Role of the Ascigerous State in the Epidemiology Of

ROLE OF THE ASCIGEROUS STATE IN THE EPIDEMIOLOGY

OF EYESPOT IN WHEAT

DANILO ISAAC VERA COELLO

A dissertation submitted in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

WASHINGTON STATE UNIVERSITY Department of Plant Pathology

MAY 2015

To the Faculty of Washington State University:

The members of the Committee appointed to examine the dissertation of DANILO ISAAC VERA COELLO find it satisfactory and recommend that it be accepted.

______Timothy D. Murray, Ph.D., Chair

______Dennis A. Johnson, Ph.D.

______Gary G. Grove, Ph.D.

______Weidong Chen, Ph.D.

ACKNOWLEDGEMENT

I would like to express my warmest gratitude to my supervisor, Professor Timothy D.

Murray for having confidence in me from the start, for his invaluable guidance and encouragement throughout this project. I would also like to thank Dr. Murray for his patience and time invested in giving me pieces of advice and preparing me for future challenges. I thank my other committee members, Professors Dennis A. Johnson, Gary G. Grove and Weidong

Chen, for their input, guidance during my graduate work and critical review of my dissertation.

I would like to thank the Plant Pathology Department at Washington State University, especially the faculty, staff and graduate students who provided encouragement and friendship during my time as graduate student. I would like a special thank Dr. Scot Hulbert, Dr. Hanu

Pappu, Cheryl Hagelganz, Debra Marsh, Mary Stormo, and Mike Adams. I would like to acknowledge all the members of the Dr. Murray research group, especially to Dr. Henry Wetzel and Dr. Hongyan Sheng for their help and support in setting up experiments in field and lab and to the graduate students in Dr. Murray lab for their friendship.

I also would like to thank the National Institute of Agricultural Research (INIAP) in

Ecuador for offer me a scholarship to obtain my Ph.D., and the Washington Grain Commission for financial support of part of my research. My special appreciation goes to Dr. Carmen Suarez, for transmitting me her spirit of adventure and excitement in regard to research. Her guidance during my initial formation encourages me to continue my preparation as a researcher. Also, I am particularly grateful to Dr. Devra Jarvis and Bioversity International for initial support of my research.

iii

A very special thanks to my beloved parents Hipolito and Isabel, and to my brother and sisters for their patience; for always being there for me, for their teaching and guidance which made me become as to what I am now. Finally, and most importantly, I would like to thanks my wife and kids for being the reason behind my smiles, my inspiration and motivation to always do my best in everything I do -without them this effort would have been worth nothing.

ROLE OF THE ASCIGEROUS STATE IN THE EPIDEMIOLOGY

OF EYESPOT IN WHEAT

Abstract

by Danilo Isaac Vera Coello Washington State University May 2015

Chair: Timothy D. Murray

Eyespot is a chronic disease of winter wheat, caused by Oculimacula yallundae (OY) and

O. acuformis (OA) that results in premature ripening of grain, lodging, and reduced grain yield.

Discovery of the Oculimacula spp. teleomorph in the Pacific Northwest region of the United

States (PNW) is relatively recent and the role of apothecia or ascospores in the epidemiology of eyespot is unclear. Our goals were to determine the occurrence of OY and OA apothecia in commercial and inoculated field plots, to investigate when apothecia are produced and ascospores released, to determine persistence of apothecia to over summer and over winter, and to investigate factors influencing production of OY apothecia in vitro. Apothecia of OY and OA were found in spring and fall in commercial wheat fields, demonstrating that sexual reproduction occurs regularly in the PNW and may play a role as primary inoculum in the eyespot disease cycle. Apothecia survived over summer but not over winter in inoculated field plots.

Occurrence of ascospores was monitored with Burkard spore traps in inoculated field plots. Ascospores of OY and OA were trapped during spring and fall, and there were no

differences in the number of ascospores trapped from fields with a wheat crop or stubble.

Number of ascospores m-3 wk -1 was positively correlated with relative humidity and weekly accumulated precipitation. Regression models based on environmental variables accounted for

27 to 36% of the variation in number of ascospores trapped. The effect of media, host substrate, inoculation method, temperature, light and stress-shock preconditions on development of primordial and mature apothecia of OY were studied. Inoculation of winter wheat and spring barley straw segments with a suspension of conidia or mycelial plugs favored apothecia development; however, mature apothecia did not developed in treatments without host substrate.

This research provides a base-line offering new insights into the role of the sexual stage of Oculimacula spp. in the epidemiology of eyespot. Understanding the incidence, seasonality and occurrence of apothecia and ascospores provides a better understanding of the role of ascospores as an inoculum source.

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS ...... iii-iv

ABSTRACT ...... v-vi

LIST OF TABLES ...... ix-x

LIST OF FIGURES ...... xi-xii

PREFACE ...... xiii

DEDICATION ...... xiv

CHAPTER

1. INTRODUCTION AND LITERATURE REVIEW ...... 1

Wheat ...... 1

Eyespot disease ...... 2

Causal organisms ...... 5

The sexual stage ...... 11

Epidemiology ...... 15

Management ...... 23

Objectives ...... 28

Literature cited ...... 30

2. OCCURRENCE AND SURVIVAL OF APOTHECIA OF OCULIMACULA

ACUFORMIS AND O. YALLUNDAE ON WHEAT STUBBLE IN THE U.S.

PACIFIC NORTHWEST ...... 49

Introduction ...... 50

Materials and Methods ...... 52

vii

Results ...... 55

Discussion ...... 57

Literature cited ...... 62

3. SEASONAL AND TEMPORAL VARIATION OF ASCOSPORE RELEASE

BY OCULIMACULA YALLUNDAE AND O. ACUFORMIS IN THE U.S.

PACIFIC NORTHWEST ...... 73

Introduction ...... 74

Materials and Methods ...... 75

Results ...... 80

Discussion ...... 82

Literature cited ...... 87

4. PRODUCTION OF APOTHECIA BY OCULIMACULA YALLUNDAE IN

VITRO ...... 100

Introduction ...... 101

Materials and Methods ...... 103

Results ...... 106

Discussion ...... 108

Literature cited ...... 111

5. INTERPRETIVE SUMMARY ...... 120

Future Work ...... 123

Literature cited ...... 125

viii

LIST OF TABLES

CHAPTER TWO

1. Occurrence of Oculimacula yallundae (OY) and O. acuformis (OA) apothecia in

harvested winter wheat fields in Idaho (ID), Oregon (OR) and Washington (WA) from

May 2012 to June 2013 ...... 69

2. Number and percentages of apothecia of O. yallundae (OY) and O. acuformis (OA) in

harvested winter wheat fields in northern Idaho, northeastern Oregon, and eastern

Washington during spring and fall, 2012 and 2013 ...... 70

CHAPTER THREE

1. Mean number of O. acuformis (OA) and O. yallundae (OY) ascospores m -3 wk -1 trapped

at the East (PPE) and West (PPW) plots at the Plant Pathology Farm, Pullman, WA, from

10 June 2012 to 25 June 2014 ...... 92

2. Mean number of O. acuformis (OA) and O. yallundae (OY) ascospores m -3 wk -1 trapped

above winter wheat plants (crop) and stubble in the East (PPE) and West (PPW) plots at

the Plant Pathology Farm from 10 June 2012 to 25 June 2014 ...... 93

3. Spearman’s rank correlation coefficients for relationship between selected meteorological

variables and the number of O. acuformis (OA) and O. yallundae (OY) ascospores m -3

wk -1 trapped in the East (PPE) and West (PPW) plots, at the Plant Pathology Farm,

Pullman, WA, 2012 to 2014 ...... 94-95

4. Best-fit regression models describing the number of O. acuformis and O. yallundae

ascospores m -3 wk -1 in the East (PPE) and West (PPW) plots at the Plant Pathology Farm,

Pullman, WA, regressed against environmental variables. Data collected from June 2012

to May 2014 ...... 96

CHAPTER FOUR

1. Effect of temperature, light and stress-shock preconditioning on number of mature and

primordial apothecia of Oculimacula yallundae after incubation for 8 months .. 115-116

2. Effect of temperature regime, light and stress-shock preconditions on number of mature

and primordial apothecia of Oculimacula yallundae after incubation for 8 months……

...... 117-118

LIST OF FIGURES

CHAPTER TWO

1. Arrangement of straws in a field experiment to determine ability of apothecia to survive

over summer and over winter. A) Wheat straws with apothecia prior to placing them in

field plots; B) individual straws placed among standing stubble; C) straws laid on the soil

surface, and D) straw bundles standing among straws in a row ...... 71

2. Mean monthly occurrence of O. acuformis and O. yallundae apothecia in harvested field

plots previously inoculated with compatible isolates of both pathogens. Filled bars =

percent of straw showing apothecia in the West plot of the Plant Pathology Farm during

2012. Open bars = percent of straw showing apothecia in the East plot of the Plant

Pathology Farm during 2013. Asterisks = apothecia from field in second year of stubble

(East plot)...... 72

CHAPTER THREE

1. Number of O. acuformis (OA) and O. yallundae (OY) ascospores m -3 wk -1 trapped in the

East (PPE) plot at the Plant Pathology Farm, Pullman, WA. June 2012 to 2014 ...... 97

2. Number of O. acuformis (OA) and O. yallundae (OY) ascospores m -3 wk -1 trapped in the

West (PPW) plot at the Plant Pathology Farm, Pullman, WA. June 2012 to 2014 ...... 98

3. Mean number of O. acuformis (A) and O. yallundae (B) ascospores m -3 wk -1 trapped

from March through May 2014 at the Pant Pathology Farm, Pullman, WA. Filled squares

= East plot; Open squares = West plot. Means with same upper- and lowercase letters

(East and West plot, respectively) are not significant different (Tukey’s HSD, P>0.05)..

...... 99

CHAPTER FOUR

1. Effect of inoculation method, plant substrate and medium on the number of primordial

and mature apothecia of Oculimacula yallundae . Inoculation method: CS = conidial

suspension, MP = mycelial plugs. Plant substrate: NS = no substrate, SBS = spring barley

straw, WWS = winter wheat straw. Medium: S= sand , SA = wheat straw agar, SA1 = wheat

straw agar variant 1, WA = water agar, and WSA = wheat seed agar. Errors bar show the

standard deviation of the error ( P = 0.05) ...... 119

xii

PREFACE

The chapters included in this dissertation have been prepared for submission to Plant Disease.

Citations within each chapter refer to the “Literature cited” section of the chapter and follow the

format used in Plant Disease.

xiii

DEDICATION

This dissertation is dedicated to Mima, my kids, and to all my family

xiv

CHAPTER ONE

INTRODUCTION AND LITERATURE REVIEW

Wheat

Wheat ( Triticum aestivum L.) is the most widely cultivated cereal in the world (Shewry

2009), with annual production over 700 million tons (FAO 2013). It is also one of the most important food grains in the human diet, contributing calories, vitamins, minerals, amino acids, and fiber (WGC 2013). Since it was first cultivated 12,000 years ago, wheat has been considered a staple food, providing nourishment for civilizations worldwide (Dubcosvsky and Dvorak

2007). Based on the genetic relationship to its diploid (genome AA, known as einkorn ) and tetraploid (genome AABB or emmer ) progenitors, wheat originated in southeastern Turkey

(Heun et al. 1997). Both einkorn and emmer wheat were developed by domestication of wild populations selected initially by farmers because of their superior yield (Shewry 2009).

Nowadays, more than 90% of the wheat growing worldwide is hexaploid bread wheat

(AABBDD) with 5% tetraploid durum wheat (AABB) and 5% other types (WGC 2013).

In the United States (US), wheat is the principal cereal produced and one of the largest grains exported, contributing to the positive agricultural trade balance. In 2013, the US exported

55 million tons, exceeded only by the European Union (EU), China and India (FAO 2013). US wheat cultivars are classified based on the season in which they are planted.

Several hundred cultivars of wheat are produced in the US; each falls into one of six market classes (hard red winter, hard red spring, soft red winter, hard white, soft white and durum wheat), based on when they are planted, color and shape of the kernels and endosperm characteristics. Each class has different uses and their production tends to be region specific

(WGC 2009). Winter wheat is sown in fall and harvested in the following summer, and spring wheat is sown in spring and harvested in late summer or early fall of the same year (WGC 2013).

Winter wheat requires vernalization (0 - 5°C) to produce grain, whereas spring wheat, does not require vernalization. Due to is greater yield potential, more than 70% of US production is from winter wheat (WGC 2013).

The US Pacific Northwest (PNW), which includes the states of Idaho, Oregon and

Washington, is one of the most important producers of wheat, with 300 billion bushels/year, corresponding to 12% of total US production (WGC 2013). The natural fertility of the land and climate of the PNW make this area ideal for wheat production. In addition, other factors like the transportation system and market availability contribute to the development of this crop. Also, most of wheat market classes including hard white, hard red spring, hard red winter, soft white and soft white club are produced in this region. However, soft white winter wheat prevails with about 70% of the acreage (WGC 2013).

Eyespot disease

Eyespot is a stem-base disease that occurs in temperate regions of the world and affects many hosts in the family Poaceae; however, it is most economically important on wheat

(Triticum spp. ), barley ( Hordeum vulgare L.), oat (A vena sativa L.), and rye ( Secale cereale L.).

Winter wheat is its most agriculturally important host and eyespot has been found on common wheat ( T. aestivum L.), club wheat ( T. compactum L.), durum ( T. durum L. ), emmer ( T. dicoccum L.), einkorn ( T. monococcum ), and spelt ( T. spelta ) (Sprague 1936). Many species related to wheat and cultivated grasses including Aegilops sp ., Agropyron sp ., Agrostis sp .,

Alopecurus sp ., Apera sp ., Bromus sp ., Cynosurus sp ., Dactylis sp ., Festuca sp. , Holcus sp .,

Hordeum sp ., Koeleria sp , Lolium sp ., Phalaris sp ., Phleum sp ., Poa sp ., Secale sp . have been

reported to be hosts (Sprague 1936; Cunningham 1965). Due to the susceptibility of most

Agropyron species inoculated with Cercosporella herpotrichoides in the greenhouse, Sprague

(1936) suggested that A. spicatum and A. inerme were native hosts of this fungus.

Eyespot was first reported in France by G. Fron in 1912 (Sprague 1936). Since then, eyespot has been found in most wheat growing areas of the world, including North America

(Canada and United States) (Dyer et al. 1994), South America (Chile) (Andrade 2005), Europe

(Austria, Belgium, Bulgaria, Denmark, Finland, France, Germany, Great Britain, Greece,

Hungary, Italy, Netherlands, Norway, Poland, Romania, Sweden, Switzerland, United Kingdom,

USSR and Yugoslavia), Africa (Morocco, South Africa and Tunisia), southern Australia (New

South Wales, South Australia, Victoria and Tasmania Island ) and New Zealand (King 1991;

Robbertse et al. 1994; Jalaluddin and Jenkin 1996; Hunter 1989; Fitt 1992).

The first description of eyespot in the US came from pioneer farmers in the Peone Prairie of eastern Washington dating from 1910 (Sprague 1936). Sprague (Sprague 1936) concluded that eyespot had been in this area for a long time on native grasses. Since 1930, the occurrence of eyespot in the US has expanded from the Pacific Northwest (PNW) to the Great Plains, Midwest and Northeastern (Sprague 1931; Sprague 1936; Bruehl 1968).

Clarkson (1981) noted that even though eyespot was found chronically in most wheat fields that it was deleterious to yield only when severe epidemics occurred. Yield reductions can reach up to 50% in commercial fields (Murray 2010); however, the economic impact of eyespot is relatively difficult to quantify because of inaccurate diagnosis of the disease, the presence of other pathogens affecting the stem-base of the plants, husbandry methods and type of cultivar used (Polley and Turner 1995; Scott and Hollins 1978). Yield loss is the result of direct and indirect effects (Glynne 1944). Direct losses are related to stem lesions that interfere with

movement of liquids and nutrients through the vascular system, which decrease yield, whereas indirect losses are related to the increased difficulty of harvesting lodged plants (Glynne 1944;

Jorgensen 1964; Defosse and Rixhon 1968; Clarkson 1981). Scott and Hollins (1978) concluded that yield loss was related more to the amount of lodging than to eyespot lesions. However,

Murray and Bruehl (1986), evaluated the effect of eyespot on yield and yield components in wheat cultivars that varied in resistance and/or susceptibility to eyespot, and concluded that the direct effects of eyespot were the principal determinants of yield losses and that losses were not always associated with lodging. Hence, host resistance, genetic yield potential and the severity of the infection determine the degree of yield reduction (Lucas et al. 2000).

Early infection of wheat by eyespot pathogens affects yield losses. In an experiment inoculating conidia of Pseudocercosporella herpotrichoides (Helgardia yallundae ) to induce early infection in a susceptible wheat cultivar (Bruehl 1982), the reduction of yield was 56%. In late infection, severe lesions were observed, but the effect on yield was relatively small (lower than 10%). Bruehl states that early infections represent the effect of primary inoculum, contributing most to yield loss and late infections represent the effect of secondary inoculum that contribute to the amount of inoculum for succeeding crops.

Symptoms

Symptoms of eyespot are initially observed in early spring after winter wheat breaks dormancy (Murray 2006). The fungus infects outer leaf sheaths of wheat seedlings and grows through successive leaf sheaths and characteristic eye-shaped lesions form near the stem base

(Sprague 1936). Lesions often have dark-colored pupil-like spots in the center with yellow- brown to dark-brown margins. Lesions size varies from 15 to 30 mm in length and 1 to 5 mm width. Lesions generally occur in the first internode above the soil (Fitt et al. 1990); however,

lesions also have been found less frequently up of the third internode (Goulds and Polley 1990).

Early symptoms can appear as pin-point, water-soaked areas no longer than 2 centimeters

(Sprague and Fellows 1934), and be easily confused with other stem-base diseases like sharp eyespot ( Rhizoctonia cerealis ) and brown foot rot, caused by Fusarium or Microdochium spp .

(Turner et al. 1999). Lesions caused by sharp eyespot and Fusarium spp. are generally confined to the out leaf sheaths, whereas eyespot lesions penetrate the outer and inner leaf sheaths, making identification more conclusive (Fitt 1992).

During eyespot infection, stem bases are vulnerable to bend or break at the point of the lesion; when lesions are severe, lodging occurs (Scott and Hollins 1974). Another common symptom in severe eyespot infections is formation of “white heads” or standing dead stems, resulting from the inability of the plant to uptake water and nutrients (Murray 2006).

Causal organisms

Two species of fungi cause eyespot disease in cereals, Oculimacula yallundae (syn:

Tapesia yallundae , Wallwork & Spooner) Crous & Gams, anamorph: Helgardia herpotrichoides

(Fron) Crous & Gams and Oculimacula acuformis (syn: Tapesia acuformis , Boerema, Pieters &

Hamer) Crous & Gams, anamorph: Helgardia acuformis (Nirenberg) Crous & Gams (Crous et al. 2003).

Taxonomically, Oculimacula is in the phylum Ascomycota, class Leotiomycetes, order

Heliotiales, family Dermateaceae (Dyer et al. 2001). Initially, these pathogens were classified as one species, Cercosporella herpotrichoides (Fron) (Sprague 1936), and later renamed

Pseudocercosporella herpotrichoides (Fron) Deighton (Deighton 1973). Oort in 1936 first reported the discovery of strains of the eyespot fungi varying in virulence (cited by Cunningham

1965). Lange-de la Camp (1966), proposed separating isolates into W- and R-types based on

differential virulence to wheat and rye; thus, W-type isolates are able to cause infection in wheat, but have a limited capacity to infect rye, whereas R-types cause disease on both wheat and rye.

Later, four pathotypes W-, R-, C- and S-type isolates were identified based on pathogenicity studies; all were pathogenic to wheat but pathogenically specialized to wheat, rye, oat and goatgrass (Aegilops tauschii ) respectively (Scott and Hollins 1980). Growth rate, colony, spore morphology and pathogenicity to wheat, rye and some grasses species were the criteria used to differentiate the pathotypes (Cunninghan 1981; Cavalier et al. 1987; Nicholson et al. 1991;

Nicholson et al. 1995).

In 1981, Nirenberg (cited by Lucas et al. 2000), proposed a taxonomic system based on colony and conidial morphology: she subdivided the species into Pseudocercosporella herpotrichoides var. herpothichoides , related to W-type, and P. herpotrichoides var. acuformis , related to R-type. In 1983, Von Arx (cited in Crous et al. 2000) reallocated P. herpotrichoides to the genus Ramulispora , which was restricted to graminicolous hosts.

After discovery of the sexual stage of the eyespot pathogens (Wallwork 1987; Dyer et al.

1994) and subsequent discovery of sexual incompatibility between the W- and R-types, they were recognized as distinct species: Tapesia yallundae and Tapesia acuformis , respectively.

Further, Crous et al. (2003) reclassified the teleomorph and anamorph of both species as

Oculimacula yallundae and O. acuformis , and Helgardia yallundae and H. acuformis , respectively. This new classification was based on sequence analysis of the internal transcribed spacer 5.8S and ITS2 regions of the ribosomal DNA. The authors concluded that the teleomorphs of eyespot form a separate group distinct from other non-eyespot Tapesia species. Consequently, the new designation for the anamorph is Helgardia in honor to Dr. Helgard Nirenberg, the first to separate P. herpotrichoides in two varieties (Crous et al. 2003).

Diagnosis and pathogen identification

Diagnosis of eyespot in the field is difficult. Other stem base diseases like sharp eyespot can cause elliptical lesions similar to eyespot, leading to confusion. In addition, O. yallundae and O. acuformis have similar symptoms that cannot be differentiated in the field. Thus, it is a challenge to make a correct assessment based on plant symptoms (Turner et al., 1999).

Conventional pathogenicity tests, based on inoculation of plant seedlings, are time-consuming and the results can be variable. An accurate measurement of the disease is important for an efficient control strategy and for the development of plant breeding programs. Thus, many molecular and biochemical techniques to identify, discriminate and/or quantify the eyespot fungi at both, culture and plant levels have been developed (Lind 1990; Nicholson et al. 1991;

Takeuchi and Kuninaga 1996).

One of the first molecular methods used to differentiate the eyespot pathogens was isozyme polymorphism. Julian and Lucas (1990) used polyacrylamide gel electrophoresis

(PAGE) of specific soluble proteins to differentiate W- and R-type isolates of P. herpotrichoides ; however, the results were inconclusive because many isolates did not present isozyme patterns.

Lind (1990), comparing visual eyespot diseases score with the results obtained from an enzyme- linked immunosorbent assay (ELISA) obtained a poor correlation between the two outputs;

ELISA was incapable to distinguish between the two species of eyespot. Similar results were obtained with an indirect ELISA method (Unger and Wolf 1988).

Restriction fragment length polymorphism (RFLP) of total DNA (Nicholson et al., 1991) and ribosomal and mitochondrial DNA (Nicholson 1993; Takeuchi and Kuninaga 1996) were used to identify pathotypes of the eyespot fungi. RFLPs are more accurate than ELISA methods and provided the first molecular confirmation that W- and R-type isolates corresponded to P.

herpotrichoides var. herpotrichpoides and P. herpotrichoides var. acuformis , respectively.

(Nicholson 1993; Takeuchi and Kuninaga 1996). Pathogen-specific probes developed by Frei and Wenzel (1993) differentiated cultivars directly by using infected plant materials without isolation and culture of the fungi. Priestley et al. (1992), using isoenzyme and DNA markers obtained similar results. Dyer (Dyer et al. 1996) concluded that W- and R-types were separate species based on the reproductive incompatibility in vitro.

The first PCR assay for detection of the eyespot pathogens was based on primers developed from the internal transcribed spacer (ITS) region of ribosomal DNA to differentiate

W- and R-types isolates (Poupard et al. 1993; Gac et al. 1996).

Random amplified polymorphic DNA (RAPD) has also been used to detect and differentiate the eyespot fungi. Jones et al. (1997) questioned the reliability of RAPD (cited by

Lucas et al. 2000), but this technique has proven to be robust for differentiating O. yallundae and

O. acuformis isolates (Nicholson and Rezanoor 1994; Nicholson et al. 1997).

The correlation between visual scoring, antibody- and DNA-based systems needs to be studied to determine which method gives the most accurate indication of eyespot infection.

Albertini et al. (2003) developed a PCR assay based on polymorphic CYP51 gene encoding cytochrome P450 to discriminate O. yallundae and O. acuformis strains with and without resistance to demethylation-inhibiting fungicides.

In addition to conventional PCR, quantitative PCR assays were developed to measure the amount of pathogen present in infected plants. Nicholson et al. (1997) used a gel-based quantitative PCR assay to determine the level of colonization of wheat and rye plant seedlings by both species of eyespot disease, but the method is labor intensive involving post PCR manipulations. Recently, a more reliable alternative for quantification is real-time PCR (qPCR).

qPCR has some advantages, for example, the steps from DNA extraction to analyzing data can be automated with less intensive hand labor and allowing the manipulation of a considerable number of samples (Okubara et al. 2005; Walsh et al. 2005 ). Thus, a reliable SYBR Green- based real-time PCR method was used to discriminate and quantify the response of different wheat genotypes to O. yallundae and O. acuformis (Meyer et al. 2011). The authors concluded that qPCR assay method is an efficient procedure of screening wheat genotypes for their response against the pathogen.

Population changes

Since the sexual stage of the eyespot fungi was reported, changes in population composition of O. yallundae and O. acuformis have been studied (Bateman et al. 1995; Bierman et al. 2002; Douhan et al. 2002; Parnell et al. 2008). The relative proportion of eyespot pathogens has fluctuated by location (Leroux and Gredt 1997). For example, a study in Scotland, suggested a greatest population of O. acuformis than O. yallundae ; in southern England the populations were similar (Hardwick et al. 2001; King and Griffin 1985). In contrast, O. yallundae was the only species reported in South Africa (Robertsee et al. 1994).

O. yallundae used to be the dominant cause of eyespot worldwide (Lucas et al. 2000).

However, the relative proportions of O. yallundae and O. acuformis isolates in field populations have changed such that O. yallundae has been replaced by O. acuformis as the dominant species in some areas (King and Griffin 1985; Nicholson and Turner 2000).

O. yallundae was the predominant eyespot pathogen in the US Pacific Northwest since

1919 (Sprague and Fellows 1934). Murray (1996) conducted a survey of eyespot fungi from

1984 to 1990 and reported that only 8% of the isolates were O. acuformis . However, 10 years later, O. acuformis isolates increased to 44% (Douhan et al. 2003). The authors concluded that

the increase in frequency of O. acuformis suggested a shift in the predominant species causing eyespot, but could not explain the change. In earlier experiments in the UK, evaluating the development of both the W-type ( O. yallundae ) and the R-type ( O. acuformis ) pathotypes in wheat and barley seedlings, Goulds and Fitt (1990) found that the relative rate of development of the two pathogens changed during the course of the season. The authors attribute these changes to variation in temperature that could affect both pathogens differently.

Another potential cause for changes in frequency of eyespot pathogens is differential sensitivity to fungicides (King and Griffin 1985). It was hypothesized that the frequent use of demethylation-inhibitors (DMI) and methyl benzimidazole carbamate (MBC) fungicides used during the 1990s for control of stem base diseases could have disrupted the competitive balance between the two species and altered their relative abundance in the field (Parnell et al. 2008).

Thus, a higher frequency of O. yallundae and O. acuformis isolates showed decreased sensitivity to MBC and DMI fungicides, respectively. Bateman et al. (1995; 2000; 2002) concluded that the frequent use of prochloraz (an imidazole fungicide) between 1990 and 2000 could favor O. acuformis , resulting in alteration of the population of eyespot pathogens.

In the US Pacific Northwest, Bruehl et al. (1985) found eyespot isolates resistant to benzimidazole fungicides in experimental plots. In a succeeding survey, Murray et al. (1990) found a high proportion of benzimidazole-resistant eyespot isolates in the US Pacific Northwest.

However, based on the fewer benzimidazole-resistant O. acuformis isolates compared with O. yallundae , Murray concluded there was not strong evidence of selection pressure for O. acuformis (Murray 1996). Thus, the relative proportion of eyespot pathogens in fields is likely to continue changing as fungicide, hosts and cultural practices change.

The sexual stage

The sexual stage (apothecia) of Pseudocercosporella herpotrichoides was discovered in

Yallunda Flat, South Australia on wheat stems (straw) 75 years after it was first reported by Fron in 1912, and named Tapesia yallundae (Wallwork 1987). It was subsequently reported in several places around the world, including New Zealand (Sanderson and King 1988), England (Hunter

1989; Dyer and Lucas 1995), United Kingdom (Hunter 1989; Nicholson et al. 1991) Belgium

(Moreau and Maraite 1995), Germany (King 1990), Denmark (Sindberg et al. 1994), South

Africa (Robbertse et al. 1994), and Chile (Andrade et al. 2005). O. acuformis , has been reported in Germany (King 1990), United Kingdom (Dyer et al. 1994), Belgium (Moreau and Maraite

1995) and the U.S. Pacific Northwest (Douhan et al. 2002). O. yallundae apothecia have been found more frequently than O. acuformis; however, the presence of apothecia of both species in many places around the world suggests that the sexual stage is a fundamental part of the life cycle of eyespot (Lucas et al. 2000).

Apothecia only have been found on stem bases of straw left after harvest (Dyer et al.

1994). The ascospores are ejected into the air and may provide a long-range dispersal (Dyer et al.

1995). Apothecia were described as separate but gregarious structures, meaning they tend to occur in groups. Initially they are globose, 0.5 to 1.5 mm in diameter, dark-colored and covered with white or pale brown hairs, with 40-55 x 4-6 m m unitunicate, inoperculate cylindric asci containing septate, hyaline and fusiform 7-11 x 1.5-2.0 m m ascospores (Wallwork 1987).

However, apothecia found in the UK and Germany were slightly smaller than described by

Wallwork (Hunter 1989; King 1990).

Discovery of the teleomorph of the eyespot pathogens resulted in a several studies examining population biology, breeding system and role of the sexual stage in the development

of eyespot (Lucas et al. 2000). Consequently, a methodology for inducing formation of apothecia under laboratory conditions was developed (Nicholson et al. 1991). Dyer et al. (1993) studied the mating system of tester strains 22-432 and 22-433 of O. yallundae and demonstrated that apothecia formed when sexually compatible pairs of isolates were present. He concluded that O. yallundae exhibits a two allele heterothallic mating system (Dyer et al. 1993; Moreau and

Maraite 1996), with two complementary mating types designated in accordance to Yoder et al.

(1986) as MAT1-1 and MAT1-2. These results were confirmed when crosses between ascospore progeny from a single apothecium segregated into two mating groups (Dyer et al. 1996).

PCR-based methods were developed to identify the mating types (Dyer et al. 2001;

Moreau and Maraite 1995) and resulted in subsequent reports of their occurrence in Africa

(Robbertse et al. 1994), Europe (Dyer et al. 2001) and the US Pacific Northwest (Douhan et al.

2002). However, not all isolates could be mated effectively with those of opposite mating type, which confirms that incompatibility could be homogenic (Robbertse et al. 1994).

Studies involving crosses between W-, C-, and S-pathotypes, resulted in fertile progeny, confirming that they were all O. yallundae (Dyer et al. 1993). In subsequent studies, the failure to produce fertile progeny in crosses between R-pathotypes and W-, C-, and S-pathotypes, provided conclusive evidence that they are separate species (Dyer et al. 1996; Moreau and

Maraite 1996). Consequently, Tapesia yallundae was accepted for the W-, C- and S-pathotypes and T. acuformis for the R-pathotype (Wallwork and Spooner 1988). Based on studies of morphology and DNA banding patterns, Tapesia yallundae and T. acuformis were proposed as the teleomorphs of Ramulispora (Pseudocercosporella ) herpotrichoides var. herpotrichoides and

R. herpotrichoides var. acuformis , respectively (Robbertse et al. 1995).

Based on a survey of commercial wheat fields in the U.S. Pacific Northwest in 2000, only apothecia of O. acuformis were found in two of the eight fields surveyed (Douhan et al. 2002).

However, the authors reported the occurrence of O. yallundae apothecia in experimental plots inoculated with compatible mating types. The absence of O. yallundae apothecia in naturally infected fields contradicts the high genotypic diversity of O. yallundae populations found in the same fields (Douhan et al. 2002b).

In all surveys, the abundance of apothecia in fields was relatively low and in most cases, apothecia were detected on 1 to 3% of the stems evaluated (King 1991; Dyer and Lucas 1995;

Dyer and Bradshaw 2002; Douhan et al. 2002). Speculation as to why the frequency of apothecia is low includes the predominance of one mating type that would limit sexual reproduction, a situation observed in other pathogens (Dyer et al. 1996). This hypothesis has been partially refuted, because surveys in Europe, New Zealand, UK, and the US confirmed that both mating types were present equally; one exception was in New Zealand, where 86% of the isolates tested were MAT1-1 and 14% were MAT1-2 (Dyer et al. 2001, Douhan et al. 2002).

Another hypothesis explaining the low frequency of the sexual stage is low fertility of the pathogen and the absence of specific environmental triggers necessary for sexual reproduction

(Dyer et al. 1996). Protocols for induction of apothecia of O. yallundae in vitro were developed to better understand eyespot biology (Dyer et al. 1993). Temperature and light regimes were studied in controlled environment conditions for their ability to induce apothecia development

(Dyer et al. 1996; Moreau and Maraite 1996). Although apothecia were produced, results were inconclusive because of the low production rate and period of time needed; their production varied from six to eight months. In addition, fewer apothecia of O. acuformis were produced than O. yallundae (Dyer et al. 1996; Moreau and Maraite 1996). Also, the low genetic variation

observed in some isolates could support the hypothesis that O. acuformis may reproduce almost exclusively by asexual means (Nicholson and Rezanoor 1994). A study attempting to produce sexual structures in vivo on artificially inoculated sites also produced a low number of O. yallundae apothecia (Dyer et al. 1994). Lucas et al. (2000) state that even though the low occurrence of sexual reproduction could not provide major source of inoculum, it could be important for long-range dispersal and genetic diversity in eyespot populations.

Role of the sexual stage

The relative contribution of sexual and asexual reproduction to population genetic structure in fungi that reproduce both sexually and asexually is difficult to determine (Shyang

Cheng and McDonald 1996). Studies of ascospore dispersal in eyespot confirm that wind is the major means of dispersal over long distances (Dyer and Lucas 1995). In contrast, several studies of conidial dispersal demonstrated that conidia were mostly dispersed by rains splash over short distances and not wind. (Rowe and Powelson 1973; Fitt and Bainbridge 1986). Thus, sexual reproduction is a benefit to eyespot pathogens in providing a mechanism for long distance dispersal, which is crucial to maintenance of genetic connectivity among populations

(Trakhtenbrot et al. 2005).

Recombination during sexual reproduction results in maintenance of genetic diversity

(Dyer et al. 1996). In sexually reproducing pathogens, new combinations of alleles occur in the progeny, whereas in pathogens that reproduce mostly by asexual means some clonal lineages can persist for several generations (Shyang Chen and McDonald 1996). Therefore, understanding the genetic structure of eyespot populations can be a useful tool for plant breeders to develop a gene deployment strategy.

Production of ascospores increases the sources of inoculum and could supply a different and long-term niche for spore production (Dyer et al. 2001). Since the first report of sexual reproduction in the eyespot pathogens, apothecia were observed on stems of other plants in the genera Bromus , Hordeum and Holcus (Wallwork 1987; Dyer and Bradshaw 2002). The occurrence of apothecia on alternative hosts may provide an important reservoir of inoculum for eyespot disease. Consequently, research on the epidemiology of the eyespot pathogens focusing on the role of the sexual stage, the relative importance of ascospore inoculum and the influence of the sexual reproduction on population dynamics and genetics of these pathogens are essential to facilitate the development of new management strategies.

Epidemiology

Eyespot is considered a monocyclic disease, (Rowe and Powelson 1973), although effective secondary inoculum is produced, epidemics fit the equation for a simple interest disease, sensu Vanderplank (1963) (Fitt and White 1988). Hollins and Scott (1980) identified two distinct portions of eyespot epidemics; the first is related to initial sporulation, dispersal and infection, and the second is a period of many weeks during lesion development. However, Fitt et al. (1988) considered sporulation, spore dispersal, infection and lesion expansion as the stages in the development of eyespot epidemics. Interestingly, some differences in these stages have been observed for O. yallundae and O. acuformis (Hunter 1989; Daniels 1991) and are discussed below.

Sporulation

O. yallundae and O. acuformis produce two different types of spores, ascospores and conidia, as a result of the sexual and asexual reproduction, respectively (Hunter 1989; Douhan et

al. 2002). Even though both spores are infective (Daniels et al. 1995), conidia are the predominant form of inoculum under field conditions (Lucas et al. 2000).

Sprague (1931) obtained conidia in pure cultures of a fungus isolated from lesions in wheat. He confirmed that the lesions occurring on stem-bases of wheat plants were caused by

Cercosporella herpotrichoides , the causal agent of eyespot identified previously in France by

Fron. Sprague and Fellows (1934) were able to produce large quantities of conidia by growing the fungus on corn meal agar and leaving the cultures outside in late fall when temperature was between -4 and 16°C (Sprague and Fellows, 1934). Glynne (1953) found that conidia were produced when hyphae growing on water agar touched water. Experiments on the effect of temperature on sporulation in naturally infected wheat found that sporulation occurred from 1 to

20°C, with an optimum at 5°C (Glynne 1953; Higgins and Fitt 1984). In culture, the optimum temperature for production of conidia was 10 to 15°C (Sprague and Fellows 1934). Rowe and

Powelson (1973) developed the “Daily thermal sporulation coefficient” (DTSC), based on the total hours of favorable and unfavorable temperature and concluded that 2-3 weeks with soil near saturation, air temperature above freezing and DTSC above 50, resulted in the greatest sporulation.

Water availability is also essential for sporulation. Glynne (1953) reported that sporulation only occurred on straw after water was absorbed, i.e. not on dry straw. Rowe and

Powelson (1973a) observed similar results. Studies on the effect of light on eyespot sporulation are contradictory. Glynne (1953) stated that daylight did not influence spore production.

Sporulation of pure culture of the eyespot fungus only occurred when exposed to near ultraviolet light (Bateman and Taylor 1976). In another study using colonized straw, sporulation was induced by incubating straws in darkness (Rowe and Powelson 1973). However, most studies

concluded that sporulation of the eyespot pathogens is enhanced by exposure to near ultraviolet light (Leach 1967; Ward and Friend 1979).

Another factor influencing sporulation is nutrient level of the media. Glynne (1953) induced production of conidia of the eyespot fungi only on media with low nutrient level.

However, Chang and Tyler (1964) induced greater sporulation on nutrient-rich media (potato dextrose agar) than on low nutrient media (water agar) by incubation at temperatures of 18-21 and 9°C, respectively. Reinecke and Fokkema (1979) also induced conidia on high-nutrient media; however, experiments in field related the conidia occurrence to the increase of temperature (Sprague and Fellows 1934; Glynne 1953; Holling and Scott 1980; Higgins and Fitt

1984).

There is little information on factors influencing apothecia and ascospore production.

King (1991) studied the seasonal occurrence of apothecia in Germany and determined that apothecia were produced in early- to mid-March, which corresponds to the beginning of the spring. No apothecia were found in autumn and the author stated that ascospores could serve as inoculum only in spring. Similar results were obtained in the UK (Bateman et al. 1995; Dyer and

Lucas 1995). In the US, apothecia of O. acuformis were observed during the spring (Douhan et al. 2002).

Spore dispersal

Spore dispersal by the eyespot fungi has been studied extensively, but most information is related to conidia (Lucas et al. 2000). Conidia have been assumed to be the primary inoculum for most disease outbreaks. They are dispersed short distances by rain splash from sporulating stubble infected in previous seasons (Fatemi and Fitt 1983; Fitt et al. 1989). Conidia are produced on hyphae on lesions or pseudoparenchymatous tissue on the surface of infested

residue, enveloped by a mucilage layer known as the extracellular matrix (ECM) that protects spores from desiccation, aids in adhesion to the plant and prevents dispersal by wind alone (Fitt et al. 1989). Conidia are dispersed in splash droplets produced when raindrops strike the conidial mass (Daniels et al. 1991). Glynne (1953) passed dry air over straws infested with the eyespot pathogen and found no conidia were detached; however, a large number of conidia were observed when water drops fell on the infested straws. Similar results were observed in subsequent experiments simulating raindrops on infested debris (Fatemi and Fitt 1983; Fitt and

Bainbridge 1984). In an experiment comparing the number of conidia trapped during light and heavy rains, Fitt and Bainbridge (1983) concluded that the ECM must dissolve before the conidia are released and that moderate to heavy rains are most effective for spore dispersal. Soleimani et al. (1995) using simulated rain confirmed that splash dispersal in P. herpotrichoides was effective over short distances less than 60 cm when no crop cover was present.

There are few studies regarding liberation and infectivity of ascospore of eyespot pathogens. Daniels et al. (1995) induced production of O. yallundae ascospores from apothecia under lab conditions and studied the capacity of ejection of the ascospores, and their infectivity to wheat seedlings. In another study, production of ascospores was related to occurrence of apothecia on field (Dyer et al. 2001); however, no more studies related to occurrence of ascospores of eyespot pathogens have been generated.

Infection

The process and conditions under which infection by the eyespot pathogens occur has been studied in detail (Deacon 1973; Bateman and Taylor 1976; Daniels et al. 1991). However, several of these studies did not distinguish between the O. yallundae and O. acuformis , but it is assumed to be O. yallundae . During infection, spores germinate and infection hyphae penetrate

coleoptiles and outer leaf sheaths through epidermal cells or stomatal openings (Sprague and

Fellows 1934), then mycelia ramify leaf tissue and penetrate inner leaf sheaths (Bateman and

Taylor 1976). In resistant plants, the initial infection process is slower and fewer leaf sheaths are colonized, compared with susceptible plants, where infection is more rapid and more leaf sheaths become infected (Lucas et al. 2000).

Based on controlled environment experiments, inoculated plants develop lesions from 6 to 18°C (Scott 1971; Bateman and Taylor 1976a; Higgins and Fitt 1985); however, these studies did not describe the infection process (Fitt et al. 1988). In a more comprehensive study, Bateman and Taylor (1976b) found that the coleoptiles and leaf sheaths were most susceptible to infection.

During the infection process, spores penetrate the coleoptile and outer leaf sheath at the base of the stems (Sprague and Fellows 1034; Bateman and Taylor 1976b).

The effect of early and late infection by eyespot fungi on wheat cultivars also has been studied. Bateman and Taylor (1976b), inoculated coleoptiles and leaf sheaths in wheat plants and found that coleoptiles were most important. The authors state that even though the coleoptile is most susceptible to infection, wheat plants remain susceptible to eyespot disease throughout their lives. Later, Hollins and Scott (1980) studying the infection period by eyespot fungi and confirmed the assumptions of Bateman and Taylor (1976b). They demonstrated that plants exposed in the field as seedlings or as plants of comparable age as the surrounding crop develop lesions (Hollins and Scott 1980). Murray and Ye (1986) studied infection of coleoptiles and leaf sheaths and found more lignified papillae with hypersensitive reactions formed in the leaf sheaths and epidermal cells of resistant than susceptible wheat cultivars (Murray and Ye 1986).

The authors concluded that lignified papillae were important in reducing penetration by the eyespot pathogen.

The first comparative study of infection by O. yallundae and O. acuformis was made by

Daniels et al. (1991), who found differences between O. yallundae and O. acuformis in the position of the appressoria on the coleoptile, resulting in different patterns of coleoptile infection.

In O. yallundae , germ tubes extend along the anticlinal cell wall between epidermal cells of the host, forming appressoria at intervals. In contrast, O. acuformis , penetration of the coleoptile and epidermal cell walls occurs directly beneath the appressorium. In an experiment using ELISA to measure eyespot pathogen growth, Poupard et al. (1994) confirmed that O. yallundae isolates colonized coleoptiles and leaf sheaths faster than O. acuformis . Gac et al. (1996) confirmed the results obtained by Poupard using PCR. The differences between O. yallundae and O. acuformis in growth rate during infection observed in previous experiments could be explained by differences in penetration patterns of the two eyespot pathogens (Daniels et al. 1991).

In a subsequent study by Daniels et al. (1995) measuring ascospore infection on 23-day- old wheat seedlings, the authors concluded that the infection process from spore adhesion to lesion formation was similar to infection by conidia. Additionally, a multicellular plaque was formed on leaf sheaths during the infection process, and these plaques differ between the eyespot fungi. For O. yallundae , the plaques tend to be asymmetrical and extended along vascular grooves, whereas in O. acuformis , the plaque tends to be more circular and compact, not larger in diameter than the width of the vascular groove (Daniels et al. 1991). Infection plaques have several functions, including protection against desiccation and survival of the pathogen, but its main role is the penetration of the host (Daniels et al. 1991). Inside the plaque, appressorial cells in contact with the host secrete ECM mucilage to produce a seal. Thus, thickening of the fungal cell wall and penetration occurs when tips of infective hyphae puncture the host cell wall (Lucas et al. 2000). As observed in other fungi, the presence of melanized appressoria suggests that both

eyespot pathotypes use physical force to penetrate the host (Deising and Werner 2000; Butler et al. 2009). In an experiment using melanin-deficient mutants of O. yallundae , Perry and Hocart

(1998) found a reduction in pathogenicity of the mutants compared with the parental strains.

Thus, the authors suggested that melanin plays an important role in pathogenesis of the eyespot fungi.

Lesion development

After infection of the coleoptile or outer leaf sheath, the eyespot fungi colonize the leaf tissue and form stromata on the inner surface next to the inner leaf sheath. It is from this tissue that penetration of the next leaf sheath occurs; this process continues as successive leaf sheaths are penetrated (Fitt et al. 1988). The time required for lesion development depends upon environmental conditions and varies from 2 to 8 weeks (Murray 2010). Another report stated that conidia of O. yallundae germinated about 12 hours after inoculation on wheat coleoptiles, appressoria formed 3 days later, and development of the stroma and coleoptile penetration occurred 8 and 30 days after the inoculation, respectively (Blein et al. 2009). After colonization, the typical eye-shaped lesions of eyespot become visible at the base of the stem (Lucas et al.

2000). Blein et al. (2009) suggests a biotrophic stage of O. yallundae occurs during coleoptile infection before switching to a necrotrophic stage once the leaf sheaths are reached. The authors also suggested considering the eyespot fungi as hemibiotroph instead of necrotrophs. Murray and

Ye (1986), studying infection in seedling wheat plants confirmed that O. yallundae colonized the coleoptile and first leaf sheaths before the appearance of visual symptoms. Nicholson et al.

(1997) supported this statement by detecting the pathogen by PCR before appearance of visible symptoms.

Glasshouse and controlled environment studies have demonstrated that the number of infected leaf sheaths increases with increasing temperature from 6 to 18°C (Scott, 1971; Higgins and Fitt 1985). In a study of disease progress in winter wheat, Fitt and White (1988) demonstrated that severity of eyespot in both leaf sheaths and stem lesions increased linearly with time and accumulated temperature. They also concluded that eyespot epidemics could be interrupted by the death of infected leaf sheaths early in the season, resulting in a reduced incidence of leaf sheath lesions.

Differences in virulence of O. yallundae and O. acuformis have been studied intensively.

Fitt et al. (1987) reported more severe lesions caused by O. yallundae compared with O. acuformis . Goulds and Fitt (1990) observed that O. acuformis was more virulent at temperatures below 7°C, whereas O. yallundae was more virulent from 10 to 15°C. Poupard et al. (1994) measured infection by O. yallundae and O. acuformis in susceptible wheat cultivars using

ELISA and reported slower penetration, infection and colonization of leaf sheaths by O. acuformis than O. yallundae .

In field experiments with winter wheat, a linear relationship between O. yallundae and O. acuformis infection in leaf sheaths with thermal time (°C days) was observed (Wan et al. 2005).

The authors also observed greater incidence during early stages, but O. acuformis developed more severe lesions in later stages. Similar results were obtained in a study of the relationship of development of eyespot pathogens with thermal time (Bock et al. 2009), where the development of the lesions was greater in the plots inoculated with O. yallundae than O. acuformis inoculated plots

Survival

Glynne (1942) made one of the first reports regarding survival of eyespot pathogens and concluded that the occurrence of eyespot in a field increased with successive wheat cropping and suggested that survival of the pathogen occurred in harvest residue. Deacon (1973) reported that survival of the eyespot fungi was only 19 weeks in buried wheat straw. The author concluded that even though Oculimacula species are not able to produce sclerotia, the development of specialized structures like pseudoparenchyma on harvested residues could contribute to survival of the pathogens. In a study of spore production by the eyespot pathogens on wheat straw,

Jalaludin and Yenking (1996) reported that the fungus was isolated from infested tissue for up to

84 weeks. Kelly et al. (2008) reported that mycelia of the eyespot fungi can survive up to 3 years on infested stubble, grasses and volunteer wheat and/or barley plants.

Limited information exists about the role of apothecia and ascospores in survival of eyespot pathogens. Wiese (1987), cited by Matusinsky (2009) who worked with both eyespot pathogens concluded that Oculimacula spp are most active during autumn and spring and dormant or least active during summer.

Management

Several different strategies have been used to limit the impact of eyespot on yield

(Herrman and Wiese 1985; Fitt et al. 1990). The most important strategies for the management of eyespot disease are discussed in this section chapter.

Cultural control

Cultural methods were especially important in the management of eyespot before the advent of effective fungicides (Fitt et al. 1990). Crop rotation, minimum tillage and sowing date have been the primary tools recommended to reduce eyespot (Herrman and Wiese 1985).

Crop rotation has the potential to reduce inoculum in soil because the eyespot pathogens only survive in residue of plants that were infected while alive (Fitt et al. 1990). Glynne and slope (1959) observed that crop rotation using a single year break was not long enough to ensure low levels of disease. They found that a two-year break with non-susceptible cultivars and controlling volunteer cereals reduced the incidence and severity of eyespot. Murray (2006) agreed that this practice could reduce the amount of inoculum and therefore be effective where eyespot severe, but was not completely effective in controlling eyespot because even a small amount of inoculum can result in severe disease when environmental conditions are favorable.

Kelly et al. (2008) stated that the eyespot fungi are able to survive on buried stubble for as long as 3 years, so a break from cereals will not necessarily reduce eyespot risk in following crops.

Delaying sowing date in autumn can reduce development of eyespot. Colbach and Saur

(1988) tested different cultural practices and concluded that sowing date had the strongest effect on eyespot disease incidence. In the PNW, delayed seedling combined with fungicide application in early spring was the most common practice used to control eyespot (Bruehl et al. 1982).

Murray (2006) reported that older plants are more susceptible to infection by the fungus, so delaying seedling could reduce disease, but also may result in more soil erosion and the increase of winter injury. In the UK, Fitt et al. (1990) concluded that control of eyespot by later sowing can substantially reduce the risk of eyespot epidemics. However, these authors also noted that even though late sowing could reduce the risk of eyespot epidemics, early sowing may be practiced due to other agronomics reasons.

Pre-plant tillage has been used to reduce the effect of eyespot disease (Herrman and

Wiese 1985); however, its use may be controversial due to increased soil erosion. Herrman and

Wiese (1985) compared different tillage systems and concluded that reduced-tillage and no-till

had significant less eyespot than conventional tillage. The authors state that soil coverage by harvest residues due to tillage could interfere with spore splashing; Murray (2010) corroborated this result. Burning stubble did not significantly decrease the incidence of eyespot (Slope et al.

1970; Shipton 1972). Murray (2006) came to a similar conclusion in the US and noted that the fungus survives on stem bases that are below the soil surface and therefore protected from burning, so enough colonized straw survives to initiate new infections in the next season. Jenkin et al. (1994) studying the effect of debris found that eyespot incidence was less when straw was chopped and incorporated than when burned. The authors also mentioned that microorganisms developing on the decaying straw or stubble might compete with the eyespot fungus, affecting survival and sporulation.

Several papers mention seed density and high levels of nitrogen fertilizer as favoring eyespot disease (Glynne and slope 1959; Fitt et al. 1990). Murray (2006) attributed an indirect effect of fertilizer on eyespot epidemics due to an increase in plant growth that results in increased humidity that favors disease development.

Chemical control

Methyl-benzimidazole carbamate (MBC) fungicides were introduced for eyespot control in the mid-1970s. Carbendazim (Fitt et al. 1990) and Benlate (Murray 1996) were the primary fungicides in this group used specifically for control of eyespot in the UK and US, respectively.

Since then, fungicides have played an important role worldwide in the control of eyespot (Ray et al. 2004; Parnell et al. 2008). MBC fungicides including benomyl, carbendazim, thiabendazole and thiophanate-methyl were used widely due to their relatively low cost and effectiveness.

The use of MBC fungicides was drastically reduced in the 1980s, when resistance to them developed in Oculimacula populations in UK and Europe (King and Griffin 1985). In a

survey of benzimidazole resistance in England and Wales, 37 and 52% of O. yallundae and O. acuformis isolates were resistant to benomyl, respectively (King and Griffin 1985). Similar results were reported by Bateman et al. (1990). In the US, in a survey for resistance to benzimidazole in wheat commercial fields during 1989 and 1990, 24 and 19% of the fields had resistance and a high percentage of isolates collected in those fields were resistant, respectively

(Murray 1996). In the UK, no differences were found in resistance to carbendazim between O. yallundae and O. acuformis (Creighton 1989). However in the US, there were fewer resistant to

O. acuformis than O. yallundae isolates relative to their proportion of the total of isolates collected (Murray 1996). Albertini et al. (1999) attributed benzimidazole resistance to a mutation in the beta-tubulin gene.

After MBC fungicide resistance appeared, prochloraz, a demethylation inhibitor (DMI) fungicide was used in combination with MBC fungicides (Dawson and Bateman 2001; Jones

1994). However, resistance to prochloraz was found in the UK (Bateman et al. 1995) and France

(Leroux and Gredt 1997). Cyprodinil, an anilinopyrimidine fungicide, was used for eyespot control after the appearance of prochloraz-resistant eyespot pathogens developed. Cyprodinil inhibits subcuticular growth of the fungus (Kunz et al. 1998). Babij et al. (2000) found reduced sensitivity in both O. yallundae and O. acuformis isolates in the UK and concluded there was a high risk of development of resistance.

Several disease forecasting programs based on crop history, weather conditions, cultivar planted and others variables have been developed to estimate the best time for fungicide application (Fitt et al. 1988). Burnett et al. (2000) recommend the use of fungicide when a visual threshold of 20% of infected shoots at stem extension stage; however, the authors also

commented on the difficulty in visual diagnosis at this growth stage made forecasting less effective.

Host resistance

The development and use of resistant cultivars to minimize eyespot disease in wheat has been considered a primary management tool because is economically beneficial to growers and environmentally friendly (Fitt et al. 1990). Resistant cultivars enable growers to pay a fixed cost for disease control and decrease the need for chemical applications that may affect non-target organisms.

One of the first studies of host resistance was made by Sprague (1936), who evaluated the susceptibility of a wide range of cereals and grasses to O. yallundae and identified two resistant wild relatives, Aegilops ventricosa and Haynaldia villosa (syn. Dasypyrum villosum L.). Several attempts were made (Doussinalt et al. 1983; Worland et al. 1988; Lind 1999) to transfer this resistance resulted in partial results. Doussinalt et al. (1983) transferred eyespot resistance from

A. ventricosa into the hexaploid wheat line VPM-1 (Ventricosa x Persicum x Marne), using tetraploid wheat as a bridge species. This source of resistance was identified as a major dominant

v gene on the long arm of chromosome 7D and named Pch1 (Worland et al. 1988). Pch1 does not confer immunity but significantly reduces the rate of disease development (Lind 1999). VPM-1 has been used extensively as a source of Pch1 in wheat breeding programs (Doussinault et al. 1983).

Pch1 was incorporated into PNW winter wheat cultivars with the first release in 1987 (Allan et al. 1989). Pch1 has been used successfully since then in many winter wheat cultivars in the US

Pacific Northwest (Leonard et al. 2008). In contrast, Pch1 as not been used widely in commercial cultivars in the UK due to linkage drag that limits yield (Koen et al. 2002)

Another widely used source of resistance is Capelle Desprez (Vincent et al. 1952). A gene associated with resistance was located on chromosome 7A and named Pch2 (De la Peña et al.

1996). Pch2 confers partial durable resistance and is considered less effective than Pch1

(Johnson 1992). Pch2 provides effective resistance against O. acuformis , but is significantly less effective to O. Yallundae (Daniels et al. 1991). Murranty et al. (2002) concluded that Pch2 is ineffective toward O. yallundae at the adult plant stage. Scott et al. (1989) recommends its use in commercial fields with low disease pressure. However, t he moderate resistance of Cappelle

Desprez to eyespot has remained durable for nearly 30 years (Muranty et al. 2002).

Anatomy of basal stem internodes is considered an important component of resistance to eyespot (Murray and Bruehl 1983). Murray and Bruehl (1983) found a high correlation between cultivar resistance with hypodermis width and number of hypodermal cell layers in the first elongated internode of the stem. They stated that cell wall thickening and lignification occurred earlier in resistant wheat cultivars and non-lignified parenchyma cell walls were more susceptible to damage than lignified cell walls. A correlation between field resistance and penetration sites with papillae in seedlings was found in a subsequent study (Murray &Ye 1986).

Objectives

The overall goal of this project is to develop a better understanding of the role of the sexual stage of Oculimacula yallundae and O. acuformis in the disease cycle of eyespot of wheat in the United States Pacific Northwest (PNW). Based on the literature, apothecia of eyespot form in spring (April to June) and presumably release ascospores during the same time frame; however, this timing does not coincide with the primary infection period for winter wheat yield loss (Bateman and Taylor 1976; Daniels et al. 1991), which occurs from October to January.

Furthermore research by Douhan et al. (2002) demonstrated large genotypic diversity of both eyespot pathogens isolated from wheat plants in spring. Therefore, the overarching question addressed in this project is how does genotypic diversity generated during recombination become incorporated into the disease cycle.

Research was conducted to determine when apothecia are present in commercial wheat fields and when ascospores are produced (spring and/or fall seasons). This information will improve our understanding of the epidemiology of eyespot that may lead to improved management measures.

Specific objectives of this research are to:

a. Develop an efficient method to induce rapid and consistent production of

Oculimacula sp. apothecia in vitro.

b. Verify the occurrence of apothecia of Oculimacula spp . in commercial fields in the

U.S. Pacific Northwest.

c. Determine the seasonal periods when apothecia are produced and ascospores

released.

d. Determine the persistence of apothecia over summer and over winter.

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agent of eyespot of wheat in southern Chile. Agricultura Técnica 65:306–311.

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eyespot, Tapesia yallundae and Tapesia acuformis to the anilinopyrimidine fungicide,

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CHAPTER TWO

OCCURRENCE AND SURVIVAL OF APOTHECIA OF OCULIMACULA ACUFORMIS AND O. YALLUNDAE ON WHEAT STUBBLE IN THE U.S. PACIFIC NORTHWEST

ABSTRACT

Eyespot is a chronic disease of wheat, caused by Oculimacula yallundae and O. acuformis that results in premature ripening of grain, lodging, and reduced grain yield. Discovery of the teleomorph of these Oculimacula spp. in the Pacific Northwest (PNW) United States is relatively recent and the role of apothecia in the epidemiology of eyespot is unclear. Our goals were to determine whether and when apothecia of these Oculimacula species are found in the

PNW, and their ability to survive over summer and over winter. Seventy-three harvested wheat fields were surveyed for apothecia during spring and fall 2012 and spring 2013. Apothecia of both species were found in spring and fall in 19% of the fields. Apothecia survived on straws placed on the soil surface the over summer but not the winter. This is the first report of O. yallundae apothecia occurring in commercial wheat fields in the PNW. Occurrence of apothecia in spring and fall demonstrates that sexual reproduction of both species occurs regularly in the

PNW and may play a role in the development of eyespot disease. This study provides a baseline for understanding the influence of sexual reproduction on population dynamics and genetics of both pathogens.

INTRODUCTION

Eyespot disease, caused by two species of the ascomycete fungus Oculimacula yallundae

(Wallwork & Spooner) Crous & Gams and O. acuformis (Boerma, Pieters & Hamers) Crous &

Gams is one of the most important diseases of wheat in the PNW (Clarkson 1981; Scott and

Hollins 1978). Both species have similar life cycles and frequently coexist in the same field; however, the species differ in pathogenicity (Fitt et al. 1987; Scott et al. 1975), cultural characteristics (Hollins et al. 1985; Nicholson et al. 1991), sensitivity to fungicides (Bateman et al. 1995, 2000; Murray 1990, 1996; Parnell et al. 2008), isozymes (Julian and Lucas 1990;

Priestley et al. 1992), ribosomal ITS sequence (Crous et al. 2003) and are reproductively incompatible (Dyer et al. 1996). Eyespot was found affecting cereals and grasses in the PNW more than a century ago (Sprague 1931), and its current distribution extends to the Great Plains,

Midwestern and Northeastern. In the PNW, eyespot can be found chronically in most winter wheat fields (Clarkson 1981) and reduce yield by up 50% (Murray 2006).

The anamorph of the eyespot pathogens was first described by Fron in 1912 (cited by

Chang and Tyler 1964) and 75 years later, Wallwork (1987) identified apothecia of O. yallundae in Australia. Since then, the teleomorphs of both species have been reported in several places around the world (Hunter 1989; King 1990; Robbertse et al. 1994). In vitro crosses of O. yallundae have demonstrated a bipolar mating system governed by a single gene, named MAT1 with two idiomorphs MAT1-1 and MAT1-2 (Dyer et al. 1993; Moreau and Maraite 1996). Field observations have demonstrated that both MAT1-1 and MAT1-2 isolates often are found in the same field (Douhan et al. 2002; Dyer et al. 1996).

Apothecia of O. acuformis were discovered in a survey of commercial wheat fields in the

PNW, but not of O. yallundae (Douhan et al. 2002). The absence of O. yallundae apothecia was surprising given the analysis of molecular markers of O. yallundae isolates that suggested sexual reproduction was likely occurring in the PNW (Douhan et al. 2002). It is possible that apothecia of O. yallundae were overlooked because they occur at low frequency or not found because they are not present in the PNW.

A study in Germany demonstrated that apothecia of O. yallundae were present in low frequency in commercial wheat fields, and that they were produced in early and mid March

(beginning of the spring) (King 1991). Other studies found similar results (Dyer et al. 2001;

Dyer and Lucas 1995; Dyer et al. 1996) and the low incidence of apothecia was attributed to the predominance of one mating type in a population, low sexual fertility and unfavorable environmental requirements necessary to trigger sexual reproduction. Studies of sporulation of the Oculimacula anamorph Helgardia , have demonstrated that temperature, light, water and nutrient requirements all influence sporulation (Chang and Tyler 1964; Glynne 1953; Higgins and Fitt 1985; Hollins and Scott 1980; Sprague and Fellows 1934; Ward and Friend 1979), and are assumed to influence production of apothecia, but studies are absent.

Additional studies on the occurrence, distribution, and persistence of apothecia in commercial fields in the PNW are needed to determine the epidemiological role of ascospores in the disease cycle of the eyespot pathogens. The overall goals of the research described here was to develop a better understanding of the population biology of Oculimacula spp . and epidemiology of eyespot in PNW, which is essential to improve crop protection strategies for the management of eyespot disease. The specific objectives of this study were to determine when

apothecia of O. acuformis and O. yallundae are present in commercial fields, their relative abundance, and their ability to persist over summer and winter.

MATERIALS AND METHODS

Survey for apothecia in commercial wheat fields. Seventy-three harvested wheat fields in northern Idaho, northeastern Oregon and eastern Washington were surveyed for presence of

O. yallundae and O. acuformis apothecia during May to June and September to October of 2012 and June to July 2013. The number of fields surveyed in each location was based on the availability of fields with standing wheat stubble. The location of each field was identified by global positioning system coordinates (GPS) taken with a Garmin etrex handheld GPS device

(Garmin Corp. Schaffhausen, Switzerland.

Fields were surveyed three or more months after harvest to allow time for apothecia to develop (Dyer et al. 2001), which was estimated based on coloration and appearance of the straws; therefore, fields with breakable yellowish-brown straw were preferred as samples.

Approximately, 100-150 straws per field were collected randomly along two transects 30m long each, with a sample interval of 50m. Samples of standing stubble were collected by digging the standing straw, carefully removing loosely adhering soil from the crown and roots, and placing the straw in a paper bag. Samples were labeled and stored at 22°C until examination.

Apothecia assessment. Wheat stems with intact lower leaf sheaths were examined individually for the presence of apothecia using a dissecting microscope at 10X magnification.

When apothecia were found, morphological observations were used for identification of the genus Oculimacula . The presence of one or more apothecia identified as Oculimacula species was designated as a positive occurrence for that location and sample. Apothecia were removed

from the straw with a dissecting needle and stored dry in 2ml Eppendorf tubes at -20°C for DNA extraction, and species determination.

DNA extraction. Apothecia were disrupted by agitation in 400 µl of 0.1% Nonidet P-40, with 0.2 g acid-washed, 425–600 μm diam Glass beads (Sigma-Aldrich St. Louis, MO, USA).

Agitation was performed for 3 min at 6 ms −1 in a Mini Beadbeater (Biospec Products, INC,

Bartlesville, OK, USA), which resulted in disruption of approximately 99% of the apothecia and ascospores, based on observation of samples under a microscope. DNA was extracted from apothecia and ascospore suspensions using the method adapted from Dellaporta et al. (1983), modified by the addition of 20μg glycogen (Thermo Scientific, Pittsburg, PA, USA) as a carrier for the DNA during isopropanol precipitation and placing the product in an incubator at -20°C for 30 min to improve DNA recovery (Williams et al. 2001).

Species determination. Quantitative PCR assay was performed in an iCycler iQ Real-

Time PCR system (Bio-Rad, Lab, Hercules, CA, USA) using SYBR Green I fluorescent dye detection. All reactions were performed in iCycler iQ PCR plates 96 well reaction plates (Bio-

Rad) in a reaction volume of 20 m l containing 1 m l of DNA extract; 10 m l of iQ SYBR Green

Supermix (Bio-Rad, Hercules, CA, USA), 0.5 m l of each purified desalted primers (20 nM)

(Sigma) and 8 m l of double distilled sterile water.

Species differentiation was made using real time PCR as described by Walsh et al.

(2005). Three primers were used in the experiment; two species-specific primers and one common primer designed from the internal transcribed spacer (ITS) region of the rDNA (Walsh et al. 2005).

Thermocycling conditions consisted of an initial denaturation at 94°C for 4 min, followed by 40 cycles at 94°C for 15 s and annealing for 45 s at 62°C. After the final amplification cycle,

a melting curve profile was obtained by heating to 95 oC, cooling to 55 oC and incrementally heating to 95 oC at the rate of 0.5 oC per 10 s to detect nonspecific products or primer-dimers, as indicated by more than one peak in the profile. A cycle threshold (Ct) line was scored positive between 14 and 35, and negative above 35, which was the limit of detection of the lowest concentration of ascospores tested (Vaerman et al. 2004). The reactions were performed using three replicates for each sample. Genomic DNA extracted from O. acuformis and O. yallundae isolates and distilled water were used as positive and negative controls, respectively.

Temporal occurrence of apothecia. The presence of Oculimacula spp. apothecia was monitored in field plots at the Washington State University Plant Pathology Farm near Pullman,

WA during May to August 2012 and April to November 2013. The west and east plots were seeded uniformly with a grain drill to the cultivar Hill 81 in September 2010 and 2011, respectively, so alternating years of winter wheat stubble were present. Plants in each plot were inoculated at Zadok’s growth stage of 20 (Zadoks et al. 1974) with conidia of compatible mating types of both species in November 2010 and 2011 as previously described (Douhan et al. 2002).

Standing stubble was maintained for 1 year after harvest in each plot and then replanted with winter wheat. In 2013, standing stubble was maintained along the edge of the east plot for a second year to observe apothecia.

Observations of apothecia were performed as described previously, except that 100 straws were collected monthly using the random point sampling method to determine the incidence of apothecia.

Apothecia survival. Straws bearing open apothecia collected from inoculated field plots were photographed, labeled and placed in a field containing newly harvested wheat stubble to determine the survival of apothecia to over summer. The experiment was carried out at the Plant

Pathology Farm, Pullman, WA between June and September 2012. Straws were placed in one of three arrangements simulating how they are found after harvest under field conditions: laying on the soil surface, standing in rows of stubble among other straws, and in a bundle of approximately 30 straws standing in a row simulating and intact harvested plant (Fig. 1). Three replicates containing four straws each were distributed randomly in the field. The labeled straws were collected after 3 months, immersed in water for 2 min to hydrate the apothecia and photographed. Pre- and post-incubation photos were compared and apothecia were counted.

Only the rehydrated apothecia (open cup) were counted and compared with the photo taken at the beginning of the experiment. Two mature apothecia selected randomly from each straw were placed on slides, dissected and observed with a compound microscope for the presence of asci and ascospores. The experiment was repeated from June to September 2013, and a similar experiment was conducted from November 2013 to March 2014 to determine whether apothecia were able to survive winter.

Data analysis. Survey data was summarized to represent the main wheat growing areas of the PNW. Samples were pooled to determine the incidence and species distribution across the surveyed area. Incidence was expressed as the number of samples bearing apothecia out of the total samples collected. An RCB design was used to test the effect of straw position on survival of apothecia. Homogeneity of the standard error from the individual experiments was conducted and data where combined to perform analysis of variance.

RESULTS

Survey for apothecia in commercial wheat fields. A total of 7,825 wheat straws were collected from 73 harvested wheat fields in northern Idaho, northeastern Oregon and eastern

Washington. Apothecia of O. yallundae and/or O. acuformis were found in 19.2% of the fields, from the counties of Benewah and Latah, ID, Umatilla, OR, and Adams, Asotin, Columbia,

Garfield, Walla Walla, and Whitman, WA. Apothecia occurred in 21.4 and 21.7% of ID and

WA fields, respectively, and 7.7% in OR (Table 1). Apothecia of both O. yallundae and O. acuformis occurred in 28.6% of the fields where apothecia were found, whereas apothecia of O. yallundae or O. acuformis only occurred in 35.7% of the fields with apothecia (Table 1). The incidence of apothecia in fields was low, ranging from 1 to 3% of the straws collected (data not shown).

A greater percentage of fields with apothecia were found in 2012 than in 2013 with

22.2% and 16.2%, respectively (Table 2). In 2012, more fields with apothecia were found in spring (38%) than in fall (10%). In spring 2013, 16.2% of surveyed fields held apothecia; unfortunately, very few harvested winter wheat fields with standing stubble were found in fall

2013 and thus, no data are available (Table 2).

Temporal occurrence of apothecia . Mature apothecia of Oculimacula spp. were present in both field plots at the Plant Pathology Farm during all sampling time periods. The

West plot was sampled from May to August 2012 and the greatest incidence of apothecia occurred in May (45%) and the least in July and August (17% each) (Fig. 2). The East plot was sampled from April to November 2013 and apothecia were present on about 22% of stems in

April through June and decreased to less than 15% from July to September. In contrast to the

West plot, stubble was allowed to remain standing into the second year after harvest so it could be observed through November, when incidence of apothecia was 52%, which was significantly greater than all other months (Fig. 2).

Apothecia survival. Apothecia were able to survive the over summer in all the three arrangements of straw tested. Survival of apothecia was greater than 70% in all three arrangements, but greater numerically on straw in bundles (85.1%) than standing in rows of stubble (77.4%), or straw laid on the soil surface (71.3%); however, they did not differ statistically (Fig. 3). In a similar experiment carried out during the winter 2013, viable apothecia were no recovered from any of the configurations tested (results not shown).

DISCUSSION

The presence of apothecia of O. yallundae and O. acuformis in wheat fields in the PNW during spring and fall demonstrates that sexual reproduction in these fungi is occurring and that ascospores may have a more role in the epidemiology of eyespot in this area than previously believed. These results expand upon the study by Douhan et al. (2002), where apothecia of O. acuformis but not O. yallundae were found in commercial PNW wheat fields. The presence of

O. yallundae apothecia under commercial field conditions was not surprising since a previous population genetics study based on molecular markers concluded that recombination was occurring in O. yallundae and apothecia of this fungus were found in an experimental plot inoculated with compatible mating types of the O. yallundae (Douhan et al. 2003).

The presence of apothecia in 19% of the commercial fields surveyed in the PNW is epidemiologically important; at the regional level, the presence of apothecia during fall, when winter wheat is planted, suggests that apothecia could provide ascospores that serve as primary inoculum initiating the disease cycle. The development of new infections by ascospores also offers an alternative means of long-range dispersal, providing higher efficiency in the spatial distribution of the disease (Daniels et al. 1995). At the field level, recombination during sexual

reproduction results in maintenance of genetic diversity in the population of O. yallundae and O. acuformis pathogens (Dyer et al. 1996).

The incidence of apothecia was low within the 14 fields in which they were found, never exceeding the 3% of stems. Other studies in Europe and North America found similar results

(Douhan et al. 2002; Dyer et al, 1996, 2001). Dyer et al. (1996) hypothesized that the main reasons for limited sexual reproduction were due to the predominance of one mating type, the low fertility of the pathogens and the absence of specific environmental triggers necessary for sexual reproduction. However, in a study carried out in the PNW, Douhan et al. (2002) determined that both mating types of O. yallundae and O . acuformis were widely distributed in equal proportion across all wheat fields sampled, suggesting that mating system is well established and might not affect production of apothecia. The fact that apothecia are forming, albeit in low percentages of infested stems, suggests that environmental conditions are not limiting and that isolate fertility and/or spatial distribution (or density) of mating types within fields is a more likely explanation for the low incidence of apothecia.

This is the first report of apothecia of O. yallundae and O. acuformis occurring in commercial wheat fields during fall in the PNW and their presence, along with the production of viable ascospores, demonstrates the potential to incorporate the genotypic variation resulting from recombination that was observed by Douhan et al. (2002) into the disease cycle. The presence of apothecia in commercial wheat fields in the fall contrasts with previous studies in

UK and Germany (Hunter 1989; King 1991; Nicholson et al. 1997) in which apothecia were only found during spring. However, Hunter (1989) and King (1991) consider the fall as the period of more suitability for occurrence of new eyespot infections because of it is the season when winter wheat is planted.

Although the incidence of Oculimacula spp. apothecia was lower in fall than spring, environmental conditions might compensate and result in a greater chance of infection than for ascospores released in spring (Kirisits et al. 2012). A study in the UK, came to a similar conclusion (Dyer et al. 1994); based on their findings, the authors suggested that ascospores may represent a potential source of inoculum for up to 9 months and that moisture, and to a lesser extent, temperature might influence the development and discharge of ascospores.

The occurrence of both O. yallundae and O. acuformis in same fields is another aspect of the disease that has been discussed in several studies (Bock et al. 2009; Hunter 1989). Bierman et al. (2002) reported that differences in competitive ability between the pathogens might favor

O. yallundae infections during fall. Other studies in controlled environment supports this conclusion based on differences in mycelial growth rate between O. yallundae and O. acuformis

(Hollins et al. 1985; Waldner-Zulauf and Gisi 1991), penetration of leaf sheaths (Bierman et al.

2002; Poupard et al. 1994) and ability for sexual reproduction (Moreau and Maraite 1995).

Based on hour results, apothecia of O. yallundae and O. acuformis occurred at similar times and frequency during 2012 and 2013, suggesting that both species, despite having small niche differences, can coexist in the same field on the same host (Fitt et al. 2006)

Based on survival of apothecia of O. yallundae on naturally colonized straws in field plots, apothecia are able to survive over summer, but not over winter. Straws were arranged in three different configurations simulating how they occur following wheat harvest and over summer survival was greater than 70% in all treatments. This is the first report of apothecia survival between seasons and demonstrate that apothecia produce in spring and surviving over summer could contribute to production of ascospores during fall and consequently, can serve as a source of primary inoculum (Bierman et al. 2002; Dyer et al. 2001). Survival over winter was

only tested in only one year and further study needs. However we hypothesize that Oculimacula spp. survives the winter asexually as hyphae in colonized stem bases, apothecia develop during spring and release ascospores, then survive over summer and release ascospores again during fall when winter wheat seedlings are present. Additional research is needed to determine the effect of environmental factors on over summering survival of apothecia. For example, the inability of apothecia to survive over winter may be due to the inability to tolerate low temperatures or the prolonged wet conditions that predominate.

The occurrence of apothecia during all the months of evaluation in the harvested West

(2012) and East plots (2013) (Fig. 2) suggests a high predisposition of Oculimacula spp. to produce sexual structures when compatible mating types are present. However, the monthly percentage of occurrence of ascospores is considered relative, since it is difficult to determine whether the founded apothecia were produced during the month of evaluation or in previous months. More studies are needed to determine the period of initial development of apothecia and their temporal capacity to produce ascospores. Also, it would be interesting to compare the incidence of apothecia in parts/sections of harvested commercial fields with previous report of incidence of eyespot disease.

In summary, apothecia of O. yallundae and O. acuformis occur widely in the PNW during spring and fall on winter wheat stems infested while the plants were alive; ascospores are produced in both seasons and constitute a source of primary inoculum in addition to conidia produced on colonized straw.

ACKNOWLEDGEMENTS

We thank the National Institute of Agricultural Research (INIAP) in Ecuador, the

Washington Grain Commission and Department of Plant Pathology, College of Agricultural,

Human, and Natural Resource Sciences Agricultural Research Center, Project No. 0670,

Washington State University, Pullman, WA 99164-6430 for financial support of this project. The authors acknowledge the technical assistance provided by Dr. Paul Carter, Columbia County

Director, Washington State University.

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TABLE 1 . Occurrence of Oculimacula yallundae (OY) and O. acuformis (OA) apothecia in harvested winter wheat fields in Idaho (ID), Oregon (OR) and Washington (WA) from May 2012 to June 2013.

Fields with Species Incidence d State County na apothecia b OY OA OY+OA c (%) ID Benewah 8 2 - + + ID Clearwater 1 0 - - - ID Idaho 1 0 - - - ID Latah 3 1 - - + ID Nez Perce 1 0 - - - 14 21.4 OR Umatilla 10 1 + - - OR Union 1 0 - - - OR Wallowa 2 0 - - - 13 7.7 WA Adams 5 1 + - - WA Asotin 5 1 + - - WA Columbia 10 2 + - + WA Franklin 1 0 - - - WA Garfield 6 1 - - + WA Walla Walla 5 2 - ++ - WA Whitman 14 3 + ++ - 46 21.7 Total 73 14 5 5 4 Frequency 19.2 35.7 35.7 28.6 a Number of fields surveyed. b Fields in which Oculimacula spp. apothecia were found. (+) One field with apothecia. (++) two

fields with apothecia. c Counties with apothecia of O. yallundae and O. acuformis in the same field. d Incidence of apothecia by state.

TABLE 2 . Number and percentages of apothecia of O. yallundae (OY) and O. acuformis (OA) in harvested winter wheat fields in northern Idaho, northeastern Oregon, and eastern Washington during spring and fall, 2012 and 2013.

Number of apothecia b Season Samples a %d OY OA OY+OA c 2012 Spring 16 2 2 2 37.5 Fall 20 0 1 1 10.0 2013* Spring 37 3 2 1 16.2 a Number of fields surveyed. b Number of fields with apothecia in which Oculimacula spp. apothecia were found. c Number of fields with apothecia of O. yallundae and O. acuformis in the same field. d Incidence of fields with apothecia.

* No data are available in fall 2013 due to limited number of harvested winter wheat fields with

standing stubble.

FIGURE 1 . Arrangement of straws in a field experiment to determine ability of apothecia to survive over summer and over winter. A = Wheat straws with apothecia prior to placing them in field plots; B= individual straws placed among standing stubble; C= straws laid on the soil surface, and D= straw bundles standing among straws in a row.

FIGURE 2 . Mean monthly occurrence of O. acuformis and O. yallundae apothecia in harvested field plots previously inoculated with compatible isolates of both pathogens. Filled bars = percent of straw showing apothecia in the West plot of the Plant Pathology Farm during 2012. Open bars = percent of straw showing apothecia in the East plot of the Plant Pathology Farm during

2013. Asterisks = apothecia from field in second year of stubble (East plot).

CHAPTER THREE

SEASONAL AND TEMPORAL VARIATION OF ASCOSPORE RELEASE BY OCULIMACULA YALLUNDAE AND O. ACUFORMIS IN THE U.S. PACIFIC NORTHWEST

ABSTRACT

Eyespot disease of wheat is caused by Oculimacula yallundae (OY) and O. acuformis

(OA). The teleomorphs of these fungi were recently reported in the U.S. Pacific Northwest, but their role in the epidemiology of eyespot is unknown. Occurrence of ascospores of both pathogens was monitored with Burkard spore traps in two inoculated field plots from 2012 to

2014 at the WSU Plant Pathology Farm. Genomic DNA was extracted from spore trap tapes and real-time PCR used to quantify DNA, which was used to enumerate ascospores. More OY than

OA ascospores were captured ( P = 0.05) during this study. There were no differences in the number of OA and OY ascospores trapped from fields with a wheat crop or stubble. More ascospores were captured from 0000 to 0600 h than at other times of the day. Number of ascospores m-3 wk -1 was positively correlated ( P < 0.05) with relative humidity and weekly accumulated precipitation. Regression models including environmental variables accounted for

27 to 36% of the variation in number of ascospores trapped in both sites. Two main seasonal ascospore release periods occur; one each in spring and fall during and after rainfall events.

Ascospores released in fall may have an important role as primary inoculum in addition to conidia.

INTRODUCTION

Eyespot disease, caused by Oculimacula yallundae (Wallwork & Spooner) Crous &

Gams and O. acuformis (Boerma, Pieters & Hamers) Crous & Gams, is one of the most damaging diseases of wheat grown in temperate regions of the world (Clarkson 1981; Murray

2006). Disease is initiated when these fungi infect the coleoptile and outer leaf sheaths of wheat seedlings and subsequently grow through the inner leaf sheaths forming eye-shaped lesions on the stem base (Daniels et al. 1991). Lesions interfere with movement of water and nutrients in the vascular system, which results in decreased yield (Clarkson 1981). Indirect losses due to lodging of plants also occur (Glynne 1944; Scott and Hollins 1974).

Oculimacula spp . produce both sexual and asexual spores (Hunter 1989; Daniels et al.

1995); however, since their initial description 75 years ago, the eyespot fungi were thought to reproduce only by asexual conidia and consequently, most epidemiological studies have focused on infection, colonization, sporulation and dissemination by the anamorph (Bateman and Taylor

1976; Fatemi and Fitt 1983; Glynne 1953; Higgins and Fitt 1984; Rowe and Powelson 1973).

Although discovery of the teleomorph (Wallwork 1987) brought new insights about the population biology of these fungi (Daniels et al 1995; Dyer et al. 1995), there is relative little information on factors influencing production of apothecia and ascospores or of their role in the disease cycle. Eyespot is considered a monocyclic disease (Fitt et al. 1988; Rowe and Powelson

1973); although secondary inoculum production occurs, its role in the infection process is considered unimportant (Dyer and Lucas 1995; King 1991).

Conidia are dispersed short distances by rain splash when raindrops strike colonized stems (Fatemi et al. 1989; Daniels et al. 1991). In contrast, wind is the major means of ascospore dispersal and provides long distance dissemination (Daniels et al. 1995; Dyer et al. 1995). Dyer

and Lucas (1995) speculated that ascospores are not a major source of inoculum due to limited production and mismatch in timing when apothecia form in the spring and primary infection occurs in the autumn. Several European studies report the occurrence of apothecia of O. yallundae and O. acuformis in spring (Burnett and Hughes 2004; Dyer et al 2001). However, in a recent survey carried out in the Pacific Northwest region of the United States (PNW), we documented the presence of mature apothecia in both spring and fall (Vera and Murray 2014).

Based on this finding, we hypothesize that ascospores of O. acuformis and O. yallundae are discharged in spring and fall, and contribute to primary inoculum.

The specific objectives of this study were to determine the seasonal and temporal occurrence of ascospores in inoculated field plots when a wheat crop or stubble was present and identify the meteorological variables associated with their occurrence.

MATERIALS AND METHODS

Ascospore trapping. Ascospores of OY and OA were monitored in two inoculated field plots, West (PPW) and East (PPE) (approx. 22 m x 14 m each), located at the Washington State

University Plant Pathology Farm near Pullman, WA during May to July and October to

November 2012; March to July and October to November 2013, and March to June 2014. These sample periods represent the times when most rainfall occurs and persistent snow cover is not present.

Field plots were seeded with winter wheat cultivar Hill 81, which is susceptible to eyespot, using a grain drill in September each year of this study. PPW was sown in 2010 and

2012, and PPE in 2011 and 2013, respectively, to obtain alternating years of wheat crop and stubble. Plots were inoculated when wheat plants were about Zadok’s growth stage 20 to 25

(tillering) (Zadoks et al. 1974) with conidia of compatible mating types of both OY and OA in

November 2010 and 2011, respectively, as previously described (Douhan et al. 2012). Following harvest each year, standing stubble was maintained until the following August, when the plot was disked in preparation for planting in September. In 2013, a strip of standing stubble 1.5 m wide was maintained along the edge of the PPE plot for a second year to observe apothecia formation.

Ascospores were trapped in each plot with a 7-day recording volumetric spore sampler

(Burkard Manufacturing Co. Ltd., Rickmansworth Hertfordshire, UK) placed 1.5 m above ground level on a small table to reduce the chance of trapping asexual spores. Air was drawn into a 14 mm x 2 mm orifice in the main body of the spore trap at 10 liters per minute. Airborne particles were trapped by impact on a Melinex tape lightly coated with Vaseline (Unilever products, Trumbull, CT, USA) and placed on an internal drum that rotates at the rate of 2 mm h -

1. Tapes were removed and replaced when the drum completed one full revolution every 7 days.

Tapes were stored in an incubator at 4°C until processing. In 2012 and 2013, each tape was cut into seven pieces each representing 24 h. In 2014, tapes were cut into 28 pieces each representing

6 h to determine the period of daily ascospore dispersal. Thus, section 1 represents 0000 to 0600 h, section 2 6001 to 1200 h, section 3 1201 to 1800 h, and section 4 1801 to 2400 h.

Each segment of tape was cut in half lengthwise. One half was fixed onto a microscope slides, stained with a saturated solution of basic fuchsin and examined with a microscope to observe the presence of asexual spores of O. yallundae and O. acuformis . If conidia were found, the other half of that segment was discarded. If conidia were not present, the other half-segment was placed into a 2-ml Eppendorf tube and stored at -20°C for DNA extraction and quantification.

DNA extraction. To determine an appropriate DNA extraction procedure for airborne ascospores of OA and OY pathogens from the Melinex tapes from the Burkard spore traps, several maceration techniques and DNA extraction procedures were tested (Calderon et al. 2002;

Dellaporta et al. 1983; Kaczmarek et al. 2009; Williams et al. 2001). Thus, Eppendorf tubes containing the half segments of tapes were disrupted by agitation in 400 m l of 0.1% Nonidet P-40 and 0.2 g acid-washed, 425-600 m m diam glass beads (Sigma-Aldrich St. Louis, MO, USA).

Agitation was performed for 3 min at 6 ms -1 in a Mini Beadbeater (Biospec Products, INC,

Bartlesville, OK, USA). DNA was extracted using the method from Dellaporta (Dellaporta et al.

1983), modified by the addition of 20 μg glycogen (Thermo Scientific, Pittsburg, PA, USA) as a carrier for the DNA during isopropanol precipitation and placed in an incubator at -20°C for 30 min to improve DNA recovery (Williams et al. 2001).

Species determination and ascospore quantification. Three primer sets previously determined as specific to differentiate OA and OY pathogens (Gedye and Murray, unpublished);

Walsh et al. 2005; Nicholson et al. 1997) were tested. The primer set YALL-1(5´-GGG GGC

TAC CCT ACT TGG CAG-3´), AC-1(5´-GCC ACC CTA CTT CGG TAA-3´), and FAM-1(5´-

ATT CAA GGG TGG AGG TCT GRA C-3´) designed from the internal transcribed spacer

(ITS) region of the rDNA (Walsh et al. 2005) was selected based on the fewest false-positives obtained during preliminary tests.

A 20-m l PCR reaction containing 1 m l of DNA extract, 10 m l of iQ SYBR Green

Supermix (Bio-Rad, Hercules, CA, USA), 20 nM of forward and reverse primers and 8 m l of double-distilled sterile water was performed in iCycler iQ PCR 96 well reaction plates (Bio-

Rad). PCR amplification was done using an iCycler iQ Real-Time (RT) PCR system (Bio-Rad,

Lab, Hercules, CA, USA) using SYBR Green I fluorescent dye.

Thermal cycling parameters for detection of O. yallundae and O. acuformis were: initial denaturation at 94°C for 4 min, followed by 40 cycles at 94°C for 15 s and annealing for 45 s at

62°C. After the final amplification cycle, a melting curve profile was obtained by heating to

95°C, cooling to 55°C and incrementally heating to 95°C at the rate of 0.5°C per 10 s to detect nonspecific products or primer-dimers, as indicated by more than one peak in the profile. A cycle threshold (Ct) line was scored positive between 14 and 35, and negative above 35, which was the limit of detection of the lowest concentration of ascospores tested (Vaerman et al. 2004).

Reactions were performed using three replicates for each sample. Genomic DNA extracted from

O. acuformis and O. yallundae isolates and distilled water were used as positive and negative controls, respectively.

The relative quantification of ascospores was based on standards prepared by PCR amplification from five-10 fold serial dilutions of known concentrations of ascospores ranging from 1x10 5 to 1 x 10 2 spores ml -1. Aliquots (250 m l) of the diluted suspensions were each deposited on pieces of coated Melinex tape, allowed to dry and then placed individually in

Eppendorf tubes for DNA extraction and RT PCR quantification. A standard curve and an equation for the linear regression were constructed with the logarithm of the initial ascospore number of the standards and their Ct values to determine the number of ascospores in the unknown sample (Schena et al. 2004). The same DNA extraction and thermal cycling parameters described above were used to quantify ascospores and melting curve analysis determined the specificity of the reaction (Okubara et al. 2005).

Ascospore counts were corrected to number of ascospores m -3 of air sampled and expressed as number of ascospores m -3 wk -1. Data from 3 March to 25 May 2014 was used to determine whether there was a diurnal pattern of ascospore release.

Weather data. Weather data were obtained from June 2012 to 2014 from the Pullman

NE automated weather station of the Washington Agricultural Weather Network

(AgWeatherNet), which is approximately 400 m and 600 m from the PPE and PPW plots, respectively.

Data analysis. The number of OA and OY ascospores trapped over time in crop or in stubble in PPE and PPW were compared using the PROC GLM procedure of SAS software

(SAS release 9.2; SAS Institute, Cary, NC). Spearman’s rank correlation test was conducted to determine the relationship between ascospore number and meteorological factors including average, maximum and minimum temperature (AvrTemp, MaxTemp and MinTemp, respectively), dew point (Dpoint), relative humidity (RH), wind speed (Wspeed), solar radiation

(Srad), Average, accumulated and frequency of precipitation (AvrPrec, AccPrec, and Frprec, respectively), and soil temperature (Stemp). The diurnal variation in ascospore number during each quarter of the day was analyzed using the PROC GLM procedure of SAS.

Multiple regression analysis was used to examine the influence of selected meteorological variables (independent variables) on the relative number of OA and OY ascospores (dependent variables). Model development was iterative to reduce the number of models examined and find the best fit. Mallows CP was used as the criterion for goodness of fit of regression equations by assessing the fit of the regression model with meteorological variables. The variance inflation factor (VIF), which measures the increase in variance of an estimated regression coefficient due to collinearity between two or more predictor variables, was used to measure the effect of multicollinearity between independent variables used for model selections. Coefficients lower than 2 and 5 obtained from Mallows CP and VIF, respectively, were considered as acceptable for fit of the model (Mallows 1973; O’Brien 2007).

PROC REG, SAS statistical software (SAS institute) was used to perform these criteria.

Both the dependent (number of ascospores) and independent (meteorological) variables were adjusted to a weekly basis to get a better fit. Data were transformed to square root of the response + 1 to reduce overdispersion of the data and satisfy normality.

RESULTS

Spore trapping and ascospore discrimination. A total of 321 24 h tape segments were collected from spore traps, in 2012-2013 and 504 6 h segments in 2014. The number of ascospores trapped of both species varied by month and season (data not shown). A greater number ( P = 0.017) of OY than OA ascospores m -3 wk -1 were collected from 2012 to 2014 in both PPE and PPW plots; however, no significant differences were observed between the weekly number of OA and OY ascospores m -3 wk -1 when PPE and PPW plots were evaluated independently (P = 0.078 and P = 0.091, respectively) (Table 1).

The number of OA and OY ascospores m -3 wk -1 collected over a wheat-crop and stubble varied in both plots (Table 2). Significant differences ( P = 0.042) in the number of OY ascospores m -3 wk -1 occurred in the PPW plot, but not in the PPE plot (Table 2). Differences in the number of OA ascospores m -3 wk -1 over a wheat crop and stubble were not significantly different in either plot (Table 2).

Trapping of OA and OY ascospores began as soon as the spore traps were established in both plots on 4 June 2012 (Figs. 1, 2). In PPE, two peaks of OA and OY ascospores, preceded by precipitation events, were observed in 2012 and 2013. The first peak occurred around the last week of May and continued through the last week of June in both years. A second smaller peak of ascospores began in the second week of October and continued for about 3 weeks (Fig. 1) in

2012 and into November in 2013. In 2014, one peak of ascospore release was observed because the trap was only in the field through June; this peak began in the second week of March and continued to the second week of May (Fig. 1). A similar pattern of ascospore discharge was observed in the PPW plot as in PPE, except that the number of ascospores m -3 wk -1 trapped for both OY and OA varied more widely (Fig. 2). In 2014, the relative weekly abundance of OA ascospores was low and never exceeded 0.2 ascospores m -3 wk -1 in PPE and PPW; however, OY ascospores were more abundant.

Diurnal ascospore release. There were not significant differences between the number of OA or OY ascospores m -3 wk -1 released and the different periods of the day (Tukey’s HSD,

P>0.05) (Figure 3). However, the release of OA ascospores followed a similar pattern in both plots with the greatest number trapped from 0000 to 0600 (Fig. 3A). Fewer ascospores were trapped during other time periods of the day, but were similar to each other. Although a greater number of OY ascospores were trapped in both plots, the trend was similar for OA with the greatest number of ascospores trapped from 0000 to 1200 and then decreased from 1201 to 2400

(Fig. 3B).

Meteorological parameters. There were significant ( P < 0.05) positive correlations in both plots for the number of OA and OY ascospores m-3 wk -1 during the course of this study with relative humidity and weekly accumulated precipitation (Table 3). In addition, the number of OY ascospores m 3 wk -1 in both plots was negatively correlated with weekly average air temperature, weekly average maximum temperature and solar radiation, and positively correlated with weekly average precipitation and accumulated precipitation during the previous week (Table 3).

However, the number of OA ascospores m -3 wk -1 in both plots was only positively correlated with dew point.

Model development. Multiple regression models were developed to explain the number of OA and OY ascospores m -3 wk-1; selection of the best models was based on application of

Collin-Mallows CP and VIF criteria (Table 4). For PPE, regression equations that included two

(RH + Srad), three (RH + Srad + Avprec) and four variables (RH + Srad + Avprec + Frprec) accounted for 27, 29 and 31% (based on coefficient of determination), respectively, of the variation in number of OA and OY ascospores m -3 wk -1 (Table 4). For PPW, regression equations with two (RH + Srad ), three (RH + Srad + Avprec) and four variables (RH + Srad +

Avprec + Stem) accounted for 29, 32 and 36 %, respectively, of the variation in number of OA and OY ascospores m -3 wk -1 (Table 6). Each of the variables used in the models in PPE and PPW plots were significantly correlated to the number of OA and OY ascospores ( P < 0.05), except

Frprec and Stemp (P = 0.209 and P = 0.053, respectively).

DISCUSSION

The routine capture of OA and OY ascospores with Burkard spore traps from 2012 to

2014 in inoculated field plots demonstrates that conditions are favorable for production of ascospores of OA and OY when compatible mating types of these fungi are present. Despite the variation in number of ascospores trapped at different times during this study, two main periods of OA and OY ascospores release were identified in spring and fall (Figs. 1, 2). These results differ with a study conducted in Germany (King 1991), where OA ascospores were detected only in spring, and no ascospores of OA or OY were detected in fall. Detection of ascospores of both eyespot pathogens in fall is consistent with the results of another study (Vera and Murray, unpublished), where apothecia of both eyespot pathogens were found in spring and fall in commercial wheat fields in the US PNW.

The number of OA and OY ascospores detected in both field plots was relatively low throughout the study, and never exceeded 17 and 2 ascospores m -3 wk -1 in spring and fall, respectively. However, the occurrence of ascospores in fall is significant in terms of the disease cycle because primary infection of winter wheat occurs from October to January in the US PNW and corresponds to the growth stage when wheat is most susceptible to infection (Bruehl 1982;

Murray 2006).

No significant differences ( P > 0.05) in the number of OA and OY ascospores trapped were observed between plots when a wheat crop or stubble from the previous crop was present.

This result may appear contradictory since apothecia of the eyespot pathogens only occur on straw from the previous crop (Douhan et al. 2002; Dyer et al. 1994), and two explanations are possible. First, enough colonized stubble from the previous crop remains near the soil surface of the current wheat crop to provide a substrate for production of apothecia and ascospores, resulting in a source of local ascospore inoculum within the field. Second, since wind is the major means of long-distance ascospore dispersal of the eyespot pathogens (Daniels et al. 1995;

Dyer and Lucas 1995), ascospores from adjacent fields in the area may have been the source of those detected in these plots. Given that the plots used in these experiments were inoculated with compatible mating types of the eyespot pathogens, and apothecia of eyespot pathogens were found in nearby commercial wheat fields (Vera and Murray, unpublished), both scenarios are possible.

Although there were no significant differences in the number of ascospores m -3 wk -1 trapped during different time periods, diurnal ascospore release showed some trends with most

OA and OY ascospores trapped from 0000 to 0600 or 0000 to 1200, depending on the plot.

Although release of ascospores decreased during the remainder of the day, significant numbers

were present from 1201 to 2400. Moisture, temperature and light quality have been identified as the primary factors affecting sporulation of the eyespot fungi, but most research has been done with conidia (King 1991; Lucas et al. 2000). There is little information on factors affecting ascospore production/liberation, and most of the studies have been focused on the characterization and differentiation of isolates (Daniels et al. 1995; Scott and Hollings 1980).

The greater number of ascospores trapped during the early hours of the day in our study suggests that relative humidity has a significant effect on ascospore liberation; however, further study is needed to clarify the effect of environmental variables on diurnal ascospore release.

Weekly accumulated precipitation and average relative humidity were significantly and positively correlated with the number of OA and OY ascospores m -3 wk -1 in both field plots.

Other variables including weekly average temperature and maximum temperature were significantly and negatively correlated with the number of OY, but not OA ascospores m -3 wk -1.

When best-fit multiple regression models were developed using Mallows CP and VIF criteria to describe the relationship between the number of OA and OY ascospores m-3 wk -1, relative humidity and solar radiation were the only variables included in all models. The VIF values for weekly accumulated precipitation and total number of days with precipitation per week were relatively high (greater than 6), revealing problems with multicollinearity (when two or more predictor variables in a multiple regression are highly correlated); and therefore, they were excluded from the models. The multiple regression equations with two, three and four environmental variables for PPE and PPW sites accounted for about 27 to 31% and 29 to 36%, respectively, of the variation in the number of ascospores trapped m -3 wk -1, but also indicates that some important environmental factors may not have been considered. Further study is needed to

identify additional variables that influence the production of apothecia and liberation of ascospores of these fungi.

This study describes the seasonal and diurnal variation in release of ascospores of the eyespot fungi in two inoculated field plots in the US Pacific Northwest. Despite the seasonal variation in number of ascospores of both pathogens trapped, ascospores were present throughout much of the growing season. Based on this finding, along with the relative common occurrence of apothecia of OY and OA in commercial wheat fields in the US Pacific Northwest

(Vera and Murray, unpublished), we hypothesize that sexual reproduction in the eyespot occurs regularly in the life cycle of these pathogens in the US PNW and is not a rare event. Our results also suggest that the sexual stage of the eyespot pathogens contributes to effective primary inoculum in the disease cycle and is not only secondary inoculum, as previously reported

(Bateman et al. 1995; Dyer and Lucas 1995; King 1991). Additional air sampling involving non- inoculated, commercial fields are needed to confirm this hypothesis.

Finally, multiple regression models identified in this study demonstrate that it is possible to predict release of OA and OY ascospores using weather data, assuming compatible mating types of the pathogens are present in sufficient number. However, the relation between environmental factors and dispersal of ascospores is far from clear and additional studies on the effect of environmental factors on reproduction biology of the eyespot pathogens is needed.

ACKNOWLEDGEMENTS

The authors acknowledge the National Institute of Agricultural Research (INIAP) in

Ecuador, the Washington Grain Commission and Department of Plant Pathology, College of

Agricultural, Human, and Natural Resource Sciences Agricultural Research Center, Project No.

0670, Washington State University, Pullman, WA 99164-6430 for financial support of this project.

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apothecia of the cereal eyespot pathogen Tapesia yallundae on straw stubble in the UK.

Annals of Applied Biology 125:489–500.

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TABLE 1. Mean number of O. acuformis (OA) and O. yallundae (OY) ascospores m -3 wk -1 trapped at the East (PPE) and West (PPW) plots at the Plant Pathology Farm, Pullman, WA, from 10 June 2012 to 25 June 2014.

OA OY Pb Mean a Mean 0.427 1.779 0.017* Overall

Location 0.377 1.877 0.078 PPE PPW 0.479 1.680 0.091 a Mean number of ascospores m -3 wk -1 of OA and OY. b P value for comparing means within rows according to the Tukey’s HSD test.

* Significant difference at P = 0.05.

TABLE 2. Mean number of O. acuformis (OA) and O. yallundae (OY) ascospores m -3 wk -1 trapped above winter wheat plants (crop) and stubble in the East (PPE) and West (PPW) plots at the Plant Pathology Farm from 10 June 2012 to 25 June 2014.

Crop Stubble Species/ Pb Location Mean a Mean OA 0.330 0.421 0.757 PPE 0.696 0.250 0.357 PPW OY 3.427 0.436 0.066 PPE PPW 0.323 2.946 0.042* a Mean number of ascospores m 3 wk -1 of OA and OY. b P value for comparing means within rows according to the Tukey’s HSD test.

* Significant difference at P = 0.05.

TABLE 3. Spearman’s rank correlation coefficients for relationship between selected meteorological variables and the number of O. acuformis (OA) and O. yallundae (OY) ascospores m -3 wk -1 trapped in the East (PPE) and West (PPW) plots, at the Plant Pathology

Farm, Pullman, WA, 2012 to 2014.

Correlation Coefficient a

Variable b PPE PPW

OA OY OA OY

AvrTemp (°C) 0.065 -0.244* -0.031 -0.408**

MaxTemp (°C) -0.006 -0.293* -0.125 -0.461**

MinTemp (°C) 0.206 -0.135 0.084 -0.313*

Dpoint (°C) 0.330* -0.057 0.288* -0.167

RH (%) 0.423** 0.472** 0.500** 0.564**

Srad (w/m 2) -0.056 -0.277* -0.160 -0.437**

Avprec (mm) 0.137 0.307* 0.193 0.468**

AccPrec (mm) 0.215* 0.303* 0.376** 0.484**

AccPrec_1Week (# events) 0.176 0.355** 0.249 0.336*

Frprec (mm) 0.217 0.226 0.389** 0.472**

Stemp (°C) 0.088 -0.258 0.016 -0.435**

Wspeed (m/s) -0.051 0.166 0.030 0.344* a ** significant correlation at P = 0.01; * significant correlation at P = 0.05. b AvrTemp= Weekly average air temperature; MaxTemp= Weekly average maximum

temperature; MinTemp= Weekly average minimum temperature; RH= Relative humidity;

Srad= Solar radiation; Avprec= Weekly average precipitation; AccPrec= Weekly accumulated

precipitation; AccPrec_1Week= Accumulated precipitation during the previous week; Frprec=

Weekly precipitation frequency; Stemp= Weekly average of soil temperature at 20 cm depth;

Wspeed= Weekly average of wind speed.

TABLE 4. Best-fit regression models describing the number of O. acuformis and O. yallundae

ascospores m -3 wk -1 in the East (PPE) and West (PPW) plots at the Plant Pathology Farm,

Pullman, WA, regressed against environmental variables. Data collected from June 2012 to

May 2014.

Location CPa R2b Regression equation c

PPE

-0.278 0.27 -6.07 + 0.08 RH + 0.09 Srad

0.75 0.29 -6.01 + 0.08 RH + 0.09 Srad + 0.03 Avprec

1.241 0.31 -6.67 + 0.09 RH + 0.09 Srad + 0.05 Avprec – 0.15 Frprec

PPW

0.708 0.29 -3.55 + 0.06 RH + 0.04 Srad

0.404 0.32 -3.47 + 0.05 RH + 0.04 Srad + 0.03 Avprec

0.124 0.36 -4.17 + 0.06 RH + 0.08 Srad + 0.02 Avprec - 0.05 Stemp a Mallows CP criterion for model selection. b Coefficient of determination. c RH= Relative humidity in percentage; Srad= Solar radiation; Avprec= Weekly average

precipitation in millimeters; Frprec= Weekly precipitation frequency; Stemp= Weekly average

of soil temperature at 20 cm depth.

Figure 1 . Number of O. acuformis (OA) and O. yallundae (OY) ascospores m -3 wk -1 trapped in the East (PPE) plot at the Plant

Pathology Farm, Pullman, WA. June 2012 to 2014.

Figure 2 . Number of O. acuformis (OA) and O. yallundae (OY) ascospores m -3 wk -1 trapped in the West (PPW) plot at the Plant

Pathology Farm, Pullman, WA. June 2012 to 2014.

Figure 3. Mean number of O. acuformis (A) and O. yallundae (B) ascospores m -3 wk -1 trapped from March through May 2014 at the Pant Pathology Farm, Pullman, WA. Filled squares = East plot; Open squares = West plot. Means with same upper- and lowercase letters (East and West plot, respectively) are not significant different (Tukey’s HSD, P>0.05).

CHAPTER FOUR

PRODUCTION OF APOTHECIA BY OCULIMACULA YALLUNDAE IN VITRO

ABSTRACT

Current methods for in vitro production of apothecia of Oculimacula yallundae , one of the two causal agents of eyespot disease of winter wheat, requires several months for their formation. The objective of this study was to investigate factors influencing production of apothecia and reduce the time to formation to facilitate in vitro studies of pathogen population genetics. Cultural factors including media, host substrate, inoculation method, temperature, light and stress-shock preconditions were studied. The effect of media, host substrate and method of inoculation on development of apothecia was tested in the first round of experiments and conditions that favored apothecia development were used in subsequent experiments. Three constant and six diurnal temperature regimes, two light regimes, and two stress-shock preconditions were tested to determine their effect on development of primordial and mature apothecia. Mature apothecia were observed 35 weeks after inoculation on sterile barley/wheat straw segments on wheat straw agar, wheat seed agar and water agar, but not on sand.

Inoculation of winter wheat and spring barley straw segments with a suspension of conidia or mycelial plugs favored apothecia development ( P<0.05) compared with treatments without plant substrate, where mature apothecia did not develop. The combined effect of temperature, light and stress-shock preconditions were not significant. The number of apothecia produced in vitro was variable and relatively low. More biological studies involving analysis of fertility of the parent isolates may provide a better understanding of factors influencing apothecia development.

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INTRODUCTION

Eyespot is a stem base disease caused by the fungi O. acuformis (Wallwork & Spooner)

Crous & Gams and O. yallundae (Boerma, Pieters & Hamers) Crous & Gams that occurs in temperate regions of the world and affects wheat ( Triticum aestivum L.), barley ( Hordeum vulgare L.), oat ( Avena sativa L.), rye ( Secale cereale L.) and several grasses. This disease has been found in most temperate wheat-growing areas of the world (Andrade 2005; Dyer et al.

1994; King 1991; Robbertse et al. 1994; Jalaluddin and Jenkin 1996; Hunter 1989; Fitt et al.

1996), causing yield reductions in commercial fields up to 50% (Murray, 2010).

O. yallundae (OY), the sexual stage of Pseudocercosporella herpotrichoides (Fron.)

Deighton, was found in Australia 75 years after its first report (Wallwork 1987). The sexual stage has since been reported in most countries where eyespot occurs (Douhan et al. 2002;

Hunter 1989; King 1990; Sanderson and King 1988); however, the sexual stage is not considered a major inoculum source due to discord between the primary infection period and the time when apothecia are present on infested stems (Douhan et al. 2002; Dyer and Bradshaw 2002; King

1991; Lucas et al., 2000); consequently, most studies involving penetration, infection, colonization and sporulation have focused on the asexual stage, which is considered the primary source of inoculum for the disease cycle (Bateman and Taylor 1976; Glynne 1953; Higgins and

Fitt 1984).

Rowe and Powelson (1973), studying the effects of primary and secondary inoculum of

Pseudocercosporella herpotrichoides on wheat production, concluded that primary inoculum was the main cause of yield losses and secondary inoculum had little role in current season

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epidemics. Bruehl et al. (1982) came to the same conclusion in another study where winter wheat was inoculated with OY at different times during fall, winter, and early spring.

Despite these findings, the occurrence of apothecia of Oculimacula spp. in many places around the world suggests that the sexual stage may play an important role in the disease cycle.

Ascospores can serve as a long-distance source of inoculum capable of infecting cereal crops

(Douhan et al. 2002; Lucas et al. 2000) and contribute to greater genetic variation and flexibility to respond to diverse selection pressures such as climate change and fungicide resistance (Dyer et al. 2001a; Douhan et al. 2002). Unfortunately, there is little information explaining the relatively sparse production of apothecia in nature, and most of this is based on speculation regarding the unequal distribution of mating types that could limit sexual reproduction (Bateman and Taylor 1976). This hypothesis was later refuted because field surveys in Europe and the US confirmed that both mating types were present in equal proportions and thus, not limiting to reproduction (Douhan et al. 2002; Dyer et al. 2001b).

Researchers have developed methods to induce apothecia formation under laboratory conditions, but there has been no systematic study of the factors influencing their production

(Dyer et al. 1993; Moreau and Maraite 1995 and 1996; Nicholson et al. 1991). Nicholson et al.

(1991) reported the occurrence of O. yallundae on laboratory-inoculated material, but results were inconsistent since a large number of apothecia developed but failed to produce ascospores.

Dyer et al. (1993) confirmed heterothallism in the eyespot pathogens and were able to produce apothecia after 12 to 24 weeks of incubation. Moreau and Maraite (1995) produced OY apothecia in vitro using wheat straw and submitted to artificial light, but results were variable due to low number of apothecia produced and the long period for development (more than 30 weeks).

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The low frequency and prolonged time needed to develop apothecia (around 8 months) in vitro demonstrates the difficulty inducing apothecia; thus, a better understanding of the factors influencing formation of apothecia is needed to facilitate genetic studies of traits such as pathogenicity and genetic diversity. The objective of this study was to develop a method to induce more rapid and consistent production of apothecia of the eyespot pathogens in vitro.

MATERIALS AND METHODS

The effect of culture media, host substrate and type of inoculum on apothecia formation was studied in the first round of experiments, and the most influential factors were used in the second round of experiments to study the influence of temperature regime, light regime and stress-shock preconditioning on development of apothecia. The same fungal isolates were used in all experiments.

Fungal isolates. Two isolates of O. yallundae , Oy 90-45-7 (Mat1-1) and Oy 90-49-1

(Mat1-2) previously selected for being sexually compatible were used as inoculum. The O. yallundae isolates came from the Murray lab collection, Washington State University, Pullman,

WA. Both isolates were from infected wheat stems collected in commercial wheat fields from

1998 to 2000 in the US Pacific Northwest (Douhan et al. 2003). Isolates were maintained on

Potato Dextrose Agar (PDA) (Difco, Sparks, MD, USA) at 22°C until use.

Effect of culture medium, plant substrate and inoculum on apothecia development.

Five media were tested for development of apothecia: wheat straw agar (SA), wheat straw agar variant 1 (SA1), wheat seed agar (WSA), water agar (WA) and sand. Wheat straw agar was prepared by boiling 30 g of 60 mm pieces of mature straw (from field in stubble with winter wheat cultivar Hill 81) in 1 liter tap water for 30 min, straining it through cheese cloth to remove

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most solids, adding agar to the filtrate (20g/l) and autoclaving for 20 min at 121°C. SA1 was prepared by adding 30 g of chopped wheat straws (cultivar Hill 81) per liter of tap water containing 20 g of agar and autoclaving for 20 min at 121°C. WSA was prepared as for SA, using 100 g of dry whole wheat seed (cultivar Hill 81)/l tap water. Media were sterilized once at

121°C for 20 min and dispensed into petri plates (20 ml of medium per 9 cm diameter plate).

Sand media was prepared by autoclaving sand (previously sieved into 500 to 1000 m m) twice for

20 min at 121°C, and then adding approximately 10 g into petri plates; 5 ml of sterile distilled water added to maintain moisture.

Three pieces 50- to 60-mm long autoclaved winter wheat straw from cultivar Hill 81

(WWS) or spring barley straw from cultivar Harrington (SBS) were placed into each petri plate as a substrate for production of apothecia. No substrate (NS) served as negative control.

A conidial suspension (CS) or mycelial plugs (MP) were used to inoculate petri dishes.

O. yallundae isolates were grown separately by transferring macerated agar pieces from cultures grown on PDA to water agar (WA) (Sigma-Aldrich, St. Louis, Mo, USA). Plates were wrapped with Parafilm (Bemis, Inc., Neenah, WI. USA) and incubated at 14°C for 10 days under constant near UV light and then examined with a dissecting microscope for conidia. A CS was prepared by washing spores from plates of both strains into sterile distilled water. Conidia were counted with a haemocytometer and adjusted to 1 x 106 spores/ml. Equal volumes of CS from each isolate were mixed and used to inoculate substrates on different media by pipetting 300 m l of the mixture onto the surface of each medium-substrate treatment. A similar procedure was used with mycelial plugs, except that a 5 mm diam plug of mycelia was cut from the actively growing margin of the OY isolates using a sterile cork borer and placed next to the straw pieces in each dish. Each medium-substrate treatment also was inoculated with autoclaved sterile water as a

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negative control. After inoculation, plates were wrapped with Parafilm and placed in growth chamber (Power scientific, Inc., Pipersville, PA. USA) for up to 10 months at 10°C under constant white light.

Effect of temperature, light and stress-shock preconditioning on development of apothecia. Autoclaved winter wheat straw segments were placed in groups of 10 into petri plates containing water agar and inoculated with 300 m l of a suspension containing conidia of both Oy isolates. Plates were incubated at room temperature for 15 days until mycelia covered more than

80% of the straws and then subjected to two stress-shock preconditions; freezing and dehydration. Straws were subjected to freezing by placing them in an incubator at -20°C for 48 hours. For dehydration, colonized straws were placed in a laminar flow hood for 48 h to maintain

RH below 65%. Colonized straws not exposed to freezing temperature or desiccation were used as a negative control.

The influence of constant and interrupted temperatures and light regimes on development of OY apothecia was studied in two experiments. In the first experiment, colonized WWS straws previously exposed to stress-shock preconditioning were incubated in continuous light or an alternating 12 h photoperiod, at 10°C, 14°C and 20°C, respectively. Light was supplied with two

40 watt cool-white and four 40 watt black-light blue fluorescent tubes.

In the second experiment, colonized WWS straws subjected to the stress-shock preconditioning treatments above were incubated in one of six temperature regimes initially and then at constant temperature for the duration of the experiment: a) 10°C/10 days + 20°C/7 days +

10°C; b) 10°C/10 days + 20°C/14 days + 10°C; c) 14°C/10 days + 20°C/7 days + 14°C; d)

14°C/10 days + 20°C/14 days + 14°C; e) 20°C/10 days + 10°C and f) 20°C/10 days + 14°C.

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Each temperature regimen treatment was incubated with a 12 h photoperiod or constant light as described above.

After 18 weeks incubation, treatments were observed weekly with a stereomicroscope to record the formation of primordia and mature apothecia. The data of number of primordia and mature apothecia observed at week 38 of incubation was used for analysis. Apothecia were considered primordial when mycelium was protruding and bulging from the straw or the apothecia were closed, and mature when the apothecium was expanded and the hymenium exposed.

Data analysis. Petri plates were the experimental units in all experiments and there were five replicate per treatment. Data were analyzed using PROC GLM in SAS 9.2 software (SAS release 9.2; SAS Institute, Cary, NC).

RESULTS

Effect of culture medium, plant substrate and inoculum on apothecia development.

Apothecia primordia were observed in all treatment combinations after 25 weeks incubation, except in treatments without host substrate where primordia was only observed when combined with SA1 medium (Fig. 1A). There was no significant interaction among medium, plant substrate and inoculum, but there were significant differences among the substrates on the number of primordia. Thus, the number of primordia developed on WWS and SBS substrates was significant greater than treatment without substrate ( P = 0.013, data not shown).

Mature apothecia did not develop on media without host substrate, but developed with

CS inoculum on both substrates on SA and SA1 media after 35 weeks incubation (Fig. 1B). For

MP inoculum, mature apothecia developed on both substrates on SA1 or WA media after 37

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weeks incubation (Fig. 1B). There was no significant interaction among factors, nor were there significant differences among media, host substrate or type of inoculum on number of mature apothecia ( P = >0.05; data not shown).

Effect of temperature, light and stress-shock preconditioning on development of apothecia. Apothecia primordia developed in all treatment combinations, and were initially observed at 10°C, 14°C and 20°C after 19, 22 and 23 weeks incubation, respectively. Mature apothecia were initially observed at 10°C and 14°C after 30 weeks incubation, and only one mature apothecium developed at 20°C after 36 weeks incubation (data not shown). There was not significant interaction among temperature, light and stress-shock preconditioning for number of primordial and mature apothecia after 38 weeks incubation; however, there were significant differences among temperatures on number of primordia and mature apothecia ( P < 0.001, and P

< 0.001, respectively). There were significantly fewer primordia and mature apothecia at 20°C than at 10°C and at 14°C, respectively (Table 1). The effects of light and stress-shock preconditioning were not significant for primordia (P = 0.865 and P = 0.462, respectively) or mature apothecia ( P = 0.473 and P = 0.331, respectively) (Table 1).

Interrupted temperature regimes had a significant effect on production of primordia and mature apothecia ( P = <0.001), but light ( P = 0.460 and P = 0.611, respectively) and stress-shock preconditioning ( P = 0.268 and P = 0.610, respectively), did not significantly affect number of primordia and mature apothecia, nor were there significant interactions among factors (Table 2).

More primordia were produced in temperature regimes with 10°C and 14°C initial temperatures than those starting at 20°C (Table 2).

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DISCUSSION

Based on these results, the production of primordial and mature apothecia depends on substrate and temperature conditions. Winter wheat and spring barley straw both supported the development of apothecia and there was no difference between them. A few primordia were produced on WSA media without substrate, but they did not develop into mature apothecia twelve weeks after the appearance of primordia.

Primordial apothecia were more abundant than mature apothecia. The inability of primordia to develop into mature apothecia without a substrate indicates that host substrate is essential to the development of mature apothecia. This result agrees with previous studies of production of apothecia in vitro (Dyer et al. 1993; Moreau and Maraite 1995 and 1996).

Although primordia developed into mature apothecia on both host substrates, the total number of apothecia was low and further study is needed to identify which factors affect the development of mature apothecia.

Although there was no difference in the effect of inoculation method on the development of primordia and mature apothecia, treatments with conidial suspension produced a greater number of these sexual structures than mycelial plugs (Fig. 1). Unfortunately, there is no information about the effect of type of inoculum on development of sexual structures in eyespot pathogens. More studies about the factors that affect inoculum are needed.

Temperature had a large effect on development of apothecia with greater production at 10 and 14°C than at 20°C (Table 1). Although 20°C is near-optimal for mycelial growth of the eyespot pathogens (Higgins and Fitt 1983; Scott 1971), it was not optimal for development of apothecia in these studies, and indicates that development and maturation of apothecia of O. yallundae requires lower temperatures. This also demonstrates that optimal conditions for

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development of sexual structures of fungi may differ from those for asexual reproduction

(Daniels et al. 1995). The results from this study were consistent with those obtained by Moreau and Maraite (1996) who reported development of apothecia in vitro ranges from 8 to 12°C.

The variable temperature regimes used in this study had a significant effect on development of apothecia. Varying temperatures from low-to-high-to-low had a positive effect on production of primordial and mature apothecia than from high-to-low temperature.

Temperature cycling has been used for other fungi to induce production of sexual structures

(Jailloux 1992; Wu and Subbarao 2008), and most of them agree that varying temperatures can shorten the time required to develop sexual structures; however, there was no difference in the time required for development of primordial and mature apothecia between variable temperature regimes and constant temperature in these studies. Neither the light regimes nor the stress-shock preconditioning used in this study had a significant effect on production of apothecia (Table 1).

This study demonstrates that production of apothecia of eyespot pathogens in vitro is influenced by cultural conditions; however, the results obtained here were inconclusive since the occurrence of apothecia was low in all treatments tested and require more than 35 weeks to develop. Moreau and Maraite (1996) obtained similar results. Although useful information about the effect of temperature, medium, substrate and inoculum have been generated from this study, there is a need of additional information on the effect of environmental factors on reproduction biology of eyespot pathogens. In this study only two sexually compatible isolates were used, and results may reflect the fertility of those isolates. In future work, additional isolates should be tested for fertility and their ability to produce apothecia should be considered.

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ACKNOWLEDGEMENTS

The authors acknowledge the National Institute of Agricultural Research (INIAP) in

Ecuador, the Washington Grain Commission and Department of Plant Pathology, College of

Agricultural, Human, and Natural Resource Sciences Agricultural Research Center, Project No.

0670, Washington State University, Pullman, WA 99164-6430 for financial support of this project.

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TABLE 1 . Effect of temperature, light and stress-shock preconditioning on number of mature and primordial apothecia of Oculimacula yallundae after incubation for 8 months.

Number of apothecia a Factor Mature d Primordia e

Temperature

10°C 2.56 af 7.26 a

14°C 2.13 a 6.83 a

20°C 0.06 b 1.10 b

Light b

12 h 1.71 a 5.00 a

24 h 1.46 a 5.13 a

Stress shock c

Freezing 1.63 a 5.87 a

Dry 2.00 a 4.83 a

No-shock 1.13 a 4.50 a a Results are the mean of five replicates. Interactions among factors were not significant and

therefore, only main effects are presented. b 12 h= 12 hour photoperiod; 24 h= continuous light. Light supplied with 40 watt cool-white and

black-light blue fluorescent tubes. c Freezing= colonized substrate placed at -20°C for 48 hours prior to incubation; Dry= colonized

substrate placed in a laminar flow hood for 48 hours prior to incubation. d Mature= apothecium disc is expanded and the hymenium is exposed. e Primordia= mycelium is protruding and bulging from the substrate or the apothecium is closed.

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f Means in the same column followed by different letters are significantly different according to

Tukey Test ( P < 0.05).

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TABLE 2. Effect of temperature regime, light and stress-shock preconditioning on number of mature and primordial apothecia of Oculimacula yallundae after incubation for 8 months.

Number of apothecia a Culture conditions Mature e Primordia f

Combined temperature b

T10 – 20/7 - 10 2.2 ag 5.2 abc

T10 – 20/14 - 10 2.1 a 7.6 a

T14 – 20/7 - 14 1.7 a 5.8 ab

T14 – 20/14 - 14 1.7 a 3.4 bcd

T20 - 10 0.1 b 1.3 cd

T20 - 14 0.0 b 1.1 d

Light c

12 h 1.3 a 3.8 a

24 h 1.2 a 4.4 a

Stress shock d

Freezing 1.3 a 4.5 a

Dry 1.2 a 3.6 a a Results are the mean of five replicates. Interactions among factors were not significant and

therefore, only main effects are presented. b T10-20/7-10= 10 days at 10°C + 7 days at 20°C + 10°C; T10-20/14-10= 10 days at 10°C + 14

days at 20°C + 10°C; T14-20/7-14= 14 days at 14°C + 7 days at 20°C + 14°C; T14-20/14-14=

14 days at 14°C + 14 days at 20°C + 14°C; T20-10= 10 days at 20°C + 10°C; T20-14 = 10

days at 20°C + 14°C.

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c 12 h= 12 hour photoperiod; 24 h= continuous light. Light supplied with 40 watt cool-white and

black-light blue fluorescent tubes. d Freezing= colonized substrate placed at -20°C for 48 hours prior to incubation; Dry= colonized

substrate placed in a laminar flow hood for 48 hours prior to incubation. e Mature= apothecium disc is expanded and the hymenium is exposed. f Primordia= mycelium is protruding and bulging or the apothecium is closed. g Means in the same column followed by the different letters are significantly different according

to Tukey Test ( P < 0.05).

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25 Primordial apothecia A

5 # of of # primordial apothecia

10 B Mature apothecia 8

2 # of mature apothecia 0 S S S S S S SA SA SA SA SA SA WA WA WA WA WA WA SA1 SA1 SA1 SA1 SA1 SA1 WSA WSA WSA WSA WSA WSA NS SBS WWS NS SBS WWS CS MP Factor combination

Figure 1 . Effect of inoculation method, plant substrate and medium on the number of primordial

(A) and mature apothecia (B) of Oculimacula yallundae . Inoculation method: CS = conidial suspension, MP = mycelial plugs. Plant substrate: NS = no substrate, SBS = spring barley straw,

WWS = winter wheat straw. Medium: S= sand , SA = wheat straw agar, SA1 = wheat straw agar variant 1, WA = water agar, and WSA = wheat seed agar. Errors bar show the standard deviation of the error ( P = 0.05).

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CHAPTER FIVE:

INTERPRETIVE SUMMARY

Despite discovery of the sexual stage of the eyespot pathogens Oculimacula yallundae

(OY) and O. acuformis (OA) more than two decades ago (Wallwork 1987), questions related to how the genetic variation created during recombination is incorporated into the disease cycle and the effect of ascospore inoculum on disease development have not been answered. The overall goal of this project was to determine the role of the sexual stage of OA and OY in the development of eyespot disease of wheat in the PNW. Research was conducted to determine the occurrence of apothecia in commercial and inoculated fields, to determine the seasonal periods when apothecia are produced and ascospores released, the persistence of apothecia over summer and over winter, and investigate factors influencing production of apothecia and reduce the time to formation to facilitate in vitro studies of pathogen population.

Apothecia of OY and OA were found in spring and fall, demonstrating that sexual reproduction occurs regularly and that ascospores may have a more significant role in the epidemiology of eyespot than previously believed. During fall, when winter wheat is planted, apothecia could provide ascospores that serve as primary inoculum initiating the disease cycle, which explains the high genotypic diversity of OY and OA isolates observed by Douhan (2002).

The incidence of OA and OY apothecia in surveyed fields was relatively low, never exceeding the 3% of stems, but they occurred at similar times and frequency, suggesting that both species can coexist in the same field on the same host (Fitt et al. 2006). Dyer et al. (1996) hypothesized that mating type, low fertility of pathogens and absence of specific environmental

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triggers are the main reasons for limited sexual reproduction in these fungi. Further study is needed on this question in light of the results presented herein.

Apothecia of OA and OY survived over summer, but not over winter. We hypothesize that Oculimacula spp. survives the winter asexually as hyphae in colonized stem bases; apothecia develop during spring and release ascospores, then survive over summer and release ascospores again during fall when winter wheat seedlings are present. Additional research is needed to determine the effect of environmental factors on oversummering survival of apothecia and the amount of ascospore inoculum present during fall to determine if it provides a significant contribution to the disease cycle.

The routine capture of OA and OY ascospores with Burkard spore traps in inoculated field plots demonstrates that conditions in the PNW are favorable for production of the teleomorph of OA and OY when compatible mating types are present. Two main periods of OA and OY ascospores release were identified, spring and fall. These results differ with a study conducted in Germany (King 1991), where OA ascospores were detected only in spring. A possible very low occurrence in fall that has been undetected in Europe, and environmental conditions favoring development of apothecia during fall in US but not in Germany are the possible reasons for this difference.

The number of OA and OY ascospores detected in both field plots was relatively low.

However, the occurrence of ascospores in fall is significant in terms of the disease cycle because primary infection of winter wheat occur from October to January in the PNW and corresponds to the growth stage when wheat is most susceptible to infection (Bruehl 1982; Murray 2006). No significant differences in the number of OA and OY ascospores trapped were observed between plots when a wheat crop or stubble was present, suggesting that colonized stubble from the

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previous crop near the soil surface of the current wheat crop is providing a substrate for production of apothecia and ascospores or, since wind is the major means of long-distance ascospore dispersal of the eyespot pathogens (Daniels et al. 1995; Dyer and Lucas 1995), ascospores from adjacent fields may have been the source. There were no significant differences between the number of ascospores and diurnal ascospore release; however, most OA and OY ascospores were trapped from 0000 to 0600 or 0000 to 1200, suggesting that relative humidity has a positive effect on ascospore liberation.

Multiple regression models with two, three and four environmental variables accounted for 27 to 36% of the variation in the number of ascospores trapped m -3 wk -1, and demonstrate that is possible to predict release of OA and OY ascospores using weather data. However, the relation between environmental factors and dispersal of ascospores is far from clear and additional studies on the effect of environmental factors on reproductive biology of the eyespot pathogens are needed.

The effect of media, host substrate, method of inoculation, temperature and light regimes and stress-shock preconditions were tested to determine their effect on development of primordial and mature apothecia in vitro. The development of primordial and mature apothecia depended on substrate and temperature conditions. Winter wheat and spring barley straw supported the development of apothecia and there was no difference between them; however, mature apothecia did not develop on media without a substrate. The inability of primordia to develop into mature apothecia without a substrate indicates that host substrate is essential to the development of mature apothecia. This result agrees with previous studies of production of apothecia in vitro (Dyer et al. 1993; Moreau and Maraite 1995 and 1996).

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Inoculation of substrates with a conidial suspension produced a greater number of mature apothecia than mycelial plugs; however, the differences were not significant. More apothecia developed when inoculated straw was incubated at 10 and 14°C than at 20°C. Although 20°C is near-optimal for mycelial growth of the eyespot pathogens (Higgins and Fitt 1984; Scott 1971), it was detrimental for development of apothecia in these studies, indicating that development and maturation of apothecia of O. yallundae requires lower temperatures. This result was consistent with those obtained by Moreau and Maraite (1996), who reported development of apothecia in vitro ranges from 8 to 12°C. Despite useful information about the effect of temperature, medium, substrate and inoculum have been generated from this study, the results were inconclusive since the occurrence of apothecia was low in all treatments tested and require more than 35 weeks to develop.

This research provides a base-line that offers new insights into the role of the sexual stage of Oculimacula spp. in the epidemiology of eyespot disease. Understanding the occurrence of apothecia and ascospores provides a better understanding of the role of ascospores as an inoculum source.

Future work

Occurrence of apothecia and ascospores of OY and OA in fall suggests that ascospores could serve as primary inoculum for the disease cycle. To confirm this hypothesis, air sampling involving non-inoculated commercial fields and additional studies of the infection process of eyespot pathogens by ascospores are needed. Also the relationship between environmental factors and dispersal of ascospores is far from clear and additional studies on the effect of environmental factors on reproductive biology of the eyespot pathogens are required.

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In this study only two sexually compatible isolates were used, and results may reflect the fertility of those isolates. In future work, additional isolates should be tested for fertility and their ability to produce apothecia should be considered. The next goal is to determine the influence of the sexual reproduction on population dynamics and genetics of these pathogens by including population genetic studies. This information will improve our understanding of the epidemiology of eyespot and hopefully lead to improved management measures.

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Dyer, P. S., Nicholson, P., Lucas, J. A., and Peberdy, J. F. 1996. Tapesia acuformis as a causal

agent of eyespot disease of cereals and evidence for a heterothallic mating system using

molecular markers. Mycological Research 100:1219–1226.

Fitt, B. D. L., Huang, Y., Van Den Bosch, F., and West, J. S. 2006. Coexistence of related

pathogen species on arable crops in space and time. Annual Review of Phytopathology

44:163-182.

Higgins, S., and Fitt, B. D. 1984. Production and pathogenicity to wheat of Pseudocercosporella

herpotrichoides conidia. Journal of Phytopathology 111:222–231.

125

King, A. C. 1991. Observations of apothecia of Tapesia yallundae and the cultural phenotypes of

their progeny. Plant Pathology 40:367–373.

Moreau, J. M., and Maraite, H. 1995. Bipolar heterothallism in Tapesia yallundae for the two

varieties of the anamorph Pseudocercosporella herpotrichoides . Mycological Research

99:76–80.

Moreau, J. M., and Maraite, H. 1996. Evidence for a heterothallic mating system in Tapesia

acuformis using benomyl sensitivity and esterase isoenzyme profiles. Mycological Research

100:1227–1236.

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of eyespot of wheat. Australasian Plant Pathology 16:92–93.

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