IDENTIFICATION AND MAPPING OF RESISTANCE GENES FOR
EYESPOT OF WHEAT IN AEGILOPS LONGISSIMA
By
HONGYAN SHENG
i
A dissertation submitted in partial fulfillment of the requirements for the degree of
Doctor of Philosophy
WASHINGTON STATE UNIVERSITY Department of Plant Pathology
May 2011
© Copyright by HONGYAN SHENG, 2011 All Rights Reserved
To the Faculty of Washington State University:
The members of the Committee appointed to examine the dissertation of HONGYAN SHENG find it satisfactory and recommend that it be accepted.
______Timothy D. Murray, Ph. D., Chair
______Xianming Chen, Ph. D.
______Scot H. Hulbert, Ph. D.
______Tobin L. Peever, Ph. D.
______Stephen S. Jones, Ph. D.
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ACKNOWLEDGMENT
I would like to express my sincere gratitude and appreciation to my mentor and major advisor, Dr. Timothy D. Murray, for all his guidance, support, patience, and encouragement throughout my entire Ph. D. process at Washington State University. I am grateful to Dr. Murray for sharing his knowledge of plant pathology, providing insight into this dissertation, and leading me to the complex and fascinating world of genetics.
My grateful appreciation goes to my committee members, Dr. Tobin L. Peever,
Dr. Xianming Chen, Dr. Scot H. Hulbert, and Dr. Stephen S. Jones for their helpful advice and guidance during my graduate work and critical review of my dissertation. I would especially like to thank Dr. Deven R. See (USDA-ARS Regional Small Grains
Genotyping Laboratory at Pullman, WA) for providing techniques and equipments for marker analysis work. Most of all, I am grateful for his critical suggestion leading to successful results. From all of these individuals, I have gained the knowledge and experience to be a successful scientist.
I would also like to thank the Washington Grain Commission for financial support of my Ph.D. research, the USDA National Small Grains Collection for providing seeds of
Ae. longissima accessions, and the Wheat Genetics and Genomic Resources Center at
Kansas State University for providing seeds of Ae. longissima addition or substitution lines.
I am also grateful to all members of Dr. Murray‟s and Dr. See‟s labs. Thanks to
Dr. Kathy Klos and Dr. Henry Wetzel for their help and support in the lab and greenhouse, and to the students who assisted in this work. Special thanks to Dan
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Dreesmann for his support in the Plant Growth Facility Greenhouse. My appreciation also goes to all of the faculty, staff, and graduate students in the Department of Plant
Pathology who provided help, encouragement, and friendship. I would especially like to thank Dr. Hanu Pappu, Dr. Brenda Schroeder, Cheryl Hagelganz, Mary Stormo, and
Robin Stratton.
My deep gratitude goes to my family back in Changchun, China. I greatly appreciate my father and mother for their sacrifices, encouragement, and understanding during my studies far from home. I also am grateful to my brother, sister, and all of my relatives and friends who take care of my parents.
Finally, I would like to express my appreciation and love to my family here in the
US. Thanks to my husband, Bo Gao, for walking with me through this long journey.
Thanks also to my wonderful children, Lucy and Luke, for inspiring me all the time and understanding the time that went into this work when they grow up.
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IDENTIFICATION AND MAPPING OF RESISTANCE GENES FOR
EYESPOT OF WHEAT IN AEGILOPS LONGISSIMA
Abstract
by Hongyan Sheng, Ph.D. Washington State University May 2011
Chair: Timothy D. Murray
Eyespot of wheat, caused by two fungal pathogens Oculimacula yallundae and O. acuformis, produces elliptical lesions at the stem base that result in lodging of infected plants and yield losses up to 50% in commercial wheat fields. The objectives of this research were: 1) to identify potential new sources of resistance to O. yallundae and O. acuformis from a wild relative of wheat, Ae. longissima (2n = 14, SlSl); and 2) to understand the genetic control of the resistance and map gene(s) in the Sl genome by developing a genetic linkage map based on wheat microsatellite markers.
Forty Ae. longissima accessions and 83 Ae. longissima addition or substitution lines in Chinese Spring or Selkirk background were evaluated for resistance to eyespot.
Forty-three percent of the accessions were resistant to O. yallundae, 48% were resistant to O. acuformis, and 33% were resistant to both pathogens. This is the first evidence that
Ae. longissima contains resistance to eyespot. Ae. longissima chromosomes 1Sl, 2Sl, 5Sl, and 7Sl contributed to the resistance for both O. yallundae and O. acuformis, and indicated that eyespot resistance was controlled by multiple regions of the Sl genome.
Differential resistance reactions to O. yallundae and O. acuformis were observed in 25% of Ae. longissima accessions and 39% addition or substitution lines.
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A recombinant inbred line (RIL) population was developed from the cross of Ae. longissima accessions PI 542196 (R) x PI 330486 (S) to map eyespot resistance genes. A genetic linkage map of the Sl genome was constructed with 169 wheat microsatellite markers covering 1261.3 cM in 7 groups. F5 lines (189) were tested for reaction to O. yallundae and four QTL were detected in chromosomes 1Sl, 3Sl, 5Sl, and 7Sl. These QTL explained 44% of the total phenotypic variation by GUS scores and 63% by visual ratings. This is the first time multiple QTL conferring resistance to eyespot in the Sl genome have been identified. Markers Xcfd6, Xwmc597, Xwmc415, and Xcfd2 are tightly linked to Q.Pch.wsu-1Sl, Q.Pch.wsu-3Sl, Q.Pch.wsu-5Sl, and Q.Pch.wsu-7Sl, respectively.
The identification of these markers will facilitate QTL transfer to wheat by marker- assisted selection and broaden the genetic diversity of eyespot resistance.
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TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS ...... iii
ABSTRACT ...... v
LIST OF TABLES ...... ix
LIST OF FIGURES ...... x
CHAPTER
1. INTRODUCTION AND LITERATURE REVIEW ...... 1
Wheat Production ...... 1
Eyespot ...... 3
Host Resistance to Eyespot ...... 31
Objectives ...... 44
References ...... 45
2. IDENTIFYING NEW SOURCES OF RESISTANCE FOR EYESPOT OF WHEAT IN AEGILOPS LONGISSIMA ...... 64
Introduction ...... 64
Materials and Methods ...... 67
Results ...... 72
Discussion ...... 85
References ...... 93
3. GENETIC ANALYSIS AND QTL MAPPING OF RESISTANCE GENES TO EYESPOT OF WHEAT IN AEGILOPS LONGISSIMA ...... 98
Introduction ...... 98
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Materials and Methods ...... 102
Results ...... 108
Discussion ...... 127
References ...... 137
4. INTERPRETIVE SUMMARY ...... 143
Future work ...... 146
References ...... 148
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LIST OF TABLES
Page
CHAPTER 1
Table 1. Virulence of eyespot isolates to seedlings of five host species (modified from Scott & Hollins, 1980) ...... 11
CHAPTER 2
Table 1. GUS scores and disease ratings of 40 Aegilops longissima accessions to Oculimacula yallundae and O. acuformis ...... 74
Table 2. GUS scores and disease ratings of 83 Ae. longissima addition or substitution lines in Chinese Spring or Selkirk background to Oculimacula yallundae and O. acuformis ...... 80
CHAPTER 3
Table 1. Mean and standard deviation for GUS scores and visual ratings in three generations of PI 542196 (R) x PI 330486 (S) progeny populations inoculated with Oculimacula yallundae ...... 109
Table 2. Genetic analysis of resistance to Oculimacula yallundae in three generations of PI 542196 (R) x PI 330486 (S) progeny populations ...... 111
Table 3. Variance components of GUS scores and visual ratings and broad-sense 2 heritability (H ) for the F3 and F5 recombinant inbred lines derived from the cross of PI 542196 (R) x PI 330486 (S) inoculated with Oculimacula. yallundae...... 117
Table 4. Genetic linkage groups of Aegilops longissima based on the cross PI 542196 (R) x PI 330486 (S) constructed with wheat microsatellite markers ...... 121
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LIST OF FIGURES
Page
CHAPTER 2
Figure 1. Reaction of 40 Aegilops longissima accessions to Oculimacula yallundae and O. acuformis. Dark bars are lines that are not significantly (P > 0.05) greater than Madsen and light bars are lines that are significantly (P < 0.05) greater than Madsen based on Dunnett‟s T-test. The GUS score of each line was the mean of 24 plants in two experiments ...... 76
Figure 2. Distribution of Oculimacula yallundae and O. acuformis resistance among chromosomes of Aegilops longissima. Dark bars are number of Ae. longissima addition or substitution lines resistant to O. yallundae and light bars are number of lines resistant to O. acuformis. Data for O. yallundae is the mean of 12 plants per line in two experiments and O. acuformis is the mean of 6 plants in one experiment ...... 83
CHAPTER 3
Figure 1. Frequency distribution of GUS scores in the F2 population (108 plants) of the cross PI 542196 (R) x PI 330486 (S) inoculated with Oculimacula yallundae. The dark-colored columns are resistant plants of which GUS scores were within 95% confidence limits of the resistant parent, whereas susceptible plants are represented by light-colored columns. Kolmogorov-Smirnov test didn‟t reject the null hypothesis of normality with a value of 0.0899 (P = 0.0213)...... 112
Figure 2. Frequency distribution of GUS scores in the F3 population (789 plants from 50 lines) of the cross PI 542196 (R) x PI 330486 (S) inoculated with Oculimacula yallundae. The dark-colored columns are resistant plants with GUS scores less or not significantly (P > 0.05) greater than the resistant parent, whereas susceptible plants are represented by light-colored columns. Kolmogorov-Smirnov test didn‟t reject the null hypothesis of normality with a value of 0.0243 (P > 0.15)...... 113
Figure 3. Frequency distribution of GUS scores (A) and visual ratings (B) in F5 population (189 lines, 12 plants/line) of the cross PI 542196 (R) x PI 330486 (S) inoculated with Oculimacula yallundae. The dark-colored columns are resistant lines with GUS scores or visual ratings less or not significantly (P > 0.05) greater than resistant parent, whereas susceptible lines are represented by light-colored columns. Kolmogorov-Smirnov test didn‟t reject the null hypothesis of normality with a value of 0.0233 and 0.0505 (P >0.15)...... 114
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Figure 4. Scatter plot of visual rating vs. GUS score for 189 F5 recombinant inbred lines derived from the cross of Aegilops longissima PI 542196 (R) x PI 330486 (S). The value of each data point was the mean of 12 plants...... 116
Figure 5A. Linkage map of Aegilops longissima chromosomes 1Sl, 2Sl, 3Sl, and 4Sl. Chromosomes 1Sl and 3Sl carry QTL Q.Pch.wsu-1Sl and Q.Pch.wsu-3Sl for eyespot resistance, respectively. QTL are indicated on chromosomes as black rectangles. Q.Pch.wsu-1Sl was closely associated between markers Xcfd6 and Xcfd48 separated by 3.1 cM. Q.Pch.wsu-3Sl was closely associated between markers Xgdm72 and Xwmc597 with a 6.1 cM interval ...... 119
Figure 5B. Linkage map of Aegilops longissima chromosome 5Sl, 6Sl, and 7Sl. Chromosome 5Sl and 7Sl carry Q.Pch.wsu-5Sl, Q.Pch.wsu-7Sl for eyespot resistance, respectively. QTL are indicated on chromosomes as black rectangles. Q.Pch.wsu-5Sl was closely associated between markers Xgwm639 and Xcfd12 with a 13.1 cM interval. Q.Pch.wsu-7Sl was closely associated between markers Xgdm132 and Xcfd2 with a 12.5 cM interval ...... 120
Figure 6. QTL for eyespot resistance identified on Aegilops longissima chromosomes 1Sl, 3Sl, 5Sl, and 7Sl by GUS score and visual rating with composite interval mapping. a.) Q.Pch.wsu-1Sl on chromosome 1Sl; b.) Q.Pch.wsu-3Sl on chromosome 3Sl; c.) Q.Pch.wsu-5Sl on chromosome 5Sl; and d.) Q.Pch.wsu-7Sl on chromosome 7Sl ...... 123
Figure 7. GUS scores (A) and visual ratings (B) for RILs of PI 542196 (R) x PI 330486 (S) with different parental alleles at the markers close to each QTL. Xcfd6 and Xgdm67 are close to Q.Pch.wsu-1Sl; Xwmc597 is close to Q.Pch.wsu-3Sl; Xgwm639, Xwmc415, and Xcfd12 are close to Q.Pch.wsu-5Sl; and Xcfd2 is close to Q.Pch.wsu-7Sl. Bars represent the mean GUS scores or visual ratings of RILs with the same parental allele at the marker closet to each QTL. Different letters on the bars indicate the significant (P < 0.05) difference between lines with resistant allele and those with susceptible allele. Error bars show standard errors ...... 125
Figure 8. Resistance of 16 genotypes produced from four QTL detected in F5 RIL populations of PI 542196 (R) x PI 330486 (S) to Oculimacula yallundae. Each genotype includes 3-18 lines and each line included 12 individual plants. Bars represent mean GUS scores or visual ratings of RILs with the same genotype. A.) Mean GUS scores of RILs within each genotype. B.) Mean visual rating of RILs within each genotype. The columns with an asterisk are genotypes with significantly (P < 0.05) lower GUS scores or visual ratings than „no QTL‟. Light- colored columns are genotypes that were not significantly (P > 0.05) greater than „all QTL‟ for either GUS score or visual rating. Error bars show standard errors ...... 128
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Dedication
This dissertation is dedicated to my dear parents,
Baohuai Sheng & Guiying Li
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CHAPTER ONE
INTRODUCTION AND LITERATURE REVIEW
Wheat Production
Wheat (Triticum aestivum L.), a member of the family poaceae (Gramineae), is one of the first domesticated cereal grain and the most important food crops in the world
(Asplund et al., 2010). Wheat provides a major source of complex carbohydrates to the human diet and contains protein, dietary fiber, minerals, and vitamins that are essential for human nutrition (Gill, 2010). In recent years, world land area used for wheat production is about 200 million hectares (FAO, 2009) with 620 million tons of grain produced annually (Dubcovsky & Dvorak, 2007). Wheat is also a principal cereal crop in the United States (US), which is one of the largest wheat-producing countries of the world and exports about 50% of its wheat production. Wheat acreage in the US represents about 10% of world wheat acreage (FAO, 2009). Although wheat production is currently the greatest in history, it must increase further to feed the growing world population. Preventing losses and improving wheat yields are critical since the available land area on the earth for agriculture is limited.
The Pacific Northwest (PNW) includes Oregon, Idaho, and Washington. Each year, 85 to 90% of total wheat production in the PNW, which is about 40% of total US wheat exports, is sold to the world market (WGA, 2009). The PNW has a long history of producing wheat since it was first planted at Fort Vancouver, WA in 1825 (WGA, 2009).
The natural soil fertility and climate of the PNW are ideal for wheat growth and results in
1 average yield potential that is greater than the national average (Shepherd, 1975). Idaho leads the country in statewide wheat yield with averages of 73.8 and 79.3 bushels per acre in 2008 and 2009, respectively (WGA, 2008 & 2009). Whitman County, WA has produced more wheat than any other US county since 1978 (WGA, 2009). In 2000, the average rainfed wheat yield of Whitman County was about 75 bushels per acre
(Schillinger & Papendick, 2009), which is much greater than the national average of about 42 bushels per acre (WORG, 2002). In 2008 and 2009, the PNW contributed 11 to
13% of the total US wheat crop with 7 to 8% of the total acreage harvested nationally
(WGA, 2008 & 2009).
Five of the six wheat market classes, soft white, soft white club, hard white, hard red winter, and hard red spring, are produced in the PNW. These classes are determined by seed hardness, bran coat color, milling, and baking qualities, and each is used for different food products (WGA, 2008). Soft white wheat occupies about 70% of the PNW wheat acreage. In 2009, the PNW accounted for 85% of US soft white wheat production, making it the primary soft white wheat producer in the US (WGA, 2009).
Winter and spring type cultivars exist within each market class. Winter wheat is planted in fall and requires several weeks of vernalization (0° to 5°C) in order to produce seeds the following summer. In contrast, spring wheat is sown in spring and does not require vernalization. Approximately 80% of wheat grown in the PNW is winter habit, primarily because its yield potential is about 14 bushels per acre more than spring wheat
(WGA, 2009).
Every year, about 25 to 30% of world wheat production is lost due to various stresses at all growth stages and in storage (Gill, 2010). Approximately 20% of the losses
2 resulted from diseases caused by fungi, bacteria, viruses, and nematodes (Wiese, 1991).
Over 30 diseases impact wheat production in the PNW. The most economically important wheat diseases in the PNW are caused by fungal pathogens. Stripe rust, caused by
Puccinia striiformis Westend. f. sp. tritici Eriks., is the most important disease of wheat and continues to receive the most attention because of the favorable weather conditions in the PNW (Line, 2002). The cool, wet winters of the PNW are also favorable for the development of fungal diseases favored by low temperature, such as snow molds,
Cephalosporium stripe, and eyespot.
Eyespot
Eyespot, caused by Oculimacula yallundae (syn: Tapesia yallundae, Wallwork &
Spooner) Crous & W. Gams and O. acuformis (syn: T. acuformis, Boerema, R. Pieters &
Hamers) Crous & W. Gams (Crous et al., 2003), which often coexist in the same field, is a chronic and economically important soilborne disease in many wheat growing areas of the world with cool and wet winter climates (Murray, 2010). It first drew attention as a problem of PNW winter cereal production in 1919 (Sprague, 1931b). In the US, eyespot occurs mainly in the PNW on winter wheat (Murray, 2010), where it is also called
Cercosporella foot rot, culm rot, strawbreaker foot rot, and Columbia Basin foot rot
(Sprague, 1931a; Maloy & Inglis, 1993).
Eyespot is a stem base disease of wheat. The characteristic symptom is an eye- shaped elliptical lesion on the stem base or basal leaf sheaths. Initially, lesions appear as pin-point, water-soaked areas, and enlarge vertically to 1 to 2 centimeters long and 3 to 6 millimeters wide (Sprague & Fellows, 1934). The affected areas are weakened and result
3 in stem breakage and lodging, which inspires the name “strawbreaker foot rot”. The pathogen doesn‟t infect roots and rarely colonizes more than 4 cm above the soil surface, making it a true foot rot disease. Lesions are light brown initially and become darker with dark-gray to black fungal pseudoparenchyma in the center (Murray, 2010). White heads with standing dead stems appear in the field when diseased tillers die prematurely.
Another stem base disease, sharp eyespot, caused by Rhizoctonia cerealis, also has elliptical lesions, which can lead to confusion during diagnosis. Although similar in shape, sharp eyespot lesions have a distinct dark brown margin with light colored centers that later may rot and leave a hole in the sheath. Eyespot lesions are firm at beginning and become brittle during development. The shriveled stem bases result in lodging of infected plants. The lodging caused by eyespot differs from lodging caused by wind or storm because it is multi-directional rather than uni-directional (Maloy & Inglis, 1993; Murray,
2010).
Eyespot affects many hosts in the Gramineae but wheat, Triticum spp., is the most agriculturally important host. Eyespot also affects barley (Hordeum vulgare L.), oat
(Avena sativa L.), rye (Secale cereale L.), and other grasses. Winter cereals are more affected by eyespot than spring cereals because of favorable environmental conditions for infection during autumn and winter (Murray, 2010). All cultivated species of wheat are affected by eyespot including wheat (T. aestivun), durum (T. durum), Einkorn (T. monococcum), Emmer (T. dicoccum), and spelt (T. spelta) (Sprague, 1936). In addition, some species of following genera have been reported to be affected by eyespot: Aegilops,
Agropyron, Agrostis, Avena, Bromus, Cynosurus, Dactylis, Festuca, Holcus, Hordeum,
Koeleria, Lolium, Phalaris, Phleum, Poa, Secale, Sitanion, and other wild species of
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Triticum (Deighton, 1973; Lucas et al., 2000). Wild grasses may serve as a source of inoculum for cultivated crops and may also contain disease resistance genes. Agropyron spicatum and Ag. inerme were proposed as the native hosts of the eyespot fungi (Sprague,
1936). Due to varying susceptibility among species of Aegilops and Triticum to the eyespot pathogens, Sprague (1936) suggested that eyespot resistant varieties could be developed from hybrids of wheat and its wild relatives.
Geographic Distribution
Eyespot occurs worldwide wherever wheat is grown and the climate is favorable to the pathogen. Eyespot has been reported in almost all wheat growing areas of the world including North and South America, Australia, New Zealand, Europe, and Africa
(Lucas et al., 2000). In Europe, most studies have been done in England, France,
Germany, and Belgium. Eyespot was first reported in France in 1912. In Germany,
Denmark, and England, it was recognized in the 1930s (Glynne, 1936; Sprague, 1936).
There are records of natural infection of eyespot on wheat in Finland, Ireland, and Russia in the 1950s and 1960s (Cunningham, 1981).
In the US, eyespot occurs occasionally in the Great Plains, Midwest, and
Northeast in addition to the PNW. The history of eyespot in the PNW began in the 1900s.
Pioneer farmers in the Peone Prairie of Washington reported eyespot on wheat before
1910 (Sprague, 1936). Sprague (1936) concluded that the eyespot pathogens had been present on native grasses in the area for a long time. Eyespot was well known in north
Idaho, north central Oregon, and some parts of Washington by 1919 and reports continued to expand geographically (Sprague, 1931b; Sprague & Fellows, 1934). In eastern Washington, eyespot was found wherever wheat was grown (Bruehl et al., 1968).
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In Canada, eyespot on wheat and barley has been reported in several provinces including
Ontario, Quebec, Alberta, and British Columbia (Slopek et al., 1990). Recently, a wheat disease in the central-southern region of Poland was confirmed to be caused by
Oculimacula yallundae and O. acuformis based on conidia, culture morphology, and PCR tests (Glazek, 2009).
Economic Impact
Eyespot results in reduced tiller number, kernel number per head, and 1000-kernel weight (Scott & Hollins, 1974; Murray & Bruehl, 1986). Yield reductions up to 50% have been documented in commercial fields with severe disease (Murray, 2010). Glynne
(1944) reported total yield loss from eyespot as high as 44% in experimental fields. In the
UK, the 10-year average loss caused by eyespot was £17 million, with the greatest loss of
£22 million in 2000 (Bateman & Jenkyn, 2001). Yield losses are greater if lodging is present.
Glynne (1944) hypothesized that eyespot reduced yield of winter wheat in two ways: direct losses caused by eyespot lesions that interfered with movement of water and nutrients through the stems and indirect losses caused by lodging that interfered with harvest (Glynne, 1944). Thus, resistance to eyespot can include resistance to lodging and to the development of lesions (Scott & Hollins, 1974). Scott and Hollins (1974) found that the greatest yield losses occurred in lodging-susceptible cultivars where eyespot was the most severe and extensive. They concluded that even though both direct and indirect effects contributed to the yield losses, breeding for eyespot resistance should consider both effects. Murray and Bruehl (1986) tested both resistant and susceptible cultivars in the fields inoculated and not inoculated with eyespot isolates for three years, and they
6 supported the conclusions of Scott and Hollins (1974) and suggested that yield losses could not be explained by lodging alone. They found that the direct effects of eyespot were the primary determinants of yield, although lodging was related. In contrast, Scott and Hollins (1978) reported that yield loss was more closely related to the amount of lodging than eyespot severity. Fitt et al. (1988) predicted that final losses were due more to lodging than the direct eyespot effects because the grain quality of lodged plants was decreased. Ray et al. (2006) found that O. yallundae and O. acuformis reduced ear weight by 7 and 3% and yield by 6 and 11%, respectively, when eyespot was severe. They suggested that both O. yallundae and O. acuformis decreased the stem bending strength of plant and caused lodging, which resulted in yield losses.
Pathotypes
Four pathotypes of the eyespot pathogen have been described: wheat (W), rye (R), couch (C), and squarrosa (S). All of them are virulent to wheat (Scott & Hollins, 1980), but have varying degrees of virulence to other hosts. W- and R-type isolates were the two widely accepted major groups of P. herpotrichoides (Lucas et al., 2000); they are now known as Oculimacula yallundae and O. acuformis, respectively (Crous et al., 2003).
Variation in virulence among strains of Oculimacula spp. was first recognized by
Oort in 1936 with the discovery of some strains infecting wheat, oats, and grasses
(Cunningham, 1981). Bawden (1950) reported that an isolate from diseased oats was more virulent to oats than to wheat. Glynne (1952) described the differences between isolates from wheat and rye when they penetrated different hosts. Lange-de la Camp differentiated the isolates into two groups, W- and R-type, in 1966. W-type isolates from wheat, barley, and oats were more virulent to wheat than to rye, whereas R-type isolates
7 from rye were almost equally virulent to both rye and wheat (Scott et al., 1975;
Cunningham, 1981). Lange-de la Camp‟s distinction was confirmed by several studies on seedlings in greenhouse studies and adult plants in the field (Scott et al., 1975; Scott &
Hollins, 1980; Fitt et al., 1987).
On potato dextrose agar (PDA) at 21.5oC, W-type isolates grow faster and produce greenish-grey colonies with furry mycelium and even, smooth margins, whereas
R-type isolates grow more slowly and produce beige to brown colonies with slimy growth and irregular feathery margins (Scott et al., 1975). W- and R-type isolates were also described as fast, even (F/E) and slow, feathery (S/F), respectively (Nicholson et al.,
1991a). Cultural morphology and growth rate are not reliable methods of identifying pathotypes because of the existence of intermediate types (Hollins et al., 1985; Creighton
& Bateman, 1991). Some P. herpotrichoides var. acuformis isolates had characteristics of
W-type isolates (Creighton, 1989). Creighton (1989) discovered that W- and R-type isolates have different pigmentation when grown on maize agar (MA) under near-UV light at 13oC for 2 weeks. Intermediate types can be distinguished positively as W- or R- types under these conditions.
Biochemical and molecular techniques provide more accurate methods to identify pathotypes. Five isozyme polymorphisms clearly distinguished W- and R-types among
101 isolates of Pseudocercosporella and the results were confirmed by pathogenicity tests (Julian & Lucas, 1990). Restriction fragment length polymorphism (RFLP) in total
DNA and rDNA also had significant correlation with morphology and pathogenicity in a group of P. herpotrichoides isolates (Nicholson et al., 1991a). Thomas et al. (1992) found most DNA probes derived from a genomic library of P. herpotrichoides could distinguish
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W- and R-types. Frei and Wenzel (1993) generated pathogen-specific probes from repetitive genomic clones to discriminate the pathotypes of P. herpotrichoides in infected plant material without isolation and culture of the fungus. Based on the results of isoenzyme profiling and DNA markers, Priestley et al. (1992) suggested that W- and R- pathotypes might correspond to P. herpotrichoides var. herpotrichoides and P. herpotrichoides var. acuformis, respectively.
Poupard et al. (1993) developed a polymerase chain reaction (PCR) with primers from the internal transcribed spacer (ITS) region of ribosomal DNA to differentiate W- and R-type isolates. Gac et al. (1996a) tested 60 isolates of P. herpotrichoides with a
PCR-based assay and found good correlation between molecular markers and
Nirenberg‟s classification system. Gac et al. (1996b) used a PCR assay with restriction enzyme digestion of an amplified ribosomal DNA to differentiate W- and R-types directly from plant tissue. These studies provided initial molecular diagnostic techniques for eyespot disease.
Takeuchi and Kuninaga (1994) suggested that the two genetically isolated groups
(W- and R-type) of P. herpotrichoides were distinct species because DNA relatedness of isolates from different geographical places was greater than 82% within each group but lower than 34% between the groups. They determined DNA relatedness by reassociation kinetics of nuclear DNA. Later, they obtained similar results using RFLP analysis of mitochondrial DNA and suggested that W- and R-types were genetically distinct species
(Takeuchi & Kuninaga, 1996). Dyer et al. (1996) provided the first evidence that W- and
R- types were different biological species based on their reproductive incompatibility in vitro.
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C-type isolates were described for those isolates highly virulent to couch grass
(Agropyron repens) in addition to wheat and barley (Cunningham, 1965, 1981). Scott &
Hollins (1980) found isolates that belonged to this pathotype and reported S-type isolates that were virulent to wheat and Aegilops squarrosa. C-type isolates were only found in
Ireland, whereas S-types were isolated from both France and the United Kingdom
(Priestley et al., 1992). It is impossible to distinguish C- and S- pathotypes from W-type isolates by morphology; pathogenicity tests are needed to differentiate them (Priestley et al., 1992). Priestley et al. (1992) used isoenzyme profiling to separate C-type isolates from W- and R-type isolates. The random amplified polymorphic DNA (RAPD) profiles from P. herpotrichoides isolates were also used to analyze pathotypes (Nicholson &
Rezanoor, 1994).
In another study, Scott & Hollins (1977) concluded that pathogenic adaptation of
P. herpotrichoides was not specific to wheat cultivars but only to different cereal species.
They classified the four distinct pathogenic types in a table, which is modified below
(Table 1) (Scott & Hollins, 1980). Host-specific pathogenic adaptation could play an important role in eyespot resistance and epiphytology. For example, a resistant wheat cultivar might be susceptible to the R-type pathogen if resistance was derived from rye
(Scott et al., 1976). Because C-type isolates exist, Ae. repens has limited possibility as a source of eyespot resistance (Cunningham, 1981), as does Ae. squarrosa (syn. Triticum tauschii) because it was infected by R-, C-, and S- pathotypes (Scott & Hollins, 1980).
Thus, identifying the pathotypes of eyespot populations in a field can provide useful information for breeding programs.
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Table 1. Virulence of eyespot isolates to seedlings of five host species (modified from Scott & Hollins, 1980). Host Species Triticum Secale Agropyron Aegilops Aegilops Pathotype aestivum cereale repens squarrosa ventricosa W-type + - - - - R-type + + - + - C-type + - + + - S-type + - - + - +, pathogenic; -, non-pathogenic.
Taxonomy
The eyespot fungus was originally named Cercosporella herpotrichoides by Fron in 1912 based on conidia isolated from infected wheat straw in Northern France in 1909
(Sprague & Fellows, 1934). Based on morphology of conidia and conidiophores,
Deighton concluded that the eyespot fungus was not a Cercosporella and assigned it to the new genus Pseudocercosporella as anamorph of Mycosphaerella because of the truncate, unthickened, and inconspicuous conidial scars (Deighton, 1973).
Nirenberg (1981) divided P. herpotrichoides (Fron.) Deighton into two varieties: var. herpotrichoides and var. acuformis. This separation was mainly based on spore shape and size. P. herpotrichoides var. herpotrichoides had shorter, curved conidia whereas P. herpotrichoides var. acuformis had longer, straight conidia (Nirenberg, 1981).
In the same study, Nirenberg also described two additional species, P. anguioides and P. aestiva, related to the eyespot pathogens. Nirenberg‟s treatment was the first formal
11 proposal to divide P. herpotrichoides into two taxonomic units and associate more species with eyespot disease (Lucas et al., 2000; Crous, 2003).
Based on the observation that conidiogenesis in the eyespot fungi was similar to
Ramulispora sorghi and their conidia produced lateral branches like all species in
Ramulispora, Von Arx concluded that Pseudocercosporella was not connected to
Mycosphaerella (Crous, 2003). Therefore, the two varieties of P. herpotrichoides were transferred to Ramulispora in 1983, which is a genus containing pathogens of gramineous plants. This change was supported by Boerema et al. (1992), who listed the two varieties of R. herpotrichoides in a check-list for scientific names of common parasitic fungi.
Robbertse et al. (1995) described R. herpotrichoides var. herpotrichoides and var. acuformis as two separate species based on the low percentage of shared RAPD PCR bands and differences in spore and culture morphology, growth rate on PDA, virulence to wheat and rye, as well as the histology of infection of wheat coleoptiles. The anamorphs of the eyespot fungi have been recognized as R. herpotrichoides and R. acuformis since
1995. Based on morphology of the conidia, P. anguioides was reassigned to Ramulispora as R. anguioides; the generic status of P. aestiva remains undecided (Robbertse et al.,
1995).
The sexual stage of the eyespot fungi was first discovered as apothecia on wheat straw collected from Yallunda Flat, South Australia (Wallwork, 1987). The field where samples were collected showed severe lodging caused by P. herpotrichoides var. herpotrichoides. The teleomorph of the fungus was named Tapesia yallundae because the dermateacous structure of the ectal excipulum fits the genus Tapesia (Wallwork &
Spooner, 1988). Robbertse et al. (1995) proposed T. yallundae and T. acuformis as the
12 teleomorph name of R. herpotrichoides var. herpotrichoides and var. acuformis, respectively (Robbertse et al., 1995).
Based on sequence analysis of the internal transcribed spacer region (ITS1, 5.8S, and ITS2) of the rDNA operon, Crous et al. (2003) concluded that the anamorph of the eyespot fungi did not belong to Ramulispora and the teleomorph formed in a separate cluster from other non-eyespot Tapesia species. Thus, new anamorph and teleomorph genera were erected for the eyespot fungi, Helgardia and Oculimacula, respectively.
Helgardia is named after Dr. Helgard Nirenberg who was the first to separate P. herpotrichoides into two varieties. Oculimacula is Latin for eyespot (Crous et al., 2003).
The names presently accepted worldwide are Oculimacula yallundae (Wallwork &
Spooner) Crous & W. Gams, anamorph: Helgardia herpotrichoides (Fron) Crous & W.
Gams and acuformis (Boerema, R. Pieters & Hamers) Crous & W. Gams, anamorph:
Helgardia acuformis (Nirenberg) Crous & W. Gams (Crous et al., 2003). The currently accepted classification places Oculimacula in the phylum Ascomycota, subphylum
Pezizomycotina, class Leotiomycetes (formerly in Discomycetes), order Helotiales, family Dermateaceae (Dyer et al., 2001a).
Morphology
Cultural and spore morphology have played an important role in identifying the eyespot pathogens. Fron originally described the conidia as 3-septate, 32-38 µm long x
1.5-2 µm wide in 1912 (Deighton, 1973). Sprague (1931a) described conidia are mostly
2-7 septate, 30-80 µm long x 1.5-3.5 µm wide with slightly branched conidiophores that are swollen sometimes. In Deighton‟s description, conidia are hyaline, acicular, straight or slightly curved, 3-7 septate, and 26.5-47 long x 1-2 µm wide; condial scars are
13 truncate, unthickened and inconspicuous; producing polyblastic conidiogenous cells occasionally (Deighton, 1973). Conidia of O. yallundae are 35-80 µm long x 1.5-2.5 µm wide, sometimes curved; whereas those of O. acuformis are 42-120 µm long x 1.2-2.3
µm wide and straight (Creighton, 1989). There are two types of mycelia; vegetative mycelium is hyaline to light brown, linear-celled and branching, whereas stromatic mycelium is thick-walled, forming dark-colored masses on media and stems (Sprague,
1931a; Murray, 2010).
Wallwork (1987) and Wallwork & Spooner (1988) described the sexual structures of O. yallundae from Australia as apothecia, 0.5-1.5 mm in diameter, sessile and gregarious, seated on a subiculum, and pale grey with a slightly raised white margin; eight-spore asci are cylindric-fusoid and 42-50 µm long x 4-6 µm wide; ascospores are 7-
11 µm long x 1.5-2.0 µm wide, hyaline, fusiform, rounded-end, non-septate. Hunter
(1989) reported the apothecia, asci, and ascospores of O. yallundae in the UK were slightly smaller compared to Wallwork & Spooner‟s description. King (1990) described the apothecia of O. acuformis in Germany usually as grey to very dark brown without white/hyaline covering and ascospores being slightly narrower.
Disease Cycle and Epidemiology
Oculimacula yallundae and O. acuformis have similar life cycles during which the dormant or least active period of the pathogen occurs in summer. Fungal mycelium survives on infested stubble, volunteer wheat and barley plants, and grasses up to 3 years
(Kelly et al., 2008). However, Deacon (1973) reported survival of eyespot pathogens on buried wheat straw was only 19 weeks (Deacon, 1973).
14
Sporulation. Sporulation occurs during autumn, winter, and spring when temperatures fluctuate between 0o and 20oC with an optimum of 10oC (Sprague &
Fellows, 1934; Rowe & Powelson, 1973; Murray, 2010). Conidia are dispersed from infested debris a short distance by rain splash and infect plants with an optimum temperature 6 to 15oC (Fitt & Bainbridge, 1983). Higgins and Fitt (1984) reported that most conidia (8.1 x 106 per straw) were produced in February and least (1.9 x 106 per straw) near the end of June on naturally infected wheat straw. They found that 5oC was the optimum temperature for spore production and few spores were produced at 25oC.
The eyespot fungi do not produce conidia on most nutrient media. Because of this, the causal organism of eyespot remained unidentified for many years after the disease was recognized. After obtaining conidia from diseased plant material, Fron identified the causal agent in 1912 (Glynne, 1953). In France, Foëx and Rosella in 1931 occasionally obtained spores of C. herpotrichoides from eyespot lesions of young wheat plants but none in pure culture, so they named it „fungus x‟ (Sprague and Fellowes,
1934). Heald (1924) consistently isolated a fungus with sterile mycelia from foot rot- infected stems of wheat near Spokane, WA from 1919-1924. Eyespot was thought to be caused by a sterile fungus until Sprague (1931a) obtained spores in pure culture isolated from eyespot lesions on wheat. Based on this observation, Sprague (1931a) confirmed the fungus causing Columbia Basin foot rot in the PNW to be C. herpotrichoides, which had been identified by Fron in France. Later, Sprague and Fellowes (1934) identified „fungus x‟ cultures provided by Foëx as C. herpotrichoides.
Various methods have been used to induce rapid sporulation in pure cultures and infected straws. Sprague grew the fungus on corn meal agar and incubated the cultures
15 outside in late fall when the temperature fluctuated between - 4 and 16oC (Sprague &
Fellows, 1934). Large quantities of spores were produced on the pseudopionnotes and sporodochia. He found fluctuation of temperature was necessary for sporulation (Sprague
& Fellowes, 1934). Glynne (1953) reported that water was an important factor for inducing sporulation and conidia developed easily from young infected plants but not on mature plants. However, conidia were produced on infected straws in wet weather.
Abundant sporulation was induced when straws were soaked in water for 24 hours, drained and kept damp at fall temperature. Sporulation was increased by frost but decreased by dry weather (Glynne, 1953). In the same study, Glynne found that conidia were produced abundantly when mycelia on potato dextrose agar or water agar was touching water and temperatures fluctuated between -3 and 13oC (Glynne, 1953).
Using the same method, Chang and Tyler (1964) found that the most favorable temperature for producing spores was 9oC and an optimum of 10oC was reported later
(Rowe & Powelson, 1973; Ward & Friend, 1979). Rowe and Powelson (1973) found that sporulation was decreased or prevented if temperatures were below 0oC for more than 14 hours or above 20oC for 10 hours and wouldn‟t resume even if the temperature was favorable. Near-UV light has been reported to stimulate sporulation (Chang & Tyler,
1964; Leach, 1967; Bateman & Taylor, 1976a; Ward & Friend, 1979).
Penetration and infection. Germination of eyespot conidia and infection require moisture for many hours. Hollins and Scott (1980) reported that infection was positively correlated with length of wet period and maximum infection was between November and
March in the UK. They also found that infection was not closely related to the number of conidia and concluded that development of disease is limited by environmental factors
16 when enough conidia are present to initiate infection. In the PNW, most infections occur in November and December (Bruehl et al., 1982a). Nelson and Sutton (1988) studied epidemiology of eyespot on winter wheat in Ontario, Canada over 3 years and concluded that the peak for infection occurred in April mainly because of the cool, humid period at night, and that infections declined in June.
During infection, conidia penetrate coleoptiles and outer leaf sheaths of lower stems directly through epidermal cells (Sprague & Fellows, 1934; Bateman & Taylor,
1976b; Murray, 2010), or stomatal openings (Sprague & Fellows, 1934). Coleoptiles and leaf sheaths were the most susceptible to infection (Higgins, 1984; Fitt et al., 1988).
Murray and Ye (1986) reported that coleoptiles and leaf sheaths of susceptible cultivars were penetrated more frequently than resistant ones. They found more papillae formed in the leaf sheaths of resistant cultivars than those of susceptible cultivars. Later,
Strausbaugh and Murray (1989a) reported that early senescence of the coleoptiles and first leaf sheaths of the susceptible cultivars resulted in rapid penetration. Soulie et al.
(1985) observed in vitro infection of seedlings by scanning electron microscopy and found that susceptible lines had stronger adhesions of mycelia in contact with coleoptiles than resistant lines. Penetration also results in formation of a stroma between leaf sheaths and on the outer surface of the host over eyespot lesions (Sprague & Fellows, 1934).
Bruehl et al. (1982a) assumed that stromata were related to sporulation because they observed that a lesion with stromata always produced conidia.
Daniels et al. (1991) found different patterns of coleoptile infection for O. yallundae and O. cauformis isolates on the same host. Germ tubes of O. yallundae isolates showed orientated growth and penetrated through anticlinal cell walls by forming
17 swollen appressorium-like structures; whereas germ tubes of O. cauformis isolates had no orientated growth and were placed on the surface of coleoptiles randomly. These different patterns may explain their different growth rates during infection. Using ELISA to measure fungal growth, Poupard et al. (1994) confirmed that O. cauformis isolates colonized coleoptiles and leaf sheaths more slowly than O. yallundae isolates. A PCR assay was used to demonstrate that O. yallundae isolates penetrated plant tissue faster than O. cauformis isolates (Gac et al., 1996b). Kelly et al. (2008) indicated that O. cauformis isolates usually infect plants later and may cause less damage than O. yallundae under field conditions.
Disease development. Depending upon environmental conditions, symptoms develop 2 to 8 weeks after infection, or longer (Murray, 2010). During the early weeks following infection there are no macroscopically visible symptoms of eyespot, but fungal growth on the coleoptiles and the first leaf sheath can be observed microscopically
(Murray and Ye, 1986; Daniels et al., 1991). Blein et al. (2009) reported that conidia germinated 12 hours after inoculation with O. yallundae on the coleoptile; appressoria formed 3 days after inoculation (d.a.i.); a mycelial network was present at 5 d.a.i.; a stroma appeared at 8 d.a.i.; and penetrated the coleoptile at 30 d.a.i.. However, typical eyespot lesions were not observed until 10 weeks after inoculation. They concluded that the development of O. yallundae had both asymptomatic and symptomatic growth phases and suggested the possibility that it is a hemibiotroph instead of a necrotrophic pathogen
(Blein et al., 2009).
The eyespot fungus invades successive leaf sheaths as the disease develops. The fungus spreads from the innermost leaf sheaths to the true stem after stem elongation
18 begins (Fitt et al., 1988). Fitt (1985) observed the progress of eyespot lesions and found that eight leaf sheaths could be penetrated in 16 weeks after inoculation at controlled temperatures (night 8/day 15oC or mean 14 to 20oC). Both relative humidity and temperature affect eyespot development. At temperatures of 6 to 18oC, the number of penetrated leaf sheaths increased (Scott, 1971). At 10oC night/ 15oC day temperature, penetration to inner leaf sheaths and death of leaf sheaths was more rapid than at 5oC night/10oC day (Higgins & Fitt, 1985).
Fitt et al. (1987) reported that O. yallundae isolates caused more severe lesions than O. cauformis. Goulds and Fitt (1990b, 1991) inoculated field plots with both pathotypes and observed developmental differences between O. yallundae and O. cauformis isolates on seedling leaf sheaths and concluded that these differences were greatly affected by weather (Goulds & Fitt, 1990b). Furthermore, they found that O. yallundae isolates infected stems faster and caused more severe lesions than O. cauformis, but that there was little difference in severity between them by the end of the season (Goulds & Fitt, 1991). Poupard et al. (1994) also measured the same levels of
ELISA for both species of eyespot at the ripening stage. In an outdoor experiment, Wan et al. (2005) observed that O. yallundae had greater incidence than O. acuformis on leaf sheaths in early stages but O. acuformis developed more severe lesions by maturity. They suggested that O. yallundae penetrated through the leaf sheaths more rapidly than O. acuformis. A subsequent study by Bock et al. (2009) showed the same patterns of development between O. yallundae and O. acuformis as on stems. These two studies concluded that the incidence and severity of eyespot on leaf sheaths and stems were linearly related to thermal time (accumulated oC days) after sowing.
19
Rowe and Powelson (1973) suggested that secondary inoculum played little role in the current season epidemics. Bruehl et al. (1982a) inoculated winter wheat in different months and concluded that most yield losses were attributed to primary inoculum. The sexual stage may play an important part in the life cycle, although the role of ascospores in infection is not known. The infection process of ascospores of O. yallundae was similar to that of conidia (Daniels et al., 1995). If two different mating types exist in a field, apothecia may form on infected debris after harvest from mid-October to July but mostly from January to March (Dyer et al., 1994b & 2001b). During the season, ascospores are discharged from apothecia and disseminated by wind. The sexual stage could be a threat to nearby cereal crops (Kelly et al., 2008). Even though apothecia have been detected from wheat fields in some countries, ascospores may not be the major inoculum based on the low percentage of stems on which apothecia formed (Lucas et al.,
2000).
Mating System
Two-allele heterothallic fungi require two mating types conferred by alleles at a single locus for sexual reproduction (Dyer et al., 1992). Apothecia of the eyespot fungi were produced in vitro only when certain O. yallundae isolates were incubated together, which suggested different mating types of O. yallundae were needed for apothecia development (Nicholson et al., 1991b). The mating system of O. yallundae was studied by crossing tester strains 22-432 and 22-433 with 51 other O. yallundae isolates (Dyer et al., 1993). Thirty-three isolates formed fertile apothecia with 22-432 and 18 isolates formed apothecia with 22-433. These results demonstrated that O. yallundae was heterothallic with two mating types designated MAT1-1 and MAT1-2 (Dyer et al., 1993;
20
Singh et al., 1999). Crosses between ascospore progeny from a single apothecium showed that the progeny belonged to two mating groups, which confirmed the two-allele heterothallic mating system (Dyer et al., 1993 & 1996; Moreau & Maraite, 1995 & 1996).
A protocol for inducing apothecia of O. yallundae in vitro was developed that involves incubation of compatible isolates at 10oC under white light for about 12 weeks
(Dyer et al., 1993). O. acuformis requires different conditions than O. yallundae to induce apothecia formation in vitro. Dyer et al. (1996) incubated straw segments at 7oC or less under near UV or white light for 8 months. Moreau and Maraite (1996) incubated moist wheat straws at 8-12oC under a mixture of near UV and daylight for at least 6 months.
Knowledge of the mating system and methods of in vitro sexual reproduction were breakthroughs for the study of the eyespot fungi.
Compatibility among pathotypes of the eyespot fungi was tested in vitro and showed that W-, C-, and S-types were compatible with each other and all possessed two- allele heterothallism (Nicholson et al., 1995). No fertile crosses resulted from crossing R- type isolates and between R-type and other types. Since there were no pairings between strains of O. yallundae and O. acuformis in vitro, it was concluded that they were sexually incompatible species (Dyer et al., 1994b; Moreau & Maraite, 1995 & 1996). In
2001, a multiplex PCR test for determining mating types was developed for both O. yallundae and O. acuformis. With three primers, two PCR products of 812 bp and 418 bp were amplified for MAT1-1 and MAT1-2 regions, respectively (Dyer et al., 2001a). This multiplex PCR assay speeds up the process of identifying mating type and is useful for population genetics studies.
21
Population Structure
The eyespot pathogen was known as an asexual fungus for a century before apothecia were first identified (Wallwork, 1987). After its initial discovery, several occurrences of the sexual stage of O. yallundae were reported (Sanderson & King, 1988;
Hunter, 1989; Nicholson et al., 1991b; Moreau & Maraite, 1995). Sanderson and King
(1988) found apothecia of O. yallundae on wheat stubble in New Zealand in 1987. In the
UK, apothecia of O. yallundae were discovered separately on wheat straw by two research groups in 1989 (Hunter, 1989; Nicholson et al., 1991b), thus providing evidence that both mating types of O. yallundae exist in fields in the U.K. (Dyer et al., 1993).
Apothecia of O. yallundae were reported on wheat stubble in Belgium in 1989 (Moreau
& Maraite, 1995), in South Africa in 1994 (Robbertse et al., 1994), and in Southern Chile in 1995 (Andrade, 2005). King (1990) reported the discovery of apothecia of O. acuformis on wheat and rye stubble in Germany in 1990. This is the first report of apothecia of O. acuformis detected in the field even though eyespot field populations shifted from O. yallundae to O. acuformis in England and Germany (King, 1990;
Nicholson et al., 1991b). The first detection of apothecia of O. acuformis isolates in the
UK was in 1993 (Dyer et al., 1994a). Apothecia of O. acuformis were collected from wheat fields in Belgium even though there were just a few of them (Moreau & Maraite,
1996). Using RFLP and RAPD markers, only O. yallundae was present in South Africa
(Campbell et al., 1996).
In the US, apothecia of O. acuformis were found in two of eight naturally infected fields in 2000 (Douhan et al., 2002a). Apothecia of O. yallundae were not observed in production fields, but only in plots inoculated with compatible isolates (Douhan et al.,
22
2002a). Even though apothecia of O. yallundae were not observed under natural conditions, the high genotypic diversity of O. yallundae populations in Washington suggests that it is present and recombination occurs (Douhan et al., 2002a & 2002b). The predominant species of eyespot pathogen in the PNW of US has been O. yallundae since
1919 (Sprague & Fellowes, 1934; Douhan et al., 2002a). Based on a survey in
Washington in 1989 and 1990, only 8% of 617 isolates were O. acuformis (Murray,
1996). Ten years later, O. acuformis had increased to 44% of 817 isolates (Douhan et al.,
2002a & 2003). A similar population shift has occurred in England and Germany in the
1980s (Hollins et al., 1985; King, 1990).
Disease Management
Eyespot control generally requires a combination of cultural practices, host resistance, and fungicides. Cultural practices are important because the use of fungicides is often not economical and environmentally undesirable (Herrman & Wiese, 1985).
Cultural practices include reducing tillage, late seeding of winter wheat, and crop rotation.
Tillage. Soil management practices can have a significant effect on infection of winter wheat by eyespot and other stem-base pathogens (Matusinsky et al., 2009).
Murray (2006) suggested reducing tillage could be used to reduce the severity of eyespot and that spring tillage should be avoided because reducing tillage reduces the splashing of spores from host residue (Herman & Wiese, 1985). Brooks and Dawson (1968) found that no-till plots had less eyespot than conventionally tilled (plowed) plots. Later,
Herrman and Wiese (1985) compared conventional tillage, reduced tillage and no-till for both eyespot incidence and yield. Their results showed that reduced-tillage and no-till
23 treatments had significantly less disease than conventional tillage. Although no-till was the best to control eyespot and soil erosion, it had unacceptably lower yield than the other two methods; the yield of the reduced-tillage treatment was not significantly lower than conventional tillage with the four cultivars tested. Colbach and Meynard (1995) studied the influence of soil tillage on eyespot and found a decrease in primary infection when soil inversion buried host residues if the previous crop was a host crop. However, if the previous crop was a non-host crop, soil inversion resulted in increased primary infection.
Less eyespot was observed when straw was chopped and incorporated than when it was burned (Jenkyn et al., 1994; Prew et al., 1995; Jalaluddin & Jenkyn, 1996).
Jalaluddin and Jenkyn (1996) hypothesized that the chopped straw blocked light, which was necessary for fungal sporulation. They concluded that the effects of wheat debris on the sporulation and survivel of eyespot pathogens were large in the field. Jenkyn et al.
(2010) suggested that debris affected inoculum and suppressed the development of eyespot. Another explanation is competition with other microorganisms on the straw (Fitt et al., 1990). Burning wheat stubble was not recommended for eyespot control by Murray
(2006). He explained that burning didn‟t destory colonized straw protected by the soil, which was enough to infect host plants in the next season.
Delayed seeding. Seeding date has a significant effect on development of eyespot. Among five management techniques (sowing date, sowing density, nitrogen fertilizer dose, fertilizer form, and removal/burial of straw), the earliest and strongest effect on disease incidence was seeding date (Colbach & Saur, 1998). Glynne and Salt
(1958) suggested that late-sown crops should have less severe eyespot. Early seeding of winter wheat results in higher eyespot severity because older seedlings are more
24 susceptible to infection by the eyespot pathogens (Murray, 2010). In the PNW, delayed fall seeding combined with a foliar application of fungicide in early spring was the typical practice for controlling eyespot (Bruehl et al., 1982b; Herrman & Wiese, 1985).
Goulds and Fitt (1990b) observed that the percentage shoots infected and number of leaf sheaths penetrated were less in late-sown plots than in early-sown plots for both O. yallundae and O. acuformis isolates in three years. Since O. acuformis isolates develop more slowly than O. yallundae isolates on winter cereal crops, late sowing was thought to be more useful when O. acuformis isolates predominate (Goulds & Fitt, 1991). Nelson and Sutton (1987) in Canada found that eyespot was less severe when wheat was sown in
October compared to wheat sown in September. Although late-sowing can reduce the risk of eyespot epidemics, early sowing may be practiced for other reasons (Fitt et al., 1990).
For example, delayed seeding may increase potential for soil erosion and winter injury in the PNW (Murray, 2006). The problem of eyespot, yield, and local environmental conditions should be considered to determine the proper seeding date (Murray, 2006).
Crop rotation. Crop rotation can also reduce eyespot inoculum in infested fields.
Eyespot fungi are able to survive on buried stubble for as long as 3 years (Kelly et al.,
2008); thus, a one year break rotation is not enough to reduce inoculum. A two year break from host crops is recommended as a control for eyespot. Before effective fungicides became available in the 1970s, crop rotation was an important cultural practice (Fitt et al.,
1990). In 1959, a two year rotation with a non-susceptible crop was suggested because it reduced eyespot damage to negligible levels (Glynne & Slope, 1959). Eyespot pathogens can build-up quickly and a small amount of inoculum can result in severe damage
(Murray, 2006). In the PNW, the choice of rotation crops for control of eyespot is
25 limited; spring grains and legumes can be used for rotation because they are not affected by or not hosts for the eyespot pathogens (Murray, 2006).
Fungicides and fungicide resistance. Fungicides have played a major role in controlling eyespot worldwide. Fungicides for control of eyespot started with the introduction of Benlate (DuPont, Wilmington, DE) in 1977 (Murray, 1996). In the early
1980s, fungicides were used for eyespot control on up to 70% of the winter wheat acreage in eastern Washington and north Idaho (Herrman & Wiese, 1985). In early spring, usually mid-February to mid-Appril, a foliar application of fungicide should be made when 10% or more stems have eyespot lesions (Murray, 2006). Fungicides must be applied before stem elongation (growth stage 31) (Zadoks et al., 1974) to be effective
(Murray, 2006). Goulds and Fitt (1990a) suggested growth stage 30/31 was the proper stage for applying fungicides and found the yield response was greatest in both O. yallundae and O. acuformis plots when that recommendation was followed. However, fungicide control shouldn‟t be the first choice and isn‟t sustainable because of increased input costs for farmers and the potential for development fungicide resistance.
The three main fungicide groups now used to control eyespot are methyl benzimidazole carbamates (MBC), sterol demethylation inhibitors (DMI), and anilinopyrimidines (AP) (Russell, 2005). Benzimidazole fungicides include benomyl
(Benlate), carbendazim, thiabendazole (Mertect), and thiophanate-methyl (Topsin-M)
(Murray, 2010). They were introduced for eyespot control in the mid-1970s and were relatively inexpensive. They provided effective control and increased wheat yield before fungicide-resistant isolates of the eyespot fungi appeared in the 1980s (Murray, 1996).
26
DMI fungicides include propiconazole (Tilt), cyproconazole, epoxiconazole, flusilazole, prochloraz (Sportak), and triazoles (Murray, 2010). DMI fungicides were recommended to replace benzimidazole fungicides for eyespot control because they are effective against both benzimidazole sensitive and resistant isolates (Fitt et al., 1990).
Prochloraz can be applied between growth stage 33 to 37, which provides flexibility in spray time. DMI fungicides also were suggested to be used in the PNW (Murray, 2010).
Currently, Tilt and Topsin-M are the only registered fungicides in the PNW (Murray,
2006).
In the mid-1990s, cyprodinil, an AP fungicide, was introduced in Western Europe and its use has increased since then (Parnell et al., 2008). Cyprodinil effectively controlled both O. yallundae and O. acuformis and increased yields (Bateman et al.,
2000). Prochloraz mainly controlled O. yallundae and its effectiveness depended on rainfall events (Bateman et al., 2000). In a field in which O. acuformis was predominant, cyprodinil was most effective in reducing disease and DNA of O. acuformis among nine fungicide treatments. However, effectiveness of prochloraz was only significant late in the season (Ray et al., 2004).
Benzimidazole-resistant isolates of the eyespot pathogen were first discovered in
Germany in 1975 (King & Griffin, 1985). The first failure to control eyespot of winter wheat with benzimidazole fungicides in the U.K. was reported in 1981 (King & Griffin,
1985). In a survey of 528 commercial fields in England and Wales, 37-52% of the eyespot isolates were resistant to benomyl and more O. acuformis isolates were resistant than O. yallundae (King & Griffin, 1985). Bateman et al. (1990) reported a difference in sensitivety to the same fungicides by two O. yallundae and O. acuformis isolates. In
27
1984, a survey in New Zealand found 17% of isolates were insensitive to benomyl (King et al., 1986). Sanders et al. (1986) reported that 43% of isolates containing both O. yallundae and O. acuformis isolates were carbendazim-resistant in winter wheat fields in the Netherlands. Brown et al. (1984) found carbendazim-resistant isolates in a field that had never been treated with benzimidazole fungicides. Murray (1996) reported that benzimidazole resistant isolates could be collected from fields with no record of being treated with benzimidazoles and those that had been treated with benzimidazole fungicides. Murray (1996) found that 96 and 70% of isolates collected from fields where fungicide resistance occured were resistant to benzimidazole fungicides in 1989 and
1990, respectively. He also found that 7% and 4% of resistant isolates were O. acuformis in 1989 and 1990, respectively. Both O. yallundae and O. acuformis isolates resistant to benzimidazole fungicides were found in the early 1980s (Leroux & Gredt, 1997).
Creighton et al. (1989) studied pathogenicity of carbendazim-resistant and -sensitive isolates from France and the UK and found no difference between them (Creighton et al.,
1989). Similar results were obtained by Brown et al. (1984) when they tested the pathogenicity of carbendazim-resistant isolates.
Widespread resistance to benzimidazoles resulted in their replacement with DMI fungicides, of which prochloraz was the most widely used. Prochloraz is a broad spectrum non-systemic fungicide (King et al., 1986). Resistance to prochloraz is more complex because insensitivity to fungicides is already present in the population (King et al., 1986). A survey for prochloraz resistance in New Zealand found that 26 of 31 isolates were insensitive (King et al., 1986). A survey in France when DMI were introduced demonstrated that O. acuformis was more resistant to triazole fungicides than O.
28 yallundae and both species were intrinsically sensitive to prochloraz (Leroux & Gredt,
1997). After intensive use, O. yallundae developed resistance to triazoles and O. acuformis developed resistance to prochloraz (Leroux & Gredt, 1997). Later, polymorphism of the 14α-demethylase gene (CYP51), the target gene of DMI, was detected between O. yallundae and O. acuformis (Albertini et al., 2003). However, South
African isolates, which are O. yallundae only, haven‟t shown insensitivity to carbendazim, prochloraz, propiconazole, and flusilazole, even though fungicide application is a primary control method for eyespot (Robbertse et al., 1996a). Cyprodinil is a relatively new fungicide; both O. yallundae and O. acuformis had reduced sensitivity in plots treated with cyprodinil (Babij et al., 2000). Clearly, there is a risk of resistance to cyprodinil in eyespot.
Fungicide application has a selective influence on pathogen population structure (Babij et al., 2000). Widespread resistance to the benzimidazole fungicides coincided with a shift in the predominant population from O. yallundae and O. acuformis isolates in UK and Germany in the 1980s (King & Griffin, 1985; Hollins et al., 1985;
Nicholson et al., 1991b), with an increased proportion of O. acuformis isolates throughout the cereal growing region. One explanation was selection for resistance to benzimidazole fungicides (Hollins et al., 1985). This selective influence may also affect apothecia production. Based on a survey of the sensitivity of ascospore offspring to fungicides from 1992 to 1994 in England, half of the sites had ascospores of O. yallundae and most of ascospore isolates were resistant to benomyl but sensitive to prochloraz
(Dyer & Lucas, 1995).
29
Bateman et al. (1995b) found similar results when they treated plots with carbendazim, prochloraz or carbendazim plus prochloraz every year for over nine years starting in fall 1984. They found a greater proportion of straw with apothecia of O. yallundae were from plots treated with carbendazim, and the smallest proportion from plots treated with prochloraz, with or without carbendazim. Due to the rapid selection for resistance, eyespot was more severe after treatment with carbendazim than untreated after only 3 years (Bateman, 2002). Continuing the experiments for up to 17 years, prochloraz treatment with or without carbendazim gradually lost efficacy, but no resistance was found. The predominance of O. acuformis in many plots may explain this because the selection was in favour of O. acuformis (Bateman, 2002). About equal percentages of both species were present in unsprayed and carbendazim-sprayed plots during the first five years, but the proportion of O. acuformis was more than 80% in the plots sprayed with prochloraz or a mixture of the two fungicides (Bierman et al., 2002). This result also suggested that prochloraz selected for O. acuformis. This result may be explained by O. acuformis naturally having broad range of sensitivity to prochloraz, but the sensitivity range of O. yallundae is narrow (Bateman et al., 1995a).
In Western Europe, a long-term survey for changes in sensitivity to MBC
(carbendazim or benomyl) and prochloraz in O. yallundae and O. acuformis was conducted from 1984 to 2000 (Parnell et al., 2008). Reduced sensitivity to both fungicides with differences between the species was found over 17 years. More O. yallundae isolates had reduced sensitivity to MBC whereas more O. acuformis isolates had reduced sensitivity to prochloraz (Parnell et al., 2008). O. yallundae and O.
30 acuformis have demonstrated an ability to adapt to selective pressures after developing resistance to two generations of fungicides for a long period of time (Babij, 2000).
Host Resistance to Eyespot
Although eyespot has been controlled with fungicides over the past 30 years, limiting input costs, environmental concerns, and fungicide resistance resulted in the need for alternative disease management strategies (Murray, 1996). One strategy is to use disease-resistant wheat cultivars to minimize disease development. Planting resistant cultivars can be economically beneficial to growers with the savings from not using fungicides. Disease resistance is also an environmentally friendly method compared with fungicide application.
Eyespot is the most important component of the stem base disease complex of cereals (Bateman et al., 2000). Rhizoctonia cerealis (sharp eyespot), Fusarium spp., and
Microdochium nivale are also present in this disease complex, which can make visual disease assessment difficult (Goulds & Polley, 1990; Bateman et al., 2000). In order to select for effective eyespot resistance, a screening technique is necessary that can be reliably applied to individual plants to measure disease development. Macer (1966) developed a screening technique in which a pathogen-colonized drinking-straw cylinder was placed over the coleoptile and the depth of penetration of the leaf sheaths was examined 6-8 weeks after growing in controlled conditions. This technique has been adapted in many programs screening for eyespot resistance during the seedling stage
(Kimber, 1967; Worland et al., 1988; Strausbaugh & Murray, 1989b; Mena et al., 1992).
31
Traditionally, visual assessment has been used for disease evaluation. In France,
Jahier et al. (1979) found that the eyespot pathogens produced two types of mycelia between leaf sheaths depending on susceptibility of the host. The mycelium was spotted and dark-brown in resistant varieties whereas it was abundant and black color in susceptible wheat lines. They assessed resistance by two observation methods; the number of leaf sheaths penetrated (quantitative method) and the type of mycelium
(qualitative method) (Jahier et al., 1979). Later, it was found that the qualitative method wasn‟t reliable (Doussinault et al., 1983).
Anatomy of basal stem internodes is an important component of resistance to eyespot (Murray & Bruehl, 1983). Schaffnit in 1933 found that the hypodermis of resistant wheat lines was wider than susceptible ones (Bruehl, 1983). Murray and Bruehl
(1983) reported that resistance was highly correlated with width of the hypodermis and number of hypodermal cell layers in the first elongated internode of mature wheat. The positive correlation between field resistance and penetration sites with papillae, penetrated papille, and total penetration in seedlings was found in a subsequent study
(Murray &Ye, 1986). Strausbaugh and Murray (1989a) used epidermal cell responses including papilla formation, penetration stopped in epidermal cells, and hypersensitivity to evaluate resistance of winter wheat to eyespot. They found the most reliable temperature for differentiation was at 10oC. The rating system used in their evaluation was able to distinguish highly resistant (VPM-1) and resistant (Cappelle Desprez) cultivars (Strausbaugh & Murray, 1989a). This system also correlated well with field resistance of adult plants.
32
Enzyme-linked immunosorbent assay (ELISA) was developed to detect and quantify O. yallundae and O. acuformis (Unger & Wolf, 1988; Lind, 1992; Priestley &
Dewey, 1993). Unger and Wolf (1988) tested naturally infected plant material with indirect ELISA and found that the ELISA values correlated well with eyespot disease index. Lind (1992) found that ELISA was a more reliable method to quantitatively differentiate genotypes at growth stages 60 and 75 compared to visual eyespot scoring. In further studies, they found that environments with moderate disease severity were more suitable to measure resistance than severe disease (Lind et al., 1994). A double-antibody- sandwich ELISA test was used as a screening assay on seedlings when the disease was at a presymptomatic infected phase (Priestley & Dewey, 1993).
Bunkers (1991) transformed eyespot isolates with the β-glucuronidase (GUS) reporter gene. Expression of the GUS gene was correlated with fungal biomass without affecting pathogenicity. This provided a rapid and sensitive reporter gene system to quantify pathogen growth. de la Peña and Murray (1994) developed a test for eyespot severity at the seedling stage by measuring GUS activity fluorimetrically. During the assay, 2-week-old wheat seedlings are inoculated with a GUS-transformed strain of
Oculimacula spp. and evaluated 4- to 8-weeks later (de la Peña & Murray, 1994). The
GUS values were highly correlated with different resistance levels based on field evaluation and the method is more objective and sensitive than visual disease ratings. It also reduced the time from 11 months required for field test to 2 months for screening resistance. This improved technique has been used to screen for new sources of eyespot resistant (Yildirim et al., 1995 & 2000; Cadle et al., 1997; Figliuolo et al., 1998; Cox et
33 al., 2002) and to map resistance genes (Murray et al., 1994; de la Peña et al., 1996 &
1997; Yildirim et al., 1998; Li et al., 2004 & 2005).
Nicholson et al. (1997) developed a competitive PCR assay to quantitatively assess colonization by O. yallundae or O. acuformis on seedlings. Turner et al. (2001) found that quantitative PCR was more reliable than visual disease assessment for measuring infection of eyespot at early growth stages, especially before stem extension.
However, the two assessment methods were also correlated later in the season. Turner et al. (2001) reported that the amount of pathogen DNA at early stages couldn‟t predict disease severity at later growth stages. Usually, the amount of Oculimacula DNA amplified by quantitative PCR was consistent with cultivar susceptibility to eyespot
(Bateman, et al., 2000). Susceptible cultivars had greater amounts of DNA for both O. yallundae and O. acuformis determined by quantitative PCR (Nicholson et al., 2002).
Development of quantitative PCR allowed for differentiation and quantification of different stem-base pathogens (Bateman, et al., 2000; Nicholson et al., 2002; Ray et al.,
2004). A real-time PCR assay was developed to specifically quantify the eyespot pathogens O. yallundae and O. acuformis for large numbers of samples (Walsh et al.,
2005). Meyer et al. (2011) demonstrated that the real-time PCR method was reliable in differenciating resistant and susceptible wheat genotypes 12 weeks after inoculating with
O. yallundae.
Resistance to eyespot has been investigated since the pathogen was first described. Sprague (1936) reported that the wheat relatives Aegilops ventricosa (2n=28, genome DDMvMv) and Haynaldia villosa (2n=14, VV) were highly resistant to eyespot.
These discoveries lead to the introgression of eyespot resistance gene Pch1 into common
34 wheat (Kimber, 1967; Doussinault et al., 1983). Plant breeders have incorporated eyespot resistance into winter wheat cultivars since the 1950s (Macer, 1966). Currently, there are only two genes, Pch1 and Pch2, available in the gene pool of wheat (Triticum aestivum)
(Murray, 2010).
Pch1 is the most effective eyespot resistance gene known. The gene was introgressed to wheat by cytogenetic manipulations (Kimber, 1967; Doussinault et al.,
1983). The most popular breeding line, VPM-1 (VPM = Ventricosa x Persicum x Marne), has been used extensively as a source of Pch1 in breeding programs (Doussinault et al.,
1983). Ometz crossed Ae. ventricosa with T. persicum (genome AABB) and hybrid chromosomes were doubled to produce fertile amphidiploids (AABBDDMvMv) in 1953
(Bruehl, 1983). Reported in 1967, the amphidiploid was backcrossed with hexaploid wheat „Marne‟ twice by Ecochard, and Maia selfed the 42-chromosome line for six generations to obtain a stable hexaploid line (VPM-1) and demonstrated an increased resistance level of VPM-1 to eyespot (Bruehl, 1983). However, linkage drag was reported for VPM-1 and it needed improvement in other important traits (Jones et al., 1995).
Several commercial wheat cultivars with Pch1 have been developed from VPM-1 by the USDA-ARS winter wheat breeding program at Pullman, WA for use in the US
PNW. Madsen was the first cultivar with resistance to eyespot released in 1988 (Allan et al., 1989). It has good yield potential and broad spectrum disease resistance and consequently, was widely grown in the PNW and one of top two soft white winter wheat cultivars in Washington for about two decades (Murray, 2010). Hyak, Coda, and Chukar soft white club wheats, were released for eyespot resistance in 1989, 1998, and 2001,
35 respectively (Allan et al., 1990; Allan et al., 2000; Garland Campbell et al., 2005), and have been popular varieties in the PNW (Jones et al., 1995).
Roazon, a French cultivar developed from the cross VPM-1 x Moisson by
Doussinault et al. (1974), possesses the cytoplasm of Ae. ventricosa and was the first commercial wheat with VPM-1 resistance and good agronomic value in France (Jahier et al., 1989). However, Roazon was only grown briefly in France and never available in the
UK commercially (Hollins et al., 1988). Rendezvous, a commercial winter wheat with eyespot resistance derived from VPM-1 and Cappelle Desprez, was released in 1986 and widely grown in Europe (Hollins et al., 1988). This cultivar was more resistant than
VPM-1 and significantly more resistant than many other commercial cultivars because it is thought to possess both Pch1 and Pch2 (Hollins et al., 1988). Cultivar 92M166, a homozygous resistant spring wheat line developed from VPM-1 adapted to the South
African growing environment, was significantly more resistant to eyespot than other local spring wheat varieties (Robbertse et al., 1996b).
It was first suggested by Delibes et al. (1977) that eyespot resistance in VPM-1 was controlled by a single genetic locus. Jahier et al. (1979) used monosomic analysis and concluded that resistance to eyespot in Roazon was associated with chromosome 7D.
Jahier et al. (1989) also developed the intervarietal substitution lines for chromosome 7D and cytoplasm of Roazon into Courtot (very susceptible to eyespot) to analyze the effectiveness of resistance in both seedlings and adult plants. They found that the high level resistance was carried on wheat chromosome 7D from Ae. ventricosa and there was no cytoplasmic effect. Gale et al. (1984) found that α-amylase couldn‟t be used as a marker for Pch1 because the two characters segregated independently in F3 families of
36
VPM-1 crossed with a susceptible line. However, they suggested that the gene conferring eyespot resistance in VPM-1 was on the distal end of chromosome 7D based on the previous results showing the α-amylase locus was near the centromere (Gale et al., 1984).
Through Mendelian analysis of inheritance of eyespot resistance in parental, F1, F2, and backcross populations, Strausbaugh & Murray (1989b) concluded that a single dominant gene controlled resistance in VPM-1. Allan and Roberts (1991) confirmed this result when they studied a population with VPM-1 as one of the parents. McMillin et al. (1986) showed a close association between the resistance gene in VPM-1 with EP-D1b, an endopeptidase isozyme allele from Ae. ventricosa chromosome 7DL. Mena et al. (1992) found that eyespot resistance was not a product of EP-D locus. Using EP-D1b as a marker for Ae. ventricosa-derived eyespot resistance, Pch1 was mapped to the distal end of the long arm of chromosome 7D as a single dominant gene (Worland et al, 1988).
Kimber (1967) developed wheat line TV1F-3H-9 with resistance to eyespot from
Ae. ventricosa. T. tugidum (AABB), a tetraploid, was used for the initial hybridization resulting in the amphiploid. The resistance of TV1F-3H-9 wasn‟t as effective as Ae. ventricosa but it was much greater than Cappelle Desprez (Kimber, 1967). They concluded that the genetic control of eyespot resistance was due to a single locus in the D genome of TV1F-3H-9. Another wheat line, H-93-70, which was also obtained using
Triticum turgidum (AABB) as a „bridge‟ species, has effective resistance to eyespot from
Ae. ventricosa (Doussinault et al., 1983). The biochemical markers endopeptidase EP-1 and EP-D1b located the gene Pch1 in H-93-70 on the long arm of 7D (Vahl & Müller,
1991; Mena et al., 1992).
37
PCR-based markers have been developed for selecting eyespot resistance gene
Pch1. RFLP marker, Xpsr121, was linked to Pch1 and mapped at the same location as
EP-D1b locus in a hexaploid wheat with VPM-1 7D chromosome (Chao et al., 1989). An
AFLP-derived microsatellite marker XustSSR2001-7DL linked to Pch1 and EP-D1b was identified with 2 cM between it and EP-D1 (Groenewald et al., 2003). However, EP-D1b was more effective than XustSSR2001-7DL as a marker for Pch1 (Santra et al., 2006).
Leonard et al. (2008) identified three sequence-tagged-site (STS) markers (Xorw1,
Xorw5, and Xorw6) and three microsatellite markers (Xwmc14, Xbarc97, and Xcfd175) completely linked to Pch1. Chapman et al. (2008) also found close linkage between Pch1 and Xwmc14, Xbarc97, and Xpsr121 in a different mapping population. Lind (1999) investigated the effect of Pch1 on resistance to eyespot at four different growth stages and found that Pch1 had a stronger effect in early growth stages than at the adult stage.
Recently, it was found that Pch1 conferred resistance to both eyespot pathogens at the seedling stage (Burt et al., 2010).
Pch2 is present in the French variety Cappelle Desprez (Hollins et al., 1988). Law et al. (1988) stated that Vincent et al. (1952) found Cappelle Desprez had less infection by eyespot than other cultivars in field trials. Batts and Fiddian (1955) also noticed that
Cappelle Desprez was less affected in winter variety trials when eyespot was severe and was the most resistant variety to eyespot in further experiments. Although Pch2 is less effective than Pch1 (Johnson, 1992), Cappelle Desprez was extensively grown and officially recommended in the UK from the 1950s to the 1970s (Silvey, 1978); its resistance has been transferred to many other wheat varieties (Hollins et al., 1988). Use of Cappelle Desprez and derived cultivars resulted in eyespot becoming relative
38 unimportant in the 1960s (Bateman & Jenkyn, 2001). The moderate resistance of
Cappelle Desprez to eyespot had remained durable for nearly 30 years (Muranty et al.,
2002). Cappelle Desprez also has resistance to Puccinia striiformis (yellow rust, or stripe rust) and its resistance remained useful in the period of extensive use of Cappelle Desprez for eyespot (Johnson, 1984).
Law et al. (1976) found evidence for eyespot resistance conferred by genes on chromosomes 1A, 2B, 5D, and 7A, with a major component on chromosome 7A of
Cappelle Desprez. In addition to the main resistance detected on 7D, intermediate resistance factors were found on chromosomes 2B, 5D, and 7A of Roazon during monosomic analysis (Jahier et al., 1979). This is thought to occur because the parent of
Roazon had common ancestors with Cappelle Desprez. These studies confirmed that genetic control of resistance in Cappelle Desprez was polygenic. Doussinault and Dosba
(1977) suggested that resistance to eyespot in Cappelle Desprez was quantitative.
Quantitative resistance at the adult stage was also found in wheat genotypes with
Cappelle Desprez in their pedigree (Lind, 1999). Strausbaugh and Murray (1989b) found one semidominant gene conferring resistance in Cappelle Desprez.
Koebner and Martin (1990) found a positive association between eyespot resistance and isozyme marker Ep-A1b and confirmed the main component of resistance was located on 7A. de la Peña et al. (1996) designated the gene on chromosome 7A as
Pch2 based on mapping studies. Using chromosome 7A homozygous recombinant substitution lines, they reported that Pch2 was not linked to Ep-A1b tightly, and found the order of Pch2-Xpsr121-Ep-A1b on chromosome 7A (de la Peña et al., 1996). This relationship was confirmed by Chapman et al. (2008). Furthermore, de la Peña et al.
39
(1997) mapped Pch2 to the long arm of chromosome 7A of wheat using an RFLP linkage map and found RFLP markers Xcdo347 and Xwg380 flanked Pch2 by 29.8 cM. Since
Pch2 also was located on the distal portion of the long arm, which is a similar position to
Pch1 on 7DL, they suggested that Pch1 and Pch2 were homoeoloci (de la Peña et al.,
1997). Muranty et al. (2002) reported that the gene on Cappelle Desprez chromosome 7A was resistant at the seedling stage and an additional gene on chromosome 5A was responsible for eyespot resistance at the adult stage. This new gene on chromosome 5A showed consistent global resistance (combining resistance at the seedling and adult stage) in their experiments. Chapman et al. (2008) linked SSR markers Xwmc346, Xwmc525, and Xcfa2040 closely to Pch2 on chromosome 7A against O. acuformis. Consequently, the suggestion of Pch1 and Pch2 being homeoloci by de la Peña et al. (1997) was strongly supported by Chapman et al. (2008).
Using a cDNA-AFLP platform, Chapman et al. (2009) identified two fragments,
4CD7A8 and 19CD7A4, as candidate genes for Pch2 resistance to O. acuformis. Burt et al. (2010) found that Pch2 was significantly more effective against O. acuformis than O. yallundae at the seedling stage. Furthermore, Burt et al. (2011) reported that a resistance gene on chromosome 5A conferred effective resistance to both O. acuformis and O. yallundae at both seedling and adult stages. A single major QTL closely linked to SSR marker Xgwm639 was detected on the long arm of chromosome 5A during the same study (Burt et al., 2011).
The eyespot resistance of Cappelle Desprez didn‟t provide sufficient resistance under severe eyespot conditions (Macer, 1966) and yield loss occurred in cultivars with resistance from Cappelle Desprez (Hollins et al., 1988). Pch1 is very effective in limiting
40 eyespot development, but it does not protect wheat completely (Jones et al., 1995).
Madsen lacks the competitive yield potential of recently released soft white wheat cultivars (Jones et al., 2010). Since neither Pch1 nor Pch2 provides complete resistance to eyespot when used alone (Cadle et al., 1997), it is necessary to enhance the effectiveness of resistance.
Huguet-Robert et al. (2001) transferred Pch1 to chromosome 7A of durum wheat by homoeologous recombination to increase Pch1 copy number. Other studies have attempted to pyramid Pch1 with the multiple eyespot resistance genes in Cappelle
Desprez. The performance of Rendezvous in the field demonstrated an enhanced level of resistance due to combination of Ae. ventricosa and Cappelle Desprez genes (Hollins et al., 1988). Lind (1999) suggested that a high and long-lasting resistance could be obtained by combining Pch1 and the quantitative trait in Cappelle Desprez since their effects were at different growth stages. Allan and Roberts (1991) crossed VPM-1 and
Cerco in which the resistances were genetically different and found some progeny exceeded the parents for eyespot resistance. Although the above sources have been effectively protecting wheat from eyespot disease, additional resistance genes are desired for incorporation into adapted wheat cultivars to improve the effectiveness and diversity of disease resistance genes.
Considerable attention has been given to the wild relatives of wheat as sources of resistance genes and numerous traits have been transferred from them into common wheat (Sears, 1983; Jiang et al., 1994; Jones et al., 1995). Most of the research to identify eyespot resistance in wild wheat species has been done at Washington State University.
In addition to discovering potent resistance to eyespot in Ae. ventricosa, Sprague (1936)
41 reported another wild relative of wheat, Dasypyrum villosum (L.) Candargy (syn.
Haynaldia villosa L.), was also highly resistant to eyespot. Sixty years later, Dasypyrum villosum chromosome 4V was identified containing the resistance gene(s) against O. yallundae using D. villosum disomic addition lines at the seedling stage (Murray et al.,
1994). The single dominant gene on D. villosum chromosome 4V (Pch3 or PchDv) was mapped to the distal part of the long arm flanked by two RFLP markers, Xcdo949 and
Xbcd588 in a wheat background (Yildirim et al., 1998). However, Pch3 hasn‟t been transferred into commercial wheat cultivars. Yildirim et al. (2000) reported that all 219 accessions of D. villosum evaluated were resistant to O. yallundae and 72% of them were more resistant than VPM-1. Uslu et al. (1998) studied the resistance of D. villosum chromosome addition lines to both O. yallundae and O. acuformis and found that D. villosum was more resistant to O. yallundae than O. acuformis. They also found that resistant factors to both species were on chromosomes 1V, 2V, and possibly 3V.
Chromosome 4V and 5V of D. villosum have resistance genes for O. yallundae and O. acuformis, respectively (Uslu et al., 1998). This was the first report showing differential resistance to these pathogens.
Yildirim et al. (1995) reported that 45% of 279 T. tauschii (syn. Aegilops squarrosa, 2n=14, DD) accessions were resistant to eyespot. Assefa and Fehrmann
(1998) reported 10% of 160 T. tauschii (Ae. tauschii) accessions immune to moderate resistance reactions. Cadle et al. (1997) found T. monococcum (2n=14, AA) was a new source of resistance to eyespot that had potential to be more effective than Pch1 or Pch2.
Recently, Burt et al. (2010) demonstrated the T. monococcum accessions had significantly different resistance to both O. yallundae and O. acuformis. Three of eight
42 tetraploid wheat species, T. durum, T. dicoccoides, and T. turanicum (2n=28, AABB), were suggested for further genetic analysis because of their high resistance to eyespot
(Figliuolo et al., 1998). Börner et al. (2006) reported that 3% (42) and 10% (412) of
Triticum accessions were resistant eyespot under conditions of natural infection at the seedling stage and the adult stage, respectively. Although more than 50% of the 489
Aegilops accessions scored highly resistant to eyespot at both stages under natural infection, only 17% were still resistant under artificial infection (Börner et al., 2006).
Moderate resistance to eyespot was found in an Ae. kotschyi (2n=28, genome
UUSvSv) accession by Freier (Thiele et al., 2002). Later, eyespot resistant lines were selected in the progeny of the cross and backcross between this accession and wheat
(Thiele et al., 2002). Athough those lines do not have resistance as effective as Pch1, they had similar resistance, and greater yield, than Cappelle Desprez under disease conditions.
Meyer et al. (2008) used a doubled haploid population to study eyespot resistance in Ae. kotschyi. They concluded that different lines carried different resistance genes to O. yallundae; however, they weren‟t able to detect the location of resistance in Ae. kotschyi.
Twenty-four perennial wheat germplasm lines were tested for resistance to three major wheat diseases in the PNW, eyespot, Cephalosporium stripe, and wheat streak mosaic. Five perennial lines plus two perennial wheatgrasses Thinopyrum ponticum
(2n=70, JJJJsJs) and Th. intermedium (2n=42, StJJs), were resistant to all three diseases and 13 lines were resistant to O. yallundae (Cox et al., 2002). Since perennial wheat lines are the result of hybridization of annual wheat with either Th. ponticum or Th. intermedium, new resistance sources to O. yallundae were suggested in Th. ponticum and
Th. intermedium (Cox et al., 2002). Using cytogenetic tools, Li et al. (2004) identified a
43 single, partially dominant gene for resistance to O. yallundae on chromosome 4J of Th. ponticum. Subsequently, Li et al. (2005) located the genetic control of the resistance to O. yallundae on the short arm of chromosome 4Js of Th. intermedium. These are the first reports of eyespot resistance from the J and Js genomes. The above identified sources for eyespot resistance are not readily available in commercial wheat cultivars now. The introgression of eyespot resistance from wild species into hexaploid wheat needs to be performed. So they are available to breeding programs.
Objectives
Aegilops longissima Schweinf. & Muschl. (2n = 2x = 14, SlSl), a diploid species in the section Sitopsis of Aegilops L. (van Slageren, 1994), is a distant relative of wheat and potential donor of genes for wheat cultivar improvement, including disease resistance
(Friebe et al., 1993). Ae. longissima has not been reported to be resistant to eyespot. The overall goal of this project is: 1) to identify potential new sources of genetic resistance to
O. yallundae and O. acuformis from Ae. longissima; and 2) to understand the genetic control of resistance and map gene(s) in the genome by developing a genetic linkage map. Wheat microsatellite markers (SSR) will be used to provide linked markers for possible transfer of resistance genes into a suitable wheat background for use in breeding programs.
44
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CHAPTER TWO
IDENTIFYING NEW SOURCES OF RESISTANCE FOR EYESPOT OF WHEAT IN AEGILOPS LONGISSIMA
Introduction
Eyespot is an economically important disease of winter wheat in the US Pacific
Northwest (PNW) and other areas of the world with cool, wet winters (Murray, 2010).
Winter wheat occupies about 80% of the total PNW wheat acreage (WGA, 2009). Yield losses caused by eyespot can be up to 50% in commercial wheat fields when eyespot is severe (Murray, 2010). Eyespot is caused by the soilborne fungi Oculimacula yallundae
(syn: Tapesia yallundae, Wallwork & Spooner) Crous & W. Gams and O. acuformis
(syn: T. acuformis, Boerema, R. Pieters & Hamers) Crous & W. Gams (Crous et al.,
2003). These fungi were respectively known as the W- and R- pathotypes of
Pseudocercosporella herpotrichoides (Fron.) Deighton prior to identification of the telemorph and their separation into distinct species (Lucas et al., 2000).
Both species affect the stem base of wheat and produce elliptical lesions that result in lodging. Foliar fungicides have played a major role in control of eyespot, but the limited selection of registered fungicides in the PNW, as well as resistance in
Oculimacula spp. to carbendazim fungicides have resulted in the need for alternative disease management strategies (Murray, 1996). Planting disease-resistant cultivars is the most economical and effective strategy to control eyespot.
Currently, two resistance genes, Pch1 and Pch2, are available in the commercial wheat genome. Pch1 was transferred from Aegilops ventricosa Tausch (2n=28,
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DDMvMv) into breeding line VPM-1 (Doussinault et al., 1983) and is a single dominant gene located near the distal portion of chromosome 7DL (Worland et al., 1988). Pch1 is effective in limiting eyespot development, but does not protect wheat completely (Jones et al., 1995). Pch2, from the French variety Cappelle Desprez, acts as a single partially dominant gene (Strausbaugh & Murray, 1989). Law et al. (1976) found that chromosomes 1A, 2B, 5D, and 7A of Cappelle Desprez influenced eyespot resistance, with a major component on chromosome 7A. Later, de la Pena et al. (1996) mapped the gene to the distal portion of the long arm on 7A.
Several wheat cultivars with Pch1 have been developed. Madsen is a cultivar that was released in 1988 (Allan et al., 1989), and was widely grown in the PNW for about two decades (Murray, 2010). Cappelle Desprez was grown extensively from the 1950s to
1970s in the UK and its resistance has been transferred to many other wheat varieties
(Hollins et al., 1988). Although the resistance of Cappelle Desprez to eyespot has been durable, Pch2 is less effective than Pch1 (Johnson, 1992). More resistance genes are desired for incorporation into adapted wheat cultivars to improve effectiveness and broaden the genetic diversity of disease resistance.
The common wheat gene pool contains little resistance to soilborne pathogens (Li et al., 2008). Wild relatives of wheat are useful sources of resistance genes (Jones et al.,
1995). Wild relatives of wheat have been considered as sources of resistance genes for many diseases, and genes conferring numerous traits have been transferred from wild species into common wheat (Sears, 1983; Ceoloni et al., 1988; Jiang et al., 1994). Pch1 was the first successful example of using an alien gene for eyespot resistance in commercial wheat (Allan et al., 1989). Murray et al. (1994) found new resistance against
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O. yallundae from Dasypyrum villosum (L.) Candargy (syn. Haynaldia villosa L.)
(2n=14, VV) on chromosome 4V. Yildirim et al. (1998) mapped a single dominant gene to the distal portion of D. villosum chromosome 4VL with RFLP markers. However, this gene hasn‟t been transferred into commercial wheat cultivars. Other sources of resistance to eyespot have been identified in Triticum tauschii (syn. Aegilops squarrosa, 2n=14,
DD) (Yildirim et al., 1995), T. monococcum (2n=14, AA) (Cadle et al., 1997; Burt et al.,
2010), T. dicoccoides (2n=28, AABB) (Figliuolo et al., 1998), Ae. Kotschyi (2n=28,
UUSvSv) (Thiele et al., 2002), Thinopyrum ponticum (2n=70, JJJJsJs) (Cox et al., 2002;
Li et al., 2004), and Th. intermedium (2n=42, StJJs) (Cox et al., 2002; Li et al., 2005).
Aegilops longissima (2n=14, SlSl) Schweinf. & Muschl. is a diploid species in the section Sitopsis of Aegilops L. (van Slageren, 1994) that has been used as a donor of numerous genes for wheat improvement, including disease resistance (Friebe et al.,
1993). Ecker et al. (1990) identified Ae. longissima accessions that were highly resistant to the Septoria glume blotch of wheat. The powdery mildew resistance gene Pm13 was mapped to the short arm of chromosome 3Sl of Ae. longissima and introgressed into wheat cultivar Chinese Spring (Ceoloni et al., 1992; Cenci et al., 1999). Anikster et al.
(2005) reported that Ae. longissima carried genes for resistance to stripe rust, leaf rust, and stem rust of wheat. Prior to this study, Ae. longissima has not been examined as a source of resistance to eyespot.
It was once widely accepted that Ae. speltoides was the donor of the B genome in bread wheat (Sarkar et al., 1956; Riley et al., 1958). Feldman (1978) observed that there were more pairings between chromosomes of B genome with Ae. longissima than with
Ae. speltoides. This evidence shows that Ae. longissima is more closely related to the B
66 genome than Ae. speltoides. Feldman (1978) also found that the Sl genome of Ae. longissima paired primarily with the B genome of T. aestivum, but relatively less with the
A and D genomes. Kota et al. (1986) reported that the heterohomologous 6Bl (=6Sl) chromosome of Ae. longissima substituted for chromosome 6B of T. aestivum. Naranjo
(1995) showed that Ae. longissima chromosomes 1Sl, 2Sl, 3Sl, 5Sl and 6Sl paired with wheat chromosome groups 1, 2, 3, 5 and 6, respectively. Zhang et al. (2001) constructed a comparative genetic map of the Ae. longissima genome and found that colinearity was conserved between 1Sl, 2Sl, 3Sl, 5Sl and 6Sl and wheat chromosomes 1D, 2D, 3D, 5D and 6D, respectively. Mello-Sampayo (1971) also demonstrated that Ae. longissima could actively promote homoeologous pairing in crosses with T. aestivum. All of the above studies imply the crossability of Ae. longissima with wheat and the possibility of transferring useful genes into adapted wheat cultivars to improve wheat production.
The objectives of this research were to identify potential new sources of genetic resistance to O. yallundae and O. acuformis from Ae. longissima and find genomic locations containing genetic control of resistance to these pathogens. Since the different plant responses to O. yallundae and O. acuformis were observed in a preliminary screening (data not shown), the two species were tested separately in order to discover whether genes that confer resistance to O. yallundae and O. acuformis in Ae. longissima are independent.
Materials and Methods
Plant materials. Forty Ae. longissima accessions obtained from the USDA
National Small Grains Collection (NSGC) were screened for eyespot resistance; all were
67 winter habit and collected from central Israel with the exception of PI 542196 from Izmir,
Turkey and PI 330486 from an unknown source. Eighty nine Ae. longissima addition or substitution lines in Chinese Spring or Selkirk background were obtained from the Wheat
Genetics and Genomic Resources Center (WGGRC) at Kansas State University. After increasing the seed numbers, 83 addition or substitution lines produced sufficient seeds to be tested for eyespot resistance. Information on the parents of these lines was not available, but is assumed to be from accessions maintained in the WGGRC. The Ae. longissima addition lines include 16 Chinese Spring disomic additions (DA), 13 Chinese
Spring ditelosomic additions (DtA), and 7 Selkirk disomic additions. The Ae. longissima substitution lines include 21 Chinese Spring disomic substitutions (DS), 17 Chinese
Spring ditelosomic substitutions (DtS), and 9 Chinese Spring double ditelosomic substitutions (dDtS). Seven wheat varieties were used as controls. Madsen and Cappelle
Desprez, which contain Pch1 and Pch2, respectively, are winter wheat cultivars that are resistant to eyespot. Both the winter wheat cultivar Hill 81 and the spring wheat cultivar
Chinese Spring are susceptible to eyespot. The spring wheat cultivars „Selkirk‟ and
„Opata‟ were also tested to confirm their susceptibility to eyespot. The resistant breeding line VPM-1 was included in the experiments as well.
Growth chamber experiments. Ae. longissima accessions were screened for resistance to eyespot by inoculating them with O. yallundae and O. acuformis in separate experiments. Ae. longissima addition or substitution lines were also evaluated for differential reactions to O. yallundae and O. acuformis in separate experiments. Due to the limitation in seed quantity, O. acuformis for Ae. longissima addition or substitution lines were only tested once, but other experiments were repeated. All experiments were
68 arranged in a randomized complete block (RCB) design. There were 6 blocks and 12 plants per line in each experiment for Ae. longissima accessions, and 3 blocks and 6 plants per line in each experiment for Ae. longissima addition or substitution lines. Fifty pots were randomly arranged in a plastic flat without drain holes (54 x 27 x 6 cm) as a block because the humidity between blocks within a growth chamber differred.
Seeds were imbibed on moist filter paper in Petri dishes for 4 days at 4oC and then kept at room temperature for 2-3 days. Sprouted seeds were planted into 6.4 cm square plastic pots with commercial Sunshine Potting Mix#1/LC1 (SunGro Horticulture,
Bellevue, WA) and fertilized with Osmocote (14-14-14, w/v) (The Scotts Company LLC,
Marysville, OH). Two seeds were planted in each pot as subsamples. The flats were placed in the growth chambers at 15/13oC with a 12 h photoperiod. Relative humidity was maintained from 98 to 100%. All plants of one experiment were kept in one growth chamber and the flats were rotated every 2-3 days.
Preparation of inoculum. One week after planting, a 3.3 cm-long split drinking straw piece was put around the coleoptile of each plant at the soil surface. When the second leaf was half the size of the first leaf, at about 2-weeks-old, seedlings were ready for inoculation. Four O. yallundae isolates (tph8934-5-61, tph8934-5-62, tph8934-5-68, and tph8934-5-70) and five O. acuformis isolates (tph98-1-54AA, tph98-1-54SS, tph98-
2-34D, tph98-2-34E, and tph98-2-34M) transformed with β-glucuronidase (GUS) reporter gene were used in all experiments. The method to test eyespot severity at the seedling stage by measuring the GUS activity fluorimetrically was modified following the GUS assay developed by de la Peña and Murray (1994).
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Inoculum was produced by growing mycelia on potato dextrose agar (PDA)
(Difco Laboratories, Detroit, MI) plates for six weeks at room temperature before inoculation. One square centimeter PDA plugs containing mycelia were chopped into small pieces and spread onto 1.5% water agar (WA) (Sigma Life Science, St. Louis, MO) plates, and 3 ml sterilized distilled water was added. The plates were sealed with Parafilm
(Pechiney Plastic Packaging, Menasha, WI) and placed in an incubator with near UV light at 13oC for at least two weeks to produce enough conidia.
On the day of inoculation, conidia were collected by scraping WA plates with a bent glass rod and counted with a hemacytometer. A slurry was made by blending 1.5% fresh WA and water. Conidia of O. yallundae or O. acuformis were added to give the final concentration of conidia. The conidial concentration of O. yallundae was 3.4 x 105 and 2.0 x 105 per ml in the first and second experiments of Ae. longissima accessions, respectively. The concentration of O. acuformis was 2.4 x 105 and 2.1 x 105 per ml in the first and second experiments of Ae. longissima accessions, respectively. In studies of Ae. longissima addition or substitution lines, the concentration of O. yallundae was 2.0 x 105 and 3.0 x 105 per ml in the first and second experiments, respectively. Concentration of conidia of O. acuformis was 2.5 x 105 per ml in the only one test. During inoculation, 250
µl of the slurry was pipetted into the straw collar around each plant stem base. The same amount of inoculum was added again one or two days later.
Disease evaluation and analysis of GUS activity. Eight weeks after inoculation, approximately between growth stage 23 to 25 (Zadoks et al., 1974), plants were sampled and a 3 cm section of the whole stem was removed from around the inoculation site. The section of stem was briefly washed with tap water to remove soil. Visual disease ratings
70 were performed on a 0 to 4 scale (Yildirim, et al., 2000), where 0 = no symptoms
(healthy), 1 = a lesion only on the first leaf sheath, 2 = a lesion on the first leaf sheath and a small lesion on the second leaf sheath, 3 = a lesion covering the first leaf sheath and up to half of the second sheath, and 4 = a lesion covering the first and second sheaths (nearly dead). Only the main tiller of wheat controls and wheat genetic stocks were evaluated.
Due to the smaller size of Ae. longissima accessions, all the tillers (2 to 4) of each plant were evaluated as a whole. The stems were then wrapped with paper towels and frozen at
-20oC for GUS assay. GUS activity in stems was used as a surrogate measurement of fungal colonization on stems.
Frozen stems were placed in a leaf squeezer (Ravenel Specialties Company,
Seneca, SC) and 2.5 ml GUS extraction buffer (50 mM NaHPO4, pH 7.0, 5 mM dithiothreitol, 10 mM Na2EDTA, 0.1% sodium lauryl sarcosine, and 0.1% Triton-100) was added to each sample. About 1 ml of sap was collected in an Eppendorf tube placed on ice and frozen at -20oC until GUS activity was measured. GUS activity was determined by adding 50µl extract with 40 µl 10 mM fluorescent substrate 4- methylumbelliferyl β-D-glucoside (MUG) (Sigma Life Science, St. Louis, MO) in a 1.2 ml polypropylene microtube (USA scientific, Ocala, FL), and then incubated at 37oC for
1 h. The reaction was stopped by adding 1 ml stop buffer (0.2 M sodium carbonate) to each tube. Then, 200 µl of each sample was transferred to the well of 96-well black microtiter plate (Greiner Bio-One, Monroe, NC). Two wells were used for each sample.
In each plate, methylumbelliferone (MU) standards (Sigma Life Science, St. Louis, MO) and samples of Madsen were included as controls. The fluorescence intensity of MU was measured in a Molecular Devices SpectraMax M2 microplate reader (Molecular Devices
71
Co., Sunnyvale, CA). GUS scores, were expressed as the log10 transformed ratio
[log10(x/resistant control) +1] of GUS activity of an individual accession (x) compared to the activity of resistant control (Madsen). Thus, resistant accessions had GUS scores that were less or not significantly (P > 0.05) greater than Madsen (1.0) and susceptible accessions had GUS scores that were significantly (P < 0.05) greater than Madsen.
Statistical analysis. Homogeneity of variances of the repeated tests was tested by the F-ratio of the larger error variance to the smaller error variance (Gomez and Gomez,
1984). If the variances were homogeneous, data from the two experiments will be combined. Statistical analysis was conducted with SAS Version 9.2 (SAS Institute Inc.,
Cary, NC). Analysis of variance of individual or combined experiments and standard deviation of each accession were carried out using PROC GLM on the visual disease rating and GUS score. Pearson correlation coefficients between the visual disease rating and GUS score of combined experiments were estimated with PROC CORR. Dunnett‟s
T-test was used to compare the mean of each accession with the mean of Madsen
(resistant control) at the 95% significance level.
Results
Reaction of Ae. longissima accessions to O. yallundae. The error variances between the two experiments were not significantly (P > 0.05) different for visual disease ratings or GUS scores (Gomez and Gomez, 1984); therefore, homogeneity of variance can be established and data from the two experiments were combined for analysis of variance. There were significantly different reactions among Ae. longissima accessions to
O. yallundae (P < 0.0001).
72
The resistant controls, VPM-1, Madsen and Cappelle Desprez, had visual ratings of 0.4, 0.5 and 1.6, respectively. The susceptible controls Hill 81 and Chinese Spring had visual ratings of 3.7 and 3.2, respectively. Spring wheat cultivars Opata and Selkirk had visual ratings of 3.3 and 3.6, respectively. Visual ratings of Ae. longissima accessions ranged from 1.3 to 3.6 (Table 1). None of the accessions had ratings less than Madsen.
However, five accessions had ratings less than Cappelle Desprez. Twenty-two accessions
(55%) had visual ratings equal or lower than 2.4, which was not significantly (P > 0.05) greater than Madsen.
GUS scores and visual ratings were significantly correlated (r = 0.87, P < 0.0001).
GUS scores of accessions ranged from 0.9 to 1.7. Eighteen accessions (45%) had GUS scores less or not significantly (P > 0.05) greater than Madsen (GUS scores < or = 1.2)
(Table 1). Only one line had significantly greater visual rating than Madsen. This line was not categorized as either resistant or susceptible lines. Therefore, seventeen accessions (43%) were classified as resistant to O. yallundae because both GUS scores and visual ratings were less or not significantly (P > 0.05) greater than Madsen. Twenty- two of them were considered to be susceptible to O. yallundae. The frequency distribution of GUS scores demonstrated variation in resistance to O. yallundae among
Ae. longissima accessions (Figure 1). Cappelle Desprez had GUS score of 1.1, which was close to VPM-1 (0.9) and Madsen (1.0). Hill 81 and Chinese Spring had GUS scores of
1.7 and 1.6, respectively. Opata and Selkirk were susceptible to O. yallundae with GUS scores of 1.5 and 1.8, respectively. Six accessions had GUS scores less than or equal to
Cappelle Desprez and one accession had a GUS score of 0.9, which was less than
Madsen.
73
Table 1. GUS scores and disease visual ratings of 40 Aegilops longissima accessions to Oculimacula yallundae and O. acuformis
O. yallundae O. acuformis Accessions GUS Ratings Reactiona GUS Ratings Reactiona PI 604140 0.9 1.3 R 1.2 2.0 R PI 604116 1.0 1.3 R 1.3 2.0 R PI 604108 1.1 1.3 R 1.4 2.3 S PI 604136 1.1 1.5 R 1.2 2.3 R PI 604137 1.1 1.6 R 1.1 1.7 R PI 604128 1.1 2.4 R 1.4 2.9 S PI 542196 1.2 1.7 R 1.3 2.0 R PI 604126 1.2 1.8 R 1.3 2.2 R PI 604114 1.2 1.9 R 1.5 2.9 S PI 604119 1.2 1.9 R 1.0 2.0 R PI 604109 1.2 2.0 R 1.4 2.7 S PI 604112 1.2 2.0 R 1.2 2.3 R PI 604125 1.2 2.1 R 1.2 2.3 R PI 604133 1.2 2.1 R 1.3 1.9 R PI 604141 1.2 2.1 R 1.3 2.1 R PI 604127 1.2 2.2 R 1.3 2.1 R PI 604104 1.2 2.3 R 1.2 1.9 R PI 604134 1.2 2.6 ? 1.2 2.5 ? PI 604105 1.3 2.0 S 1.4 1.9 S PI 604142 1.3 2.7 S 1.5 3.3 S PI 604118 1.3 2.8 S 1.4 2.8 S PI 604138 1.3 2.8 S 1.1 2.2 R PI 604131 1.3 3.3 S 1.2 2.5 ? PI 604143 1.4 2.1 S 1.5 2.8 S PI 604107 1.4 2.3 S 1.4 1.8 S PI 604123 1.4 2.3 S 1.5 2.9 S PI 604135 1.4 2.4 S 1.3 1.7 R PI 604110 1.4 2.7 S 1.4 2.5 S PI 604106 1.4 2.8 S 1.5 3.4 S PI 604144 1.4 2.8 S 1.2 2.0 R PI 604103 1.4 3.0 S 1.3 2.1 R PI 604122 1.4 3.0 S 1.4 2.8 S PI 604129 1.4 3.2 S 1.5 2.8 S PI 604130 1.4 3.2 S 1.2 2.1 R PI 604115 1.5 2.7 S 1.4 2.3 S PI 604139 1.5 3.3 S 1.3 2.4 R PI 604124 1.6 3.0 S 1.4 2.7 S PI 604111 1.6 3.3 S 1.5 3.0 S PI 330486 1.6 3.6 S 1.5 3.7 S PI 604117 1.7 3.1 S 1.6 3.2 S Wheat VPM-1 (R) 0.9 0.4 R 0.9 0.5 R Madsen (R) 1.0 0.5 R 1.0 0.9 R CDb (R) 1.1 1.6 R 1.0 0.7 R Hill 81 (S) 1.7 3.7 S 1.3 2.6 ? CSb (S) 1.6 3.2 S 1.4 2.6 S Opata 1.5 3.3 S 1.2 1.8 R Selkirk 1.8 3.6 S 1.5 3.1 S
74
Table 1. continued a Data sorted by GUS scores of O. yallundae. R = resistant, GUS scores and visual ratings are not significantly greater than Madsen (P > 0.05) based on Dunnett‟s T-test. S = susceptible, GUS scores are significantly (P < 0.05) greater than Madsen. ? = GUS scores are not significantly (P > 0.05) greater than Madsen but visual ratings are. These lines were not categorized as either resistant or susceptible lines. Accessions in bold are resistant to both pathogens. The GUS score and visual rating of each line was the mean of 24 plants in two experiments. b CD = Cappelle Desprez; CS = Chinese Spring.
75
Figure 1. Reaction of 40 Aegilops longissima accessions to Oculimacula yallundae and O. acuformis. Dark bars are lines that are not significantly (P > 0.05) greater than Madsen and light bars are lines that are significantly (P < 0.05) greater than Madsen based on Dunnett‟s T-test. The GUS score of each line was the mean of 24 plants in two experiments.
76
Reaction of Ae. longissima accessions to O. acuformis. Homogeneity of error variance was confirmed at the 95% significance level for visual disease ratings and GUS scores, therefore, data from the two experiments were combined for the analysis of variance. Reactions among Ae. longissima accessions to O. acuformis were significantly different (P < 0.0001 for both GUS scores and visual ratings).
VPM-1, Madsen and Cappelle Desprez had visual ratings of 0.5, 0.9 and 0.7, respectively. Hill 81 and Chinese Spring had the same visual ratings of 2.6. Opata and
Selkirk reacted significantly differently to O. acuformis with visual ratings of 1.8 and 3.1, respectively. Visual ratings of the 40 Ae. longissima accessions ranged from 1.7 to 3.7
(Table 1). None of accessions had visual ratings less than the resistant controls. Twenty- three accessions (58%) had visual ratings that were equal or not significantly (P > 0.05) greater than Madsen (visual ratings < or = 2.4).
GUS scores and disease visual ratings were significantly correlated (r = 0.83, P <
0.0001). GUS scores of accessions ranged from 1.0 to 1.6. Twenty-one accessions (53%) had GUS scores that were less or not significantly (P > 0.05) greater than Madsen (GUS scores < or = 1.3) (Table 1). There were two lines had significantly (P < 0.05) greater visual rating than Madsen. These lines were not categorized as either resistant or susceptible lines. Therefore, nineteen accessions (48%) were classified as resistant to O. acuformis because they had both GUS scores and visual ratings that were less or not significantly (P > 0.05) greater than Madsen. Nineteen other accessions were classified as susceptible to O. acuformis. The frequency distribution of GUS scores of O. acuformis showed the range of reactions of Ae. longissima accessions to O. acuformis (Figure 1).
Cappelle Desprez had the same GUS score as Madsen (1.0), which was close to VPM-1
77
(0.9). Opata, Hill 81, Chinese Spring and Selkirk had GUS scores of 1.2, 1.3, 1.4, and
1.5, respectively.
Differential reaction of Ae. longissima accessions to O. yallundae and O. acuformis. Resistant controls VPM-1, Madsen, and Cappelle Desprez had similar reactions to O. yallundae and O. acuformis (Table 1). However, Cappelle Desprez had less severe visual ratings and lower GUS scores for O. acuformis than O. yallundae.
Although Chinese Spring and Selkirk had lower visual ratings and GUS scores when inoculated with O. acuformis, they were still classified as susceptible to both pathogens compared to Madsen. In contrast, Opata was susceptible to O. yallundae but resistant to
O. acuformis. Hill 81 was highly susceptible to O. yallundae but it wasn‟t clear for its reaction to O. acuformis because inconsistent results between GUS score and visual rating. Some Ae. longissima accessions also demonstrated differential reactions to O. yallundae and O. acuformis (Table 1); however, on average, accessions had the same
GUS scores (1.3) and visual ratings (2.4) for both pathogens. Based on the GUS scores,
13 accessions (33%) were resistant to both O. yallundae and O. acuformis (shown as
Bold in Table 1), 15 accessions (38%) were susceptible, and 10 accessions (25%) reacted differently. Accessions PI 604108, PI 604109, PI 604114, and PI 604128 were resistant to O. yallundae by both GUS scores and visual ratings, but were susceptible to O. acuformis. In contrast, accessions PI 604103, PI 604130, PI 604139, and PI 604144 were susceptible to O. yallundae by both assessments, but were resistant to O. acuformis.
Reaction of Ae. longissima addition or substitution lines to O. yallundae.
Homogeneity of variance was accepted because the error variances of the two experiments were not significantly (P > 0.05) different for GUS scores and visual ratings;
78 therefore, data from the two experiments were combined. The disease reactions of the 83
Ae. longissima addition or substitution lines to O. yallundae were significantly different
(P < 0.0001) for both GUS scores and disease ratings.
Madsen had a visual rating of 0.9. Hill 81, Chinese Spring, Opata, and Selkirk had visual ratings of 3.3, 3.2, 3.2, and 2.9, respectively. Visual ratings of the 83 genetic stocks ranged from 1.4 to 3.6 (Table 2). Forty-three (52%) lines had visual ratings less than or not significantly (P > 0.05) greater than Madsen (visual ratings < or = 2.8).
GUS scores were significantly (P < 0.0001) correlated with visual ratings (r =
0.629). GUS scores of the genetic stocks ranged from 1.2 to 1.9 (Table 2). A total of 29
(35%) lines had GUS scores less or not significantly (P > 0.05) greater than Madsen
(GUS scores < or = 1.5), and 23 of them had visual ratings less or not significantly (P >
0.05) greater than Madsen, which were less than or equal to 2.8. Therefore, these 23 lines
(28%) were classified as resistant to O. yallundae. Five resistant lines contain Ae. longissima chromosome 1Sl, 2 lines contain 2Sl, 5 lines contain 5Sl, 5 lines contain 7Sl, 2 lines contain 4Sl/7Sl translocation, and 4 lines contain unknown Ae. longissima chromosomes (Figure 2). Hill 81, Chinese Spring, Opata, and Selkirk had GUS scores of
1.6, 1.7, 1.6, and 1.6, respectively; all were susceptible to O. yallundae compared to
Madsen.
79
Table 2. GUS scores and disease ratings of 83 Ae. longissima addition or substitution lines in Chinese Spring or Selkirk background to Oculimacula yallundae and O. acuformis
O. yallundae O. acuformis Lines Description GUS Ratings Reactiona GUS Ratings Reactiona TA 3716 Selkirk DA ?Sl 1.2 1.6 R 1.5 1.7 S TA 3710 Selkirk DA ?Sl 1.3 2.5 R 1.3 1.7 R TA 3576 DA 4Sl/7Sl 1.3 2.2 R 1.6 2.5 S TA 3717 Selkirk DA ?Sl 1.3 1.4 R 1.7 2.0 S TA 6519 DS 7Sl (7D) 1.4 2.6 R 1.2 1.8 R TA 6504 DS 2Sl (2A) 1.4 3.2 ? 1.3 2.2 R TA 3599 DA ?Sl 1.4 1.9 R 1.4 1.8 S TA 3579 DA 7Sl/4Sl 1.4 2.4 R 1.4 2.5 S TA 3466 DS 5Sl (5B) 1.4 2.3 R 1.5 3.0 S TA 6523 DtS 1Sl/L (1B) 1.4 2.0 R 1.7 2.8 S TA 6503 DS 1Sl (1D) 1.5 2.8 R 1.2 1.5 R TA 7528 DtA 7Sl/L 1.5 2.2 R 1.3 2.0 R TA 6513 DS 5Sl (5A) 1.5 2.4 R 1.3 2.2 R TA 6540 DtS 5Sl/L (5D) 1.5 2.5 R 1.3 2.0 R TA 7544 DA 2Sl 1.5 2.7 R 1.3 2.2 R TA 6603 dDtS 1Sl (1D) 1.5 2.8 R 1.3 1.2 R TA 6506 DS 2Sl (2D) 1.5 3.0 ? 1.3 3.0 ? TA 7550 DA 2Sl 1.5 2.3 R 1.4 2.2 S TA 7543 DA 1Sl 1.5 2.6 R 1.4 2.3 S TA 7524 DtA 5Sl/L 1.5 2.8 R 1.4 2.8 S TA 6512 DS 4Sl (4D) 1.5 2.9 ? 1.4 3.0 S TA 6518 DS 7Sl (7B) 1.5 2.5 R 1.5 2.7 S TA 6543 DtS 7Sl/L (7B) 1.5 2.6 R 1.5 2.3 S TA 6510 DS 4Sl (4A) 1.5 3.1 ? 1.5 2.5 S TA 7547 DA 5Sl 1.5 2.3 R 1.6 2.3 S TA 3709 Selkirk DA ?Sl 1.5 3.3 ? 1.6 2.8 S TA 6502 DS 1Sl (1B) 1.5 2.4 R 1.7 2.5 S TA 6542 DtS 7Sl/L (7A) 1.5 2.4 R 1.7 2.7 S TA 3635 DA ?Sl 1.5 3.3 ? 1.7 3.5 S TA 7548 DA 6Sl 1.6 3.4 S 1.0 1.2 R TA 6505 DS 2Sl (2B) 1.6 3.1 S 1.1 1.8 R TA 7551 DtA 2Sl/L 1.6 2.8 S 1.2 2.2 R TA 7525 DtA 6Sl/S 1.6 3.2 S 1.2 2.4 R l TA 6529 DtS 2S /S (2D) 1.6 3.5 S 1.2 1.8 R TA 6537 DtS 4Sl/S (4B) 1.6 2.8 S 1.3 2.0 R TA 6545 DS 2Sl (2B) 1.6 2.8 S 1.4 2.7 S
80
Table 2. continued
TA 6517 DS 7Sl (7A) 1.6 2.8 S 1.4 1.5 S TA 6528 DtS 2Sl/L (2B) 1.6 2.8 S 1.4 1.8 S TA 6610 dDtS 6Sl (6B) 1.6 2.8 S 1.4 1.7 S TA 6508 DS 3Sl (3B) 1.6 3.0 S 1.4 2.3 S TA 6501 DS 1Sl (1A) 1.6 3.2 S 1.4 2.5 S TA 3711 Selkirk DA ?Sl 1.6 3.6 S 1.4 2.3 S TA 7515 DtA 1Sl/S 1.6 2.8 S 1.5 3.5 S TA 6541 DtS 6Sl/S (6B) 1.6 3.0 S 1.5 2.8 S TA 3714 Selkirk DA ?Sl 1.6 2.4 S 1.6 2.0 S TA 3636 DA ?Sl 1.6 2.8 S 1.6 2.8 S TA 6548 dDtS 3Sl (3D) 1.6 2.8 S 1.6 1.8 S TA 6521 DtS 1Sl/L (1A) 1.6 3.0 S 1.6 3.3 S TA 3574 DA 2Sl 1.6 3.2 S 1.6 3.0 S TA 6525 DtS 1Sl/L (1D) 1.7 3.1 S 1.1 1.8 R TA 6604 dDtS 2Sl (2D) 1.7 3.3 S 1.1 1.5 R TA 6546 DS 2Sl (2D) 1.7 2.5 S 1.2 1.8 R TA 6515 DS 5Sl (5D) 1.7 2.9 S 1.2 1.7 R TA 6538 DtS 4Sl/S (4D) 1.7 3.2 S 1.2 1.8 R TA 3465 DS 4Sl (4B) 1.7 2.9 S 1.3 2.0 R TA 7546 DA 4Sl 1.7 3.0 S 1.3 2.2 R TA 6608 dDtS 5Sl (5D) 1.7 3.2 S 1.3 2.2 R TA 7516 DtA 1Sl/L 1.7 3.3 S 1.3 2.0 R TA 7527 DtA 7Sl/S 1.7 2.7 S 1.4 2.7 S TA 6605 dDtS 3Sl (3D) 1.7 2.8 S 1.4 2.2 S TA 7517 DtA 2Sl/S 1.7 3.0 S 1.4 2.3 S TA 6544 DtS 7Sl/L (7D) 1.7 3.1 S 1.4 2.2 S TA 6509 DS 3Sl (3D) 1.7 3.2 S 1.4 1.8 S TA 6611 dDtS 7Sl (7D) 1.7 3.3 S 1.4 2.8 S TA 7545 DA 3Sl 1.7 3.4 S 1.4 2.7 S TA 3573 DA 1Sl 1.7 2.6 S 1.5 2.5 S TA 3715 Selkirk DA ?Sl 1.7 2.6 S 1.5 3.0 S TA 7518 DtA 2Sl/L 1.7 2.8 S 1.5 3.2 S TA 6606 dDtS 4Sl (4D) 1.7 2.8 S 1.5 2.0 S TA 7522 DtA 4Sl/L 1.7 3.1 S 1.5 2.8 S TA 6530 DtS 2Sl/L (2D) 1.7 2.5 S 1.6 2.2 S TA 6507 DS 3Sl (3A) 1.7 2.8 S 1.6 2.7 S TA 7523 DtA 5Sl/S 1.7 3.2 S 1.6 2.3 S TA 6533 DtS 3Sl/S (3B) 1.8 3.0 S 1.2 1.8 R TA 7593 DA ?Sl 1.8 2.7 S 1.5 2.3 S TA 6640 DS 5Sl (5D) 1.8 3.0 S 1.5 2.5 S
81
Table 2. continued
TA 7521 DtA 4Sl/S 1.8 3.2 S 1.5 2.8 S TA 7519 DtA 3Sl/S 1.8 2.9 S 1.6 2.3 S TA 6531 DtS 3Sl/S (3A) 1.8 2.9 S 1.6 3.5 S TA 6522 DtS 1Sl/S (1A) 1.8 3.0 S 1.7 3.2 S TA 6547 dDtS 1Sl (1D) 1.9 3.5 S 1.2 1.8 R TA 3575 DA 3Sl 1.9 3.1 S 1.4 2.0 S TA 6526 DtS 2Sl/S (2A) 1.9 3.3 S 1.5 2.7 S Madsen control (R) 1.0 0.9 R 1.0 0.8 R Hill 81 control (S) 1.6 3.3 S 1.4 2.2 S CSb control (S) 1.7 3.2 S 1.3 2.5 ? Opata control 1.6 3.2 S ------Selkirk control 1.6 2.9 S 1.4 3.2 S
a Data sorted by GUS scores of O. yallundae. R = resistant, GUS scores and visual ratings are not significantly (P > 0.05) greater than Madsen based on Dunnett‟s T-test. S = susceptible, GUS scores are significantly (P < 0.05) greater than Madsen. ? = GUS scores are not significantly (P > 0.05) greater than Madsen but visual ratings are. These lines were not categorized as either resistant or susceptible lines. Lines in bold are resistant to both pathogens. Data for O. yallundae is the mean of 12 plants per line in two experiments and O. acuformis is the mean of 6 plants in one experiment. b CS = Chinese Spring.
82
Figure 2. Distribution of Oculimacula yallundae and O. acuformis resistance among chromosomes of Aegilops longissima. Dark bars are number of Ae. longissima addition or substitution lines resistant to O. yallundae and light bars are number of lines resistant to O. acuformis. Data for O. yallundae is the mean of 12 plants per line in two experiments and O. acuformis is the mean of 6 plants in one experiment.
83
Reaction of Ae. longissima addition or substitution lines to O. acuformis.
Significantly different disease reactions occurred among the 83 Ae. longissima addition or substitution lines to O. acuformis for GUS score (P =0.0024) and visual rating (P =
0.0008). Madsen had the lowest visual rating (0.8). Hill 81, Chinese Spring, and Selkirk had visual ratings of 2.2, 2.5, and 3.2, respectively. Visual ratings of the 83 genetic stocks ranged from 1.2 to 3.5 (Table 2). Fifty (60%) of them had visual ratings less than or not significantly (P > 0.05) greater than Madsen (visual ratings < or = 2.4).
GUS scores were significantly correlated with disease ratings (r = 0.61, P <
0.0001). GUS scores of the genetic stocks ranged from 1.0 to 1.7 (Table 2). A total of 27
(33%) lines had GUS scores less or not significantly (P > 0.05) greater than Madsen
(GUS scores < or = 1.3) and 26 of them had visual ratings less or not significantly (P >
0.05) greater than Madsen, which were less than or equal to 2.4. Therefore, these 26 lines
(31%) were classified as resistant to O. acuformis. Five resistant lines contain Ae. longissima chromosome 1Sl, 7 lines contain 2Sl, one line contains 3Sl, 4 lines contain 4Sl,
4 lines contain 5Sl, 2 lines contain 6Sl, 2 lines contain 7Sl, and one line contains an unknown Ae. longissima chromosome (Figure 2). Hill 81, Chinese Spring, and Selkirk had GUS scores of 1.4, 1.3, and 1.4, respectively. Hill 81 and Selkirk were susceptible to
O. acuformis comparing to Madsen. Since the GUS score of Chinese Spring was not significantly greater than Madsen but visual rating was, the reaction of Chinese Spring to
O. acuformis was not categorized as either resistant or susceptible.
Differential reactions of Ae. longissima addition or substitution lines to O. yallundae and O. acuformis. Reaction of Madsen was similar to both pathogens. Hill 81,
Chinese Spring, and Selkirk had higher GUS scores when inoculated with O. yallundae
84 than O. acuformis. Ae. longissima addition or substitution lines had mean GUS scores of
1.6 and 1.4 and mean disease ratings of 2.8 and 2.3 for O. yallundae and O. acuformis, respectively. There were 62 lines with GUS scores for O. yallundae greater than for O. acuformis, but only 9 lines with higher GUS scores for O. acuformis.
Statistically, a GUS score greater than 1.5 was significantly (P < 0.05) greater than Madsen for O. yallundae. However, for O. acuformis, a GUS score greater than 1.3 was significantly (P < 0.05) greater than Madsen. Based on GUS scores, 32 lines (39%) reacted differently to O. yallundae and O. acuformis including 15 resistant lines to O. yallundae alone and 17 resistant lines to O. acuformis alone; 37 lines (45%) were susceptible to both pathogens. Only 8 lines (10%) were resistant to both O. yallundae and
O. acuformis (shown as Bold in Table 2). Lines resistant to both pathogens collectively contain Ae. longissima chromosomes 1Sl, 2Sl, 5Sl, and 7Sl (Figure 2). There were nine lines resistant to O. yallundae with low GUS scores of 1.2 - 1.4, but only two were resistant to O. acuformis (Table 2). In contrast, 13 lines had very low GUS scores (1.0 –
1.2) to O. acuformis and 11 of them were susceptible to O. yallundae.
Discussion
Among the 40 Ae. longissima accessions tested, 17 (43%) were resistant to O. yallundae, 19 (48%) were resistant to O. acuformis, and 13 (33%) were resistant to both pathogens. These results confirm the original hypothesis that Ae. longissima can be a new source for resistance genes to eyespot of wheat. This is the first evidence that Ae. longissima confers eyespot resistance. Based on consideration of both assessments and the standard deviation of each line, seven resistant lines (PI 542196, PI 604112, PI
85
604116, PI 604119, PI 604136, PI 604137, and PI 604140) and three susceptible lines (PI
330486, PI 604111, and PI 604117) with the same responses to both species were selected as parents for future genetic studies.
Among the 83 Ae. longissima addition or substitution lines tested, 23 (28%) were classified as resistant to O. yallundae including the lines contain Ae. longissima chromosomes 1Sl, 2Sl, 5Sl, 7Sl, and 4Sl/7Sl translocation. Twenty-six lines (31%) were classified as resistant to O. acuformis and the resistance was found on all Ae. longissima chromosomes with more resistant lines containing chromosomes 1Sl, 2Sl, 4Sl, and 5Sl.
Eight lines (10%) containing Ae. longissima chromosomes 1Sl, 2Sl, 5Sl, and 7Sl were resistant to both O. yallundae and O. acuformis. These demonstrated that the genetic controls of eyespot resistance located in multiple regions of the Ae. longissima genome.
This is the first report that different reactions of Ae. longissima to the two eyespot pathogens were observed. This is also the first time finding the chromosome locations of the genetic controls to eyespot and some resistance to O. yallundae and O. acuformis are in different chromosomes.
Although Ae. longissima had not been tested for resistance to eyespot previously, it has been reported to confer resistance to Septoria glume blotch, powdery mildew, and rust diseases (Ecker et al., 1990; Ceoloni et al., 1992; Anikster et al., 2005). The percentages of resistance in different accessions to stem rust, leaf rust, and stripe rust were 30%, 50%, and 90%, respectively (Anikster et al., 2005). Thus, Ae. longissima can be a potential source of resistance to eyespot and other diseases.
Genetic diversity for eyespot resistance exists within Ae. longissima even though the majority of them were collected from the same geographic area. Among the 40
86 accessions, 33% were resistant to both O. yallundae and O. acuformis, 38% were susceptible to them, and 25% reacted differently. Genetic diversity for eyespot resistance was also observed in the Ae. longissima addition or substitution lines. Even lines with the same alien chromosome composition sometimes differed in reaction to eyespot. Such as
62% lines with Ae. longissima chromosome 1Sl, 50% lines with 2Sl , 10% lines with 3Sl,
49% lines with 4Sl, 78% lines with 5Sl, 25% lines with 6Sl, and 56% lines with 7Sl, had eyespot resistance. Polymorphism among the lines tested will be useful for mapping the genes.
Some lines had different reactions to O. yallundae and O. acuformis. Ten of 40
(25%) Ae. longissima accessions and 32 of 83 (39%) Ae. longissima addition or substitution lines responded differently to O. yallundae and O. acuformis. These results support the hypothesis that genetic control of resistance to O. yallundae and O. acuformis are different in some lines. Uslu et al. (1998) first reported the differential genetic control of resistance in D. villosum to different eyespot pathogens. Recently, Burt et al. (2010) demonstrated that four of 22 T. monococcum lines had significantly different reactions to
O. yallundae and O. acuformis. Lange-de la Camp reported that W-type isolates (O. yallundae) were more virulent to wheat than to rye, whereas R-type isolates (O. acuformis) were almost equally virulent to both rye and wheat in 1966 (Cunningham,
1981). Although the two pathogens coexist in the field, cause similar symptoms, and are now considered separate species, their infection processes are different. Daniels et al.
(1991) observed different patterns of infection for O. yallundae and O. acuformis isolates on the same host. Poupard et al. (1994) confirmed that O. acuformis colonized coleoptiles and leaf sheaths more slowly than O. yallundae. Goulds and Fitt (1991) found that O.
87 yallundae isolates infected stems faster and caused more severe lesions than the R-type, but that there is little difference in severity between them by the end of the season. The different infection processes may trigger the different resistance reactions to them.
Some control cultivars reacted differentially to the eyespot pathogens, too.
Madsen was highly resistant to both pathogens, but a little less to O. acuformis than O. yallundae. In contrast, Cappelle Desprez was more resistant to O. acuformis than O. yallundae. Burt et al. (2010) also found that Pch2 was significantly more effective against O. acuformis than O. yallundae. The susceptible controls Hill 81 and Chinese
Spring were highly susceptible to O. yallundae and less susceptible to O. acuformis.
Selkirk was highly susceptible in all the tests. Opata reacted completely different to O. yallundae and O. acuformis. It showed susceptibility to O. yallundae in both accessions and genetic stock screenings and resistance to O. acuformis in accessions tests.
Unfortunately, it was not included in the tests of genetic stocks for O. acuformis. More tests should be done to confirm the resistance of Opata to O. acuformis. Since both
Chinese Spring and Selkirk are susceptible to eyespot with an exception that the susceptibility of Chinese Spring to O. acuformis wasn‟t clear in one experiment, the resistant genetic stocks identified in this study will be useful for transferring the resistance genes to an adapted wheat cultivar. TA 3710, TA 3716, and TA 3717, which are disomic addition lines in a Selkirk background, were highly resistant to O. yallundae.
TA 3710 is also resistant to O. acuformis. Identifying the alien chromosomes in these lines will be useful in locating the genes for eyespot resistance.
Since the genetic control of the resistance to O. yallundae and O. acuformis are different in some lines, it is important to identify the resistance genes for each of them to
88 develop varieties resistant to O. acuformis as well. Therefore, both O. yallundae and O. acuformis should be tested when screening for the resistance to eyespot from wild relatives of wheat. Uslu et al. (1998) tested O. yallundae and O. acuformis separately when they investigated resistance in Dasypyrum villosum. They found that resistance in
D. villosum was more effective to O. yallundae than to O. acuformis. Our results showed that most Ae. longissima accessions reacted similarly to O. yallundae and O. acuformis.
However, for most Ae. longissima genetic stocks, O. acuformis was less severe than O. yallundae. The difference between accessions and genetic stocks may be due to the different stem sizes. The stems of Ae. longissima accessions are thinner than those of the genetic stocks, which resemble wheat stems. It was reported that O. yallundae infected rapidly and more severely in early stages but there were either no differences or O. acuformis was more severe at end of the season (Goulds and Fitt, 1991; Bock et al.,
2009). Since the stems of accessions are small, penetration of pathogens may be almost finished at eight weeks after inoculation. The differences in the amount of fungal growth on most of the genetic stocks may become smaller as they near maturity.
In this study, resistance to O. yallundae was found on Ae. longissima chromosomes 1Sl, 2Sl, 5Sl, 7Sl, and two 4Sl/7Sl translocation lines, and resistance to O. acuformis was found on all Ae. longissima chromosomes, with greater resistance from chromosomes 1Sl, 2Sl, 4Sl, and 5Sl. The two translocation lines were resistant only to O. yallundae. Since no lines containing 4Sl were resistant to O. yallundae, it is possible that resistance is on the 7Sl segment in the translocation lines. If so, only Ae. longissima chromosomes 1Sl, 2Sl, 5Sl, and 7Sl carried resistance to O. yallundae. They also carried resistance to O. acuformis. Then, chromosomes 3Sl, 4Sl, and 6Sl only had controls for O.
89 acuformis. Although resistance to both eyespot pathogens in D. villosum was determined by genes on chromosomes 1V, 2V, and 3V, Uslu et al. (1998) found effective resistance for O. yallundae alone on chromosome 4V and for O. acuformis alone on 5V.
Eight genetic stocks with Ae. longissima chromosome 1Sl were resistant to either
O. yallundae or O. acuformis and two lines were resistant to both pathogens. One of the eight resistant lines with chromosome 2Sl was resistant to both O. yallundae and O. acuformis. Uslu et al. (1998) also reported eyespot resistance to both pathogens in D. villosum chromosome 1V and 2V. Law et al. (1976) found eyespot resistance on Cappelle
Desprez chromosome 2B. One line with Ae. longissima chromosome 3Sl short arm was resistant to O. acuformis only. However, resistance on chromosome 3V to both species was found in the study of Uslu et al. (1998).
Four lines with Ae. longissima chromosome 4Sl only showed resistance to O. acuformis. This finding is unexpected because resistance to O. yallundae has been found on homoeologous chromosome group 4 in other studies (Murray et al., 1994; Uslu et al.,
1998; Li et al., 2004 & 2005). Even though two 4Sl/7Sl translocation lines showed resistance to O. yallundae, the evidence of resistance to O. yallundae in 4Sl was hardly found in the genetic stocks line tested in this study.
Two of seven resistant lines with Ae. longissima chromosome 5Sl were resistant to both O. yallundae and O. acuformis. Evidence for resistance on homoeologous chromosome group 5 has been reported in other studies. Law et al. (1976) reported that
Cappelle Desprez chromosome 5D had eyespot resistance. D. villosum 5V showed resistance to O. acuformis (Uslu et al., 1998). Muranty et al. (2002) found eyespot resistance on Cappelle Desprez chromosome 5A. Recently, Burt et al. (2011) reported
90 that a resistance gene on chromosome 5A of Cappelle Desprez conferred effective resistance to both O. acuformis and O. yallundae.
Eyespot resistance found in two lines with Ae. longissima chromosome 6Sl is the first report that homoeologous chromosome group 6 carries resistance to eyespot. Two of five resistant lines with Ae. longissima chromosome 7Sl, were resistant to both pathogens.
Jahier et al. (1979) first found the genetic control of VPM-1 associated with chromosome
7D. Pch1 was located on the long arm of 7D by its linkage with an isozyme marker (EP-
D1b) (McMinnlin et al., 1986). The genetic control of eyespot resistance in Cappelle
Desprez was found on 7A (Law et al., 1976; de la Peña et al., 1996 ; Muranty et al.,
2002) and mapped to 7AL by an RFLP linkage map (de la Peña et al., 1997). de la Peña et al. (1997) suggested that Pch1 and Pch2 were homoeoloci. A conclusion supported by
Chapman et al., (2008) because of the similar positions of microsatellite markers linked to Pch1and Pch2.
Two Ae. longissima accessions and six Ae. longissima addition or substitution lines showed resistance to either O. yallundae or O. acuformis with GUS scores, but not with visual ratings. These lines need to be examined again in order to confirm their resistance to eyespot. If they were categorized as resistant lines, the percentage of eyespot resistance in Ae. longissima would be increased. Two more resistant lines would contain
Ae. longissima chromosome 2Sl and the lines that contain chromosome 4Sl and resist to
O. yallundae would be two instead of none.
Based on these results, Ae. longissima is a new source of eyespot resistance, multiple resistance genes to O. yallundae and O. acuformis are present in Sl genome, and the genetic control of resistance to both pathogens differs in some lines. This research
91 provides the first evidence for eyespot resistance in Ae. longissima. The resistance to O. yallundae and O. acuformis identified on different Ae. longissima chromosomes in wheat genetic stocks provides important information for future studies of eyespot resistance.
The introgression of these new sources to wheat will broaden the genetic base of eyespot resistance, and has the potential to improve wheat production.
92
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CHAPTER THREE
GENETIC ANALYSIS AND QTL MAPPING OF RESISTANCE GENES TO EYESPOT OF WHEAT IN AEGILOPS LONGISSIMA
Introduction
Eyespot, a chronic and economically important disease of winter wheat, is caused by the soilborne fungi Oculimacula yallundae (syn: Tapesia yallundae, Wallwork &
Spooner) Crous & W. Gams and O. acuformis Crous & W. Gams (syn: T. acuformis)
(Crous et al., 2003). These two pathogens were formerly known as the W- and R- pathotypes of Pseudocercosporella herpotrichoides (Fron.) Deighton, respectively, before the teleomorph was discovered (Lucas et al., 2000). These fungi affect the stem base of wheat, causing eye-shaped elliptical lesions that result in lodging of infected plants and yield loss (Murray, 2010). When eyespot is severe, yield loss of up to 50% can occur in susceptible cultivars (Murray & Bruehl, 1986).
Eyespot has been reported in several wheat-growing areas of the world with cool, wet fall, and winters including North and South America, Australia, New Zealand,
Europe, and Africa (Lucas et al., 2000). In the US, eyespot is a yield-limiting disease mainly in the Pacific Northwest (PNW) where winter wheat is the majority of production, but the pathogens are widespread in the US. The most economical and environmentally friendly control method for eyespot is growing resistant wheat cultivars.
Cappelle Desprez was the first source of eyespot resistance reported from hexaploid wheat. This resistance has been used extensively since the 1950s and
98 transferred to many cultivars in Europe (Hollins et al, 1988). The genetic control of eyespot resistance in Cappelle Desprez was first studied by Law et al. (1976) using chromosome substitution lines and monosomic analysis. They found that chromosome
7A was critical for eyespot resistance and chromosomes 1A, 2B, and 5D also affected resistance. Law et al. (1976) suggested that the inheritance of eyespot resistance in
Cappelle Desprez was complex. This conclusion was supported by Jahier et al. (1979) because they found multiple resistance factors in wheat cultivar Roazon, which had
Cappelle Desprez in its pedigree. The resistance gene on chromosome 7A of Cappelle
Desprez was designated Pch2 and mapped to the distal portion of the long arm by RFLP mapping (de la Peña et al., 1996, 1997). Doussinault and Dosba (1977) and Lind (1999) suggested that resistance to eyespot in Cappelle Desprez was quantitative.
Muranty et al. (2002) found that chromosome 5A of Cappelle Desprez carried a gene for eyespot resistance. Later, Burt et al. (2011) mapped a major QTL on chromosome 5AL and associated it with a simple sequence repeat (SSR) marker,
Xgwm639. Three SSR markers (Xwmc346, Xwmc525, and Xcfa2040) were closely linked to Pch2 on chromosome 7A (Chapman et al., 2008). The eyespot resistance in Cappelle
Desprez has been durable through long term exploitation (Johnson, 1984). However, the effectiveness of resistance conferred by the genes in Cappelle Desprez is not sufficient under severe eyespot conditions (Macer, 1966, Hollins et al., 1988) and fungicide application is required to prevent yield loss (Law et al., 1988).
Johnson (1992) indicated that eyespot resistance was difficult to find and exploit because it is not readily available in wheat. Consequently, wild wheat species have been evaluated as sources of resistance (Jones et al., 1995). Wild species, especially Aeligops
99 spp., can broaden the genetic diversity of cultivated wheat (Schneider, 2008). Aegilops ventricosa was reported to be highly resistant to eyespot (Sprague, 1936). The introgression of eyespot resistance gene Pch1 from tetraploid Aegilops ventricosa Tausch
(2n=28, DDMvMv) to the breeding line VPM-1 is the most successful example of utilizing eyespot resistance genes from a wild relative of wheat (Doussinault et al, 1983;
Jones et al., 1995). Several wheat cultivars with Pch1 derived from VPM-1 have been developed in the US PNW. One of them was soft winter wheat cultivar Madsen, which has been widely grown in the PNW since it was released in 1988 (Allan et al., 1989).
The genetic control of eyespot resistance in VPM-1 was reported to be a single dominant gene (Worland et al., 1988; Strausbaugh & Murray, 1989; Allan & Roberts,
1991) that was mapped to the distal portion of chromosome 7DL (Gale et al., 1984;
McMillin et al., 1986; Worland et al., 1988; Jahier et al., 1989). Three sequence-tagged- site (STS) markers (Xorw1, Xorw5, and Xorw6) and three microsatellite markers
(Xwmc14, Xbarc97, and Xcfd175) were tightly linked to Pch1 (Leonard et al., 2008).
Chapman et al. (2008) found that SSR markers Xwmc14, Xbarc97, and Xpsr121 were closely linked with Pch1. Meyer et al. (2011) reported that Xorw1, Xorw6, and Xcfd175 were the most suitable markers for marker assisted selection (MAS).
Aegilops longissima Schweinf. & Muschl. (2n = 2x = 14, SlSl) is a diploid species in the section Sitopsis of Aegilops L. (van Slageren, 1994). Species of section Sitopsis are valuable sources of genes for wheat improvement and disease resistance (Friebe et al.,
1993; Millet, 2007). Ae. longissima has provided exploitable traits in grain quality, grain weight, and drought tolerance (Levy et al., 1985; Millet et al., 1988; Millet et al., 2007).
Resistance in Ae. longissima to Septoria glume blotch, powdery mildew, and rusts
100 of wheat has been reported (Ecker et al., 1990; Ceoloni et al., 1992; Anikster et al.,
2005). In the previous study (Chapter 2). Ae. longissima was identified to be a new source of resistance to eyespot and some lines reacted differently to O. yallundae and O. acuformis. Eyespot resistance for both pathogens was associated with Ae. longissima chromosomes 1Sl, 2Sl, 5Sl, and 7Sl.
A genetic marker system is needed to map the eyespot resistance genes in Ae. longissima. To date, there have been no molecular markers developed directly from the
Ae. longissima genome. Zhang et al. (2001) used 59 RFLP probes of wheat to construct a genetic map of Ae. longissima with 7 linkage groups, but it only comprises 67 loci. They found that 62% of the markers were polymorphic between the parents, and provided evidence that wheat RFLP markers can be used in Ae. longissima.
Wheat microsatellite markers, also known as SSR markers, are tandem repeats of short (2-6 bp) DNA sequences (Röder et al., 1998). Over 1,500 SSR markers have been developed from the wheat genome to date (Röder, 1998; Somers et al., 2004; Adonina et al., 2005; Song et al., 2005). SSR markers are more polymorphic in wheat than any other marker system to date (Adonina et al., 2005). The majority of SSRs are co-dominant and chromosome-specific (Röder et al., 1998). Wheat SSRs were used to amplify DNA from wheat relatives, including Ae. longissima (Sourdille et al., 2001). Adonina et al. (2005) tested 253 wheat SSRs for their transferability to diploid Aegilops species and found that
68% of them amplified in Ae. longissima. These results demonstrated the possibility of applying wheat SSRs to Ae. longissima.
The objectives of this study were to determine the genetic control of eyespot resistance in Ae. longissima and to locate the genes in the Sl genome by developing a
101 linkage map using wheat SSR markers. This work will contribute to the long-term goal of transferring new eyespot resistance genes to wheat.
Materials and Methods
Mapping populations. In chapter 2, seven Ae. longissima resistant accessions and three susceptible accessions to both O. yallundae and O. acuformis obtained from the
USDA National Small Grains Collection (NSGC) were selected as parents. Amplified fragment length polymorphism (AFLP) analysis (Vos et al., 1995) was carried out following the protocols of AFLP Analysis System I and AFLP Starter Primer Kit
(Invitrogen Life Technologies, Carlsbad, CA) to select accessions as potential parents based on high percentages of polymorphism. Based on these results, two recombinant inbred line (RIL) populations were developed through single-seed descent from the crosses PI 542196 (R) x PI 330486 (S) and PI604136 (R) x PI 604117 (S) with 200 lines in each population; all parents showed winter habit. PI 542196 was originally collected from Izmir, Turkey, PI 330486 was from an unknown source, and the others were from central Israel. The population of PI 542196 (R) x PI 330486 (S) was used in this study.
The population of PI604136 (R) x PI 604117 (S) has not been used for phenotypic evaluation.
Phenotypic evaluation. The method to test for eyespot resistance was a modified
GUS assay developed by de la Peña and Murray (1994). F1, F2, F3, and F5 populations of
PI 542196 (R) x PI 330486 (S) were tested for resistance to O. yallundae in growth chamber experiments. PI 542196 (R), PI 330486 (S), Madsen (R), and Hill 81 (S) were included in all experiments. Three F1 plants and 108 F2 plants were tested in a completely
102 randomized experiment. Fifty F3 RIL lines were tested in a RCB design experiment with eight blocks and 16 plants per line. One hundred and eighty nine F5 RIL lines were tested twice in a randomized complete block (RCB) design experiment with three blocks and 12 plants per line.
All seeds were imbibed at 4oC for four days before planting. Two seeds were planted into a 6.4 cm square plastic pot with commercial Sunshine Potting Mix#1/LC1
(SunGro Horticulture, Bellevue, WA) and fertilized with Osmocote (14-14-14, w/v) (The
Scotts Company LLC, Marysville, OH). The plastic flats without drain holes (54 x 27 x 6 cm) holding 50 pots were used as blocks. The flats were placed in the growth chambers maintained at 15/13oC with a 12 hour photoperiod. Relative humidity was between 98 to
100%. In the F5 tests, four flats together acted as one block and were always placed in one growth chamber and rotated every 2-3 days
Plants were inoculated with slurry of conidia from β-glucuronidase (GUS) transformed O. yallundae isolates tph8934-5-61, tph8934-5-62, tph8934-5-68, and tph8934-5-70 when the second leaf was half the size of the first leaf. Conidia was produced by spreading small pieces potato dextrose agar (PDA) (Difco Laboratories,
Detroit, MI) mycelia plugs containing mycelia onto 1.5% water agar (WA) (Sigma Life
Science, St. Louis, MO) plates with 3 ml sterilized distilled water per plate. The plates placed in an incubator with near UV light at 13oC for at least two weeks. A slurry was made by blending conidia, 1.5% fresh WA, and water together. The final concentration of conidia was 2.1 x 105 per ml. During inoculation, 250 µl of the slurry was pipetted into a
3.3 cm long split drinking straw collar around each plant stem base. The same amount of inoculum was added again one or two days later.
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Eight weeks after inoculation, at approximately growth stage 23-25 (Zadoks et al.,
1974), a 3 cm section of the whole stem was removed from around the inoculation site.
The section was briefly washed with tap water to remove soil. Visual disease ratings were performed on a 0 to 4 scale (Yildirim, et al., 2000), where 0 = no symptoms (healthy), 1 = a lesion only on the first leaf sheath, 2 = a lesion on the first leaf sheath and a small lesion on the second leaf sheath, 3 = a lesion covering the first leaf sheath and up to half of the second sheath, and 4 = a lesion covering the first and second sheaths (nearly dead). All the tillers (2 to 4) of each plant were evaluated as a whole. The stems were then wrapped with paper towels and frozen at -20oC until the GUS assay was performed. GUS activity in stems was used as a surrogate measurement of the amount of fungal colonization.
Frozen stems were ground in a leaf squeezer (Ravenel Specialties Company,
Seneca, SC) with 2.5 ml GUS extraction buffer added per sample. GUS activity was determined by adding 50µl extract with 40µl 10mM fluorescent substrate 4- methylumbelliferyl β-D-glucoside (MUG) (Sigma Life Science, St. Louis, MO) in a
1.2ml testing tube, and then incubating at 37oC for 1 hour to produce fluorescent methylumbelliferone (MU). The fluorescence intensity of MU was measured in a
Molecular Devices SpectraMax M2 microplate reader (Molecular Devices Co.,
Sunnyvale, CA). GUS scores were expressed as the log10 transformed ratio [log10(x/ resistant control) +1] of GUS activity of an individual accession (x) compared to the activity of resistant control (Madsen). The GUS score of Madsen was 1.0. Therefore, resistant accessions had GUS scores that were smaller or not significantly (P > 0.05) greater than the resistant parent (PI 542196) and susceptible accessions had GUS scores significantly greater than PI542196.
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Statistical analysis. A Chi-squared (χ2) test was used for analysis of segregation data. Homogeneity of variance of two F5 experiments was tested with the F-ratio of the larger error variance to the smaller error variance (Gomez and Gomez, 1984). If the variances are homogeneous, the data of the two experiments will be combined.
Statistical analysis was conducted with SAS Version 9.2 (SAS Institute Inc.,
Cary, NC). Analysis of variance (ANOVA) for GUS score and visual rating in individual or combined experiments and standard deviation were carried out by PROC GLM procedure. Variance components were based on ANOVA for a random model generated from PROC GLM procedure with variances of lines and experiments considered random effects. The hypothesis of normality for the frequency distribution of GUS scores or visual ratings in different populations was tested by Kolmogorov-Smirnov test with
PROC UNIVARIATE. Dunnett‟s t-test was used for multiple comparisons with the least squares mean (lsmean) of each line compared with the resistant parent (PI542196).
Pearson correlation coefficients between visual ratings and GUS scores of combined experiments were calculated by PROC CORR.
Broad-sense heritability (H2) was calculated as H2 = Var(G) / Var(P), where
Var(G) is the genetic variance and Var(P) is the phenotypic variance, which is the combination of genetic variance and environmental variance [Var(E)]. Then, the genetic variance is the variance component for lines and the environmental variance was estimated by the variance components for experiments and the lines x experiment
2 2 interaction. The equation of broad-sense heritability based on entry mean is: H = σ g /
2 2 2 (σ g + σ gxe/r + σ e/rn), where r is the number of experiments and n is the number of plants per line (Hanson, 1963; Shen et al., 2003).
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DNA isolation and marker analysis. Genomic DNA was extracted from fresh leaves (growth stage 18-20) of the parents and F5 RIL of PI 542196 (R) x PI 330486 (S) as described in the protocol for monocot DNA isolation (Wheat Genetic and Genomic
Resources Center at Kansas State University, http://www.k-state.edu/wgrc). DNA extraction buffer includes 0.5 M NaCl, 0.1 M Tris-HCl (pH 8.0), 50 mM EDTA (pH 8.0),
0.84% (w/v) SDS, 0.38 g sodium bisulfate per 100 ml, and 5N NaOH to adjust pH to 8.0.
DNA was dissolved in sterile distilled H2O and quantified using a Bio-Rad Fluorescent
DNA Quantitation Kit (Bio-Rad laboratories, Hercules, CA) on a Molecular Devices
SpectraMax M2 microplate reader (Molecular Devices Co., Sunnyvale, CA).
Marker analyses were performed using multiplex PCR in which the forward primer had a 19-bp M13 tail (5`-CACGACGTTGTAAAACGAC-3`) at the 5` end and a
M13 fluorescently labeled primer added as the third universal primer (Boutin et al.,
1997). Six hundred fifteen wheat microsatellite (SSR) primer sets covering the A, B, and
D genomes were screened for polymorphism between the parents. They included 143
BARC primers (developed by Beltsville Agricultural Research Center), 31 CFA and 71
CFD primers (developed by INRA Clermont-Ferrand), 175 GWM primers and 20 GDM primers (developed by Institut für Pflanzengenetik und Kulturpflanzenforschung), and
175 WMC primers (developed by Wheat Microsatellite Consortium). All primers were provided by the USDA-ARS Regional Small Grains Genotyping Laboratory at Pullman,
WA (WRSGGL).
Polymorphic markers were used to genotype individual F5 RILs from PI 542196 x
PI 330486 with parental DNA and water as controls. The 12 µl PCR reaction mix contained 40 ng DNA, 1.2 µl of 10x PCR buffer with 15 mM MgCl2 (New England
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Biolabs, Ipswich, MA), 0.48 µl of 25 mM MgCl2 (Fermentas, Glen Burnie, MD), 0.24 µl of 250 µM each of dCTP, dGTP, dTTP, and dATP (GenScript USA Inc. Piscataway, NJ),
0.06 µl of 10 µM M13-tailed forward primer, 0.3 µl of 10 µM reverse primer, 0.24 µl of
10 µM M13 primer fluorescently labeled with 6-FAM, VIC, NED, or PET (Applied
Biosystems, Foster City, CA), and 0.6 unit Taq DNA polymerase (New England Biolabs,
Ipswich, MA). Thermocycler conditions consisted of 5 min initial denaturation at 94oC,
42 cycles of 1 min denaturing at 94oC, 1 min annealing at primer-specific temperature, 1 min extension at 72oC, and final extension at 72oC for 10 min. PCR was conducted in a
Bio-Rad iCycler with 384 well Reaction Module (Bio-Rad laboratories, Hercules, CA ).
PCR products with different fluorophores were mixed and detected on an ABI 3730 Gene
Analyzer (Applied Biosystems, Foster City, CA) in WRSGGL. Fragment analysis was performed using GeneMarker V1.50 software (SoftGenetics, State College, PA).
Linkage map construction and QTL analysis. Marker segregation for resistant
2 lines to susceptible lines in F5 was tested by Chi-squared (χ ) analysis for goodness-of-fit to the expected ratio of 17:15. Segregating markers were used to construct linkage maps with Mapmaker V3.0 (Lander et al., 1987). A minimum logarithm of odds (LOD) score of 4.0 was used as the threshold value for grouping markers into linkage groups. Three- point linkage analyses were carried out to order the linked markers with maximum recombination value of 0.5 for calculating the distance between markers. Genetic distance among markers (cM) was computed by the Kosambi map function (Kosambi,
1944). Linkage groups were assigned to the putative homoeologous chromosomes of the
Sl genome according to the wheat chromosome information provided in GrainGenes
(http://wheat.pw.usda.gov/cgi-bin/graingenes). The order of the markers on the Sl
107 genome were compared with the previously published wheat genome maps (Röder, et al.,
1998; Somers et al., 2004).
QTL analysis was performed using both genotypic and phenotypic data of F5
RILs with WinQTLCart V2.5 (Wang et al., 2006). The least squares means for GUS score and visual rating of two experiments for each line were used in the QTL analysis.
Single Marker Analysis (Wang et al., 2006) was performed to identify markers with significant effects (P < 0.05) for GUS score and visual rating and the chromosome locations of the major QTL for eyespot resistance. Composite Interval Mapping (Wang et al., 2006) was conducted to detect the QTL associated with the resistance to O. yallundae. The LOD threshold value for detecting significant QTL was 2.5 (P < 0.01) based on a 1,000-permutation test (Doerge & Churchill, 1996). The phenotypic variation
(R2) which was explained by significant QTL and additive effects were also carried out with Composite Interval Mapping.
Results
Genetic analysis. The mean GUS score and visual rating of the three F1 plants were 1.4 and 2.0, respectively. GUS scores for 108 F2 plants ranged from 0.7 to 1.9 with a mean of 1.4 (Table 1). Visual ratings of F2 ranged from 0 to 4 with a mean of 2.3. The
F2 plants were considered resistant when their GUS scores and visual ratings were within the 95% confidence limits of the resistant parent (PI 542196), which were 1.4 and 3.3, respectively.
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Table 1. Mean and standard deviation for GUS scores and visual ratings in three generations of PI 542196 (R) x PI 330486 (S) progeny populations inoculated with Oculimacula yallundae
F2 population F3 population F5 population a b Genotype (108 plants) (50 lines ) (189 lines ) GUS Rating GUS Rating GUS Rating
PI 542196 (R) 1.2 ± 0.18 1.5 ± 0.71 1.2 ± 0.11 1.5 ± 0.71 1.2 ± 0.14 1.1 ± 0.51
PI 330486 (S) 1.7 ± 0.11 3.5 ± 0.71 1.6 ± 0.14 3.6 ± 0.55 1.7 ± 0.27 3.6 ± 0.51
F1 (3 plants) 1.4 ± 0.04 2.0 ± 0 ______Min. 0.7 0 0.5 0 1.0 0.6 RILs Mean 1.4 ± 0.24 2.3 ± 1.24 1.3 ± 0.27 2.2 ± 1.24 1.4 ± 0.29 2.2 ± 1.18 Max. 1.9 4.0 2.0 4.0 1.9 4.0 Madsen 1.0 ± 0 0 ± 0 1.0 ± 0 0.3 ± 0.46 1.0 ± 0 0.4 ± 0.5 Hill 81 2.0 ± 0.18 3.5 ± 0.71 1.7 ± 0.24 3.0 ± 0.97 1.8 ± 0.28 3.3 ± 0.65
a There were 16 plants for each line. b Data for F5 populations is combined from two experiments (12 plants/line).
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When data were tested for goodness-of-fit to a single gene model, the F2 plants failed to segregate in a 3:1 (R:S) ratio for GUS score based on the Chi-squared test (P <
0.0001) (Table 2), but segregation of the F2 for visual rating fit a single gene model (P =
0.8241). GUS scores of the F2 plants fit a complementary gene action model (P = 0.1328)
(Rieseberg et al., 1999), which means two genes are required for resistance (9:7).
GUS scores for 789 F3 plants ranged from 0.5 to 2.0 with a mean of 1.3 (Table 1).
Visual ratings of F3 populations ranged from 0 to 4 with a mean of 2.2. They were considered resistant when GUS scores and visual ratings of them were less than or not significantly (P > 0.05) greater than the resistant parent. Segregation of GUS scores didn‟t fit a single gene model (P < 0.0001), but visual ratings fit a single gene model (P =
0.2989) (Table 2).
Mean GUS scores of 189 F5 lines ranged from 1.0 to 1.9 (Table 1), and mean visual ratings ranged from 0.6 to 4.0. They were categorized as resistant when GUS scores and visual ratings of them were less than or not significantly (P > 0.05) greater than the resistant parent. Segregation ratios did not fit a single gene model for GUS score
(P = 0.0475) or visual rating (P < 0.0006), respectively (Table 2). Distribution of GUS scores for all three populations and visual rating in the F5 population were continuous and normal based on the Kolmogorov-Smirnov test of normality (P > 0.01) (Figures 1, 2, and
3a, 3b).
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Table 2. Genetic analysis of resistance to Oculimacula yallundae in three generations of PI 542196 (R) x PI 330486 (S) progeny populations
Generation Observed number Model Expected χ2 R S tested ratio P-value F2 (108 plants) GUS score 61a 47 Single gene 3 : 1 < 0.0001 Complementary 9 : 7 0.1328 gene action Visual rating 82b 26 Single gene 3 : 1 0.8241 Complementary 9 : 7 <0.0001 gene action g F3 (789 plants) GUS score 580c 209 Single gene 5 : 3 < 0.0001
Visual rating 479d 310 Single gene 5 : 3 0.2989
h F5 (189 RILs) GUS score 114e 75 Single gene 17 : 15 0.0475
Visual rating 124f 65 Single gene 17 : 15 0.0006
a GUS score of the F2 population were within the 95% confidence limits of the resistant parent (PI 542196). b Visual rating of the F2 population were within the 95% confidence limits of the resistant parent (PI 542196). c, e GUS score was less or not significantly (P > 0.05) greater than the resistant parent in the tests of F3 and F5 populations, respectively. d, f Visual rating was less than or not significantly (P > 0.05) greater than the resistant parent in the tests of F3 and F5 populations, respectively. g F3 plants were from 50 lines (16 plants/line). h Each F5 RIL included 12 plants in two experiments.
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Figure 1. Frequency distribution of GUS scores in the F2 population (108 plants) of the cross PI 542196 (R) x PI 330486 (S) inoculated with Oculimacula yallundae. The dark- colored columns are resistant plants of which GUS scores within the 95% confidence limits of the resistant parent, whereas susceptible plants are represented by light-colored columns. Kolmogorov-Smirnov test didn‟t reject the null hypothesis of normality with a value of 0.0899 (P = 0.0213).
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Figure 2. Frequency distribution of GUS scores in the F3 population (789 plants from 50 lines) of the cross PI 542196 (R) x PI 330486 (S) inoculated with Oculimacula yallundae. The dark-colored columns are resistant plants with GUS scores less or not significantly (P > 0.05) greater than the resistant parent, whereas susceptible plants are represented by light-colored columns. Kolmogorov-Smirnov test didn‟t reject the null hypothesis of normality with a value of 0.0243 (P > 0.15).
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Figure 3. Frequency distribution of GUS scores (A) and visual ratings (B) in F5 population (189 lines, 12 plants/line) of the cross PI 542196 (R) x PI 330486 (S) inoculated with Oculimacula yallundae. The dark-colored columns are resistant lines with GUS scores or visual ratings less or not significantly (P > 0.05) greater than resistant parent, whereas susceptible lines are represented by light-colored columns. Kolmogorov-Smirnov test didn‟t reject the null hypothesis of normality with a value of 0.0233 and 0.0505 (P >0.15).
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Phenotypic evaluation. The error variances of two F5 experiments were not significantly different (P > 0.05) for visual ratings and GUS scores, therefore, homogeneity of variance was established between the two experiments and data were combined for analysis (Gomez and Gomez, 1984). GUS scores and visual ratings were significantly correlated (P < 0.0001) in both F2 and F3 population tests (r = 0.5027 and
0.6254, respectively). GUS scores and visual ratings were also significantly correlated (r
= 0.678, P < 0.0001) with combined data in the F5 population tests (Figure 4).
The parental lines, PI 542196 (R) and PI330486 (S) had significantly (P < 0.0078) different GUS scores and visual ratings when tested with F2, F3, and F5 populations
(Table 1). The GUS score (1.2) of PI 542196 was not significantly (P > 0.90) different from 1.0, which was the value of Madsen (control), in all the experiments, whereas the
GUS scores (1.6 or 1.7) of PI330486 were significantly (P < 0.0001) greater than 1.0 in each experiment.
Based on analysis of variance (ANOVA), there was significant variation among
RILs for both GUS score and visual rating. The genotype (RILs) had significantly (P <
0.0001) different GUS scores and visual ratings in the F3 and F5 populations (Table 3). In both populations, the effects of environment (block or block within experiment) and genotype by environment interaction (Block x RILs or Expt. x RILs) were significant (P
< 0.05). However, neither GUS scores (P = 0.23) nor the visual ratings (P = 0.83) were significantly different between the two experiments in the F5 population. Broad-sense
2 heritability (H ) based on line means in the F3 population was 85.6 and 83.7% for GUS score and visual rating, respectively (Table 3). In the F5 population, broad-sense heritability was 81.8 and 81.0% for GUS score and visual rating, respectively.
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Figure 4. Scatter plot of visual rating vs. GUS score for 189 F5 recombinant inbred lines derived from the cross of Aegilops longissima PI 542196 (R) x PI 330486 (S). The value of each data point was the mean of 12 plants.
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2 Table 3. Variance components of GUS scores and visual ratings and broad-sense heritability (H ) for the F3 and F5 recombinant inbred lines derived from the cross of PI 542196 (R) x PI 330486 (S) inoculated with Oculimacula yallundae
a b Source of F3 population F5 population variation GUS score Visual rating GUS Visual rating
Mean Mean Mean Mean df F-value F-value df F-value F-value square square square square
RILs 49 0.23 3.3**c 4.75 3.01** 188 0.27 4.54** 4.94 5.65**
Block 7 0.71 10.03** 4.62 2.93*d ______
117 Block (Expt.) ______4 2.69 46.17** 34.76 39.76**
Block x RILs 341 0.07 1.59** 1.58 1.57** ______
Experiment ______1 5.71 2.12 1.84 0.05
Expt. x RILs ______188 0.09 1.6** 1.65 1.88**
Error 391 0.044 __ 1.005 __ 1660 0.058 __ 0.87 __
H2 (line mean basis) 85.6% 83.7% 81.8% 81.0%
a F3 population = 50 lines, 16 plants/line. b F5 population = 189 lines, 12 plants/line in two experiments. c ** P < 0.01. d * P < 0.05.
Genetic linkage map. Based on 16 AFLP primers, the polymorphism between PI
542196 and PI 330486 and PI 604136 and PI 604117 was 39% and 38%, respectively.
Based on 615 wheat SSR markers, 437 (71%) of them amplified fragments in at least one of the four Ae. longissima parental lines. The polymorphism between the two pairs of parents was 76% (332 markers) and 60% (264 markers), respectively. There were 57% and 64% co-dominant markers within the polymorphic markers for each pair of parents, respectively. The 332 polymorphic markers for PI 542196 and PI 330486 were used to genotype 178 F5 RILs along with the parents. The DNA of 178 F5 RILs was chosen randomly among the 189 F5 RILs used for phenotype evaluation because of the limited number of wells in the PCR plate.
Two hundred fifteen segregating SSR markers were utilized for mapping. A linkage map of Ae. longissima was constructed with 169 (79%) wheat SSR markers covering 1261.3 cM in 7 linkage groups. The average distance was 7.46 cM between markers. One hundred one of the 169 markers (60%) were mapped on homoeologous chromosomes (1A, 1B, or 1D) in the Sl genome. Seventy-seven of 101 markers (76%) have the same order as in wheat homoeologous chromosomes. Putative homoeologous chromosomes were assigned as 1Sl, 2Sl, 3Sl, 4Sl, 5Sl, 6Sl, and 7Sl (Figures 5A & 5B).
Individual chromosomes had 15 to 39 markers and length ranged from 53.4 cM to 287.6 cM (Table 4). The percentage of markers from homoeologous chromosomes ranged from
20 to 95% and 67 to 88% of them had the same order as in the published wheat genome map (Röder, et al., 1998, Somers et al., 2004) (Table 4). This linkage map was used to detect QTL for eyespot resistance in Ae. longissima genome.
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Figure 5A. Linkage map of Aegilops longissima chromosomes 1Sl, 2Sl, 3Sl, and 4Sl. Chromosomes 1Sl and 3Sl carry QTL Q.Pch.wsu-1Sl and Q.Pch.wsu-3Sl for eyespot resistance, respectively. QTL are indicated on chromosomes as black rectangles. Q.Pch.wsu-1Sl was closely associated between markers Xcfd6 and Xcfd48 separated by 3.1 cM. Q.Pch.wsu-3Sl was closely associated between markers Xgdm72 and Xwmc597 with a 6.1 cM interval.
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Figure 5B. Linkage map of Aegilops longissima chromosome 5Sl, 6Sl, and 7Sl. Chromosome 5Sl and 7Sl carry Q.Pch.wsu-5Sl, Q.Pch.wsu-7Sl for eyespot resistance, respectively. QTL are indicated on chromosomes as black rectangles. Q.Pch.wsu-5Sl was closely associated between markers Xgwm639 and Xcfd12 with a 13.1 cM interval. Q.Pch.wsu-7Sl was closely associated between markers Xgdm132 and Xcfd2 with a 12.5 cM interval.
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Table 4. Genetic linkage groups of Aegilops longissima based on the cross PI 542196 (R) x PI 330486 (S) constructed with wheat microsatellite markers
Co- Markers with Length Homoeologous Chromosome Marker # dominant the same order (cMa) markersb markers as wheatc 1Sl 133.5 25 14 15 12
2Sl 185.9 35 19 25 19
3Sl 206.5 19 5 18 12
4Sl 287.6 15 1 3 2
5Sl 131.2 21 14 16 14
6Sl 53.4 15 10 8 6
7Sl 263.2 39 11 16 12
a Genetic distance (cM) was computed by the Kosambi map function. b The markers of homoeologous chromosomes of A, B, or D genomes. c The order of the markers on the Sl genome were compared with the previously published wheat genome maps (Röder, et al., 1998; Somers et al., 2004).
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QTL analysis. Four QTL for eyespot resistance were detected on chromosomes
1Sl, 3Sl, 5Sl, and 7Sl, respectively (Figure 6A-6D). All QTL were detected using both
GUS scores and visual ratings and were contributed by the resistant parent, PI 542196.
These QTL were designated as Q.Pch.wsu-1Sl, Q.Pch.wsu-3Sl, Q.Pch.wsu-5Sl, and
Q.Pch.wsu-7Sl.
Q.Pch.wsu-1Sl was detected on chromosome 1Sl with LOD values of 5.2 and 7.1 for GUS score and visual rating, respectively (Figure 6A). Q.Pch.wsu-1Sl explained 11 and 15% of the phenotypic variation with GUS score and visual rating, respectively, and is flanked by SSR markers Xbarc119 and Xcfd83, including 12 markers and covering a
25.7 cM interval. All 12 markers were significantly associated with Q.Pch.wsu-1Sl (P <
0.001). Markers Xcfd6, Xgdm67, Xgwm642, and Xcfd48, which were clustered at a 3.1 cM interval on chromosome 1Sl, were most closely linked to Q.Pch.wsu-1Sl-1 (P <
0.0001). Three of them are co-dominant. For both GUS score and visual rating, the mean of lines with the resistant allele (PI 542196) was significantly less than the susceptible allele (PI 330486) (P < 0.0005) at markers Xcfd6 and Xgdm67 (Figure 7). An additive effect (0.06 for GUS score and 0.19 for visual rating) was contributed from the resistant parent PI 542196.
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Figure 6. QTL for eyespot resistance identified on Aegilops longissima chromosomes 1Sl, 3Sl, 5Sl, and 7Sl by GUS score and visual rating with composite interval mapping. A.) Q.Pch.wsu-1Sl on chromosome 1Sl; B.) Q.Pch.wsu-3Sl on chromosome 3Sl; C.) Q.Pch.wsu-5Sl on chromosome 5Sl; and D.) Q.Pch.wsu-7Sl on chromosome 7Sl.
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Figure 7. GUS scores (A) and visual ratings (B) for RILs of PI 542196 (R) x PI 330486 (S) with different parental alleles at the markers close to each QTL. Xcfd6 and Xgdm67 are close to Q.Pch.wsu-1Sl; Xwmc597 is close to Q.Pch.wsu-3Sl; Xgwm639, Xwmc415, and Xcfd12 are close to Q.Pch.wsu-5Sl; and Xcfd2 is close to Q.Pch.wsu-7Sl. Bars represent the mean GUS scores or visual ratings of RILs with the same parental allele at the marker closet to each QTL. Different letters on the bars indicate the significant (P < 0.05) difference between lines with resistant allele and those with susceptible allele. Error bars show standard errors.
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Q.Pch.wsu-3Sl explained 14 and 9% of the phenotypic variation with GUS score and visual rating, respectively. Its LOD values were 5.3 and 3.4 for GUS score and visual rating, respectively (Figure 6B). This QTL was associated with both GUS score and visual rating on chromosome 3Sl in a 39.2 cM interval between markers Xwmc169 and
Xwmc231. Markers Xgdm72 and Xwmc597, in an interval of 6.1 cM, had a significant (P
< 0.0001) effect on both GUS score and visual rating. Both GUS score and visual rating were significantly (P < 0.0001 and P = 0.0005, respectively) different between resistant and susceptible lines at the closest marker Xwmc597 (Figure 7). The additive effects from the resistant parent were 0.21 and 0.05 for GUS score and visual rating, respectively.
Q.Pch.wsu-5Sl had LOD values of 4.3 and 17.1 for GUS score and visual rating, respectively. It explained 10 and 28% of the phenotypic variation with GUS score and visual rating, respectively (Figure 6C). Q.Pch.wsu-5Sl was flanked between markers
Xgwm293 and Xgwm271 in a 26.5 cM interval. Nine markers fell in that range, and all were significantly (P < 0.0001) associated with Q.Pch.wsu-5Sl by GUS score and visual rating. Xgwm639, Xwmc415, and Xcfd12, in a 13.1 cM interval, were the most closely linked markers. At these markers, GUS score and visual rating were significantly less for the resistant parental alleles (P < 0.0085 and P < 0.0001, respectively) (Figure 7).
Additive effects were 0.06 for GUS score and 0.32 for visual rating, which came from the resistant parent.
Q.Pch.wsu-7Sl was detected from both visual rating and GUS score near one end of chromosome 7Sl, flanked by Xgdm132 and Xcfd2, in a 12.5 cM interval (Figure 6D).
Both markers were significantly (P < 0.003) associated with the QTL. Q.Pch.wsu-7Sl had
LOD values of 3.3 and 4.8 and explained 9 and 11% of the phenotypic variation of GUS
126 score and visual rating, respectively. At marker Xcfd2, both GUS score and visual rating were significantly lower (P = 0.0024 and 0.0017, respectively) for the resistant parental types (Figure 7). The resistant parent had additive effects with values of 0.05 and 0.24 for
GUS score and visual rating, respectively.
Sixteen genotypes with three to eighteen lines each were produced from 178 RILs based on the presence of four QTL. The genotypes were defined by combinations of the resistant parental allele at the closest marker to each QTL. Markers Xcfd6, Xwmc597,
Xwmc415, and Xcfd2 are the closest markers to Q.Pch.wsu-1Sl, Q.Pch.wsu-3Sl,
Q.Pch.wsu-5Sl, and Q.Pch.wsu-7Sl, respectively. The mean GUS score and visual rating for each genotype were calculated (Figure 8). Both GUS score and visual rating were significantly (P < 0.0001) different among the 16 genotypes. Lines with no QTL had the highest GUS score (1.6) and visual rating (3.1), and the lines with four QTL had the lowest GUS score (1.2) and visual rating (1.4). GUS scores and visual ratings for genotypes with one QTL were not significantly different (P > 0.15 and P > 0.14, respectively) from lines with no QTL. All of them were significantly (P < 0.0001) greater than the lines with all QTL. However, within the single QTL genotypes, lines with
Q.Pch.wsu-5Sl had higher mean GUS score (1.5) than lines with other individual QTL even though the difference was not significant (P > 0.50).
127
Figure 8. Resistance of 16 genotypes produced from four QTL detected in F5 RIL populations of PI 542196 (R) x PI 330486 (S) to Oculimacula yallundae. Each genotype includes 3-18 lines and each line included 12 individual plants. Bars represent mean GUS scores or visual ratings of RILs with the same genotype. A.) Mean GUS scores of RILs within each genotype. B.) Mean visual rating of RILs within each genotype. The columns with an asterisk are genotypes with significantly (P < 0.05) lower GUS scores or visual ratings than „no QTL‟. Light-colored columns are genotypes that were not significantly (P > 0.05) greater than „all QTL‟ for either GUS score or visual rating. Error bars show standard errors.
128
Six genotypes had combinations of two QTL; only one genotype (Q.Pch.wsu-3Sl
+ Q.Pch.wsu-5Sl) was not significantly different than lines with all QTL for either GUS score or visual rating (P = 0.81 and P = 0.49, respectively). All of the two QTL combinations had significantly (P < 0.05) lower GUS scores, and four of them had significantly (P < 0.05) lower visual ratings than the „no QTL‟ genotype. Genotypes with combinations of three QTL had significantly lower GUS scores and visual ratings (P <
0.0006 and P < 0.0002, respectively) than „no QTL‟. With one exception for visual rating, all were not significantly greater than the „all QTL‟ for both GUS score and visual rating (P > 0.21 and P > 0.12, respectively).
Discussion
Four QTL contributing resistance to O. yallundae were detected on Ae. longissima chromosomes 1Sl, 3Sl, 5Sl, and 7Sl. These four QTL explained 44% of the phenotypic variation in GUS scores and 63% of the variation in visual ratings. These results demonstrate that genetic control of eyespot resistance in Ae. longissima RILs of PI
542196 x PI 330486 is polygenic and controlled by QTL. This is the first time that multiple QTL conferring resistance to eyespot in Ae. longissima have been reported. To date, QTL for eyespot resistance have been characterized only on the long arm of chromosome 5A of Cappelle Desprez, which explains 34% of the phenotypic variation
(Burt et al., 2011).
The two commercially available eyespot resistance genes, Pch1 and Pch2, both have been characterized as single genes (Worland et al., 1988; de la Peña et al., 1996). At the beginning of this study, one of the questions was to determine the inheritance of
129 eyespot resistance in Ae. longissima. Based on the phenotypic data, only visual ratings in the F2 and F3 populations fit a single gene model. GUS scores in F2, F3, and F5 populations and visual ratings in the F5 population failed to fit a single gene model. The inconsistency in F2 and F3 visual ratings may be due to the lack of separation between the visual rating scales (0 - 4). Johnson (2000) mentioned that resistance to eyespot was a difficult characteristic to score. The continuous distributions of GUS scores in F2, F3, and
F5 populations and visual ratings in the F5 population showed that the classical genetic analysis was not suitable for eyespot resistance; therefore, a QTL mapping approach was conducted. Based on both phenotypic and genotypic data of the F5 population, identification of multiple QTL confirmed that eyespot resistance in Ae. longissima behaved in this study as a trait with quantitative inheritance rather than single gene segregation.
Law et al. (1976) detected eyespot resistance on chromosomes 1A, 2B and 5D of
Cappelle Desprez in addition to the major resistance on 7A in cytogenetic study.
Strausbaugh and Murray (1989) found one semidominant gene for eyespot resistance in
Cappelle Desprez and suggested the possibility of more genes involved in resistance during plant development since their samples were collected at four weeks after inoculation. Burt et al. (2011) identified a QTL (QPch.jic-5A) for eyespot in Cappelle
Desprez in both seedling and adult plants. However, Law et al. (1976) wasn‟t able to detect resistance on chromosome 5A of Cappelle Desprez seedlings. Muranty et al.
(2002) evaluated eyespot resistance in Cappelle Desprez at both the seedling and adult stages. They found a major gene on chromosome 7A only at the seedling stage, as well as another gene on 5A only at the adult stage. Resistance of Cappelle Desprez has remained
130 durable for more than 30 years (Muranty et al., 2002). Durable resistance does not usually fit single gene model, and the phenotype that identifies it is not typical of main genes (Johnson, 2000). In our study, phenotypic data were collected at 8 weeks after inoculation, which was approximately between the growth stage 23 to 25 (Zadoks et al.,
1974), and four QTL were detected. Eyespot resistance in Ae. longissima may be inherited in a similar manner to that of Cappelle Desprez, rather than the single dominant gene in VPM-1.
In QTL mapping, phenotypic evaluation is a key prerequisite. In this study, all
QTL were identified with both GUS scores and visual ratings. de la Peña and Murray
(1994) developed the GUS assay to more accurately assess the eyespot resistance phenotype. GUS scores reflect eyespot pathogen growth and were highly correlated with eyespot resistance based on field evaluation; it is more objective and sensitive than visual ratings (de la Peña & Murray, 1994). During the GUS assay, the inoculation was conducted similar to Macer‟s straw-cylinder technique (Macer, 1966), in which inoculum slurry was pipetted into a straw collar around the stem base. This technique provides both uniformity and accuracy of visual rating, because other pathogens rarely attack the stem base (Doussinault & Dosba, 1977).
Significant effects of environment and genotype by environment interaction existed in phenotypic tests in this study. The block factor was the different growth chamber environments. Although the experiments were under the similar controlled growth chambers and blocks were rotated in order to minimize environmental effects, the humidity varied in different growth chamber and in different locations within one chamber. Another factor contributing the large block effect was the evaluation period.
131
Since the sample size was large and it took about one week to complete one experiment.
During this time, continuous fungal growth would result in block variation. However, high broad-sense heritabilities in GUS scores (82%) and visual ratings (81%) indicated the reproducible phenotypic measurement.
Q.Pch.wsu-1Sl was significantly associated with 12 SSR markers on chromosome
1Sl. Nine of the 12 markers are co-dominant, and Xcfd6 and Xgdm67 are closely linked to
Q.Pch.wsu-1Sl. Xcfd6 is not co-dominant, but co-dominant markers Xwmc156, Xwmc149,
Xgdm67, and Xgwm642 are 6.9, 2.7, 2.1, and 3.1 cM away from Xcfd6, respectively.
When lines had resistant alleles for either Xcfd6 or Xgdm67, both GUS scores and visual ratings were significantly lower than the lines with susceptible alleles. Resistance to eyespot has been detected in homoeologous chromosome Group 1 in other studies. Law et al. (1976) reported that Chromosome 1A of Cappelle Desprez was implicated in resistance to eyespot. Uslu et al. (1998) found resistance in Dasypyrum villosum chromosome 1V to both O. yallundae and O. acuformis. Resistance to both pathogens was also identified in Ae. longissima chromosome 1Sl when Ae. longissima addition and substitution lines were tested (Chapter 2).
Q.Pch.wsu-3Sl was flanked between Xgdm72 and Xwmc231. Xwmc597 was about
7 cM from the QTL peak. The co-dominant marker Xwmc231 was about 8.4 cM on the other side. Q.Pch.wsu-3Sl plays critical role in eyespot resistance since GUS scores and visual ratings were significantly lower when a line had the resistant allele of Xwmc597.
Finding a major QTL conferring resistance to O. yallundae on chromosome 3Sl is not consistent with the results of Ae. longissima addition or substitution lines study, in which only one substitution line containing 3Sl was resistant to O. acuformis (Chapter 2). This
132 may be due to different Ae. longissima accessions used in that study. However,
Dasypyrum villosum chromosome 3V was associated with resistance to both O. yallundae and O. acuformis (Uslu et al., 1998). The powdery mildew resistance gene Pm13 was located on Ae. longissima chromosome 3Sl (Ceoloni et al., 1992).
Q.Pch.wsu-5Sl was the most significant QTL identified in this study; it had an
LOD value of 17.1 and explained 28% of the phenotypic variation in visual ratings. The
LOD value for the GUS score was relatively low (4.3), but the markers were significantly associated with Q.Pch.wsu-5Sl. Xwmc415 is a dominant marker flanked between
Xgwm639 and Xcfd12, which are 6.7 and 6.4 cM away, respectively. The other flanking marker, Xcfd12, is co-dominant. Q.Pch.wsu-5Sl significantly affected resistance; lines with resistant alleles of Xgwm639, Xwmc415, or Xcfd12 had significantly lower GUS scores and visual ratings than lines with susceptible alleles. Homoeologous chromosome
Group 5 has been reported to contain eyespot resistance in several studies (Law et al.,
1976; Uslu et al., 1998; Muranty et al., 2002; Burt et al., 2011). When Burt et al. (2011) detected resistance to eyespot on chromosome 5AL of Cappelle Desprez, SSR marker
Xgwm639 was the closest marker. A QTL for Fusarium head blight resistance was detected in two different winter wheat populations and linked to Xgwm639 (Gervais et al., 2003; Paillard et al., 2004). Thus, Xgwm639 may be a critical marker for disease resistance. SSR marker Xcfd12 was closely linked to an adult plant stripe rust resistance
QTL from the diploid A genome species, Triticum boeoticum, on chromosome 5A
(Chhuneja et al., 2008). Resistance to O. yallundae was also identified in Ae. longissima chromosome 5Sl when Ae. longissima addition and substitution lines were tested
(Chapter 2).
133
Q.Pch.wsu-7Sl was identified at the distal end of chromosome 7Sl. This may be a homoeolocus of Pch1 and Pch2, which are located in the distal portion of chromosome
7DL of VPM-1 (Worland et al., 1988) and 7AL of Cappelle Desprez (de la Peña et al.,
1997), respectively. It has been suggested that Pch1 and Pch2 are homoeoloci (de la Peña et al., 1997; Chapman et al., 2008). The linked markers of Q.Pch.wsu-7Sl, Xgdm132 and
Xcfd2, are different than the markers linked to Pch1 and Pch2. Xwmc14, Xbarc97, and
Xcfd175 are linked to Pch1 (Chapman et al., 2008; Leonard et al., 2008; Meyer et al.,
2011) and Xwmc346, Xwmc525, and Xcfa2040 are linked to Pch2 (Chapman et al., 2008).
Only Xcfa2040 was mapped on chromosome 7Sl in this study, and it was located at the other end of the chromosome. Q.Pch.wsu-7Sl should be located near the end of the long arm if it is a homoeolocus of Pch1 and Pch2. More markers should be added in order to see the other side of Q.Pch.wsu-7Sl. Resistance to eyespot was found in Ae. longissima addition and substitution lines with Ae. longissima chromosome 7Sl (Chapter 2).
The QTL in Ae. longissima exhibited additive effects ranging from 0.05
(Q.Pch.wsu-7Sl) to 0.32 (Q.Pch.wsu-5Sl). In total, the additive effect was 0.38 for GUS scores and 0.80 for visual ratings. During QTL analysis, epistatic effects were detected between some QTL, but they were not significant. The results of different genotype combinations confirmed that the QTL in Ae. longissima are additive.
One hundred seventy eight RILs were grouped according to different QTL combinations, in order to determine the contributions of each QTL to phenotype. No single QTL had significantly greater contribution to resistance than the others. Two QTL combinations resulted in greater eyespot resistance than one QTL. The combination of
Q.Pch.wsu-3Sl and Q.Pch.wsu-5Sl was as effective as three QTL combinations, but not
134 significantly different than all four QTL. With the exception of the combination of
Q.Pch.wsu-1Sl, Q.Pch.wsu-3Sl and Q.Pch.wsu-7Sl for visual rating, all other combinations of three QTL reduced disease more than most two QTL combinations, but the difference were not always significant. When combined with other QTL, Q.Pch.wsu-5Sl played a critical role in reducing eyespot. Ten lines that had all four QTL had the lowest GUS scores and visual ratings, and were significantly lower than most other genotypes. Each
QTL contributed to reducing GUS scores and visual ratings, but the combination of four
QTL provided the best control. The ten lines with four QTL will be very useful for breeding programs because the multiple genes are already pyramided together. Polygenic eyespot resistance has been confirmed in Cappelle Desprez (Law et al., 1976; Jahier et al., 1979), but not all genes in Cappelle Desprez have been characterized and mapped.
Although SSR markers are highly polymorphic, they are not always useful in related genera (Röder et al., 1995). Adonina et al. (2005) reported that the transferability of wheat SSR markers to Ae. longissima was 68% and the polymorphism within Ae. longissima was 75%. In our study, the transferability was 71% with 60 - 76% polymorphism in Ae. longissima. Thus, wheat SSR markers were useful for mapping genes in Ae. longissima and enabled the first genetic linkage map of the Sl genome using wheat SSR markers to be constructed covering seven linkage group in 1261.3 cM.
Polymorphism between the parents of two mapping populations PI 604136 x PI 604117 and PI 542196 x PI 330486 were 43 and 54% out of 615 SSR markers, respectively. The wheat SSR markers will be useful in transferring these QTL to wheat.
QTL mapping with SSR markers has been used for resistance to Fusarium head blight (Gervais et al., 2003; Shen et al., 2003; Paillard et al., 2004; Yang et al., 2005),
135 powdery mildew (Lillemo et al., 2008), and stripe rust (Carter et al., 2009; Lin & Chen,
2009) of wheat. With the same approach, Leonard et al. (2008) linked markers to eyespot resistance gene Pch1 and a major QTL conferring resistance to eyespot on chromosome
5A of Cappelle Desprez was detected (Burt et al., 2011). In our study, four QTL conferring resistance to O. yallundae were mapped in the genome of Ae. longissima. The markers tightly linked to these QTL can be used in breeding programs for marker assisted selection. This is the first study to use wheat microsatellite markers for genetic dissection of disease resistance QTL in a wild relative of wheat. In the near future, the established
RIL populations and genetic map of Sl genome will be used to map QTL for resistance to
O. acuformis. The genes conferring eyespot resistance from Ae. longissima will be introgressed into a suitable genetic background for use in breeding programs.
136
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CHAPTER FOUR
INTERPRETIVE SUMMARY
Wheat (Triticum aestivum L.) is one of the most important human food crops in the world. The US Pacific Northwest (PNW) exports 40% of total US wheat to the world market each year and approximately 80% of wheat grown in the PNW is winter wheat
(WGA, 2009). However, the chronic and economically important soilborne disease, eyespot, caused by Oculimacula yallundae and O. acuformis Crous &W. Gams (Crous et al., 2003), can cause yield losses up to 50% in commercial wheat fields (Murray, 2010).
Planting disease-resistant wheat cultivars is the most economical and environmentally friendly strategy to control eyespot. Currently, Pch1 and Pch2 are the only two eyespot resistance genes available in commercial wheat. Thus, additional genes are desired to develop new wheat varieties with broadly based, effective and stable eyespot resistance in wheat. Ultimately, this will improve winter wheat production in the
PNW and contribute to the world economy. However, the common wheat gene pool contains little resistance to soilborne pathogens including eyespot (Li et al., 2008). In this study, Aegilops longissima Schweinf. & Muschl. (2n = 14, SlSl), a wild species of wheat was evaluated as a potential source of eyespot resistance.
Forty Ae. longissima accessions and 83 Ae. longissima addition or substitution lines were tested for resistance to O. yallundae and O. acuformis and chromosome locations of genetic control in the Sl genome. Among Ae. longissima accessions tested,
43% were resistant to O. yallundae, 48% were resistant to O. acuformis, 33% were
143 resistant to both pathogens, and 25% reacted differently to O. yallundae and O. acuformis. These results provided the first evidence that Ae. longissima can be a new source for resistance genes to eyespot of wheat and that the genetic control of resistance to the two pathogens can differ in some lines. The first report of differential resistance to different eyespot pathogens in D. villosum was presented by Uslu et al. (1998). Burt et al.
(2010) also reported that T. monococcum had significantly different reactions to O. yallundae and O. acuformis.
Among the Ae. longissima addition or substitution lines tested, 28% were resistant to O. yallundae, 31% were resistant to O. acuformis, 10% were resistant to both O. yallundae and O. acuformis, and 39% reacted differently to the two species. Ae. longissima chromosomes 1Sl, 2Sl, 5Sl, and 7Sl were associated with genetic control of resistance to both pathogens. Chromosomes 3Sl, 4Sl, and 6Sl only were associated with resistance to O. acuformis, with an exception of two 4Sl/7Sl translocation lines that were found to be resistant to O. yallundae. These results provide evidence that Ae. longissima contains multiple genes for eyespot resistance, and that some genes control resistance to only one of the pathogens. Uslu et al. (1998) also found effective resistance to both eyespot pathogens on D. villosum chromosomes 1V, 2V, and 3V. Other genes on chromosome 4V and 5V controlled resistance to either O. yallundae or O. acuformis alone, respectively.
A recombinant inbred line (RIL) population was developed from the cross between Ae. longissima accessions PI 542196 (R) and PI 330486 (S) to map the genes. PI
542196 is resistant to both O. yallundae and O. acuformis, and PI 330486 is susceptible to both of them. A genetic linkage map of the Sl genome was constructed with 169 wheat
144 microsatellite markers covering 1261.3 cM in 7 groups. Putative homoeologous
l l l l l l l chromosomes were assigned as 1S , 2S , 3S , 4S , 5S , 6S , and 7S . F5 RILs (189) were tested twice with GUS-transformed isolates for resistance to O. yallundae. Four QTL were detected in chromosomes 1Sl, 3Sl, 5Sl, and 7Sl with LOD values ranging from 3.3 to
5.3 for GUS scores and from 3.4 to 17.1 for visual ratings. Together, these QTL explained 44% of the total phenotypic variation in GUS scores and 63% in visual ratings.
These results demonstrate the genetic control of O. yallundae in Ae. longissima is polygenic. This is the first time multiple QTL conferring resistance to eyespot in Ae. longissima have been reported. The analysis of genetic stocks showed that Ae. longissima chromosomes 1Sl, 2Sl, 5Sl, and 7Sl were associated with the genetic control of eyespot resistance. This was consistent with the QTL analysis in three locations (1Sl, 5Sl, and
7Sl).
Q.Pch.wsu-1Sl is closely linked with markers Xcfd6, Xgdm67, Xgwm642, and
Xcfd48. Eyespot resistance was reported on chromosome 1A of Cappelle Desprez (Law et al., 1976 and chromosome 1V of Dasypyrum villosum (Uslu et al., 1998). Q.Pch.wsu-
3Sl was significantly associated with markers Xgdm72 and Xwmc597. Uslu et al. (1998) found that Dasypyrum villosum chromosome 3V was associated with eyespot resistance.
Q.Pch.wsu-5Sl was closely linked with Xgwm639, Xwmc415, and Xcfd12. Homoeologous chromosome group 5 has been reported to contain eyespot resistance in several studies
(Law et al., 1976; Uslu et al., 1998; Muranty et al., 2002; Burt et al., 2011). Q.Pch.wsu-
7Sl was flanked by Xgdm132 and Xcfd2 and may be a homoeolocus of Pch1 and Pch2 because all are located in the distal portion of homoeologous chromosome group 7.
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Markers Xcfd6, Xwmc597, Xwmc415, and Xcfd2 are tightly linked to Q.Pch.wsu-
1Sl, Q.Pch.wsu-3Sl, Q.Pch.wsu-5Sl, and Q.Pch.wsu-7Sl, respectively. For both GUS scores and visual ratings, the mean values of lines with the resistance allele (PI 542196) was significantly less than the susceptible allele (PI 330486) at these markers. When
RILs were grouped into different QTL combinations, generally lines with more QTL had greater eyespot resistance than those with fewer QTL. Those lines will be useful for breeding programs because the multiple genes are already pyramided together.
This research provides a new source of eyespot resistance and will broaden the genetic diversity of eyespot resistance. The genetic control of eyespot resistence that was identified on different Ae. longissima chromosomes will provide important information for future studies. The markers tightly linked to the QTL with resistance to O. yallundae will be useful in marker-assisted selection for introgressing identified genes into a suitable genetic background for use in breeding programs.
Future Work
Evaluation of accessions and genetic stocks showed that there are different resistance genes in Ae. longissima to O. yallundae and O. acuformis. Therefore, it is logical to detect QTL(s) conferring resistance to O. acuformis by using the available mapping population and linkage map of Ae. longissima. The identification of molecular markers tightly linked to resistance genes for both pathogens will facilitate their transfer to wheat through marker-assisted selection. The next goal is to introgress the genes conferring eyespot resistance from Ae. longissima into a suitable genetic background for use in breeding programs. This will broaden the genetic diversity of eyespot resistance
146 and facilitate development of new varieties containing genes for resistance to both O. yallundae and O. acuformis, resulting in improved control of eyespot.
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