlorosis toxin, has been isolated from cultures of P. tritici-repentis obtained from
the Turkish-Syrian border, the center of diversity for wheat (43). Wheat nuclear
genes control the reaction to each race and the toxins they produce (25).
Additional virulence factors apparently influence disease severity, but these
factors have not been well characterized (22-23, 43).
The reaction of commercial soft red winter wheat cultivars grown in Ohio
to P. tritici-repentis has not been determined. Field assessments of resistance
are confounded by co-infection of leaves by S. nodorum, which produces a
similar lesion type. Since sensitivity to S. nodorum (45) and P. tritici-repentis (4,
23, 40, 42, 50) toxins is inherited as dominant factors, seedling reactions are well suited for cultivar resistance characterization. Xu et al. examined 120 elite synthetics and 35 durum wheat genotypes for their reaction to P. tritici-repentis
race 1 and the S. nodorum isolate Sn2000 at the seedling stage (88). Their results indicated that durum wheat was susceptible to both pathogens and some of the elite synthetic hexaploid lines had partial resistance (88). Resistance for
both of these pathogens in the elite synthetic hexaploid lines was hypothesized to be on the D genome contributed from the Aegilops tauschii parent. Reactions
of wheat seedlings have been used to characterize resistance in other wheat-
pathogen systems, such as leaf rust (Puccinia triticina) (84, 58) and powdery
mildew (Blumaria graminis f. sp. tritici) (8, 63).
Objectives The overall objective was to determine the seedling response of soft red winter wheat cultivars and lines to S. nodorum and P. tritici-repentis. This 50 objective was further divided into evaluating the reaction of seedlings of winter wheat cultivars and lines to S. nodorum isolates producing higher levels of
SnTox1 or SnTox2 or for reaction to race 1 or race 2 of P. tritici-repentis.
Materials and Methods
Genotypes and seedling production
Thirteen soft red winter wheat cultivars were chosen from the cultivars evaluated in the Ohio Wheat Performance Trial based on their Stagonospora leaf blotch reactions over 3 years (Table 2.1). These cultivars ranged in reaction from moderately resistant to susceptible, with their rank sometimes varying across years. The soft red winter wheat cultivar AGRA GR863 and the hard red spring wheat cultivar ND2375 were included as susceptible checks, while the
Ohio breeding line OH708 was included as a S. nodorum moderately resistant check.
Seed of the 15 cultivars was obtained from seed companies and stored at
3.5°C until use. OH708 seed was obtained from plots maintained by the Ohio
State University Wheat Breeding Program. Seed of the cultivars and lines were planted into 11.5- x 3.5-cm conetainer trays (11.5 x 21.5 x 35.5 cm). In each conetainer, 2 seed of each cultivar or line was planted to obtain a total of 19 cones of each cultivar or line in each tray. Ten cones of the susceptible cultivar,
AGRA GR863, were planted in each tray. The trays were maintained in the greenhouse under the following conditions until inoculation.
51 The 44.5-m2 greenhouses averaged 20.8°C during the day and 19.2°C at night, with a 12-hour light period provided by one 1000-watt metal halide lamp and four 1000-watt high pressure sodium lights (Rudd lighting Co., Solon, OH).
Pots were fertilized weekly with 100 ml of a triple 20 Peter’s® fertilizer solution (N
20%, P 20%, K 20%; 4.8 g/liter water) (Scotts, Inc., Marysville, OH). For insect control, plants were sprayed weekly with S-kinoprene (Enstar®, Wellmark,
Bensenville, IL) (0.01 g ai/liter water) or abamectin (Avid®, Syngenta Crop
Protection, Inc., Greensboro, NC) (0.01 g ai/liter water).
Inoculum and Inoculation
Seedlings were inoculated at the three-leaf stage, approximately 3 weeks after planting. Two trays were planted for each experiment, one tray to be inoculated with S. nodorum isolate SN2000, a high SnTox1 and low SnTox2 toxin producer, and one tray to be inoculated with isolate SN69-1, a high SnTox2 and low SnTox1 toxin producer. These two isolates were obtained from Dr. Tim
Friesen from the USDA-ARS Nothern Cereal Laboratory (USDA-APHIS Permit
No.64574). Plants used for P. tritici-repentis inoculations were arranged in the same configuration in the conetainer trays. One tray was inoculated with race 1 and one tray was inoculated with race 2. P. tritici-repentis isolates were also obtained from Dr. Time Friesen (USDA-APHIS Permit No.61766).
Inoculum was produced by growing isolates on V8 agar modified with 50 mg/liter of streptomycin sulfate (Sigma-Aldrich, St. Louis, MO). The agar cultures from 8 cm diameter petri dishes of each S. nodorum isolate were inverted onto a
52 15-cm cheese cloth square (four layers) and dipped into a glass beaker of 100-ml tap water and strained to obtain pycnidiospores of S. nodorum. Conidial
suspensions were enumerated using a Spotlite Hemacytometer® (American
Scientific Products, McGaw Park, IL) according to the protocol for counting small
spores (80), then diluted to obtain a 1x106 pynidiospores/ml of S. nodorum. For
P. tritici-repentis inoculum, 5-ml tap water was added to the surface of each Petri
dish and conidia were dislodged from the agar surface with a rubber policeman
then strained through two layers of cheesecloth. The protocol for enumerating
large spores (80) was used during hemacytometer counts and the suspensions
were diluted to obtain a 3x103 conidia/ml. Plants were inoculated by atomizing
either conidial suspension onto the leaf surface until run-off.
After inoculation seedlings were placed into a mist chamber. The mist
chamber consisted of a 2.3- x 1.4- x 1.0-m PVC skeleton enclosed by
polyethylene sheeting with adjustable sides. An air atomizer with a fluid nozzle
(#2850 SS, Spraying Systems, Co., Spokane, WA) (0.56 kg/cm3 water) and an
air nozzle(#70) (4.2k g/cm3 air) misted 30 ml of water for 30 seconds every 3 minutes above a polyethylene canopy over the plants. The temperature in the mist chamber was maintained at approximately 22°C and relative humidity was approximately 100%. Inoculated plants were incubated in the mist chamber for
48 hours. During this incubation period, a 12 hour photoperiod was maintained by one 1000-watt metal halide lamp and one 1000-watt high pressure sodium lamp suspended above the chamber. After incubation, the seedlings were returned to the greenhouse.
53
Cultivar reaction assessments
Seven days post-inoculation, seedlings were rated for lesions type using
the rating scale developed by Liu et al for S. nodorum toxin sensitivity (44).
Seedlings inoculated with P. tritici-repentis were visually assessed daily for symptom development and a final rating was obtained 7 days after inoculation using the Lamari and Bernier scale (41). Both scales range from 0
(insensitive/resistant) to 5 (sensitive/susceptible). These lesion reaction rating scales account for both lesion type and disease severity.
Experimental design and statistical analysis
Inoculation assay of the two pathogens were considered separate experiments. An experimental repeat of either pathogen was 2 trays of conetainers planted with the same cultivars or lines in each tray inoculated with one of the pathogen isolates. There were four complete experimental repeats of the inoculations of S. nodorum isolates. Five experimental repeats were
conducting using P. tritici-repentis isolates. The data was analyzed using a marginal effects nonparametric analysis as described in Shah and Madden (77) with experimental repeat and replicate plant being random effect factors and isolate and cultivar being fixed effect factors (77). The median rating for each cultivar by race or isolate interaction was calculated in SAS (SAS Institute Inc.,
Cary, NC version 9.1). The ANOVA-type test statistic, p, was calculated using ranks, and relative marginal effects were estimated. These marginal effect
54 estimates were then compared to determine differences in cultivar reactions to
the different isolates of either pathogen (77). For example in Table 2.1, relative
marginal effects values can be compared directly by taking the cultivar with the
higher p value (e.g. ND2375 p=0.82), subtract the cultivar with the lower p value
(e.g. Coker 9663 p=0.4), then taking the result and adding the constant, 0.5. The
resulting value of 0.92 is the probability of overlap in obtained lesion type ratings.
If the resulting value is less than 0.50, then the obtained ratings may not be
significantly different.
Results
S. nodorum reactions
Analysis of variance indicated a significant interaction (P < 0.0001) of cultivar and S. nodorum isolates SN69-1 and SN2000, producing primarily
SnTox2 and SnTox1, respectively. Statistical analysis also indicated the main effects of S. nodorum isolate was significant (P = 0.009), as well as cultivar or line reaction (P < 0.0001). The majority of cultivars had a moderately resistant to moderately susceptible reaction (median rating of 3.0) when inoculated by either isolate (Table 2.1). Seven of the sixteen cultivars or lines had a significantly different (P < 0.05) reaction based on relative marginal effects analysis. Bouillon,
Coker 9025 and Coker 9474 had significantly greater median ratings and larger relative marginal effects after inoculation with SN2000, the higher SnTox1 producer. Cultivars AGRA GR863, Bravo, OH708 and Patterson had significantly greater median ratings and larger relative marginal effects after
55 inoculation with SN69-1, the higher SNTox2 producer. The susceptible check
cultivar, ND2375, had the highest median rating of 5.0 and relative marginal
effects when inoculated with either isolate.
P. tritici-repentis reactions
Analysis of variance indicated a significant interaction effect (P = 0.0001) of cultivars and races of P. tritici-repentis for sensitivity to the toxins produced.
The main effect of P. tritici-repentis race was also significant (P < 0.0001) as was the main effect of cultivar or line reaction (P < 0.0001). The highest rating obtained after inoculation with either race was 3.0 (moderately resistant to moderately susceptible) for cultivars Coker 9474 and Hopewell. Analysis of relative marginal effects indicated that Coker 9474 had the largest effect after inoculation of either race. There were significant differences between the marginal effects after inoculation of the cultivars, with race 2 having higher marginal effects on 9 of the 16 cultivars tested than race 1. Cultivars Bravo and
OH708 had the lowest observed relative marginal effects (p = 0.30) after race 1 inoculation. Cultivars OH708, Patterson, Patton and Sisson had the lowest observed relative marginal effects (p = 0.37) after race 2 inoculation.
Discussion
For some wheat disease screening programs, seedling reactions have been found to be a cost-effective way of evaluating germplasm due to the high correlation of seedling and adult reactions. For example, leaf rust (8, 84) and
56 powdery mildew (58, 63) reactions can be efficiently determined in the seedling
stage. These seedling reactions appear to be effective only for factors controlled
by major genes. Sensitivity to toxins produced by S. nodorum (45) and P. tritici-
repentis (4, 23, 40, 42, 50) has been shown to be highly specific and dominantly
inherited. Therefore, seedling inoculations by either S. nodorum or P. tritici-
repentis are useful to screen for toxin sensitivity since the reaction obtained on seedling leaves is representative of adult plant reactions.
Inoculation with S. nodorum isolates producing predominately SnTox1 or
SnTox2 indicated that sensitivity levels of soft red winter wheat genotypes differed to these phytotoxins. Twenty isolates of S. nodorum obtained in Ohio in
2002 and 2003 that were preliminarily screened for toxin activity indicated that most isolates produced predominately SnTox1 and only low amounts of SnTox2
(data not presented). Cultivars examined for resistance to S. nodorum are apparently being exposed to the Snox1 toxin and negligible levels of SnTox2.
These results indicate that breeding lines are primarily being selected for insensitivity to SnTox1 in the Ohio breeding program with little selection for insensitivity to other phytotoxins.
Resistance to S. nodorum is not only conferred by toxin insensitivity (45), but also by other resistance components, such as the incubation period, lesion size and number of pycnidia per lesion. Various components of resistance have been estimated to be controlled by 1 to 4 genes (20, 32). Previous researchers have found that, in general, winter wheat cultivars have higher levels of resistance than spring wheat cultivars (2, 17, 66, 68). Additionally, epistatic
57 effects have been shown to be important in resistance to S. nodorum (45, 54),
while cytoplasmic effects have not been observed in resistance studies (7, 32).
Toxin sensitivity is only a part of the reaction response to S. nodorum, therefore
breeding cultivars insensitive to fungal phytotoxins may improve cultivar
resistance levels only to a limited degree and obtaining higher levels of
resistance may require incorporation of other resistance components.
Cultivar reaction to each P. tritici-repentis toxin has been shown to be
controlled by a single locus at all ploidy levels (4, 22, 24-25, 79). Toxin
production appears to not be associated with the initial (< 24 hours) resistance
response, although when intracellular growth of the pathogen reached the
mesophyll cells (72 hours after infection) differences in resistance level were
observed (13). The majority of P. tritici-repentis isolates characterized from Ohio
were race 1 or race 2 (Chapter 1). Nine of the 16 cultivars tested were
significantly more sensitive to inoculation with race 2 than race 1 as indicated by
relative marginal analysis. The cultivars were resistant to toxins produced by
race 1, with most cultivars having a moderately resistant reaction. These results
indicated that all soft red winter wheat cultivars tested were sensitive to the
phytotoxins produced by races 1 and 2 of P. tritici-repentis. However, most
cultivars had moderate reactions to infection to either race.
Resistance to P. tritici-repentis has been shown to be associated with a
reduction in conidial production due to restriction of the lesion size (19).
Supplementary virulence factors have not been well characterized in P. tritici- repentis (22-23, 43), and should be evaluated in future studies. The moderate
58 reactions of the cultivars tested to races producing the different toxins indicate
that toxins may not be the main factors responsible for susceptible reactions of
cultivars. Results of this study confirm that soft red winter wheat cultivars have
toxin sensitivity loci, but factors controlling partial resistance to the other
virulence factors of P. tritici-repentis may be important as well. Screening of
bread wheat genotypes from in China, Brazil and Mexico indicated that juvenile
plant reactions could separate the more resistant lines from the susceptible ones,
although some discrepancies occurred between adult reactions and juvenile
reactions (60). Screening early generation breeding lines at the seedling stage
for P. tritici-repentis toxin sensitivity will likely assist in identifying resistant lines.
The soft red winter wheat cultivars tested were representative of lines
available for commercial planting from public and private breeding programs.
Results of this study indicate that screening seedling reactions of soft red winter
wheat germplasm is effective in determining toxin sensitivity of S. nodorum and
P. tritici-repentis. Interestingly, the cultivars tested in this study were sensitive to
toxins produced by S. nodorum isolate SN69-1 and P. tritici-repentis race 2.
Sixty-five of the 120 elite synthetic hexaploid synthetics examined at the seedling stage by Xu et al. for reaction to P. tritici-repentis race 1 and the S. nodorum
isolate Sn2000 were moderately resistant to both pathogens (mean reactions of
<2.00) (88). The similarity of S. nodorum and P. tritici-repentis reactions of
cultivars tested in this study and previous results (88) may indicate a link
between certain toxin specific sensitivity loci. Results of this test indicate that the
relatively small set of cultivars varied from moderately resistant to susceptible
59 based on sensitivity to S. nodorum isolates producing predominately SnTox1 and
SnTox2. However, all cultivars tested were moderately resistant based on relatively low sensitivity to toxins produced by P. tritici-repentis race 1 and 2.
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76. Shah, D. and Bergstrom, G. C. 1993. Assessment of seedborne Stagonospora nodorum in New York soft white winter wheat. Plant Dis. 77:468-471.
77. Shah, D. A. and Madden, L.V. 2004. Nonparametric analysis of ordinal data in designed factorial experiments. Phytopathology 94:33-43.
78. Shaner, G. and Buechley, G. 1995. Epidemiology of leaf blotch of soft red winter wheat caused by Septoria tritici and Stagonospora nodorum. Plant Dis. 79:928-938.
79. Sykes, E. E. and Bernier, C. C. 1991. Qualitative inheritance of tan spot resistance in hexaploid, tetraploid, and diploid wheat. Can. J. of Plant Pathol. 13:38-44.
68 80. Tuite, J. 1969. Plant Pathological methods fungi and bacteria. Burgess Publishing Company Minneapolis, Minn.
81. van Ginkel, M. and Rajaram, S. 1999. Breeding for resistance to the Septoria/Stagonospora blights of wheat. Pages 117-126 in: Septoria and Stagonospora Diseases of Cereals: A Compilation of Global Research. http://www.cimmyt.org/Research/Wheat/pdf/Septoria/contents.htm.
82. Verreet, J. A. and Hoffmann, G. M. 1990. A biologically oriented threshold decision model for control of epidemics of Septoria nodorum in wheat. Plant Dis. 74:731-738.
83. Walther, H. and Bohmer, M. 1992. Improved quantitative-genetic selection in breeding for resistance to Septoria nodorum (Berk.) in wheat. J. of Plant Dis. and Protect. 99:371-380.
84. Wamishe, Y. A. and Milus, E. A. 2004. Seedling resistance genes to leaf rust in soft red winter wheat. Plant Dis. 88:136-146.
85. Wicki, W., Messmar, M., Winzeler, M., Stamp, P. and Schmid, J. E. 1999. In vitro screening for resistance against Septoria nodorum blotch in wheat. Theor. Appl. Genet. 99:1273-1280.
86. Wiese, M. V. ed. 1987. Compendium of wheat diseases. 2nd ed. The American Phytopathological Society, APS Press. St. Paul, Minn.
87. Wright, K. H. and Sutton, J. C. 1990. Inoculum of Pyrenophora tritici- repentis in relation to epidemics of tan spot of winter wheat in Ontario. Can. J. of Plant Pathol. 12:149-157.
88. Xu, S. S., Friesen, T. L. and Mujeeb-Kazi, A. 2004. Seedling resistance to tan spot and Stagonospora nodorum blotch in synthetic hexaploid wheats. Crop Sci. 44:2238-2245.
89. Zhang, W. and Jin, Y. 1998. Sensitivity to Ptr ToxA and tan spot infection responses in Aegilops/Tritcum complex. Can. J. of Plant Pathol. 20:415- 418.
69
90. Zhang, W. and Pfender, W. F. 1993. Effect of wetting-period duration on ascocarp suppression by selected antagonistic fungi in wheat straw infested with Pyrenophora tritici-repentis. Phytopathology 83:1288-1293.
70 Table 2.1: Reaction of soft red winter wheat cultivars to seedling inoculation with
Stagonospora nodorum isolates SN2000 (higher SnTox1 producer) and SN69-1
(higher SnTox2 producer).
a = The main effect of cultivar was significant (P < 0.0001) from F-tests obtained in a linear mixed model with the Satterthwaite approximation for degrees of freedom.
b = The main effect of isolate was significant (P = 0.009).
c = Relative summary statistic. Differences in p values indicate the probability
that ratings in one group are larger than in another group.
d = Groups were assigned based on pairwise comparisons of relative marginal
effects. Means followed by the same letter are not significantly (P = 0.05) different. e = The interaction of cultivar by isolate was significant (P < 0.0001). P values presented were obtained from pairwise comparisons of relative marginal effects. f = The medians were based on 19 replicate seedlings in each of four experimental repetitions. Seedlings were visually rated on a 1 – 5 scale with 1 indicating insensitivity and 5 indicating highly sensitive.
71
72
Table 2.2: Reaction of soft red winter wheat cultivars to seedling inoculation with
race 1 and 2 isolates of Pyrenophora tritici-repentis.
a = The main effect of cultivar was significant (P <0.0001) from F-tests obtained in a linear mixed model with the Satterthwaite approximation for degrees of freedom.
b = The main effect of race was significant (P <0.0001).
c = Relative summary statistic. Differences in p value indicate the probability that
ratings in one group are larger than in another group.
d = Groups were assigned based on pairwise comparisons of relative marginal
effects. Means followed by the same letter are not significantly (P = 0.05) different. e = The interaction of cultivar by race was significant (P = 0.0001). P values presented were obtained from pair-wise comparisons of relative marginal effects. f = The medians were based on 19 replicate seedlings in each of five repetitions.
Seedlings were visually rated on a 1 – 5 scale with 1 indicating insensitivity and 5 indicating highly sensitive
73 . 74 CHAPTER 3
REACTION OF THIRTEEN SOFT RED WINTER WHEAT CULTIVARS TO
PYRENOPHORA TRITICI-REPENTIS IN THE FIELD AND GREENHOUSE
Abstract
Pyrenophora tritici-repentis, the cause of tan spot, occurs in the eastern soft red winter wheat growing areas and in Ohio it occurs primarily as race 1 and
2 and more rarely race 3. Historically, cultivar resistance screening in the east has focused on S. nodorum and little is known about the reaction of soft red winter wheat cultivars to P. tritici-repentis. This study examined thirteen
commercially available wheat cultivar reactions to P. tritici-repentis. In the
greenhouse, the reaction of the cultivars were evaluated with inoculum consisting
of a combination of race 1 and 2 isolates obtained in Ohio and individual
inoculations using a race 1 and a race 2 isolate obtained from North Dakota. The
highest mean tan spot severity obtained in the greenhouse using the combination
of race 1 and 2 isolates was observed on cultivars Sisson and Bouillon (6.6% flag
leaf area affected (FLAA)). There was a significant interaction (P < 0.0001)
between race and cultivar. Results of individual race inoculations indicated a
greater sensitivity to race 1 by most cultivars, but mean disease severities
75 were low regardless of race used to inoculate plants. The highest observed mean severity caused by race 1 was 12.7% FLAA for cultivar Hopewell. The highest mean severity caused by race 2 was 12.5% FLAA for cultivar Coker
9474. Relative marginal effect analysis indicated Freedom, Hopewell, Roane and Sisson had higher reactions after race 1 inoculation. Regardless of race inoculated, Patton had the lowest marginal effect of all cultivars tested. These cultivars were also evaluated for tan spot resistance in two field plots in Ohio during 2003. Disease severities were generally greater in the field than in the greenhouse, with a maximum of 35% FLAA for the cultivar Sisson, but due to variability in cultivar reactions there was no statistical difference among cultivars.
The amount of seed infestation was assessed by determining the percentage of harvested seed from this plot yielding P. tritici-repentis. Seed infestation intensity was low (0.6 to 3.0%) with Coker 9663 having the highest percentage of infested seed (3.0%). These results indicated that most cultivars tested had moderately resistant leaf reactions to P. tritici-repentis and seed infestation in Ohio appears to be relatively low, even in inoculated plots.
Introduction
Tan, or yellow leaf spot, of wheat caused by Pyrenophora tritici-repentis
(Died) Drechs. (syn. P. trichostoma (Fr.) Fckl.), (anamorph Drechslera tritici- repentis (Died.) Shoem. (syn. Helminthosporium tritici-repentis Died.)) has a world-wide distribution (2, 18, 30, 37, 45). In the Northern Great Plains of North
76 America, tan spot is considered one of the major foliar diseases of wheat with
increased occurrence in recent decades after the adoption of conservation tillage
(1, 35-36, 41, 47-48). Tan spot has caused significant losses in yield, due to
reduced thousand-kernel weight and lower numbers of grains produced per spike
(5, 12, 41). P. tritici-repentis infects the seed causing the disease ‘red smudge’
(13). The most common symptom of ‘red smudge’ is a pink or red discoloration
of the seed that becomes black over time (13). Infection of the seed results in
loss of grade, increased potential for sprouting in the field under moist conditions,
reduced seedling vigor, reduced emergence and possible transmission of the
fungus from the seed to emerging leaves (5, 13, 40).
This pathogen exists as eight distinct races and produces three
phytotoxins (1, 7, 18, 30). The most abundant races found in the United States have been race 1 and race 2 with some occurrences of races 3, 4 and 5 (1, 17).
The remaining races exist in other areas of the world (1). There are numerous graminaceous hosts of P. tritici-repentis which allow for overwintering of the fungus (1, 20, 22-23). Wheat residue left on the soil surface is another reservoir for saprophytic growth and survival of P. tritici-repentis (49). Under favorable conditions, wet weather for approximately 2 weeks after flag leaf emergence, disease severities on the flag leaf accounted for approximately 84.7% of the variation in yield with a potential of decreasing yield by 30% in Australia (5). Tan spot is currently managed by at least 1-year non-host crop rotation, mold-board plowing to bury wheat stubble and fungicide application to seed and foliage (6,
37-38).
77 Genetic resistance in commercial cultivars is considered the most
environmentally safe and economical means of control (14, 16, 36-37). Fourteen
hundred bread wheat genotypes were evaluated for race-specific resistance to P.
tritici-repentis in one study with no cultivar expressing complete resistance (36).
Resistance has been shown to be associated with a reduction in conidial
production due to a reduction in lesion size (15). Independence of resistance to
the leaf phase and the seed phase of this disease has been established (13). An
increase in disease severity at later plant maturities has been demonstrated by
inoculating plants at the boot and flowering stages (41).
There are two distinct symptoms expressed by susceptible cultivars, tan
necrosis and/or chlorosis, which are caused by the plant response to phytotoxins
(28-30, 48). The P. tritici-repentis toxins have been shown to disrupt mesophyll
tissue resulting in electrolyte leakage (2, 9, 21, 24, 28-29). The toxin eliciting the
necrotic response, Ptr ToxA, has been shown to be secreted by the fungus in an
inactive form that the plant enzymatically cleaves into an active form (30, 32, 34).
Ptr ToxA has been shown to be localized to the chloroplasts in plant cells (32).
Ptr ToxB (6.61 kDa protein) causes a chlorotic response (17, 30, 33). Production of this toxin has been found to be associated with multiple loci in the fungal genome and non-functional copies have been detected in the non-pathogenic race 4 (33). The chlorosis response to toxin production has been shown to be associated with the wheat A and D genomes (48). Genes encoding both Ptr
ToxA and B have been cloned and characterized (30, 33). The third toxin, Ptr
ToxC also a chlorosis toxin, has been isolated from cultures of P. tritici-repentis
78 obtained from the Turkish-Syrian boarder, the center of diversity for wheat (30).
Genes encoding this toxin have not been cloned, but has been partially characterized (30). All three toxins have been shown to be deactivated by temperatures above 30ºC and seedlings inoculated after plants have been exposed to temperatures above 30ºC expressed no toxin sensitivity regardless of previous reaction type (24, 25).
In disease resistance inheritance studies, the susceptible reaction has been shown to be dominant over the resistant reaction (19). Results of most studies indicated that a few major genes condition resistance and expression of resistance is qualitative (19). Quantitative trait loci (QTL) for resistance to P. tritici-repentis have been located on chromosomes 1A, 3B, 5A and 5B, with most
of the variation being explained by the 1AS marker (10). Wheat nuclear genes
control the reaction to all races and toxins produced (18), and a single locus at all
ploidy levels controls the reaction of a cultivar to either PtrToxA or PtrToxB (2,
16, 18, 43). Specific receptors encoded by single dominant genes for toxin
sensitivity have been found in the wheat genome (2, 17, 26, 29, 32). Additional
virulence factors have been shown to influence disease severity, but these have
not been well characterized (16-17, 30).
One study examined the reactions of bread wheats and synthetic
hexalpoids at the seedling and adult stages with a mixture of races (37). This
study found that the synthetic hexalpoids had a median rating of 1-2 (moderately
resistant) on the Lamari et al. rating scale in the seedling and adult stages (37).
In Western Australia, moderate levels of resistance have been observed in field
79 studies (46). This resistance appeared to be durable and some of these cultivars also had resistance to Stagonospora glume blotch caused by Stagonospora nodorum (46). Screening of bread wheats from breeding programs in China,
Brazil and Mexico indicated that juvenile plant reactions could better distinguish the more resistant cultivars from those that were susceptible, but some genotypes had adult reactions that did not correspond to juvenile plant reactions
(36). Discrepancies between reactions resulting from greenhouse and field studies were observed in Oklahoma (12) on adult plants. These studies used P. tritici-repentis isolates that had not been characterized for race according to the currently accepted race concept.
Tan spot has not been considered a major disease in Ohio, but races 1, 2 and 3 have been found in Ohio (11). The economic occurrence of P. tritici- repentis in the U. S. has been mainly in the Great Plains region. The majority of wheat grown in the Great Plains is hard red winter, hard red spring and durum wheat, while soft red winter wheat dominates the Midwest. This has lead to a lack of knowledge of this pathogen in most Eastern and Midwest wheat producing states. The similarity in lesion type with those caused by S. nodorum has confounded disease assessments in field trial aimed at evaluating cultivar resistance in many areas of the world (5, 28).
Objectives
The main objective of this study was to determine the reaction of common commercially grown cultivars of soft red winter wheat to P. tritici-repentis. The
80 second objective was to determine cultivar reaction to a composite of isolates
that were race 1 and 2 as well as independent inoculations of either race on the
flag leaves in the greenhouse. The third objective was to compare the reaction
of cultivars obtained by inoculating adult plants in the greenhouse and with those
obtained in inoculated field plots in Ohio.
Materials and Methods
Thirteen soft red winter wheat cultivars were chosen from the Ohio Wheat
Performance Trial based on Stagonospora leaf blotch reactions over 3 years
(Table 3.1). Cultivars were chosen based on Stagonospora leaf blotch reactions
because differences in cultivar reactions to Stagonospora leaf blotch across
years and locations were hypothesized to be a result of contaminate P. tritici-
repentis infections. This was possible because of the similarity in lesion types and the lack of knowledge concerning tan spot resistance reactions of cultivars grown in Ohio. The cultivars represented genotypes from public (Freedom,
Hopewell, Roane, Sisson, Patterson), and private (Honey, Bravo, Bouillon, Coker
9025, Coker 9474, Coker 9663, AGRA GR535, Patton) wheat breeding programs and one advanced line from the Ohio State University Breeding program
(OH708). Seed of the genotypes from private and public breeding programs has been available for commercial planting in Ohio.
81 Greenhouse Experiments-Plant growth
Seed of the 13 cultivars was obtained from seed companies and stored at
3.5°C until use. In the fall of 2003, seed of the 13 cultivars were germinated on folded 12.7- x 50.8-cm moistened germination paper (Anchor Paper Company,
St. Paul, MN) placed in clear 10.2- x 30.5- x 5.1-cm plastic bags with 5 ml of water. The bags were maintained in the laboratory (21°C ± 2°C) under a 12 hour photoperiod using ultraviolet and white fluorescent lights. After 1 week, healthy germinated seeds were placed into flats (5.1 x 25.4 x 50.8 cm) of autoclaved
(115°C for 6 hours) Wooster silt-loam soil and put into a temperature controlled chamber (3.5°C) for a 65 day vernalization period. Each tray was treated with granular mefenoxam (Ridomil®, Novartis, Inc., Greensboro, NC, 1 g ai/tray), fertilized with pellets of triple 14 Osmocoat® (N 14%, P 14%, K 14%; 12 g/tray)(Scotts, Inc., Marysville, OH) and watered. Lights in the chamber were set to provide an 8 hour photoperiod and trays were watered as needed.
After the vernalization period, 32 plants of each cultivar were transplanted
individually into 12.7 cm pots of 3M Metro Mix (Conrad Fafard, Inc., Agawam,
MA). Leaves of plants were clipped to approximately 5 cm above the soil line 2
days after transplanting and pots were randomly arranged on benches in a 44.5-
m2 greenhouse. The greenhouses averaged 20.8°C during the day and 19.2°C
at night, with a 12 hour light period provided by one 1000-watt metal halide lamp
and four 1000-watt high pressure sodium lights (Rudd lighting Co., Solon, OH).
Through the course of the experiment plants were loosely tied with Sturdy
82 Stretch Tie® (California Plastic Products, Tustin, CA) to 30-60 cm long bamboo
stakes for support.
Pots were fertilized weekly with 100 ml of a triple 20 Peter’s® fertilizer
solution (N 20%, P 20%, K 20%; 4.8 g/liter water) (Scotts, Inc., Marysville, OH).
For insect control, the plants were sprayed weekly with S-kinoprene (Enstar®,
Wellmark, Bensenville, IL) (0.01 g ai/ liter water) or abamectin (Avid®, Syngenta
Crop Protection, Inc., Greensboro, NC) (0.01 g ai/ liter water). Applications of
triadimefon (Bayleton®, Bayer Corp., Kansas City, MO) (2.2 g ai/ liter water)
were applied 2 weeks after transplanting seedlings into pots and 2 weeks
thereafter for control of powdery mildew (Blumaria graminis f. sp. tritici ((DC.) E.
O. Speer) for 1 month.
Greenhouse multiple isolate inoculation
When the main tiller of each plant had a fully emerged flag leaf (Feekes
growth stage 9 (31)), the plant was inoculated. Inoculations were made by
atomizing a suspension of 3x103 conidia/ml on each fully expanded flag leaf until the point of run-off. Bulk suspensions were made with conidia from ten isolates of P. tritici-repentis obtained from wheat leaves collected in 2002 at different locations in Ohio. Six of these isolates were race 1 and the remaining four were race 2. Isolates were grown for 1 week on V8 agar modified with 50 mg/ liter of streptomycin sulfate (Sigma-Aldrich, St. Louis MO). Conidia were scraped from the agar surface of petri dishes using a rubber policeman after flooding the dish with tap water. After the conidia had been dislodged from the agar surface,
83 suspensions were strained through two layers of cheesecloth to separate conidia
from mycelial fragments. Suspensions of conidia were combined prior to
enumeration. Suspensions were quantified using a Spotlite Hemacytometer®
(American Scientific Products, McGaw Park, IL) according to the protocol for counting large spores (44).
Each individual plant in a pot was considered a replication. Each complete experimental repeat had a total of 30 replications for each cultivar.
Experimental repeats were separated over time with approximately a month in between planting dates. There were a total of five independent experimental repeats, but not all cultivars were represented in each experimental repeat.
Experimental repeats two and five had numerous missing plants for some
cultivars because of seedling death during vernalization. For each complete
repetition, 20 plants were inoculated and 10 plants were not inoculated and
served as control plants.
After inoculation plants were placed into a mist chamber. Control plants
were placed at the opposite end in the mist chamber from inoculated plants. The
mist chamber consisted of a 2.3- x 1.4- x 1.0-m PVC skeleton enclosed by
polyethylene sheeting with adjustable sides. An air atomizer with a fluid nozzle
(#2850 SS, Spraying Systems, Co., Spokane, WA) (0.56 kg/cm3 water) and an
air nozzle(#70) (4.2 kg/cm3 air) misted 30 ml of water for 30 seconds every 3 minutes above a polyethylene canopy over the plants. The temperature in the mist chamber was maintained at approximately 22°C and the relative humidity
84 was approximately 100%. Inoculated plants were incubated in the mist chamber
for 24 hours with misting and 24 hours without misting.
After the 48 hour incubation period in the mist chamber, plants were
returned to the greenhouse. Care was taken to prevent contamination of the
non-inoculated plants by the inoculated plants. Plants were randomly arranged
on greenhouse benches by inoculation date. The flag leaves were visually
assessed for percentage diseased leaf area 7, 14, 21 and 28 days after inoculation.
Greenhouse inoculations with race 1 and race 2
Seeds of the thirteen cultivars were germinated and vernalized as
previously described. Instead of placing the seedlings into pots after
vernalization, plants were placed into 20.5- x 4-cm conetainers (Stuewe and
Sons, Inc., Corvallis, OR). A single seedling was placed into each conetainer
and considered a replication. A block consisted of a total of 30 plants for each
cultivar. In each block, 15 random plants per cultivar were chosen for inoculation
of each race. There were a total of four blocks (experimental repeats) separated
in time.
Race 1 and race 2 inoculum and inoculations
Isolates were obtained from Dr. Tim Friesen from the USDA-ARS
Northern Cereal Laboratory (USDA-APHIS Permit No.61766). Inoculum was
grown and enumerated for each race as previously described. The plants were
85 inoculated at the boot stage (Feekes growth stage 10 (31)). A 3x103 conidia/ml suspension was applied to the flag leaf of each plant with a hand mister to the point of run-off. After inoculation, plants were placed into the misting chamber previously described for 48 hours with continuous misting and a 12 hour photoperiod. After incubation, plants were returned to the greenhouse.
The plants were randomly placed on greenhouse benches organized by inoculation date. Care was taken to separate plants inoculated with race 1 and race 2. Plants were visually assessed for lesion type 7, 14, 21 and 28 days using the scale developed by Lamari and Bernier (27). On the final assessment day a visual estimation of percentage flag leaf area affected (FLAA) was also recorded.
Field experiments
Seed of the thirteen cultivars were planted in field plots on 7 October 2003 in Wayne County and 9 October 2003 in Wood County, OH. The experiment was arranged as a randomized complete block with three replicate blocks. The
Stagonospora leaf blotch susceptible cultivar, AGRA GR863, and a moderately resistant check, OH708, were included to evaluate the impact of S. nodorum on the test. Approximately 3 g of seed of each cultivar were planted into each hill plot, 0.46 m apart. The fields had previously been cropped with soybeans. The fields were prepared by moldboard plowing and a fall (22.8 kg/ha) and spring
(56.8 kg/ha) application of nitrogen.
Plots were inoculated twice. The first inoculation consisted of colonized oat kernels spread on the soil surface around plots. The oat kernel inoculum was
86 made by soaking 150 g of oat kernels in 130 ml of distilled water in autoclaveable
plastic containers (30). After 24 hours of soaking, oat kernels were sterilized by
autoclaving for 90 minutes. The next day each jar was inoculated with two
isolates of P. tritici-repentis from a total of ten isolates collected from Ohio wheat fields in 2002. The inoculum was maintained in the laboratory for 2 weeks before being mixed then broadcast (900 g per 9.3 m2 ground surface) in both field plots on 12 November 2003.
Plots were inoculated a second time by spraying a conidial suspension of
P. tritici-repentis conidia on the flag leaves (Feekes growth stage 10 (31)). The ten isolates used to produce oat kernel inoculum were grown on V8 agar in petri dishes in the laboratory as previously described. Conidial suspensions were created by scraping the agar surface using a rubber policeman to dislodge conidia after flooding the dish with tap water. Suspensions were strained through two layers of cheesecloth to separate conidia from mycelial fragments.
Suspensions were enumerated as previously described. Plots were visually assessed weekly for percentage leaf area affected from flag leaf emergence through senescence.
Seed was hand harvested from plots at both sites. The seed was thrashed using a small bundle thrasher (ALMACO, Nevada, IA). The seed samples were then aspirated (Fractionating Aspirator, CFZ1 model, Carter Day
International, Inc., Minneapolis, MN) to remove light-weight seed from the
samples. From each of these sites, 105 randomly chosen seed of each cultivar
were used to assess presence of seed borne P. tritici-repentis. This was done by
87 placing 21 harvested seed on moistened filterpaper (9 cm Fisher Scientific) in
each of five 9-cm Pyrex Petri dishes. The dishes were placed under the light
banks previously described for fungal spore production. Petri dishes were
maintained under a 12 hour light period for 5 days then examined under a
dissecting scope for conidia and conidiophores of P. tritici-repentis. The
percentage of seed of each cultivar in each site yielding P. tritici-repentis was determined.
Statistical analysis
Analysis of variance (ANOVA) was conducted using the linear mixed
model in SAS (SAS, Inc., release 8.3, Cary, N.C.). The model was fitted using
the restricted maximum likelihood method and the Satterthwaite approximation
was utilized to determine degress of freedom for F-tests (42).
Least squares mean disease severity 28 days after inoculation with 10 P.
tritici-repentis isolates was analyzed with cultivar as fixed and block as random
effects, respectively. Control plants were excluded from analysis since these
were all zero. Pairwise comparisons (P = 0.05) of least squares means disease
severity for all cultivars were done.
Least squares mean disease severity and median lesion type rating 28
days after inoculation with race 1 or 2 was used for ANOVA. The mean disease
severity or median lesion rating was analyzed using a linear mixed model with
cultivar and race as fixed effect factors and block as a random effect factor. The
nonparametric marginal effects analysis (42) was used to determine effects of
88 cultivar and race on ordinal rating for lesion type. The interaction of cultivar by race was examined using pairwise comparisons of relative marginal effects values. For example, in Table 3.3 race 1 lesion type ratings, relative marginal effects values can be compared directly by taking the cultivar with the higher p value (e.g. Hopewell p=0.76), subtract the cultivar with the lower p value (e.g.
Coker 9663 p=0.56), then taking the result and adding the constant, 0.5. The resulting value of 0.7 is the probability of overlap in obtained lesion type ratings.
If the resulting value is less than 0.50, then the obtained ratings may not be significantly different.
Data obtained from the two inoculated field sites were combined for statistical analysis. The cultivar factor was fixed, whereas location and block were random effect factors. Pairwise comparisons of cultivars for mean disease severity and percentage seed infestation were used for mean separation.
Results
Greenhouse experiments
Multiple isolate inoculation
Inoculation using a mixture of 10 P. tritici-repentis isolates obtained in
Ohio produced a maximum mean percentage flag leaf area affected (FLAA) of
6.6% on cultivars Sisson and Bouillon (Table 3.2). The lowest mean disease severity was observed on the cultivar Honey, (3.8%). The main effect of cultivar mean disease severity was significant even with the relatively low disease severities. Pair-wise comparisons separated cultivars into three main groups; the
89 more susceptible cultivars (Bouillon and Sisson) with 6.6% FLAA, a more
resistant cultivar (Honey) with 3.8% FLAA and the remaining ten cultivars with
intermediate reactions ranging from 4.2% to 5.6% FLAA. However, some
cultivars in the intermediate group had severities that did not differ statistically
from the more resistant cultivar, Honey.
Race 1 and race 2 inoculations
There were significant interactions between race and cultivar for mean
disease severity. The highest mean disease severity was observed on Hopewell
(12.7% FLAA) when inoculated with race 1, while Honey had the lowest severity
(4% FLAA, Table 3.3). The highest mean disease severity after inoculation with
race 2 was 12.5% FLAA for Coker 9474, while Patton had the least amount,
3.8% FLAA. A highly significant difference (P < 0.0001) occurred between the
response to race 1 and 2 inoculations for Sisson in mean disease severity. While
most cultivars had higher mean disease severities when inoculated with race 1 than race 2, cultivars Coker 9474, Coker 9663 and Patterson had significantly higher disease severities when inoculated with race 2.
There were significant (P < 0.0001) interaction between race and cultivar for median lesion type and relative marginal effects. The highest median lesion type rating was observed on Hopewell (4.0) and greatest marginal effect (p =
0.76) in response to inoculation with race 1 and on Coker 9025 (median 3.7, p =
0.64) in response to race 2. The lowest median lesion type rating was observed
90 on Patton for both races (2.6 for race 1, 2.4 for race 2) and lowest marginal
effects (p = 0.30 for race 1, p = 0.24 for race 2).
Field reactions
Higher tan spot severities were obtained on cultivars in the field than in the
greenhouse. Sisson had the highest observed severity, 35.3% FLAA, while
Patton had the lowest (6.0% FLAA, Table 3.4). The S. nodorum susceptible
cultivar, AGRA GR863, had an intermediate reaction with 19.2% FLAA, indicating
that possible co-infection by S. nodorum had not substantially inflated disease
ratings. OH708, a line with moderate resistant to S. nodorum, had an
intermediate reaction to P. tritici-repentis as well, 15.3% FLAA. The variability of
the cultivar reactions resulted in a non-significant main effect of cultivar mean
disease severity (P = 0.578).
Seed infestation of P. tritici-repentis in the field was generally low. The
highest percentage of infested seed was 3.0% for Coker 9663 (Table 3.4). Less
than 1.0% of the seed were found to be infested on cultivars AGRA GR863 and
Roane. Although statistical analysis indicated that there was a significant effect
of cultivar on amount of seed infestation, levels of seed infestation were probably too low to indicate substantial differences among cultivars.
Correlations
Mean disease severities of the cultivars tested after inoculation with a mixture of Ohio isolates was not correlated with results of race 1 and 2
91 inoculations (r = 0.37, P = 0.21 for race 1, and r = 0.13, P = 0.68 for race 2). The
mean disease severities obtained in the field were also not correlated with
disease severities obtained using the mixture of Ohio isolates in the greenhouse
(r = 0.17, P = 0.58).
Discussion
Mean disease severities of commercial cultivars grown in Ohio were low
(highest observed 13% FLAA) after inoculations of P. tritici-repentis in the greenhouse. These low disease severities were observed regardless of inoculating with a mixture of isolates representing race 1 and 2 or inoculating with single races. Possible virulence factors, other than toxin production, did not increase disease severities to a great extent even with the diverse origins of test isolates. An increase in disease severity has been observed after inoculation of plants at later maturities with the majority of injury occurring at the boot and flowering stages in Kansas (41). In this study, plants were inoculated at either flag leaf fully expanded (Feekes growth stage 9 (31)) or boot stage (Feekes growth stage 10 (31)) in the greenhouse. While there was an increase in leaf area affected after inoculating at the boot stage in this study, the disease intensity was still very low.
There was no correlation between disease severities of these greenhouse tests inoculating with either a mixture of isolates obtained in Ohio or individual race 1 or race 2 inoculations. This may have occurred due to the possible confounding response of cultivar to the inoculum containing isolates of both of
92 races 1 and 2. The cultivars had higher disease severities after inoculation with
either individual race, but generally severities were higher in response to race 1
inoculations (Table 3.3). Additionally, the cultivars could respond to additional
virulence factors expressed by specific races of P. tritici-repentis, other than
production of certain phytotoxins (16-17, 30). These virulence factors could be
variously expressed by isolates from different origins or there may be different
alleles associated with the geographically separated populations of isolates used.
Tan spot development in field plots resulted in moderate intensity of
disease on most cultivars, but the variability in cultivar reactions resulted in a
non-significant effect of cultivar. Variability in cultivar response may have been
caused by either potential variability in inoculum levels, environmental effects
and other unknown variables. Oat kernel inoculum has been shown to be less
consistent than applying conidial suspensions to enhance disease development
in field plots (30), but in this study a conidial suspension was applied in addition
to the oat kernel inoculum in the field plots. Another possible explanation is the
expression of partial resistance by cultivars to other virulence factors which were
not examined in this study. These additional virulence factors have not been well
characterized (16-17, 30), therefore quantification of cultivar reaction is difficult to determine with full certainty.
Cultivar mean disease severities were not correlated between the
greenhouse and field tests. Discrepancies between evaluations in greenhouse
and field studies have been previously observed (12, 36). Evans et al. concluded
that while the reaction could be determined in greenhouse experiments, more
93 complex interactions that also impact yield could only be determined in field trials
(8). Yield was not measured in this study due to the small plot size. Regardless
of the lack of significant correlation between greenhouse and field studies, there
was an observed trend. Cultivars Sisson and Hopewell tended to have more leaf
area affected and cultivars Patton and Honey tended to have less leaf area
affected than the other cultivars tested. The low disease severities from both the
greenhouse and field tests indicated that the cultivars tested had moderate
resistance responses to inoculation with P. tritici-repentis. These results concur
with previous reports that moderately resistant cultivars could be selected based
on appropriate screening techniques (36). However, several different techniques
should be used to adequately evaluate resistance to P. tritici-repentis.
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99
Leaf blotch rating Glume blotch 2000 2001 2002 rating Cultivar Ohioa Ohiob Penn.c 2000 Ohiod AGRA GR535 7.0 7.0 15.0 Bouillon 5.0 7.3 6.0 Bravo 8.0 6.0 5.0 17.0 Coker 9025 5.0 4.7 Coker 9474 3.0 8.0 6.0 6.0 Coker 9663 2.0 3.3 5.0 1.0 Freedom 7.0 6.3 3.3 7.0 Honey 4.0 7.3 4.7 0.0 Hopewell 3.0 7.0 5.0 2.0 Patterson 8.0 7.0 23.0 Patton 6.0 7.0 3.7 12.0 Roane 2.0 6.3 4.3 1.0 Sisson 6.7 4.3 Total no. lines 53 43 36 53 High 10.0 9.7 7.3 28.0 Average 6.0 7.4 4.8 12.0 Low 2.0 3.3 2.7 0.0 LSD 2.0 1.5 11.0
Table 3.1: Stagonospora nodorum field reactions of selected soft red winter wheat cultivars during 2000 and 2001 in Ohio and 2002 in Pennsylvania. a = 0 (resistant) - 10 (susceptible) visual scale rated on 8 June 2000 in Wayne County, Ohio. (3) b = 0 (resistant) - 10 (susceptible) visual scale rated on 14 June 2001 in Pickaway County, Ohio. (4) c = 0 (resistant) - 10 (susceptible) visual scale from one field site. (39) d = Percentage glume blotch rated on 8 June 2000 in Wayne County, Ohio. (3) 100
Cultivarb Meanc Groupd AGRA GR535 4.6e E D Bouillon 6.6 A Bravo 4.2 E D Coker 9025 5.6 A B C D Coker 9474 5.0 E B C D Coker 9663 6.0 A B C Freedom 5.1 E B C D Honey 3.8 E Hopewell 6.5 A B Patterson 6.0 A B C Patton 5.4 A B C D Roane 4.8 E C D Sisson 6.6 A
Table 3.2: Mean disease severity of 13 wheat cultivars after inoculation of flag leaf with Pyrenophora tritici-repentis isolatesa from Ohio in the greenhouse. a = A conidial suspension from a composite of 10 P. tritici-repentis isolates. b = The effect of cultivar was significant (P = 0.03) using the F test based on a linear mixed model fitted to the data, with the Satterthwaite approximation for degrees of freedom. c = Least squares means were determined from the fitted model using restricted maximum likelihood. d = Groups were assigned based on pairwise comparisons of the cultivar least sqaures means (P = 0.05). Means followed by the same letter are not significantly different. e = Means were based on five repetitions of 18 replicate plants per cultivar. Control plants (10/cultivar) were excluded from data analysis.
101 Table 3.3: Mean disease severity, median lesion rating, and relative marginal
effects of adult plants 28 days after inoculation with race 1 or race 2 of
Pyrenophora tritici-repentis isolates.
a = The effect of cultivar was significant (P < 0.0001) using the F test in a linear mixed model for both disease severity and lesion type with the Satterthwaite approximation for degrees of freedom. b = The effect of race was not significant for visual estimation of leaf area affected (P = 0.21). c = The effect of race was significant for lesion type rating and relative marginal effects (P < 0.0001). d = Least squares means were approximated using restricted maximum likelihood. e = The interaction of race by cultivar was significant (P < 0.0001) for both disease severity, lesion type rating and relative marginal effects. f = Median lesion type rating based on a 0 (resistant) – 5 (susceptible) scale. g = Each mean or median was calculated based on reaction of 15 plants for each race of each cultivar in 4 experimental repeats separated in time.
102
103
Disease severitya Seed infestationb Cultivar or line Meanc Mean Groupd AGRA GR535 20.0e 1.1f CD AGRA GR863 19.2 0.8 C D Bouillon 26.2 1.7 A B C D Bravo 16.7 2.9 A B Coker 9025 20.8 1.2 C D Coker 9474 28.3 1.8 A B C D Coker 9663 33.3 3.0 A Freedom 18.3 1.0 C D Honey 28.7 1.6 B C D Hopewell 17.8 2.1 A B C OH708 15.3 1.5 C D Patterson 12.8 1.0 C D Patton 6.0 1.6 B C D Roane 18.2 0.6 D Sisson 35.3 1.5 C D
Table 3.4. Mean tan spot severity and percentage seed infestation of 13 winter wheat cultivars evaluated in two inoculated field plots in Ohio during 2004. a = The effect of cultivar was not significant (P = 0.578) using the F test in a linear mixed model, with the Satterthwaite approximation for degrees of freedom. b = The effect of cultivar was significant (P = 0.026). c = Least squares means were approximated using restricted maximum likelihood. d = Groups were assigned based on pairwise comparisons of the cultivars (P≤0.05). e = Means were based on three repetitions of each cultivar at two sites. Each repetition consisted of a hill plot planted with 3 g of seed spaced 0.46 m apart. f = Means were based on 105 seed from each of three repetitions of each cultivar at two sites.
104 CHAPTER 4
REACTION OF COMMERCIAL SOFT RED WINTER WHEAT CULTIVARS TO
STAGONOSPORA NODORUM IN THE GREENHOUSE AND FIELD
Abstract
Stagonospora nodorum has world wide distribution and affects all classes
of wheat. Resistance to this pathogen is quantitative in nature. Thirteen cultivars
available to be grown commercially in Ohio were evaluated in greenhouse and
field studies for leaf and glume blotch resistance. Greenhouse experiments
indicated that Coker 9663 had the highest leaf and glume blotch resistance of the
cultivars tested. Inoculated field plots consistently indicated that Coker 9663 and
OH708 had high leaf and glume blotch resistance reactions. Naturally infested
field plots indicated that Coker 9663 had leaf blotch resistance and Bouillon had higher glume blotch resistance of the cultivars tested. All cultivars had a relatively high percentage of seed infested with S. nodorum from both inoculated
and naturally infested field plots, but Coker 9025 had the least amount of seed
infection in either plot. Correlations between reactions from greenhouse and field
plots indicated that the most resistant and susceptible genotypes could be
identified in both environments. There were moderately high correlations
105 between leaf blotch and glume blotch severities obtained in the greenhouse and
in inoculated field plots. Inoculated field plots produced similar disease levels as
the greenhouse. The amount of S. nodorum resistance expressed by cultivars
was dependent on plant organ assessed, but some cultivars, (Coker 9663 and
OH708) had moderate levels of resistance to both leaf blotch and glume blotch.
Cultivars reacted similarly based on mean leaf blotch or glume blotch severities
in inoculated and naturally infested field plots. Mean cultivar leaf blotch and
glume blotch severities in the greenhouse and naturally infested field plots were
not correlated (P > 0.05). These results indicate Stagonospora leaf and glume
blotch resistance can be identified in naturally infested plots in epidemic years,
but cultivar reactions should be confirmed in inoculated plots or greenhouse
testes due to possible interference from contamination of other leaf blotching
pathogens.
Introduction
The wheat (Triticum aestivum L.) pathogen Stagonospora nodorum
(Berk.) (Castellani and E. G. Germano) (teliomorph Phaeosphaeria nodorum (E.
Muller (Hedjaroude))) causes Stagonospora leaf and glume blotch (46). This disease became one of the major leaf spotting diseases in Indiana during the late
1980’s, coinciding with the increase of conservation tillage (55). Stagonospora leaf and glume blotch affect both grain quality and yield. Yield may be reduced as the result of lower test weights (29, 42). Foliar disease reduces the photosynthetic area of the flag leaf and decreases the amount of carbohydrates
106 available for seed fill (16, 60). The milling quality of grain may be reduced by
infection of the developing seed causing shriveling and reduced flour yield (13).
S. nodorum can infect wheat plants at any stage of development (43, 58)
and it has many grass host species (17, 21, 25-27, 63). Current control of this
disease is achieved through cultural practices, fungicide application and the use
of resistant cultivars (24, 35-36, 38, 57). A primary management practice utilized is crop rotation (2, 24, 35-36, 38, 57). Burying infested residue by tillage is recommended to reduce the survival of the fungus from one crop to the next (2,
24, 35-36, 57). Control of the fungus on seed is achieved through fungicide seed treatments to prevent the seedling phase of the disease (2, 19, 35-37, 38, 52-53).
Application of foliar fungicides can be effective in managing Stagonospora leaf blotch to prevent yield loss in epidemic years, but the cost can be economically prohibitive when the price of wheat is low (33, 35-36, 38, 58). Moderately resistant cultivars are available, but there are currently no cultivars with high levels of resistance to this pathogen (33).
Resistance to S. nodorum is quantitatively inherited (9, 11, 15, 32, 39-41,
43, 46-50). Epistatic effects are important in resistance (32, 39), while
cytoplasmic effects have not been observed in resistance studies (8, 22).
Components of resistance are estimated to be controlled by 1 to 4 genes for
each component (14, 22) and quantitative trait loci (QTL) have been determined
for some resistance components. QTL for S. nodorum resistance to the
seedling, leaf blotch and glume blotch phases were examined in resistant
germplasm sources (18, 51, 57). QTLs for resistance to leaf blotch were found
107 on chromosomes 3A, 4A and 3BS, and glume blotch resistance was located at those same loci as well as on chromosomes 7A and 4BL (18, 51) although there were other significant QTL that were different for each phase of the disease.
Resistance reactions at different stages of wheat development appear to be independent and not highly correlated (1, 8, 15, 18, 25, 43, 61). Aggressiveness of S. nodorum isolates were shown to be a main determinate in resistant reactions with little separation of genotypes using low aggressive isolates (47).
Use of highly aggressive isolates allows better estimation of reaction and QTL loci. To avoid confounding affects of plant height in greenhouse resistance studies and QTL analysis, it is recommended to evaluate resistance to S. nodorum by inoculating and assessing plants at the same physiological stage
(10). Previous researchers have found that, in general, winter wheat cultivars have higher levels of resistance than spring wheat cultivars (1, 12, 45, 47) in both field and greenhouse studies.
In addition to resistance QTL, loci for sensitivity to toxins produced by S. nodorum have also been identified. A single nuclear gene on chromosome 1BS has been identified as the sensitivity locus for the proteinaceous toxin, SnTox1, that elicits a necrotic response (31). This toxin appears to be a major virulence factor (31), although it is not required for disease development (32). This locus has been named Snn1 and is inherited as a dominant factor (32). Various other toxins have been previously reported, but never fully characterized (20).
In general, the glume blotch reaction of cultivars has had a significantly higher correlation between greenhouse and field studies than leaf blotch
108 reactions (59). The majority of the resistance components measured in controlled environments, such as incubation period and percentage leaf area affected of adult plants and juvenile detached leaf assays, have not consistently correlated with field disease severity (1, 14-15, 45, 47). The most susceptible genotypes can be differentiated from moderately resistant genotypes in greenhouse and growth chamber studies (1, 14-15, 28, 39, 45, 47). Studies examining segregating populations have concluded there is potential for early generation selection for Stagonospora glume blotch (11, 39). A confounding variable in field studies is mixed infection with other leaf spotting pathogens, such as Pyrenophora tritici-repentis (tan spot) and Septoria tritici (Septoria blotch), which may produce similar lesion types on cultivars (34).
Evaluating resistance response in the field may be complicated by such factors as environment, amount of natural inoculum and competition between pathogens on the plant surface. Determining the resistance reaction of cultivars and lines to Stagonospora leaf blotch requires multiple year and location studies
due to the quantitative nature of resistance and environmental effects. The
response of a cultivar or line in a resistance screening nursery over multiple
years may be confounded by other pathogens, leading to incorrect estimations of
the resistance reaction (7). The similarity of S. nodorum lesions with P. tritici-
repentis lesions frequently require the assessor to group disease ratings into a
leaf blotch ‘score’ for cultivars and lines. While research plots can be managed
to create disease epidemics, large scale cultivar performance trials at multiple
locations across a state can not be maintained economically. Producers depend
109 on state-wide performance trials to obtain relative performance evaluations of
cultivars and lines for specific areas of a state, including disease reactions.
When producers know that a field has a history of Stagonospora leaf and glume
blotch, they will examine the disease reactions in the performance trials to select
resistant cultivars that perform well in their area of the state. Since there is a similarity between S. nodorum lesions and P. tritici-repentis lesions, the use of a
leaf blotching disease rating may lead to inaccurate selection of resistant
cultivars. Unfortunately, specific reactions to individual pathogens are frequently
not available to wheat producers.
Objectives
The objective of this study was to determine the leaf blotch and glume blotch reactions of thirteen commercially grown soft red winter wheat cultivars to isolates of S. nodorum obtained in Ohio. A secondary objective was to determine if cultivars responded similarly to inoculation in the greenhouse, under natural infestations in field plots and in inoculated field plots.
Materials and Methods
Cultivar information
Thirteen soft red winter wheat cultivars were chosen from the Ohio Wheat
Performance Trial based on field reaction ratings for Stagonospora leaf blotch
over 3 years (Table 4.1). These cultivars had reactions ranging from moderately resistant to susceptible, with reaction differences across years in previous
110 studies. The cultivars represented genotypes from public (Freedom, Hopewell,
Patterson, Roane, Sisson), private (Honey, Bravo, Bouillon, Coker 9025, Coker
9474, Coker 9663, AGRA GR535, Patton) wheat breeding programs, and one
line from the Ohio State University Breeding program (OH708). Seed of the
genotypes from the private and public breeding programs was available for
commercial planting in Ohio.
Greenhouse experiments
Plant production
Seed of each of the 13 cultivars was obtained from seed companies and
stored at 3.5°C until use. In the fall of 2002, seed of each of the 13 cultivars
were germinated on folded 12.7- x 50.8-cm moistened germination paper
(Anchor Paper Company, St. Paul, MN) placed in clear 10.2- x 30.5 -x 5.1-cm
plastic bags with 5 ml of water. The bags were maintained in the laboratory
(21°C ± 2°C) under a 12-hour photoperiod using ultraviolet and white fluorescent
lights. After 1 wk, healthy germinated seeds were placed into flats (5.1 x 25.4 x
50.8 cm) of autoclaved (115°C for 6 hours) Wooster silt-loam soil and maintained in a temperature controlled chamber (3.5°C) for 65 days for vernalization. Each
tray was treated with granular mefenoxam (Ridomil®, Novartis, Inc., Greensboro,
NC, 1 g ai/tray), fertilized with pellets of triple 14 Osmocoat® (N 14%, P 14%, K
14%; 12 g/tray) (Scotts, Inc., Marysville, OH) and watered. Lights in the chamber
were set to provide an 8-hour photoperiod and trays were watered as needed.
111 After the vernalization period, 30 plants of each cultivar were transplanted
individually in 12.7-cm pots of 3M Metro Mix (Conrad Fafard, Inc., Agawam, MA).
Leaves of plants were clipped to approximately 5 cm above the soil line 2 days after transplanting and pots were randomly arranged on benches in the greenhouse. The greenhouses averaged 20.8°C during the day and 19.2°C at night, with a 12-hour light period provided by one 1000-watt metal halide lamp and four 1000-watt high pressure sodium lights (Rudd lighting Co., Solon, OH) in each 44.5-m2 greenhouse. Through the course of the experiment plants were
loosely tied with Sturdy Stretch Tie® (California Plastic Products, Tustin, CA) to
30-60-cm long bamboo stakes for support.
Pots were fertilized weekly with 100 ml of a triple 20 Peter’s® fertilizer
solution (N 20%, P 20%, K 20%; 4.8 g/liter water) (Scotts, Inc., Marysville, OH).
For insect control, plants were sprayed weekly with S-kinoprene (Enstar®,
Wellmark, Bensenville, IL) (0.01 g ai/liter water) or abamectin (Avid®, Syngenta
Crop Protection, Inc., Greensboro, NC) (0.01 g ai/liter water). Applications of
triadimefon (Bayleton®, Bayer Corp., Kansas City, MO) (2.2 g ai/liter water) were
applied 2 weeks after transplanting into pots and 2 weeks thereafter for control of
powdery mildew (Blumaria graminis f. sp. tritici ((DC.) E. O. Speer) for the first 4
weeks after vernalization.
Inoculum production and inoculation
Plants were inoculated when the main tiller of each plant had a fully
emerged flag leaf (Feekes growth stage 9 (30)). Inoculations were made by
112 atomizating a 1x106/ml pynidiospore suspension on the fully expanded flag leaf
to the point of run-off. Bulk suspensions were made from ten isolates of
Stagonospora nodurm obtained from wheat leaves collected at different locations in Ohio in 2002. Isolates were grown on V8 agar modified with 50mg/liter of streptomycin sulfate (Sigma-Aldrich, St. Louis, MO). The agar cultures from 8-
cm diameter petri dishes of each isolate were inverted onto a 15-cm cheese cloth
square (four layers) and dipped into a glass beaker of 100-ml tap water and
strained to obtain pycnidiospores. No attempt was made to have equal numbers
of pycnidiospores from the individual isolates in the inoculum suspension.
Suspensions were enumerated using a Spotlite Hemacytometer® (American
Scientific Products, McGaw Park, IL) according to the protocol for counting small
spores (56).
After inoculation plants were placed into a mist chamber. The mist
chamber consisted of a 2.3- x 1.4- x 1.0-m PVC skeleton enclosed by
polyethylene sheeting with adjustable sides. An air atomizer with a fluid nozzle
(#2850 SS, Spraying Systems, Co., Spokane, WA) (0.56 kg/cm3 water) and an
air nozzle(#70) (4.2 kg/cm3 air) misted 30 ml of water for 30 seconds every 3 minutes above a polyethylene canopy over the plants. The temperature in the mist chamber was maintained at approximately 22°C and the relative humidity was approximately 100%. Inoculated plants were incubated in the mist chamber for 48 hours.
Each individual plant in a pot was considered a replication. A repetition
was considered an experimental repeat. Experimental repeats were separated
113 over time with approximately one month in between planting dates. Four experimental repeats were conducted in total. For each repetition, 20 plants of each cultivar were inoculated and 10 plants were not inoculated and maintained as control plants. Control plants were placed in the mist chamber with the inoculated plants at separate ends of the mist chamber. After 48 hours in the mist chamber, plants were returned to the greenhouse. Care was taken to prevent contact between the control plants and the inoculated plants. Plants of different cultivars were randomly arranged on greenhouse benches by inoculation date. The flag leaves were visually assessed for percentage diseased leaf area 7, 14, 21 and 28 days after inoculation.
When spikes had fully emerged on the main tiller of each plant (Feekes
stage 10.3 (30)), the spikes were inoculated. The plants were removed form the
greenhouse and inoculated in a separate room. A spore suspension of 1x106 /ml pycnidiospores was atomized onto the fully emerged spike as earlier described.
The previously inoculated flag leaves were protected by placing a plastic bag over the leaf during inoculation. After inoculation of the spike was completed, a clear plastic bag was loosely placed over the spike and secured using a twisty tie. The plants were returned to the greenhouse and randomly placed on the greenhouse benches according to spike inoculation date rather then leaf inoculation date. After a 48 hour incubation period, the plastic bags were removed from the inoculated spikes. Care was taken to limit damage to the spikes from the presence of the plastic bag. The spikes were visually assessed for percentage diseased glume area 7, 14, 21 and 28 days after inoculation.
114
Field experiments
Inoculated plots
Seed of the thirteen cultivars and the S. nodorum susceptible check
AGRA GR863, and in 2003 the resistant check OH708, were planted in
inoculated field plots in 2002 and 2003. The experiments were arranged as a
randomized complete block with three replicate blocks. One field plot was
planted in Wayne County, Ohio, on 17 October 2002. In 2003, field plots were
established in Wayne County and Wood County, Ohio, on 10 October and 9
October 2003, respectively. Approximately 3 g of seed of each cultivar were
planted into each hill plot and hill plots were spaced 0.46 m apart, in a field that
had previously been cropped with soybeans. The fields were prepared by
moldboard plowing and a fall (22.8 kg/ha) and spring (56.8 kg/ha) application of
nitrogen was made. Three rows of GR863 were planted between hill plots using
small plot drill to promote spread of inoculum.
Field inoculum and inoculations
Plots were inoculated two ways. Plots were inoculated by spreading
wheat straw on the ground surface in the spring before flag leaf emergence. The
straw was obtained from plots infested with S. nodorum the preceding year. The
plots were inoculated again by spraying a 1x106 /ml pycnidial suspension of S. nodorum when 50% of the plants in the plot had fully emerged flag leaves
(Feekes stage 9 (30)). The same composite of ten isolates of S. nodorum that
115 was used in greenhouse experiments were used to inoculate the field plots.
Conidial suspensions were collected and enumerated as previously described.
After inoculation, plots were misted continuously for 3 minutes out of every 10 minutes for 1 week from 7 PM to 7 AM to maintain a high relative humidity to favor disease development. A second inoculation was applied when 50% of the plants in the plot had fully emerged spikes (Feekes stage 10.3 (30)). The inoculum was produced as previously described. Flag leaves and glumes were visually assessed weekly from flag leaf emergence through senescence for leaf or glume area affected.
Naturally infested plots
The thirteen cultivars were also planted in the Ohio Wheat Performance
Trial at five locations across the state during 2002 and 2003. Each site had four completely randomized replications where a replication was a 6.2 m long by 1.6 m wide drill strip containing seven rows. The plots were maintained according to wheat production recommendations for Ohio and only exposed to natural inoculum (5-6). Leaf blotch on flag leaves was visually assessed weekly from flag leaf emergence through early senescence for disease severity. Glume blotch on spikes was visually assessed from spike emergence through early senescence. Of the five locations planted in each year, only the Pickaway
County site had adequate amounts of glume blotch to compare cultivar reactions both years and the Wayne County site in 2003. Leaf blotch severities were
116 obtained from Wayne, Pickaway, and Darke County sites in 2003, and Wayne,
Pickaway and Crawford Counties in 2004.
Throughout the assessment period, samples of flag leaves were obtained from the naturally infested plots for confirmation of leaf blotching organisms.
Flag leaves were harvested during each weekly visual assessment, placed in a paper coin envelope, and dried for 2 days in an exhaust fume hood in the laboratory. After drying, flag leaf samples were stored at room temperature. To determine leaf blotching pathogens, flag leaves were incubated in moisture chambers. The moisture chambers consisted of 9-cm Pyrex Petri dishes containing 9-cm filter paper (Fisher Scientific) moistened with 5 ml of water underneath a square of wire mesh that suspended the glass slide above the moistened filter paper. The glass slide had two pieces of double sided sticky tape placed evenly on the top of the slide for adherence to leaves. This prevented the flag leaf from rolling during incubation in the moisture chamber.
Visual assessment of fungal fruiting structures on incubated flag leaves were made 48 and 72 hours after placing the leaf sample in the moisture chamber.
In 2002, Pickaway, Wayne and Darke County field sites had measurable amounts of Stagonospora leaf blotch and the Pickaway and Wayne County sites had measurable amounts of glume blotch. In 2003, Pickaway, Wayne and
Crawford County field sites had measurable amounts of leaf blotch and the
Pickaway County site had measurable amounts of glume blotch. Examination of leaf blotching pathogens from field samples in 2002 indicated that over 80% of leaf samples had S. nodorum (data not presented). In 2003, the leaf blotching
117 pathogens were more diverse, with disease being caused by S. nodorum, P. tritici-repentis and B. sorokiniana (data not presented). The most prevalent pathogen (63% of leaf samples) in 2003 plots was S. nodorum. Pearson’s correlations coefficient between disease parameters across years and sites in the naturally infested plots were highly variable due to disease intensity. Since
Wayne and Pickaway County sites had consistent measurable amounts of leaf blotch and glume blotch, these sites were used in data analysis.
Seed infestation
At the end of the 2002 and 2003 growing seasons, 0.9-kg seed samples of combine harvested grain were obtained from plots at the Pickaway and Wayne
County sites. Each cultivar was replicated in each of four blocks at all locations and a seed sample from each of these replicate plots was examined for S. nodorum infestation. From each of these samples, 100 seeds were used to assess the presence of seed-borne S. nodorum. Seed appearing to be infected by Fusarium graminearum were not selected. The seed was placed on basal oxgall medium in petri dishes (37). The seed were screened for fluorescence under an ultraviolet lamp (2.5 AMP, Ultra-Violet Products, Inc., San Gabriel, CA)
4 days after placing the seed in the petri dishes. The percentage of seed with associated fluorescence, typical of those infected by S. nodorum, was calculated for each seed sample.
Seed was hand-harvested from the inoculated field plots. The seed was thrashed using a small bundle thrasher (ALMACO, Nevada, IA). The seed
118 samples were then aspirated (Fractionating Aspirator, CFZ1 model, Carter Day
International, Inc., Minneapolis, MN) to remove light-weight seed from the
samples. From each of these samples, 100 random seeds were used to assess
the presence of seed borne S. nodorum. These seed samples were placed on basal oxgall medium plates to determine S. nodorum infestation. Again, the percentage of infected seed was calculated for each sample.
In 2003, seed harvested from the inoculated field plots and naturally
infested plots at Wayne and Pickaway County sites were examined for the
presence of Pyrenophora tritici-repentis (Died) Drechs. and Bipolaris sorokiniana
(Sacc. Shoem). A total of 100 seeds from each replicate plot for each cultivar
from both sites were examined. This was done by placing 21 harvested seed on
moistened filterpaper (9 cm Fisher Scientific) in each 9-cm Pyrex Petri dish. The
dishes were placed under the light banks previously described for fungal spore
production. Petri dishes were maintained under a 12-hour light period for 5 days
before being examined. Five plates for each seed sample were examined under
a dissecting scope for the conidia and conidiophores of P. tritici-repentis and B.
sorokiniana.
Statistical analysis
Disease severity assessments for leaf blotch and glume blotch taken 28
days after inoculation were analyzed for studies conducted in the greenhouse. In
the inoculated and naturally infested field plots, the last assessment of disease
severity on flag leaves and glumes before senescence was used for analysis.
119 The percentage of harvested seed infested with S. nodorum, P. tritici-repentis or
B. sorokiniana from the field plots was used for analysis. Analysis of variance
(ANOVA) was conducted by means of a linear mixed model in SAS (SAS, Inc.,
release 8.3, Cary, N.C.) using the restricted maximum likelihood (REML) method
of model fitting and the Satterthwaite approximation for degrees of freedom
calculation (54). Wheat cultivar was considered a fixed effect and blocks a
random effect. The least squares mean for disease severity was used for the
pairwise comparisons of cultivars. Data from the inoculated plots in 2002 and
2003 were combined for statistical analysis by making both year and location
random effect factors in the mixed model in order to increase power of the F-test.
Results
Greenhouse inoculations
Inoculations of the adult plants in the greenhouse produced a maximum
disease severity of 42.6% flag leaf area affected (FLAA) on Patterson and 98.5%
glume area affected (GAA) on AGRA GR535 (Table 4.2). Coker 9663 had the
least amount of FLAA (7.2%) and GAA (26.1%). Cultivars had significantly different least squares mean estimates of disease severity as indicated by using a mixed ANOVA model. All pairwise comparisons among cultivars were examined to determine statistically significant differences in disease severities.
Most of the intermediate cultivar groups overlapped in severity. Only Coker 9663 and Roane had less than 10% FLAA, but this did not differ significantly from the severity of FLAA on Coker 9025, Coker 9474 or Sisson. Coker 9663 and Sisson
120 had less than 30% GAA and this GAA severity was significantly less than that of
other cultivars tested.
Field experiments: inoculated plots
There were significant differences in the reactions of the cultivars
evaluated according to the pairwise comparisons for leaf blotch, glume blotch
and percentage seed infested with S. nodorum. The susceptible check, AGRA
GR863, was most severely diseased, with a mean percentage of 77.3% FLAA
(Table 4.3), 54.4% GAA, and a higher amount (45.8%) of seed infested as
compared to most other cultivars. Coker 9663 had the least amount of FLAA
(3.6%) and GAA (6.0%), but had similar seed infestation (33%) levels as the
other 15 cultivars tested. Sisson had the least percentage of infested seed
(25.1%), but this amount was not significantly different from five of the other
cultivars. Interestingly, Coker 9474 had intermediate FLAA (19.8%), but high
GAA (32.5%) and the highest seed infestation (60.1%). The cultivars in
intermediate groups created from the pairwise comparisons had considerable variation for all disease severities measured.
Naturally infested plots
Disease intensity in naturally infested plots was high enough and the
range in cultivar reactions broad enough to statistically evaluate the cultivars in
Wayne and Pickaway Counties. Leaf blotch severities ranged from a high of
58.9% FLAA (Patterson and Honey) to a low of 19.8% FLAA for Coker 9663
121 (Table 4.4). Glume blotch severities ranged from a high of 24.1% GAA for
Sisson to a low of 5.4% GAA for Bouillon. There were fewer cultivars represented in the intermediate reaction groups for both leaf blotch and glume blotch tested in naturally infested plots than in the inoculated field plots (Tables
4.3 and 4.4). The percentage of infested seed was greatest for Patterson
(47.8%, Table 4.4) and most cultivars had seed infestation amounts that were not statistically different from Coker 9025 (19.3%), that had the least seed infestation. While the intensity of seed infestation was similar in the inoculated and naturally infested plots, cultivars varied in relative position between the two types of plots for the level of seed infestation (Tables 4.3 and 4.4).
Visual examinations of seed presumed to be infested by S. nodorum from naturally infested plots in 2004 were observed to be also infested with P. tritici- repentis and B. sorokiniana. Another random sample of 100 seeds per cultivar from the 2004 naturally infested plots was examined to determine overall infestation intensity. The amount of seed infested by P. tritici-repentis was determined from both Wayne and Pickaway County sites. The greatest amount of seed infestation by P. tritici-repentis was on cultivar Roane (0.4%, Table 4.5), but this fungus was not recovered from seed of cultivars Patton, Patterson and
Sisson. While the presence of this pathogen in seed samples can be detected, the low intensity of infestation did not allow for clear separation of the cultivars examined based on statistical tests.
The amount of B. sorokiniana infestation was negligible (< 3%) on seed from the Pickaway County site, so only seed from the Wayne County site were
122 analyzed. ANOVA indicated a statistical effect of cultivar in the amount of seed
infested by B. sorokiniana. Bouillon had 8.8% seed infested while Patton 1.5%
(Table 4. 5) had the least seed infestation. Infestation of these pathogens was
not surprising since leaf samples from naturally infested plots collected in 2003 had a higher prevalence of these pathogens (38%) than the previous year (20%).
Correlations
Pearson’s correlation between the least squares mean estimates of disease severities and percentages of seed infested were calculated (Table 4.6).
Mean leaf blotch severities from the greenhouse and field plots were all significantly correlated, with the highest correlation between the inoculated field plots and naturally infested field plots (r = 0.91, P < 0.0001). Cultivar mean glume blotch severities obtained in the greenhouse were marginally significantly correlated with the inoculated field plot (r = 0.54, P = 0.055), but not statistically correlated with mean cultivar glume blotch severities in the naturally infested field plot. Mean cultivar glume blotch severities from the inoculated field plot were significantly correlated with the percentage of S. nodorum infested seed (r = 0.57,
P = 0.028), but mean cultivar glume blotch severities were not correlated with percentage S. nodorum infested seed in the naturally infested field plot.
Greenhouse leaf blotch severities were moderately correlated with greenhouse glume blotch severities (r = 0.56, P = 0.046). Mean leaf blotch severities from inoculated plots were moderately correlated with glume blotch severities from inoculated plots (r = 0.65, P = 0.008). In the naturally infested
123 field plots, mean leaf blotch severities were not significantly correlated with glume
blotch severities from the naturally infested field plots (r = 0.52, P = 0.07). The percentage of infested seed from the inoculated plots had a moderately high correlation with the percentage of infested seed from naturally infested plots (r =
0.76, P = 0.003).
Discussion
Inoculation of the various cultivars with S. nodorum pycnidiospores in the
greenhouse produced only moderate Stagonospora leaf blotch severities (Table
4.2), yet significant statistical differences were detected among the reactions of
the cultivars. The low leaf blotch severities of Coker 9663 and Roane (7.2 and
9.0% FLAA, respectively) compared to the susceptible check, Patterson (42.6%
FLAA) indicated these cultivars had moderately high resistance reactions (Table
4.2). In inoculated field plots over 2 years and two locations, Coker 9663 consistently had a moderately high resistance reaction (Table 4.3). Although its reaction was the lowest observed of the tested cultivars, it was still considered only moderately resistant. The cultivar Roane (19.0% FLAA) had higher leaf blotch severities similar to Bravo and Sisson (34% FLAA, Table 4.3) when tested in the inoculated field plot indicating that, under very high disease pressure,
Roane may exhibit less resistance than expected. By comparison, the Ohio
State University advanced breeding line, OH708, expressed a moderately high resistance reaction in the inoculated field plot, with 6.7% FLAA (Table 4.3).
These results indicate that Coker 9663 and OH708 have moderate resistance to
124 S. nodorum and resistance reactions in the greenhouse and inoculated field plots consistently identified the resistant from the susceptible genotypes.
In naturally infested field plots over 2 years and two sites, Coker 9663 had higher leaf blotch severity (19.8% FLAA, Table 4.4) than in inoculated field plots and greenhouse inoculations. Several factors could contribute to this variability, including differences in the environment or inoculum amount. The possibility of differences in aggressiveness of S. nodorum isolates across locations exists, but a study on differences in S. nodorum aggressiveness in several regions of Ohio indicated that there were no major differences in the aggressiveness among the isolates from where the naturally infested plots were located in Ohio (Chapter 2).
Some inconsistency between field plots and greenhouse studies could have been caused by a portion of the observed severity being due to infections by other leaf blotching pathogens.
Difficulty in assessing cultivar reactions to Stagonospora leaf blotch has been observed in other areas of the world due to competition from other leaf pathogens (7). Both P. tritici-repentis and B. sorokiniana have been isolated from wheat flag leaves collected in most wheat growing regions of Ohio (Chapter
1) and leaf sampling to determine predominant leaf blotching pathogens indicated their presence in the naturally infested field plots. In some cases individual flag leaves have been observed to have S. nodorum, B. sorokiniana and P. tritici-repentis within lesions (Chapter 1). These pathogens were also recovered from harvested seed from the naturally infested plots (Table 4.5). The most resistant cultivar tested, Coker 9663, had mean disease severities of 6 to
125 12% after inoculation of P. tritici-repentis on flag leaves in a greenhouse study
(Chapter 3). Due to similarity between lesion types produced by P. tritici-repentis and S. nodorum (7), confusion of lesion types could cause an observer to classify
the reaction of this cultivar as moderately susceptible. The reaction of Coker
9663 to inoculation with B. sorokiniana was not determined. It is likely that the higher leaf blotch severities of Coker 9663 from the naturally infested plots may be influenced by mixed infections of S. nodorum, B. sorokiniana and/or P. tritici- repentis, regardless of care to estimate severities of Stagonospora leaf blotch alone. Regardless of the possibility of infection by multiple pathogens in naturally
infested plots, it appears that Coker 9663 had a moderately resistant reaction.
Inoculations in the greenhouse produced high cultivar mean severities of
Stagonospora glume blotch on some cultivars but not others (Table 4.2), allowing
the tested cultivars to be statistically separated for disease reaction. Coker 9663
had the lowest observed glume blotch severity (26% GAA, Table 4.2), which
indicated this cultivar had moderately high resistance to Stagonospora glume
blotch. Glume blotch severities were less extreme in the inoculated field plot at
two sites in 2 years than those obtained in the greenhouse (Table 4.3). The
cultivar Coker 9663 had a moderately high resistant reaction for glume blotch in
the inoculated field plot (6.0% GAA, Table 4.3). A mean of 10.8% GAA was
observed on the breeding line OH708, indicating it also had a moderately high
resistant reaction to Stagonospora glume blotch. In the naturally infested field
plots at two sites in 2 years, glume blotch severities were relatively low for the
more susceptible cultivar Sisson (24% GAA, Table 4.4). These lower severities
126 were probably due to limited inoculum for the glume blotch phase of the disease.
Coker 9663 had a mean glume blotch severity of 7.5% (Table 4.4) and was not
statistically different from cultivar Bouillon, which had the lowest observed glume
blotch severity (5.4% GAA, Table 4.4). These results indicated that the cultivar
Coker 9663 and breeding line OH708 had moderately high resistance to
Stagonospora glume blotch.
S. nodorum seed infestation was not determined after greenhouse
inoculations because of higher amounts of disease which caused floret sterility.
In inoculated field plots, the susceptible cultivar Sisson and the more resistant
cultivar Coker 9663 had 25% and 33% seed infestation by S. nodorum,
respectively (Table 4.3). Statistically, the amount of seed infestation of Coker
9663 was not significantly different from that of Sisson. Both of these levels of
infestation could initiate epidemics (53). The correlation between glume blotch
severity and percentage seed infestation for the naturally infested plot was
significant, but moderate (r = 0.57, P = 0.03). In naturally infested plots, Coker
9025 had 19% S. nodorum seed infestation while Coker 9663 had 21% (Table
4.4). These infestation intensities were not significantly different. There was no significant correlation between glume blotch severity and percentage seed infestation among cultivars in the naturally infested field plot. The moderately high percentage of seed infestation may be explained by an increase in inoculum during several wet periods at the later stages of seed fill and maturation (after
Feekes growth stage 10.5.4 (30)), which occurred after glume blotch severities were recorded. Resistance to seed infestation by S. nodorum appears to be
127 correlated with glume blotch resistance (r = 0.57, P = 0.028, Table 4.6) in inoculated field plots. All cultivars had higher than expected seed infestation levels, indicating that resistance to seed infestation in the cultivars tested may not be adequate.
Seed infestation by other leaf blotching pathogens was also examined on seed harvested from the naturally infested field plots. The percentage of seed yielding P. tritici-repentis was low, with all cultivars having less then 1% seed infestation (Table 4.5). The percentage of seed infested with B. sorokiniana was examined on seed from the Wayne County site in 2004 (Table 4.5). The cultivar
Patton had the lowest percentage of seed infestation (mean 1.5%), while Coker
9663 had 8.5%. The cultivar Bouillon had the highest percentage of infested
seed (8.8%), which was not significantly different from Coker 9663. These
results indicated that seed infestation by foliar pathogens other than S. nodorum
is likely to be lower than seed infestation amounts by S. nodorum in Ohio.
In conclusion, cultivar reactions to S. nodorum can be determined in
naturally infested plots such as performance trials, but confirmation in either
inoculated field plots or greenhouse studies is necessary for determining
accurate resistance reactions. The similarity of lesion types produced by S.
nodorum and P. tritici-repentis may compromise the accuracy of cultivar
reactions obtained from naturally infested field plots. Inoculated cultivars
resulted in a wide range of Stagonospora glume blotch severities in greenhouse
inoculations, indicating sufficient spore concentrations may be needed to assess
the range of cultivar reactions. The relative level of S. nodorum seed infestation
128 in relation to the resistance levels to leaf blotch or glume blotch of the cultivars tested indicated that leaf or glume blotch resistance may not be limiting transmission of the fungus to the seed. The cultivar Coker 9663 and the breeding line OH708 appear to have moderately high resistance to Stagonospora leaf and glume blotch of the cultivars tested. Coker 9663, and possibly OH708, may be excellent candidates for more detailed analysis to determine the components of partial resistance and chromosomal location of resistance components by quantitative trait loci analysis.
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136
Leaf blotch rating Glume blotch 2000 2001 2002 rating Cultivar Ohioa Ohiob Penn.c 2000 Ohiod AGRA GR535 7.0 7.0 15.0 Bouillon 5.0 7.3 6.0 Bravo 8.0 6.0 5.0 17.0 Coker 9025 5.0 4.7 Coker 9474 3.0 8.0 6.0 6.0 Coker 9663 2.0 3.3 5.0 1.0 Freedom 7.0 6.3 3.3 7.0 Honey 4.0 7.3 4.7 0.0 Hopewell 3.0 7.0 5.0 2.0 Patterson 8.0 7.0 23.0 Patton 6.0 7.0 3.7 12.0 Roane 2.0 6.3 4.3 1.0 Sisson 6.7 4.3 Total no. lines 53 43 36 53 High 10.0 9.7 7.3 28.0 Mean 6.0 7.4 4.8 12.0 Low 2.0 3.3 2.7 0.0 LSD 2.0 1.5 11.0
Table 4.1: Field reaction of 13 soft red winter wheat cultivars to Stagonospora nodorum during 2000 and 2001 in Ohio and 2002 in Pennsylvania. a = 0 (resistant)-10 (susceptible) visual scale rated on 8 June 2000 in Wayne County, Ohio (3). b = 0 (resistant)-10 (susceptible) visual scale rated on 14 June 2001 in Pickaway County, Ohio (4). c = 0 (resistant)-10 (susceptible) visual scale from one field site (44). d = Percentage glume affected rated on 8 June 2000 in Wayne County, Ohio (3).
137
Leaf blotch Glume blotch Cultivara Meanb Groupc Meanb Groupc AGRA GR535 16.6d CD 98.5d A Boullion 14.6 CD 58.5 D Bravo 24.1 B 85.8 A B C Coker 9025 13.7 CDE 59.1 D Coker 9474 11.5 DEF 72.3 B C D Coker 9663 7.2 EF 26.1 E Freedom 17.0 C 74.6 B C D Honey 24.7 B 89.6 A B Hopewell 26.1 B 79.9 A B C Patterson 42.6 A 82.2 A B C Patton 16.2 CD 79.2 B C Roane 9.0 EF 68.5 C D Sisson 11.4 DEF 30.8 E
Table 4.2: Leaf and glume blotch reactions of commercial soft red winter wheat cultivars inoculated with a composite of ten Stagonospora nodorum isolates in the greenhouse. a = The effect of cultivar was significant (P < 0.0001) using the F test in a linear mixed model (with Satterthwaite approximation for degrees of freedom) for both leaf and glume blotch. b = Least squares means of percentage flag leaf or glume area affected 28 days after inoculation were approximated using restricted maximum likelihood. c = Groups were assigned based on pairwise comparisons of the cultivar means, based on standard error of mean differences determined in the mixed model. Means followed by the same letter are not significantly different at P ≤ 0.05. d = Means were based on four repetitions of approximately 20 replicate plants per cultivar inoculated. Un-inoculated control plants (10) were excluded from data analysis.
138 Table 4.3: Reaction of the fifteen cultivars in inoculated plots in Wayne County
2003 and Wayne and Wood Counties in 2004. a = The effect of cultivar was significant (P ≤ 0.0001) using the F test in a linear mixed model (with Satterthwaite approximation for degrees of freedom) for leaf and glume blotch and percentage seed infested. b = Least squares means of diseased are visually rated on the last assessment date were approximated using restricted maximum likelihood. c = Groups were assigned based on pairwise comparisons of the cultivar means, based on standard error of mean differences determined in the mixed model.
Means followed by the same letter are not significantly different at P ≤ 0.05. d = Means were based on three repetitions of each cultivar at each site in each year. Each repetition was a hill plot planted with 3 g of seed spaced 0.46 m apart.
139
140 Table 4.4: Mean Stagonospora leaf and glume blotch severities of the thirteen
cultivars planted in naturally infested plots in Wayne and Pickaway Counties
2003 and 2004.
a = The effect of cultivar was significant (leaf = P < 0.0001, glume = P = 0.001, seed = P = 0.0003) using the F test in a linear mixed model (with Satterthwaite approximation for degrees of freedom). b = Least squares means of disease severities and percentage seed infestation were approximated using restricted maximum likelihood. c = Groups were assigned based on pairwise comparisons of the cultivar means, based on standard error of mean differences determined in the mixed model.
Means followed by the same letter are not significantly different at P ≤ 0.05. d = Means were based on four repetitions of each cultivar. Each repetition was a drill strip 8 rows by 6.1 m long. e = Means were based on 105 seed for each cultivar in each of four repetitions.
141 142
P. tritici-repentis B. sorokinianac Cultivar Meana Groupb Meand Groupe AGRA GR535 0.1f A B 4.7f CBD Bouillon 0.1 A B 8.8 A Bravo 0.1 A B 3.3 C D Coker 9025 0.3 A B 7.8 A B Coker 9474 0.1 A B 4.5 C D Coker 9663 0.3 A B 8.5 A Freedom 0.1 A B 2.0 D Honey 0.1 A B 2.0 D Hopewell 0.1 A B 2.0 D Patterson 0 B 3.3 C D Patton 0 B 1.5 D Roane 0.4 A 5.3 C B Sisson 0 B 3.5 C D
Table 4.5: Percentage of seed infested with Pyrenophora tritici-repentis and Bipolaris sorokiniana of the thirteen cultivars harvested from naturally infested field plots in Pickaway and Wayne Counties in 2004. a = Means were based on 105 seeds from each of four repetitions of each cultivar at both sites. b = Groups were assigned based upon an unprotected Fisher’s LSD of 0.4 using the general linear model. c = The effect of cultivar was significant (P < 0.0001) using the F test in a linear mixed model (with Satterthwaite approximation for degrees of freedom). Only seed from Wayne site examined. d = Least squares means were approximated using restricted maximum likelihood. e = Groups were assigned based on pairwise comparisons of the cultivar means, based on standard error of mean differences determined in the mixed model. Means followed by the same letter are not significantly different at P ≤ 0.05. f = Means are based on four repetitions of each cultivar. Each repetition was a drill strip seven rows by 6.1 m long.
143
Leaf blotchb Inoculated FPa Nat. Infest. FP r P value r P value GHa 0.78e 0.002 0.70 0.008 NIFPa 0.91 <0.0001 Glume blotchc r P value r P value GHa 0.54 0.055 -0.08 ns NIFPa 0.13 ns %seed infestation by glume blotch severityd r P value r P value IFP 0.57 0.028 0.76 0.002 NIFP -0.28 ns -0.009 ns
Table 4.6: Pearson’s correlation coefficients between mean cultivar assessments in the greenhouse and field plots. a = GH = greenhouse; IFP = inoculated field plot, NIFP = naturally infested field plot. b = Leaf blotch assessments were visual estimations of percentage leaf area affected. Greenhouse assessments were taken 28 days post-inoculation. Field plot assessments were the last assessment before senescence. c = Glume blotch assessments were visual estimations of percentage glume area affected. Greenhouse assessments were taken 28 days post-inoculation. Field plot assessments were the last assessment before senescence. d = Percentage of seed infested with S. nodorum was assayed on 100 seed for each cultivar in each repetition. There were three repetitions in two plots of inoculated field sites and four repetitions in naturally infested field plots. e = Pearson’s correlation coefficients were based on mean cultivar disease assessments in each study.
144 CHAPTER 5
REACTIONS OF WHEAT GNEOTYPES TO ISOLATES OF STAGONOPORA
NODORUM FROM OHIO
Abstract
Resistance to Stagonospora nodorum in wheat has been studied for over
40 years in the United States, with no immune or resistant genotypes identified to
date. This study examined older wheat genotypes reported to vary in resistance
for S. nodorum to identify potential sources for resistant cultivar development.
Genotypes were examined in greenhouse inoculation studies and in an
inoculated field plot. Leaf blotch severities in greenhouse tests ranged from
4.4% to 35.8% flag leaf area affected (FLAA) with genotypes Coker 68-8, Cvl-22-
4 and Redhart having 10% FLAA or less leaf blotch. In the field, genotypes Atlas
66, Attila, Cvl-22-4, Gasta, Harvest Queen, Honor, Kansas no. 43B337, Moking,
Moro, Redhart and RedMay had mean leaf blotch severities of 10% FLAA or
less. Glume blotch severities were also recorded in field plots. Coker 68-15,
Coker 68-8, Holly E, Kansas no. 43B337, Moro and Oasis had glume blotch
severities greater than 18% glume area affected (GAA), while all other genotypes
had less than 10% GAA. These results indicate that potentially effective
145 resistance is available in older genotypes. Resistance may have been
incorporated into newer cultivars, but some genes for partial resistance may not
have been transferred to newer cultivars. Higher levels of partial resistance to
Stagonospora leaf and glume blotch than what is in current commercial germplasm may be obtained from older genotypes.
Introduction
The wheat (Triticum aestivum L.) pathogen Stagonospora nodorum
(Berk.) (Castellani and E. G. Germano) (teliomorph Phaeosphaeria nodorum (E.
Muller (Hedjaroude))) causes Stagonospora leaf and glume blotch (37, 49).
Stagonospora leaf and glume blotch affects both grain quality and yield. S. nodorum can infect wheat plants at any stage of development (34, 47) and has many grass host species (10, 14, 18-20, 50). Current control of this disease is achieved through cultural practices, fungicide application and the use of resistant cultivars (17, 27-29, 46). Moderately resistant cultivars are available, but there are currently no commercially acceptable cultivars with high levels of resistance to this pathogen (24).
Resistance to S. nodorum has been shown to be quantitatively inherited
(4, 6, 9, 26, 30-32, 35, 37-41, 43). Epistatic effects have been shown to be important in resistance (26, 30), while cytoplasmic effects have not been observed in resistance studies (2, 16). Components of partial resistance that have been examined include toxin sensitivity, incubation period, latent period and pathogen sporulation, with evidence that they were durable (5, 12). Each of
146 these components of resistance was estimated to be controlled by 1 to 4 genes
(8, 16). Resistance reactions at different stages of wheat development appear to be independent and not highly correlated (1-2, 9, 11, 15, 35, 48).
Aggressiveness of S. nodorum isolates has been shown to be a main determinate in resistant reactions with little difference between reactions of the more resistant and susceptible genotypes using less aggressive isolates (38). In
general, the most susceptible genotypes can be differentiated from moderately
resistant genotypes in greenhouse and growth chamber studies (1, 8-9, 21, 30,
36, 38). Previous researchers have reported that, in general, winter wheat cultivars have higher levels of resistance than spring wheat cultivars (1, 7, 36,
38). Studies examining segregating populations for resistance indicate effective early generation selection for Stagonospora glume blotch resistance may be possible(6, 30).
Resistance has been determined to be quantitative in nature with oligo- or polygenic inheritance pattern (9, 16, 31-32, 42). Quantitative trait loci (QTL) for
S. nodorum resistance for the seedling, leaf blotch and glume blotch phases have been examined in resistant germplasm sources (11, 26, 42, 46). QTLs for resistance to leaf blotch were found on chromosomes 3A, 4AL and 3BS, and glume blotch resistance was located on those same chromosomes as well as on chromosomes 7A and 4BL (11, 42). Flag leaf resistance QTLs were determined on chromosomes 3A, 3BL and 4AL by comparing the area under the disease
progress curve (AUDPC) for monosomic lines after a resistant by susceptible
cross (11). Similarly, Stagonospora glume blotch QTLs were identified on
147 chromosomes 3BS, 4BL, and 3AS by comparing the AUDPC for F5:7 lines of a resistant by susceptible cross (42).
In addition to resistance genes that condition one or more components of
partial resistance, loci for sensitivity to toxins produced by S. nodorum have also
been identified. A single nuclear gene on chromosome 1BS has been identified
as the sensitivity locus for the proteinaceous toxin SnTox1, which elicits a
necrotic response (25). This toxin appears to be a major virulence factor (25),
although it is not required for disease development (26). This locus has been
named Snn1 and has been shown to be dominantly inherited (26). Genotypes
‘Erik’, ‘Opata85’ and ‘BR34’ responded with an insensitive reaction after
inoculation of isolates producing SnTox1, while ‘W-7984’, ‘Kulm’, ‘ND495’,
‘Grandin’ and ‘CS’ had sensitive reactions (26). Other toxins have been
previously reported, but never fully characterized (13).
Genotypes react differently under different inoculation procedures, plant
maturity and plant organ inoculated. Results of seedling inoculation tests
indicated that genotypes Coker 68-8, Harvest Queen, Moro, Moking and Red
May had a mean of 5% leaf area affected (LAA), a highly resistant reaction (39).
A moderately high resistant reaction was observed on Chalk (36), Coker 68-15
(36), Hadden (39), Knox 62 (39), Moking (21), Red Chief (39) and Redhart (36)
with means of 5-10% LAA after seedling inoculation. A mean of 10-15% LAA
was observed on genotypes Gasta (21, 39), Harvest Queen (21), Red Chief (21)
and Redhart (39) after seedling inoculation indicating moderate resistance.
Moderately resistant to moderately susceptible reactions were observed on
148 Baldrock (21), Hadden (21, 36), Oasis (36, 39), Red Chief (36) and Redhart (21),
with mean seedling leaf blotch severities of 15-25%. Seedling reactions of the
cultivar Oasis had a mean of 27% LAA (31) indicating a moderately susceptible
reaction. There have been discrepancies with the resistance reaction observed
on seedling leaves and adult plant inoculations within studies and between
studies.
Adult leaf blotch severities with a mean of less than 10%, indicating
moderately high resistance, have been observed on Knox 62 and Redhart in
inoculated fields (22). In the same study, a mean of less than 30% flag leaf area
affected (FLAA) was recorded on Gasta and Cvl-22-4, indicating moderate
susceptibility. A susceptible reaction was Moking, Moro and Red Chief with
greater than 30% FLAA (22). Oasis had a resistant reaction with a value of 14.8 for AUDPC for flag leaf diseased area 27 days after inoculation, while the
susceptible check Holley had an AUDPC value of 100 (5). Most researchers
concur that genotypes with reactions of 10% or less FLAA are considered
resistant. The varying results from these studies indicate that genotype reactions
were variable and differences in reactions on seedling leaves versus flag leaves
should be expected.
Variability in glume blotch ratings from inoculated and non-inoculated field
plots have also been reported (31, 33, 36). The cultivar Oasis had a mean of 3.6
to 4.5 (0 resistant to 9 susceptible scale) in greenhouse studies (31, 32), but 3-yr
averages in field studies were 63% glume area affected (GAA) (36). Susceptible
reactions have been observed on genotypes Chalk, Coker 68-15 and Redhart
149 with more than 38% GAA in inoculated field plots, while Hadden and Red Chief
had means greater than 40% GAA (36). In a non-inoculated field plot in Georgia,
genotypes Coker 68-8, Coker 68-15 and Knox 62 were reported to have less
than a 25% yield reduction based on unreported glume blotch ratings (33). The
inconsistency between the resistance reactions of the tested genotypes in the
previous studies hinders genotype selection by other researchers.
The variability in reactions of the genotypes tested in previous studies
indicates that the resistance response may be influenced by plant growth stage
when inoculated or by use of different inoculum, isolates or inoculation
procedures. Variability in aggressiveness of S. nodorum isolates have been observed among geographically separated areas (40), which may have influenced variability in resistance response in previous studies. Determining genotype reactions using locally obtained isolates and recording partial resistance reactions in field plots may be the most conclusive way to evaluate disease resistance.
Objective The objective was to re-evaluate the reactions of genotypes previously reported resistant to Stagonosproa leaf and glume blotch with isolates of S. nodorum obtained in Ohio.
Materials and Methods The literature on S. nodorum resistant genotypes was reviewed to compile a list of genotypes for this study (Table 5.1). Seed of the genotypes were
150 obtained from Dr. H. E. Bockelman at the National Small Grains Collection in Aberdeen, Idaho. All seed was stored at 3.5°C until use.
Greenhouse inoculations-Plant production
In the fall of 2002, seed of the 26 genotypes were germinated on folded
12.7- x 50.8-cm moistened germination paper (Anchor Paper Company, St. Paul,
MN) placed in clear 10.2- x 30.5- x 5.1-cm plastic bags with 5 ml of water. The
bags were maintained in the laboratory (21°C ± 2°C) under a 12-hour
photoperiod of ultraviolet and white fluorescent lights. After 1 week, healthy
germinated seeds were placed into flats (5.1 x 25.4 x 50.8 cm) of autoclaved
(115°C for 6 hours) Wooster silt-loam soil and put into a temperature controlled
chamber (3.5°C) for 65 days for vernalization. Each tray was treated with
granular mefenoxam (Ridomil®, Novartis, Inc., Greensboro, NC, 1 g ai/tray), fertilized with pellets of triple 14 Osmocoat® (N 14%, P 14%, K 14%; 12 g/tray)(Scotts, Inc., Marysville, OH) and watered. Lights in the chamber were set to provide an 8-hour photoperiod and trays were watered as needed.
After the vernalization period, 15 plants of each cultivar per repetition were
transplanted individually into 12.7-cm pots of 3M Metro Mix (Conrad Fafard, Inc.,
Agawam, MA). Leaves of plants were clipped to approximately 5 cm above the
soil line 2 days after transplanting and pots were randomly arranged on benches
in the greenhouse. The 44.5-m2 greenhouses averaged 20.8°C during the day
and 19.2°C at night, with a 12-hour light period provided by one 1000-watt metal
halide lamp and four 1000-watt high pressure sodium lights (Rudd lighting Co.,
151 Solon, OH). Through the course of the experiment plants were loosely tied with
Sturdy Stretch Tie® (California Plastic Products, Tustin, CA) to 30-60 cm long
bamboo stakes for support.
Pots were fertilized weekly with 100 ml of a triple 20 Peter’s® fertilizer
solution (N 20%, P 20%, K 20%; 4.8 g/liter water) (Scotts, Inc., Marysville, OH).
For insect control, the plants were sprayed weekly with S-kinoprene (Enstar®,
Wellmark, Bensenville, IL) (0.01 g ai/liter water) or abamectin (Avid®, Syngenta
Crop Protection, Inc., Greensboro, NC) (0.01 g ai/liter water). Applications of
triadimefon (Bayleton®, Bayer Corp., Kansas City, MO) (2.2 g ai/liter water) were
applied 2 weeks after transplanting into pots and 2 weeks thereafter for control of
powdery mildew (Blumaria graminis f. sp. tritici ((DC.) E. O. Speer))) for the first 6
weeks.
Inoculum production and inoculation
Plants were inoculated when the main tiller of each plant had a fully
emerged flag leaf (Feekes growth stage 9 (23)). Pycnidiospores (1x106 /ml) in a water suspension were atomized until run-off on fully expanded flag leaves.
Inoculum consisted of a mixture of pycnidiospores from ten aggressive isolates of
S. nodorum obtained from wheat leaves collected at various locations across
Ohio in 2002. Isolates were grown on V8 agar modified with 50 mg/liter of streptomycin sulfate (Sigma-Aldrich, St. Louis, MO). The agar cultures from 8- cm diameter Petri dishes of each isolate were inverted onto a 15-cm cheese cloth square (four layers) and dipped into a glass beaker of 100-ml tap water and
152 strained to release pycnidiospores. Suspensions were enumerated using a
Spotlite Hemacytometer® (American Scientific Products, McGaw Park, IL)
according to the protocol for counting small spores (45). There was no attempt
to obtain equal amounts of pycnidiospores from each isolate.
After inoculation, plants were placed into a mist chamber consisting of a
2.3- x 1.4- x 1.0-m PVC skeleton enclosed by polyethylene sheeting with
adjustable sides. An air atomizer with a fluid nozzle (#2850 SS, Spraying
Systems, Co., Spokane, WA) (0.56 kg/cm3 water) and an air nozzle(#70) (4.2
kg/cm3 air) misted 30 ml of water for 30 seconds every 3 minutes above a polyethylene canopy over the plants. The temperature in the mist chamber was
maintained at approximately 22°C and the relative humidity was approximately
100%. Inoculated plants were incubated in the mist chamber for 48 hours.
After the 48 hour incubation period in the mist chamber, plants were returned to the greenhouse. Care was taken to prevent contact between the non-inoculated control plants and the inoculated plants. Plants were randomly arranged on greenhouse benches by inoculation date. The flag leaves were visually assessed for percentage diseased leaf area 7, 14 and 21 days after inoculation by visual assessment.
Experimental design and statistical analysis
Each individual plant in a pot was considered a replication. A repetition
was considered an experimental repeat separated over time with approximately a
month in between planting dates. Each experimental repeat had 15 replicate
153 plants for each genotype. Two random pots of each cultivar were not inoculated
and used as controls. There were a total of four repetitions.
Analysis of variance (ANOVA) was conducted using a linear mixed model
in SAS (SAS, Inc., release 8.3, Cary, N.C.) for the mean severity assessment.
The leaf blotch assessments taken 21 days after inoculation were analyzed with
genotype as a fixed effect factor and repetition as a random effect factor (44).
The restricted maximum likelihood method was used to fit the model, and least
squares means of disease severity were then calculated (44). The Satterthwaite
approximation for degrees of freedom was utilized for F-tests and significance
testing.
Field plots
In the fall of 2003, seed of the 26 genotypes were planted in a field
previously cropped with soybeans, on 10 October in Wayne County, OH.
Approximately 3 g of seed were planted into each hill plot of each genotype, and hill plots were 0.46 m apart. The field was prepared by moldboard plowing and a fall (22.8 kg/ha) and spring (56.8 kg/ha) application of nitrogen. Three rows of a susceptible cultivar, AGRA GR863, were also planted between hill plots using a small plot drill. This cultivar was used to promote spread of inoculum throughout the plot.
154 Field inoculations
The field plot was inoculated two ways. Before flag leaf emergence, plots were inoculated by spreading wheat straw on the soil surface. The straw was obtained from plots infested with S. nodorum the preceding year. The plots were
also inoculated by spraying a 1x106/ml pycnidiospore suspension onto leaf surfaces when 50% of the plants in the plot had fully emerged flag leaves
(Feekes growth stage 9 (23)). The same ten isolates of S. nodorum that were used in greenhouse experiments were used to inoculate the field plot.
Pycnidiospore suspensions were collected and enumerated as previously described. After inoculation, plots were misted continuously for 3 minutes out of every 10 minutes from 7 PM to 7 AM for 1 week to maintain high relative humidity to favor disease development. A second pycnidiospore inoculation was applied when 50% of the plants in the plot had fully emerged heads (Feekes growth stage 10.3 (23)). The inoculum was produced in the same manner as previously described. Flag leaves and glumes were visually assessed weekly after inoculations through senescence and percentage diseased area was assessed on flag leaves and glumes.
Field experimental design and statistical analysis
The field plot was arranged in a randomized complete block design. Each
genotype was planted in one hill plot of each block and there was a total of three
blocks in the field plot. The genotypes were randomly assigned within each
block. ANOVA was conducted using the linear mixed model in SAS (proc mixed)
155 (SAS Institute Inc., Cary, NC version 9.1). The last assessment of leaf and
glume blotch were analyzed with genotype as a fixed effect factor and repetition
as a random effect factor. The restricted maximum likelihood method was used
to fit the model and least squares means of disease severity were calculated
(44). The Satterthwaite approximation was utilized for degress of freedom
calculation for F-tests.
Results
Greenhouse reactions
During the course of the experiment, powdery mildew (Blumaria graminis
f. sp. tritici ((DC.) E. O. Speer)) became established in the greenhouse, limiting the number of plants that could be used as replications per experimental repeat for the majority of the genotypes. Genotypes with less than 15 replicates for all four repeats of the experiment were excluded from data analysis. This resulted in five genotypes being excluded. The remaining 21 genotypes were analyzed for leaf blotch reactions.
The highest leaf blotch severity was observed on Holly E (35.8% FLAA) while Cvl-22-4 had the lowest disease severity (4.4% FLAA, Table 5.2). There was clear separation of the genotypes into high and low leaf blotch severities
based on multiple comparisons of least squares means. Four genotypes had
high severities of leaf blotch, ranging from 25.8% to 35.8% FLAA, and their
severities were not significantly different from each other. Nine genotypes, with
leaf blotch severities ranging from 4.4% to 16.5% FLAA, had lower severities
156 than the rest, and their severities were not significantly different from each other.
The other genotypes had intermediate reactions, but the disease severity
observed on some of these genotypes did not differ statistically from the
responses of the more resistant or more susceptible genotypes tested.
Field Nursery
Two of the 26 genotypes, Hadden and Navarro, did not survive the winter.
Severe powdery mildew prevented the accurate assessment of cultivar Iohardi
reducing the number of genotypes that could be adequately evaluated. Leaf
blotch severities of the remaining 23 genotypes ranged from 3 to 67% FLAA
(Table 5.3). The susceptible check, AGRA GR863, had a mean of 66.7% FLAA
followed by the awned segregate of Coker 68-8, with 40% FLAA. Coker 68-8 was segregating for the presence or absence of awns and therefore disease assessments for awned and non-awned types were analyzed separately due to the possibility of physiological differences that could affect disease reactions.
The lowest amount of leaf disease in the field was observed on Atlas 66 and
Harvest Queen, both with 3.0% FLAA. While there were significant differences observed in pairwise comparisons of least squares means among the genotypes, there was no significant difference (P = 0.05) among 20 of the genotypes. Leaf blotch severities in greenhouse reactions were not significantly correlated (r =
0.173, P = 0.508) with leaf blotch severities obtained in the field plot.
Statistical analysis indicated a significant difference among genotypes for glume blotch severity (Table 5.3). AGRA GR863 had the highest glume blotch
157 severity, 51.0% GAA, followed by Coker 68-15 with 45% GAA. The lowest observed glume blotch severity occurred on Atilla (0.5% GAA) followed by
Kansas no. 43B337, Minhardi and Harvest Queen, each with 2.3% GAA. There was no statistically significant difference among the 20 genotypes with less than
25% GAA in the field.
Discussion
The genotypes tested in this study had been listed in the National Small
Grains Collection as varying from resistant to susceptible to S. nodorum. These reactions were based upon different tests and timing of inoculation (adult or seedling leaf blotch severity or glume blotch severity) and with different isolates of S. nodorum. The genotype reactions recorded in this study generally concurred with the National Small Grain Collection listing of genotypes reported to have resistant reactions for leaf and glume blotch. Previous studies that examined reactions on inoculated seedling leaves (21, 36, 39) reported Coker
68-8, Harvest Queen, Moking, Moro and Red May as being resistant. The greenhouse flag leaf inoculations in this study indicated that of these genotypes, only Cvl-22-4 was highly resistant with a leaf blotch severity of less than 5%
(Table 5.2). However, in the field, genotypes Harvest Queen, Moking and Red
May appeared as resistant as Cvl-22-4 and several other genotypes (Table 5.3).
The low correlation between greenhouse and field severities may have been due to differences in expression of partial resistance between the greenhouse and the field. Partial resistance expression is heavily influenced by plant
158 development stage and environmental conditions, such as soil moisture levels, nutrient levels and light intensity (3).
Stagonospora glume blotch reactions of certain cultivars have varied between studies (33, 36) possibly due to different field conditions and measurements used to assess disease severities. Glume blotch reactions reported in previous studies were above 38% GAA for genotypes that had resistant seedling reactions (36), but in this study the 13 more resistant genotypes had a mean percentage glume area affected of 5% or less in the field
(Table 5.3). These high disease severities on moderately resistant genotypes were not confirmed by inoculation experiments in the present greenhouse study using a more controlled environment. Nonetheless, the susceptible check,
AGRA GR863, used in this study had a mean of 51% GAA, indicating that sufficient inoculum was present and environmental conditions were conducive for higher disease severities required to assess genotype reactions.
Resistance to S. nodorum has been shown to be quantitatively inherited
(4, 6, 9, 26, 30-32, 35, 37, 38-41, 43) and epistatic effects appear to be important in resistance (26, 30). While some of these genotypes may have undesirable characteristics, such as late maturity and susceptibility to other diseases, they may have either different alleles or different genes for resistance than current commercial cultivars. Examining individual components of resistance, such as toxin sensitivity or incubation period, in segregating populations and conducting
QTL analysis from this group of genotypes may uncover resistance factors not present in today’s commercial cultivars. For example, Schnurbusch et al (42)
159 detected two Stagonospora glume blotch resistance QTLs that did not overlap
with QTLs for heading time or height. These QTL markers, QSng.sfr-3BS and
QSng.sfr-4BL (42), could be used to examine segregating populations of resistant by susceptible crosses of genotypes tested in this study. The potential desired alleles for Stagonospora leaf and glume blotch resistance in the genotypes tested in this study could be transferred into breeding lines utilizing marker assisted selection based on these QTL markers.
In conclusion, the genotypes that had been previously reported as
Stagonospora leaf and/or glume blotch resistant were confirmed, in general, in
this study using isolates of S. nodorum obtained in Ohio, indicating they have
potential for use in a breeding program for resistance to S. nodorum in Ohio.
Presence of sensitivity loci to S. nodorum produced phytotoxins are other components of resistance that have yet to be tested on these genotypes. Minor
QTLs contributing to epistatic effects of partial resistance from these genotypes or alternate alleles of major QTLs could be intercrossed into breeding lines for cultivar development for use in Ohio.
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166
Accession Date of S. nodorum Growth numbera Name Originb release reactionc habit CItr 17427 16-52-2 Brazil 1975 Resistant Spring CItr 12561 Atlas 66 North Carolina 1948 Susceptible Winter PI 351590 Attila Germany 1969 Intermediate Winter CItr 11538 Baldrock Michigan 1931 Intermediate Winter PI 357305 Chalk England 1971 Susceptible Winter CItr 17663 Coker 68-8 South Carolina 1976 Intermediate Winter CItr 15291 Coker 68-15 South Carolina 1971 Resistant Winter CItr 13554 Cvl-22-4 Maryland 1953 Resistant Winter CItr 11398 Gasta Georgia 1931 Intermediate Winter CItr 13488 Hadden/Coker 59-11 South Carolina 1962 Susceptible Facultative CItr 5314 Harvest Queen Kansas 1897 Susceptible Winter CItr 17431 Holly E Georgia 1977 Intermediate Winter CItr 6161 Honor New York 1920 Intermediate Winter CItr 12510 Iohardi Iowa 1948 Intermediate Spring CItr 12752 Kansas no. 43B337 Kansas 1950 Resistant Winter CItr 13701 Knox 62 Indiana 1962 Susceptible Winter Citr 5149 Minhardi Minnesota 1915 Susceptible Winter CItr 12556 Moking Kansas 1946 Resistant Winter CItr 13740 Moro Oregon 1965 Resistant Winter PI 191165 Navarro 183 Spain 1950 Intermediate Winter CItr 13550 Nebr. Sel. 513585 Nebraska 1960 Resistant Winter CItr 6962 Nittany Pennsylvania 1918 Susceptible Winter CItr 15929 Oasis Indiana 1974 Resistant Winter CItr 12109 Red Chief Kansas 1940 Resistant Winter CItr 8898 Redhart South Carolina 1921 Resistant Winter CItr 5336 Red May Missouri 1926 Susceptible Winter
Table 5.1: Characteristics of the wheat genotypes used in this study. a =Accession numbers of the genotypes used from National Small Grains Collection in Aberdeen, Idaho. b = Country or state where the genotype was developed. c = Reactions listed in accession descriptions from National Small Grains Collection in Aberdeen, Idaho. 167
Genotypea Plantsb Meanc t groupd 16-52-2 37 35.0 A Atlas 66 22 22.3 D B C Coker 68-15 27 18.7 D B C E F Coker 68-8 (Awned)e 28 23.3 B C Coker 68-8 (Awnless) 40 9.2 F G Cvl-22-4 29 4.4 G Gasta 22 11.3 D E F G Hadden 44 18.8 D B C E Harvest Queen 17 21.7 D B C E Holly E 46 35.8 A Iohardi 14 25.8 A B C Kansas no. 43B337 12 16.5 D B C E F G Knox 62 32 19.3 D B C E Moking 16 12.0 D C E F G Moro 17 26.9 A B Navarro 183 29 16.8 D B C E F Neb. Sel. 513585 23 23.7 B C Nittany 18 16.1 D B C E F G Oasis 42 10.7 E F G Red Chief 37 13.9 D C E F G Red Hart 22 10.4 E F G
Table 5.2: Least squares means of Stagonospora leaf blotch severity 21 days after inoculating flag leaves of wheat genotypes in the greenhouse. a = The effect of genotype was significant (P < 0.0001) using the F-test of the mixed model fitted to the data, with the Satterthwaite approximation for degrees of freedom. b = Total number of plants tested in four repetitions of the experiment. c = Least squares means of percentage leaf area affected were determined after use of restricted maximum likelihood to fit the mixed model to the data, with repetition as a random factor. d = Groups were based on pairwise comparisons of each cultivar pair of means (P = 0.05). Means followd by the same letter are not significantly different. e = Genotype was segregating for awned and non-awned types and separately evaluated.
168
Leaf blotch Glume blotch Genotypea Blocksb Meanc Groupd Meanc Groupd AGRA GR863 3 66.7 A 51.0 A Atlas 66 2 3.0 E 3.5 E Attlia 2 4.5 E 0.5 E Baldrock 3 15.0 C D E 5.3 E Chalk 3 10.0 C D E 2.7 E Coker 68-15 3 31.7 B C 45.0 A B Coker 68-8 (Awned)e 3 40.0 B 21.7 D C E Coker 68-8 (Awnless) 3 12.7 C D E 18.3 D C E Cvl-22-4 3 6.0 D E 5.7 E Gasta 3 10.0 C D E 2.7 E Harvest Queen 3 3.0 E 2.3 E Holley E 3 12.3 C D E 26.7 D B C Honor 3 3.7 E 3.3 E Kansas no. 43B337 3 6.7 D E 2.3 E Knox 62 3 11.7 C D E 33.3 A B C Minhardi 3 28.3 B C D 2.3 E Moking 3 3.3 E 3.3 E Moro 3 10.0 C D E 26.7 D B C Neb. Sel. 513585 3 25.0 B C D E 4.0 E Nittany 3 20.0 B C D E 3.3 E Oasis 3 25.0 B C D E 28.3 B C Red Chief 3 21.7 B C D E 7.3 D E Red Hart 2 4.0 E 3.0 E Red May 3 5.0 E 4.3 E
Table 5.3: Least squares mean estimate of leaf blotch and glume blotch severity for genotypes inoculated in a field plot at Wooster, OH in 2004. a = The effect of genotype was significant (leaf blotch, P = 0.0003, glume blotch, P < 0.0001) using the F-test of the mixed model fitted to the data, with the Satterthwaite approximation for degrees of freedom. b = Total number of hill blocks that were evaluated in the inoculated field plot. c = Least squares means of percentage leaf area affected were determined after use of restricted maximum likelihood to fit the mixed model to the data, with repetition as a random effect factor. d = Groups were based on pairwise comparisons of each cultivar pair of means (P = 0.05). Means followed by the same letter are not significantly different. e = Genotype was segregating for awned and non-awned types, and separately evaluated.
169
APPENDIX A.
NUMBER OF FIELDS WITH LEAF BLOTCHING PATHOGENS IN OHIO
COUNTIES SAMPLED IN 2002
170
Region County Fields S. nodoruma P. tritici-repentisb C. sativusc 7 Allen 15 2 3 1 6 Ashland 7 2 3 0 1 Ashtabula 2 0 0 0 7 Auglaize 9 8 2 0 5 Clark 3 2 1 0 5 Clinton 3 3 1 0 2 Crawford 14 7 5 2 4 Darke 15 10 3 2 3 Defiance 6 2 2 0 5 Fayette 5 4 4 0 7 Hancock 15 4 8 0 7 Hardin 10 6 4 0 3 Henry 15 2 6 2 5 Highland 1 1 0 0 2 Huron 11 8 4 0 4 Logan 10 7 5 0 6 Lorain 5 3 3 0 5 Madison 7 4 0 0 4 Marion 10 5 5 0 7 Mercer 9 5 0 0 2 Morrow 8 6 2 1 3 Paulding 13 3 9 1 5 Pickaway 15 8 5 0 4 Preble 3 3 0 2 3 Putnam 15 3 7 0 2 Richland 8 3 0 0 5 Ross 12 7 7 0 3 Sandusky 2 0 0 0 7 Seneca 6 4 1 0 4 Shelby 10 5 3 1 1 Trumbull 15 4 5 0 4 Union 12 9 4 0 7 Van Wert 15 6 5 0 6 Wayne 14 9 2 0 3 Williams 8 4 2 0 3 Wood 18 4 6 0 7 Wyandot 15 6 7 0 Total 359 167 121 12
Table A.1: Number of fields with leaf blotching pathogens in Ohio Counties sampled in 2002. a = Stagonospora nodorum b = Pyrenophora tritici-repentis c = Cochliobolus sativus
171
APPENDIX B.
NUMBER OF FIELDS WITH LEAF BLOTCHING PATHOGENS IN OHIO
COUNTIES SAMPLED IN 2003
172
Region County Fields S. nodoruma P. tritici-repentisb C. sativusc 7 Allen 15 14 1 0 6 Ashland 9 7 1 0 1 Ashtabula 3 3 0 0 7 Auglaize 15 15 2 0 5 Clinton 4 3 0 0 2 Crawford 14 15 4 1 4 Darke 13 12 0 0 3 Defiance 8 2 4 0 5 Fayette 4 4 0 0 7 Hancock 13 11 3 0 7 Hardin 10 10 1 0 3 Henry 15 10 4 0 5 Highland 3 1 1 0 2 Huron 11 9 4 0 4 Logan 5 1 2 0 5 Madison 10 8 0 0 4 Marion 10 8 0 0 7 Mercer 13 11 0 0 2 Morrow 11 10 0 0 3 Paulding 15 8 9 2 5 Pickaway 14 14 3 1 4 Preble 8 6 0 0 3 Putnam 15 9 0 0 2 Richland 6 4 3 0 5 Ross 15 13 10 1 3 Sandusky 3 1 1 0 7 Seneca 13 10 0 1 4 Shelby 14 12 0 0 1 Trumbull 6 6 0 0 4 Union 11 8 0 0 7 Van Wert 15 11 2 1 6 Wayne 14 12 2 1 3 Williams 9 2 4 0 3 Wood 15 12 6 0 7 Wyandot 15 14 2 0 Total 368 298 70 8
Table B.1: Number of fields with leaf blotching pathogens in Ohio Counties sampled in 2003. a = Stagonospora nodorum b = Pyrenophora tritici-repentis c = Cochliobolus sativus.
173
APPENDIX C
DESIGNATION OF THE POPULATIONS OBTAINED FROM A PARTIAL
DIALLELE CROSS OF THIRTEEN COMMERCIAL CULTIVARS SCREENED
FOR STAGONOSPORA NODORUM AND PYRENOPHORA TRITICI-
REPENTIS RESISTANCE
174
175
Table C.1: Designation of the populations obtained from a partial diallel cross of thirteen commercial cultivars
screened from Stagonospora nodorum and Pyrenophora tritici-repentis resistance.
APPENDIX D
GENERAL CONCLUSIONS
176 GENERAL CONCLUSIONS
The survey of diseased flag leaves in major wheat producing counties of
Ohio in 2002 and 2003 indicated that the most prevalent leaf blotching pathogen was Stagonospora nodorum. Pyrenophora tritici-repentis, tan spot of wheat, was also isolated from flag leaves across the state. P. tritici-repentis had previously been considered a minor pathogen with sporadic occurrence across Ohio. A third pathogen, Bipolaris sorokiniana, was also found, but the occurrence and distribution of this pathogen was erratic. The state was arbitrarily divided into seven regions for analysis of occurrence and distribution of S. nodorum and P. tritici-repentis. Analysis of occurrence and distribution indicated that there was no positive or negative association between these two pathogens. This result indicates that occurrence of S. nodorum and P. tritici-repentis is independent of each other. The occurrence of each pathogen in the seven regions was also calculated with no region by pathogen association found. This result indicates that the environmental conditions in the seven sampled regions does not preclude the occurrence of either pathogen.
A sub-set of the S. nodorum isolates collected in 2002 and 2003 was examined for differences in aggressiveness by region where the isolate was
177 obtained. Isolates from North Eastern Ohio, geographically separated from the
rest of the state, were significantly less aggressive than isolates from other sampled areas of the state. This area of Ohio has a low number of fields planted with wheat annually. S. nodorum isolates from Central West Ohio were significantly more aggressive than isolates examined from other regions of Ohio.
The Central West Ohio region is characterized by a large number of fields planted to wheat annually. These results indicate that there may be environmental or other conditions that are either enhancing or hindering changes in pathogen aggressiveness. One hypothesis is that the environmental conditions in the regions with significantly different isolate aggressiveness are influencing pathogen overwintering and/or sexual reproduction. An alternative hypothesis is that the number of fields annually planted to wheat may be affecting sexual reproduction with lower wheat production slowing the rate of genetic recombination in the pathogen population.
A sub-set of the P. tritici-repentis isolates collected in 2002 and 2003 was
race characterized. The most common race found was race 1 (60% of the sub-
sampled isolates) followed by race 2 (35% of the sub-sampled isolates). Race 3
was also found, but it was infrequent in occurrence (5% of the sub-sampled
isolates). Races 1 and 2 had no pattern of distribution in the seven sampled
regions, while the occurrence of race 3 limited distribution characterization.
These results are the first reported along the East Coast using the currently
accepted race characterization for P. tritici-repentis. Since race 1 and 2 were
178 common, current breeding efforts may need to focus on breeding for specific
resistance to these races.
Seedlings of commercially available wheat cultivars were screened for
sensitivity to toxins produced by S. nodorum. Two isolates, SN2000 higher
SnTox1 producer and SN69-1 higher SnTox2 producer, were individually inoculated onto seedlings and visually assessed for lesion type. Results indicated that in general, the cultivars were more sensitive to the isolate producing more SnTox2. Cultivars Bouillon, Coker 9025, and Coker 9474 were the only cultivars with a significantly more sensitive reaction to SnTox1.
Regardless of which toxins the inoculated isolates produced, all of the cultivars tested had moderately resistant to moderately susceptible reactions. This is the first study examining the reaction of soft red winter wheat cultivars to specific toxins produced by S. nodorum at the seedling stage.
Seedlings of commercially available wheat cultivars were also screened for sensitivity to toxins produced by races 1 and 2 of P. tritici-repentis. Each race was individually inoculated onto the seedlings and visually rated for lesion type severity. The cultivars generally had a more sensitive reaction to race 2, which produces Ptr ToxA, than race 1, which produces Ptr ToxA and Ptr ToxC.
Cultivars had a moderate response regardless of race inoculated, indicating sensitivity to the toxins, but that there are also other variables involved in the
resistance reaction.
Flag leaf reactions of commercially available cultivars to P. tritici-repentis
were evaluated in the greenhouse and in field plots. These reactions were
179 determined by inoculating these cultivars with either a composite of Ohio races or
individual races from North Dakota. The composite isolate reactions had low
disease severities, yet the cultivars were able to be statistically separated.
Honey was found to be the most resistant cultivar using mean disease severities
after inoculation with the composite of P. tritici-repentis races, but was not
statistically different from Bravo, AGRA GR535, Coker 9474, Freedom and
Roane. After inoculation with the race 1 isolate, Honey had a resistant reaction
again, but was not statistically different from cultivars Bravo, Patterson and
Patton. The mean disease severities of Bouillon, Bravo, Honey, Patton, Roane
and Sisson were not statistical different, although Patton had a resistant reaction
to race 2. Using a lesion type rating scale and calculated relative marginal
effects analysis, Patton was identified as the most resistant cultivar tested in this
study regardless of race inoculated. In the field, variability in cultivar reactions
did not allow for mean separation of the cultivars tested. Seed infestation from
harvested seed was determined, with low infestation regardless of the fact that
the plot was inoculated. This result indicates that even under high inoculum
levels, seed infestation by P. tritici-repentis does not appear to be a concern in
Ohio. The low disease severities obtained from flag leaf inoculations indicate that P. tritici-repentis alone is probably not economically limiting in cultivars currently grown in Ohio.
Flag leaf reactions of commercially available cultivars to S. nodorum were
evaluated in the greenhouse and in field plots. These reactions were determined
by inoculating these cultivars with a composite of 10 aggressive isolates from
180 Ohio. In the greenhouse after flag inoculation, Coker 9663 had a resistant reaction, but the mean disease severity was not statistically different from cultivars Coker 9474, Roane and Sisson. After spike inoculation, Coker 9663 had the lowest observed glume area affected, but was not statistically different from the mean disease severity of Sisson. In inoculated field plots, the lowest observed leaf area affected was on Coker 9663, although it was not statistically different from the mean disease severities observed on AGRA GR535, Bouillon,
Coker 9474, OH708, Patton and Roane. Coker 9663 also had the lowest observed glume blotch severity, but was not statistically different from eight other cultivars tested. Seed infestation from harvested seed from the inoculated field plot indicated all cultivars tested had higher than expected levels of S. nodorum on seed.
Leaf blotch reactions from naturally infested field plots also indicated that
Coker 9663 was the more resistant cultivar tested, although mean leaf area affected was higher than observed in inoculated plots. The higher disease severities on Coker 9663 may indicate co-infection with P. tritici-repentis. Glume blotch reactions were low in naturally infested field plots, with little mean separation between the tested cultivars. The percentage of infestation on harvested seed was also determined from naturally infested plots. All cultivars once again had higher than expected S. nodorum seed infestation, with Coker
9025 having the least amount of seed infestation. The results from these tests indicate that resistance to leaf and glume blotch is available in cultivars commercially grown in Ohio. The co-infection of S. nodorum and P. tritici-
181 repentis may be increasing disease severity ratings in naturally infested field plots, decreasing the accuracy of specific pathogen resistance reactions.
Another concern these results raise is the economic impact of co-infection by
both pathogens in production fields.
Genotypes that had been previously cited as resistant to S. nodorum but replaced in production were also screened for resistance reaction in the greenhouse and field. The majority of genotypes previously reported as resistant were confirmed in this study as having either leaf, glume or both types of resistance. While these genotypes have detrimental traits, such as maturity and low disease resistance to other wheat pathogens, they may have major and/or minor QTL for S. nodorum resistance. Further studies on these cultivars need to be completed for accurate characterization.
In conclusion, 13 commercial planted cultivars were studied for multiple
traits. These traits were: seedling resistance to toxins produced by S. nodorum;
seedling race reactions to P. tritici-repentis; flag leaf and glume blotch reactions
in the greenhouse and field to S. nodorum; and flag leaf reactions to P. tritici-
repentis in the greenhouse and field. These studies indicate that some of these
cultivars have resistance to these pathogens during different phases of maturity
and different magnitudes to each phase and pathogen. Further studies to
examine the genetic nature of the identified resistance are necessary for
complete understanding of these resistance reactions. These results also
indicate that cultivars currently in production in Ohio have resistance to either
182 pathogen, but future breeding efforts may need to focus on producing genotypes with resistance to both pathogens.
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