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2019 Some Studies on Leaf Spot of Oats and Triticale Mohammed Abdullah Lashram South Dakota State University

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BY

MOHAMMED ABDULLAH LASHRAM

A thesis submitted in partial fulfillment of the requirements for the

Master of Science

Major in Plant Science

South Dakota State University

2019

iii

ACKNOWLEDGEMENTS First and foremost, I wish to pay my sincere regards to the Almighty, who provided me with an opportunity, to begin with, my research for my MS and making me conduct it successfully. My most heartfelt thanks to my advisor Dr. Shaukat Ali for providing me a platform for working on this project as a master thesis. He has been my source of inspiration and encouragement throughout my stay at SDSU.

I especially want to thank Dr. Sunish Kumar Sehgal, my committee member, for his continuous support and guidance to accomplish my degree. I also like to thank Dr.

Byamukama Emmanuel, my committee member, for his valuable insights and comments while conducting my research and beyond. I would like specially to thank Dr. Xiangming

Guan, Graduate Faculty representative, for his sweet harmony and serve on my committee.

It is an honor for me to express my deep indebtedness to Dr. Madhav Nepal as well as

Surendra Neupane and Ethan Andersen (his students) allowing me to work in his laboratory and learned some molecular techniques.

I highly appreciate Mr. Richard Geppert for his technical help in samples collection, lab, and greenhouse work. Thank you to all my colleagues: Navjot Kaur, Girma Tadesse

Ayana, Jytirmoy Halder, Rami Altameemi, Sidhu Jagdeep, Collins Bugingo, Zunera

Shabbir and Mustafa Aljadi who have provided their utter encouragement in making this research a wonderful experience and my stay at SDSU. Also, I am thankful to them for their continuous support in performing my lab work, data collection, and working with statistical software for data analysis.

I am grateful to the Department Head Dr. David Wright who provided me an excellent opportunity to join the department as a graduate student and accomplish my MS degree. I like to thank my family and friends for their inspiration and patience during my iv

MS at SDSU and studying abroad. At the last but not least, I appreciate Jazan University,

Kingdom of Saudi Arabia, South Dakota Wheat Commission, and SDSU AES for their financial support for completing my MS degree.

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CONTENTS

LIST OF TABLES ...... viii LIST OF FIGURES ...... ix ABSTRACT ...... x CHAPTER 1 (Literature review) ...... 1 Oat ...... 1 Origin and History ...... 1 Health benefits ...... 2 Production ...... 2 Biotic stresses in oat production ...... 2 Drechslera avenae leaf spot ...... 2 Drechslera avenae history ...... 2 Drechslera avenae distribution and significance ...... 4 Host range ...... 6 Host resistance ...... 6 Drechslera avenae leaf spot symptoms and disease cycle ...... 6 Role of fungal effectors (toxins) in disease development ...... 7 Disease management ...... 8 Wheat ...... 10 Origin, history, and domestication of wheat ...... 10 Triticale ...... 12 Origin, and history of triticale ...... 12 Uses of triticale ...... 13 Tan spot ...... 15 Importance ...... 15 History ...... 15 Distribution ...... 16 Host range ...... 17 Symptoms and significance ...... 18 Virulence variation in the pathogen ...... 19 vi

Role of Host-Selective toxins in Disease development ...... 20 Disease cycle ...... 22 Disease management ...... 23 Rational, Significance and Objectives ...... 26 Literature cited ...... 29 CHAPTER 2 ...... 51 Survey of oat leaf spot pathogens in South Dakota and evaluation of a worldwide collection of oats germplasm against Drechslera avenae leaf spot ...... 51 ABSTRACT ...... 51 Introduction ...... 53 Materials and methods ...... 54 Recovery and characterization of Drechslera avenae isolates from oat...... 54 Plant Materials ...... 55 Inoculum preparation, plants inoculation, and disease rating ...... 56 Data analysis ...... 57 Results ...... 57 Recovery and characterization of Drechslera avenae ...... 57 Reaction of oat genotypes to leaf spot ...... 58 Discussion ...... 63 Literature cited ...... 66 CHAPTER 3 ...... 70 Characterization of a global collection of triticale genotypes to Pyrenophora tritici- repentis (tan spot) race 1, race 5, and Ptr ToxA ...... 70 ABSTRACT ...... 70 Introduction ...... 72 Materials and methods ...... 74 Plant material and seedlings production ...... 74 Inoculum preparation, plants inoculation, and disease rating ...... 75 Date analysis ...... 76 Results ...... 76 Reaction of triticale genotypes to tan spot (race 1) and Ptr ToxA ...... 76 Reaction of triticale genotypes to tan spot (race 5) ...... 80 Discussion ...... 83 vii

Literature cited ...... 86 General Conclusion ...... 90 APPENDIX ...... 93

viii

LIST OF TABLES

Table 1.1: Races of Pyrenophra tritici-repentis and their symptoms on tan spot wheat differentials set (Lamari et al., 2003) ...... 24 Table 2.1: Fungi associated with oat leaf samples collected from various locations in South Dakota in 2017 and 2018 ...... 60 Table 2.2: ANOVA of Drechslera avenae leaf sopt ...... 60 Table 2.3: Reaction of 155 oat genotypes from six continents to Drechslera avenae ...... 60 Table 2.4. Reaction of oats genotypes to Drechslera avenae leaf spot from six continents (33 countries) ...... 60 Table 3.1: ANOVA of tan spot (race 1) ...... 78 Table 3.2: Reaction of 366 triticale genotypes from six continents to Ptr race 1 and Ptr ToxA ...... 78 Table 3.3: Reaction of 366 triticale genotypes from 21 countries to Ptr race 1 and Ptr ToxA ...... 78 Table 3.4: ANOVA of tan spot (race 5) ...... 81 Table 3.5: Reaction of 366 triticale genotypes from six continents to Ptr race 5 ...... 81 Table 3.6. Reaction of oats genotypes to Ptr race 5 from 21 countries...... 82 Table A 2.1. Disease reaction of 155 oat genotypes to Drechslera avenae leaf spot ...... 93 Table A 3.1. Disease reaction of 366 triticale genotypes to Pyrenophora tritici-repentis (tan spot) races 1 and 5 and Ptr ToxA ...... 97

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LIST OF FIGURES

Figuer 1.1: Global distribution of P. chaetomioides ...... 12 Figure 1.2: Tan necrotic leaf spots on two-week-old seedling leaf of a susceptible oat genotype ...... 12 Figuer 1.3: Life cycle of P. chaetomioides ...... 13 Figure 1.4 : World map depicting the global occurrence of tan spot in the wheat growing countries ...... 27 Figure 1.5: Tan necrotic leaf spots on a susceptible triticale genotype inoculated with race 1 ...... 27 Figure 1.6: Schematic representation of the life cycle of Ptr ...... 28 Figure 2.1: Countries of origin of oat genotypes screened in this study ...... 59 Figure 2.2: Range of oat genotypes reaction (lesion types 1- 5) induced by Drechslera avenae...... 60 Figure 2.3. D. avenae conidia on an infected leaf segment ...... 65 Figure 2.4: D. avenae conidia under microscope ...... 65 Figure 2.5. Tan necrotic leaf spots on two-week-old seedlings of a susceptible oat genotype (top) and small tan necrotic spot on a resistant genotype (bottom) inoculated with D. avenae in greenhouse ...... 65 Figure 3.1. Origin of 366 triticale genotypes evaluated in this study ...... 78 Figuer 3.2. Reaction of genotypes to Pyrenophora tritici-repentis race 1 and Ptr ToxA. R = resistant; S = susceptible; I= insensitive to Ptr ToxA; Sen = sensitive to Ptr ToxA...... 82 Figure 3.3: Two triticale genotypes inoculated with race 1 (susceptible-top leaf and resistant-bottom leaf)...... 83 Figure 3.4. Two triticale genotypes infiltrated with Ptr ToxA (sensitive-top leaf and insensitive bottom leaf)...... 83 Figure 3.5: Two susceptible triticale genotypes inoculated with race 5 and exhibiting chlorosis symptoms (top two leaves). Two triticale race 5 resistant genotypes exhibiting resistant lesion type 1 (black spots without chlorosis) reaction (Bottom two leaves) ...... 85

x

ABSTRACT

SOME STUDIES ON LEAF SPOT OF OATS AND TRITICALE

MOHAMMED LASHRAM

2019

Oat is an important cereal crop, and it is considered to be among the highest used in cereal crops. It is considered among the healthiest grains due to the rich source of the soluble fiber, β-glucan that helps in lowering the cholesterol. Oat production and demand have increased considerably in the past few years due to its health benefits. South Dakota is ranked in the top three largest oat-producing states in the USA. Oat is relatively less susceptible to pest and diseases except for leaf diseases such as crown rust, Drechslera avenae leaf spot, and Stagonospora avenae leaf blotch. Leaf spot diseases like Drechslera avenae leaf spot and Stagonospora avenae leaf blotch can cause high yield losses in oats under conducive environment for disease development. However, no information is available on the prevalence of leaf spot diseases in oats and reaction of oat cultivars to

Drechslera avenae leaf spot in South Dakota. Triticale is another cereal crop that plays an important role by serving as a bridge between the rye and wheat in transferring good agronomic traits like yield, winter hardiness, and pest and disease resistance genes to improve wheat quality. Additionally, it is used as a forage and a rotational crop in improving soil health. It is generally considered less prone to pest and diseases; however, it has been reported to be susceptible to some foliar diseases including tan spot, an important disease of wheat. Also, it can serve as an additional inoculum source of tan spot causing pathogen, Pyrenophora tritici-repentis, inoculum/virulence variation when cultivated in the vicinity of wheat. Information on the reaction of triticale to commonly xi prevalent P. tritici-repentis races1 and 5 is unknown. The objectives of this study were to,

1) conduct a disease survey of leaf spot pathogens associated with oat in South Dakota, 2) characterize a global collection of oat genotypes including commercial cultivars grown in

South Dakota, against D. avenae leaf spot in greenhouse; and 3) evaluate a worldwide collection of triticale genotypes for their reaction to tan spot using the pathogen,

Pyrenophora tritci-repentis which causes tan spot, race1, race 5, and host selective toxin

Ptr ToxA. To achieve these objectives, the diseased leaves samples displaying leaf spots were collected from different oat fields in 2017 and 2018 in South Dakota. The leaf samples were examined for leaf spot pathogens using standard protocols. One hundred fifty-five oat genotypes representative of 33 countries and 366 triticale genotypes from 21 countries were evaluated for their reaction to D. avenae leaf spot and tan spot, respectively at seedlings stage in the greenhouse. Four leaf spot pathogens D. avenae and Stagonospora avenae,

Bipolaris victoriae, and Colletotrichum graminicola were observed on the collected oat leaves samples; however, D. avenae and Stagonospora avenae had hhigher prevalence as compared to B. victoriae, and C. graminicola. The 155 evaluated oat genotypes varied in their reaction to D. avenae leaf spot in ranging from susceptible (41.3%), moderately susceptible (28.4%), moderately resistant (15.5%), and resistant (14.8%). All commercial cultivars exhibited a susceptible reaction to D. avenae leaf spot. The results of the 366 triticale genotypes screened for their reaction against P. tritici-repentis race 1 and race 5 exhibited variability in their responses to both races 1 and 5. The majority of genotypes

(53%) that evaluated were found either resistant or moderately resistant to race 1. The sensitive reaction against Ptr ToxA observed in only 68 (18.55%) genotypes. In this study, the correlation between susceptibility and Ptr ToxA sensitivity or resistance and Ptr ToxA xii insensitivity of the genotypes was not observed. Most of the genotypes (64.75%) screened against race 5 developed resistant to– moderately resistant reactions. Our results showed that most of the triticale genotypes harbor resistance against tan spot, races 1 and 5 both at the continent or country level. Ptr ToxA seems not playing a significant role in disease development as reported in previous studies in wheat and rye. The outcomes of this study warrants observing leaf spot pathogens in the state at regular bases and incorporate of resistance to Drechslera avenae leaf spot in oat cultivars grown in future to circumvent any chance of occurring the disease epidemic in the region. Further, oat genotypes exhibited resistance to the disease can be used as sources of resistance in the oat breeding program.

Oat genotypes with D. avenae leaf spot resistance should further be tested against S. avenae leaf blotch, another leaf spot pathogen prevalent in the state. Also, triticale genotypes with tan spot resistance should be used when planted nearby wheat area, and in transferring good agronomic traits in wheat to avoid transferring accidental susceptibility to tan spot.

xiii

General Introduction Oat is considered as a vital crop, and it is viewed as among the most important cereals (Murphy and Hoffman, 1992). It has been demonstrated that it is among the most advantageous crop. In this way, demand and production of oat have picked up impressive progress (Whitehead, et al., 2014). Globally, the 2018/2019 projection of oat to be produced is 22.27 million metric tons (USDA, 2019). A production of 49.3 million bushels was reported in 2017; However, in 2018, its production was again raised to 56.1 million bushels. A major oat production has happened in the USA in 2016 which was around 9 million bushels. In South Dakota, a decrease in oat production 4.2 million bushels was seen in 2017. In 2018, the production was increased up to 7.7 million bushels and the state was announced as its second largest producer in the country (USDA, 2018).

Oat is generally less susceptible to pests and crop diseases aside from foliar diseases

(Rosentrater and Evers, 2018). Drechslera avenae is the causal agent of leaf spot that is a sexual phase of a necrotrophic parasitic pathogen named Pyrenophora chaetomioides, the reddish-brown spots with purple edges symptoms produced by Drechslera avenae (Clark, et al., 2008). These spots cause a diminishing in the leaf green area; thus, the process of photosynthesis in plants is negatively impacted that translate it to a reduction in energy and starch hence, reduced the tillering process (Dennis, 1933).

Drechslera avenae is a globally found pathogen and in 1984, it has been reported to causes a critical problem in the southern part of the USA, Western Europe, Japan, and

India (Simmons, 1985). When epidemics of leaf spot were accounted, the losses were estimated about 30-40%, for Germany and the southern US. 3-5% of yield losses can be caused if the seed has 40-70% seed-borne infestation (Gough and McDaniel, 1974; xiv

Olofsson, 1976). For the development of Drechslera avenae leaf spot, warm temperature and high humidity are ideal (Turner and Millard, 1931).

Triticale (× Triticosecale Wittm. ex A. Camus.) was developed through the hybridization of wheat and rye (Wilson, 1875). Triticale has been regarded as a minor crop that is utilized for fodder and forage (Ayalew, et al., 2018); however, triticale has a high return leading to an expansion in ubiquity and making it an important crop. Globally, the production of triticale is averaging 17 million metric tons annually leading by Germany,

France, Poland, and Belarus (Ayalew, et al., 2018).

In breeding programs of wheat, triticale has been utilized broadly as a bridge which facilitated transferring beneficial genome from rye to wheat. The rye genome has some genes which can help wheat to resist biotic and abiotic stress (Saulescu, et al., 2011).

Pyrenophora tritici repentis is a pathogen which causes the disease “tan spot” in wheat and could infect triticale, as well (De Wolf et al. 1998; Krupinsky, 1992; Wakulinski, et, al.,

2005).

In wheat, tan spot is a serious disease with real consequences to worldwide food security, as yield could decrease of up to 50% and in severe cases up to 70% (Sharp, et al.,

1976; Shabeer and Bockus, 1988; Moreno and Perelló, 2010). It is the most observed leaf spot disease in South Dakota (Byamukama, 2013). The fungus P. tritici repentis induces oval shaped tan necrotic spots surrounded by a chlorotic halo with a little dark spot in the center (Hosford, 1971; De Wolf, et al., 1998; Moreno and Perelló, 2010). Three host toxins are produced by P. tritici repentis are Ptr ToxA, Ptr ToxB, and Ptr ToxC. These toxins cause necrosis and chlorosis symptoms in toxins sensitive genotypes. Eight races have been identified in P. tritici repentis isolates based on their ability to produce two distinct xv symptoms, necrosis and chlorosis, in wheat differential genotypes, and races are 1 and 5 are the most prevalent in the US and elsewhere (Lamari et al. 2005; Ali et al. 2010;

Abdullah et al. 2016; Ali and Francl 2003; Lamari et al. 2003).

In the present study, I chose the following objectives 1) recover and characterize the causal agent(s) of leaf spot of oats in South Dakota. 2) evaluate oat germplasms for its reaction to Drechslera avenae leaf spot, and 3) characterize triticale genotypes for their reaction to P. tritici-repentis races 1 and 5 and sensitivity to Ptr ToxA. 1

CHAPTER 1 (Literature review)

Oat

Origin and History Oat (Avena sativa L.) is an important crop (domesticated weed), cultivated worldwide. (Murphy and Hoffman, 1992). The origin of the present-day oat was discovered back to its presence as a contaminant in wheat and barley in southwest Asia, especially

Mesopotamia (Murphy and Hoffman, 1992; Zhou, et al., 1999). It probably reached in

Europe when transported with primary crops, during the spread of agriculture, in the

Neolithic age (Murphy and Hoffman, 1992; Pinhasi, et al., 2005).

Molecular phylogenetics using random amplified polymorphic DNA (RAPD) studied the variations in the distribution of polymorphic DNA markers, have found that it was domesticated roughly 2000–3000 years ago in northern or central Europe (Zhou, et al.,

1999). Its spread to the Mediterranean region was hypothesized to have given rise to Avena byzantina by introgression (Ladizinsky, 1988).

The absence of pre-Columbian evidence suggests that it was probably introduced to North America during colonialization (Murphy and Hoffman, 1992). Oat spread to North

America is thought to be through two ways from different parts of Europe: Avena sativa was introduced in northeastern USA, Canada, and Newfoundland by the English, while the

Spanish introduced A. byzantina in the southern USA (Coffman, 1977; Murphy and

Hoffman, 1992). The spring-sown germplasm in North America has been traced to the northern European A. sativa, and the fall-sown oat to the Mediterranean A. byzantina

(Coffman, 1977; Zhou, et al., 1999). 2

Health benefits Oat (Avena sativa) is widely utilized as both food and fodder (Zhou, et al., 1999).

Oat is abundant in the soluble fiber, β-glucan that lowers cholesterol and may diminish the risk of heart disease (Whitehead, et al., 2014). The protein content of oat is estimated to be equal to soy protein and egg protein (Lasztity, 1999). It is rich in several vitamins of the

B-complex and minerals such as manganese (Ahuja, et al., 2018). 100 g of oats also give

389 calories in the form of carbohydrates and fats (Ahuja, et al., 2018). Due to healthful nutritive value, oat is broadly used as a healthy diet, but care must be taken by individuals who have celiac disease (Ciacci, et al., 2015; La Vieille, et al., 2016). Consequently, oat production and demand have increased substantially after its beneficial effects were confirmed (Whitehead, et al., 2014).

Production Globally, the 2018/2019 projection of oat production is 22.27 million metric tons

(USDA, 2019). The USA is among the leading oat producers (USDA, 2018). In the USA, the production of oat was approximately 64.7 million bushels in 2016. The production was diminished in 2017 and 2018 roughly 49.3 and 56.1 million bushels, respectively (USDA,

2018). South Dakota had the highest oat production, 9 million bushels, in the USA in 2016.

In 2018, South Dakota was ranked as the second largest oat producer after North Dakota in the USA producing 7.7 million bushels (USDA, 2018).

Biotic stresses in oat production

Drechslera avenae leaf spot Drechslera avenae history Oats are relatively less affected by pests and diseases except for foliar diseases

(Rosentrater and Evers, 2018). The diseases such as crown rust, stem rust, Stagonospora 3 avenae leaf blotch, and Drechslera avenae leaf spot are prevalent in the oat producing areas worldwide (Fig 1.1). This includes all of North and South America, Europe, Russia,

Australia, New Zealand, the Indian subcontinent, parts of Africa, and even spreading into

China, Korea, Japan, and Malaysia (CABI, 2018; Carmona, et al., 2004; Filipas,et, al,1997;

Ariyawansa, et, al, 2014; Hetherington, et, al,2002; Kim,et, al.,1995; Morrissey,et,al.,2000;

Mundt, et, al.,1985). (Leonard, 2003; Martens, 1985; Cunfer, 2000). Leaf spot, sometimes called leaf blotch, is a significant foliar disease of oats caused by Drechslera avenae

(teleomorph: Pyrenophora avenae S. Ito &Kurib.) a necrotrophic fungal pathogen that belongs to the phylum Ascomycota, class Dothideomycetes, subclass Pleosporomycetidae, order Pleosporales, and family Pleosporaceae (Carmona, et al., 2004, Ariyawansa, et, al,

2014).

Drechslera avenae leaf spot of oat was for the first time observed in Italy in 1889

(Briosi and Cavara, 1889), and the fungus was first called Helminthosporium teres (var.) avenae-sativae. Two years later, the same fungus was independently classified as a pathogen that was specific only to oat, but not wheat or barley, and was given the name H. avenae (Eidam, 1891). Helminthosporium infection of oats was also reported in Holland in

1900, in Denmark the next year, and in the USA in 1914 (Johnson, 1914; Turner and

Millard, 1931). Based on the fungus (pathogens) recovered from Italy, Holland, Denmark, and the USA similarity, the two-fungal species were concluded to be the same and was named H. avenae (Dennis, 1933).

The general practice of mycologists and plant pathologists, who were interested in biosystematics, was to club together various unrelated and partially characterized species under the genus name Helminthosporium (McGinnis, et al., 1986). Extensive studies were 4 required to resolve the confusion and divide unrelated and partially characterized species into distinct genera, such as Bipolaris, Drechslera and Exserohilum from the genus

Helminthosporium (McGinnis, et al., 1986), and H. avenae renamed as Drechslera avenae

(Ito and Kuribayaski, 1930).

Drechslera avenae name came to be strongly associated with the asexual phase

(anamorph) of this fungus, even though, based on morphological similarity and other characteristics, the teleomorph was grouped with other fungal pathogens of Ascomycota that affect cereal grains, called Pyrenophora (Shoemaker, 1962). From Pyrenophora avenae, the name of the teleomorph changed to P. chaetomioides, after comparison of morphological characters of the ascocarp (Sivanesan, 1987), but the anamorph stage remained as Drechslera avenae, until the recommendation of the 18th International

Botanical Congress in 2011 to use only one single name, whereupon both the anamorph and the teleomorph came to be referred to as P. chaetomioides (Braun, 2012).

Drechslera avenae distribution and significance Oats require temperate climate, cooler, and wetter areas because they need lower temperatures for their optimal growth and are more tolerant to rain and frost when compared to wheat, barley, and rye (Contreras-Govea and Albrecht, 2006). Cool temperatures and high moisture are also optimal for the growth of D. avenae and infection process (Turner and Millard, 1931). In particular, the pathogen was reported mostly in regions where the environment was cool and rainy during the planting time (de Tempe,

1964). The optimum temperature for the pathogen is approximately 20°C, with a minimum temperature of 2–3°C, while the mycelium can survive at −14°C for at least 56 days under lab conditions (Dennis, 1933). 5

Drechslera avenae is the widespread pathogen of oats and has caused considerable harm to oats in southern USA, Western Europe, Japan, and India in 1984 (Simmons, 1985).

It was of economic importance in Denmark (Turner and Millard, 1931) and was the most critical oat disease in Canada in the 1920s (Drechsler, 1923) and Scotland in the following decade (O'Brien and M'Naughton, 1933). When epidemics of leaf spot occurred in

Germany and the southern USA, with losses estimated to be about 30–40% (Gough and

McDaniel, 1974). Up to 10% yield losses were reported due to the disease in Finland and

Sweden, where it is a regularly observed seed-borne pathogen, while in India, the disease was detected affecting oats from the seedling stage till the maturity of the plant (Harder and Haber, 1992). Drechslera avenae was seen as the primary pathogen of oat kernels in

Brazil, even though severe infestations were not common (Blum, 1997). Typical incidences of seed-borne infection around 14% have been documented (Kunovski and Breshkov,

1981), but even about 40–70% seed-borne infection was reported resulting in yield losses of only 3–5% (Olofsson, 1976). The reason behind this low yield loss was because of effective control through seed treatment in modern times. The disease is still widespread and important in oats producing regions because the pathogen can survive on crop residue; but the use of effective fungicides could minimize the yield losses (Amelung, 1990;

Paveley, et al., 1996).

Drechslera avenae is a necrotrophic fungus and diminishes the green leaf area of for photosynthesis in affected plants, reducing energy and starch production. Infection of the first 3–4 leaves of the emerging seedling, drives to stunting and decrease in tillering

(Dennis, 1933). When the grain spikelets emerge, they are sometimes dry, reflecting the 6 nutrient deprivation in the affected plant, which might even die in severe infection (Turner and Millard, 1931; Dennis, 1933).

Host range Drechslera avenae infection is extensively documented in oats, and the pathogen is primarily specific to various species of oats (Eidam, 1891; Turner and Millard, 1931;

Dennis, 1933). Nonetheless, researches have documented the occurrence of the fungus on seeds of wheat (Sisterna and Carranza, 2004). Furthermore, the pathogen is recorded as a minor pathogen of barley (Braithwaite, Alexander, and Adams, 1998) and infrequently in some grasses (Chełkowski, 1995).

Host resistance Studies have heeded that many oats genotypes, such as B1-47-67 and Wisconsin hybrid X279-1 (Earhart and Shands, 1952), Flamingsweiss II, Omeko Clinton, Bontram,

2411 (Müller, 1963), 7930-6, 8172-2, 8184-14, 8184-18 (Frank and Christ, 1988), Kasadra,

Roxton, Orlando, Rodney M, Vermiou and Melys exhibit partial or substantial resistance to D. aveanae leaf spot when tested in the greenhouse, and they can be used as a source of resistant (Sebesta, et al., 2001; Petrova, et al., 2006; Šebesta et al., 1995; Cegiełko et al.,

2011)

Drechslera avenae leaf spot symptoms and disease cycle Leaf spots first begin as little oblong spots with white centers that are circled by a reddish-brown halo (Ellis, 1971). Later they become dark with sometimes sunken centers, often purplish brown or gray, with a reddish-brown margin surrounded by a lighter halo that slowly merges with the normal green coloration of the leaf (Fig1.2) (Turner and

Millard, 1931). These individual spots also combine, forming striped lesions that are laterally restricted by the leaf veins (Turner and Millard, 1931). 7

Leaf spot was found to develop in two well-marked phases: a primary seedling infection that appears from the seed-borne pathogen, and the secondary adult infections that emerge from spores of the fungus (Fig 1.3) (Turner and Millard, 1931; Drechsler,

1923; Butler and Jones, 1956; Rekola, et al., 1970; Gair, et al., 1978), both of which need cool and wet conditions (Turner and Millard, 1931). The most obvious and characteristic symptom of leaf spot is the outcome of the secondary adult infection (Turner and Millard,

1931; Drechsler, 1923).

Moreover, the infection can spread to the grain sheath letting the spikelet to droop

(Ivanoff, 1963) affecting the kernels that become shriveled, darkened, lightweight (Blum,

1997) and of inadequate quality (Bocchese, et al., 2006), as a result of the presence of the mycelium of the fungus, and the color variation depends on its density and enzyme activity

(Clark, et al., 2008).

The infection also results in darkening of nodes next to infected leaves due to the spread of the fungus, producing in a symptom termed “black-stem” (Luke, et al., 1957;

Jones and Clifford, 1983). Infected stems are weak, likely to easily breaking, and in severe situations, the stem cavity contains the fungal mycelia (Luke, et al., 1957; Clark, et al.,

2008).

Role of fungal effectors (toxins) in disease development Although oat possesses a natural protective barrier of saponins (Cegiełko, 2008),

β-esterase in Drechslera avenae aids in invasion (Bocchese, et al., 2003). Furthermore,

Drechslera avenae produces secondary metabolites like macrodiolides dihydropyrenophorin, pyrenophorin and pyrenophoral (Sugawara and Strobel 1986;

Kastanias and Chrysayi-Tokousbalides 1999; 2000), along with anthraquinone derivatives

(Engström, et al., 1993; Cegiełko, 2011). The cytokinins and secondary metabolites 8 produced by this pathogen were suspected to decrease seed germination (Graniti and

Puglia, 1984). It is now known that the fungus indeed produces a substance named KM-

01, which is like bipolaroxin of Bipolaris cynodonti. It is a selective inhibitor of the plant hormone brassinolide, which controls plant growth (Kim et al. 1995; 1998). Due to its phytotoxic nature to susceptible plants, this fungus has been tested for utility as a biocontrol agent for bromegrass (Lawrie, et al., 2010).

Disease management Drechslera avenae infection and leaf spot disease can be kept under control to limit substantial yield loss using various disease management strategies (Amelung, 1990;

Paveley, et al., 1996), and these include conventional tillage, crop rotation utilizing non- host species, and elimination of crop debris from previous harvest (Shaner, 1981; Harder and Haber, 1992); fungicide seed treatment (De Tempe, 1964; Jones and Clifford, 1983;

Harder and Haber, 1992) and foliar sprays with fungicides (Margot, et al., 1998); and utilization of resistant cultivars (Šebesta, et, al., 1995). Resistant genotypes to D. avenae leaf spot have been documented (Earhart and Shands, 1952; Müller, 1963; Frank and

Christ, 1988; Sebesta, et al., 2001; Petrova, et al., 2006) and their use presents a sustainable approach. In particular, Cc 3678 and Suregrain have shown promise in field tests (Jalli,

2004) An additional technique is biocontrol agents utilizing common microbes of the rhizosphere, such as Pseudomonas chloraphis, as antagonists, which provided positive outcomes if the seed in infected/infested (Ronquist, 1994; Maude, 1998). 9

Figure 1.1: Global distribution of P. chaetomioides, indicated by green circles (image credit: CABI, 2018)

Figure 1.2: Tan necrotic leaf spots on two-week-old seedling leaf of a susceptible oat genotype 10

Figure 1.3: Life cycle of P. chaetomioides (image credit: Clark, et al., 2008)

Wheat Origin, history, and domestication of wheat Wheat is the most cultivated crop in the world (IWGSC, 2018). It provides around

20% of the total calories consumed by humans (IWGSC, 2018). The prediction of the world population is to be more than 9 billion people by 2050, so wheat production needs a substantial increase to feed these increased population (Figueroa et al. 2018). Wheat was domesticated from wild grass as landraces, more than 10,000 years ago, in the Fertile

Crescent of Mesopotamia during the Neolithic agricultural age (Shewry, 2009; Faris,

2014). Archeological evidence from Levantine in Turkey, dating about 8650–7950 BCE, shows that the wild grasses initially domesticated were einkorn (diploid) and emmer 11

(tetraploid) wild wheat (Feldman and Kislev, 2007; Colledge and Conolly, 2007; Weiss and Zohary, 2011).

Hexaploid wheat first appeared about 9,000 years ago, possibly because of hybridization of wild varieties of emmer and einkorn, as cultivation spread further into the

Middle East (Dvorak, et al., 1993). Eventually, wheat spread to Europe through Greece from Turkey 8,000 years ago, and then to Italy, Spain, and France 7,000 years ago, reaching

Scandinavia and the UK 5,000 years ago. Later spread to Central Asia, China and Africa about 3,000 years ago (Feldman, 2001).

It was first introduced to the North American continent in Mexico, via Spaniards in

1529 (Feldman, 2001). Nonetheless, it was only in 1602 that wheat was first grown in trials by Bartholomew Gosnold in the geographical region that later become the USA (Brigham,

1910). These trials paved the way for wheat production, first, in Virginia in 1607, and then, throughout most of the East coast by 1800 (Brigham, 1910). Wheat cultivation expanded into the large fields, and the USA has become one of the global leaders in wheat production since then (Clay, 2004).

During the domestication process, artificial selection pressure may have been applied unintentionally, selecting for traits such as larger grains, improved yield

(toughened rachis to prevent spikelet dispersal) and free-threshing characters (Tanno and

Wilcox, 2006). This method might have led to the evolution of contemporary domesticated wheat. The contemporary dwarf wheat grown globally, nevertheless, originates from Japan

(Borojevic and Borojevic, 2005).

Wheat is the most traded crop in the world (Curtis, et al., 2002). Approximately

750 million tons of wheat is produced every year globally (FAO, 2018). Wheat ranks as 12 the third field crops in the USA (NASS, 2018), and the total production of wheat in the

USA was estimated 1.88 billion bushels in 2018. The production of winter wheat ranks first by 1.18 billion bushels, then spring wheat and Durum wheat by 623 and 77.3 million bushels, respectively (USDA, 2018). In South Dakota, wheat total production in 2018 was estimated 72.2 million bushels. Winter wheat and spring wheat productions were estimated

31.6 and 40.5 million bushels respectively (USDA, 2018).

Triticale Origin, and history of triticale Triticale (× Triticosecale Wittm. ex A. Camus.) is a human-made crop. It was first announced in 1875, from Scotland and acquired through the interspecific hybridization of female hexaploid wheat (Triticum) and male diploid rye (Secale) (Wilson, 1875); however, it was sterile and unsuccessful (Stace, 1987). Later, a fertile, true-breeding hybrid was reported from Germany in 1888 (Rimpau, 1891).

In plants, interspecific or intergenic hybridization followed by chromosome doubling, happens in nature, leading to the development of a new species (Gupta and

Priyadarshan, 1982). Indeed, the cytogenetic analysis in 1935 showed that these fertile hybrids and the ones similarly produced in Russia (Meister, 1930) were amphidiploid

(allotetraploid, 2n = 56), including an entire diploid set of chromosomes from each parent

(Lindschau and Oehler, 1935).

The discovery of colchicine-induced polyploidy increased the ease of producing fertile amphidiploid triticale (Kostoff, 1938; Stace, 1987). Crossing within these amphidiploid strains provided the secondary, octaploid triticale that is still grown, (Stace,

1987). However, most contemporary triticale genotypes are hexaploid, received from recombined primary triticale produced via crossing tetraploid wheat and diploid rye, 13 utilizing colchicine (Derzhavin, 1938; Stace, 1987). Tetraploid triticale has also been developed using hexaploid wheat and diploid rye, followed by self-crossing (Krolow,

1973). Nevertheless, the strong cross-pollinating tendency of rye creates genetic instability in the absence of substantial copies of the wheat genome to give tolerance to self- fertilization (Muentzing, 1979; Gupta and Priyadarshan, 1982).

Uses of triticale The primary usage of triticale had been in wheat breeding programs, as a bridge to transfer useful genes from rye (Saulescu, et al., 2011). This use includes the genes for resistance to biotic and abiotic stress, such as diseases and pests, adaptability, and the ability to grow in marginal soil through better nutrient utilization (Jessop, 1996; Blum,

2014; Ayalew, et al., 2018). Its hardy nature, combined with its extensive root system, makes it suitable for planting as a cover crop to prevent soil erosion, soil nutrient depletion, and nitrogen leaching (Ayalew, et al., 2018). It is cultivated as part of crop rotation, to control weeds and reduce soil pathogen load (Mergoum, et al., 2009). It is also being used, to a limited extent, in food products, due to its high protein quality (Meyer and Barnett,

2011; Nakurte, et al., 2012). Additionally, it is being explored for use as an energy crop in biofuel production and molecular biology (Mergoum, et al., 2009).

It is currently a minor crop, grown for fodder and forage, but its popularity is increasing, due to the better biomass yield and performance, endowed by its large canopy cover that helps utilize more sunlight and increases photosynthetic output, even in suboptimal conditions (Ayalew, et al., 2018). Globally, the production of triticale is averaging 17 million metric tons yearly under the area of 4 million hectares. The production and area for triticale was amplified to 17% and 8% receptively in 2014 compared to previous years; out of which Poland is the leader, contributing to more than a 14 third of the world production with Belarus, France, Germany, and Russia being the other significant producers (Ayalew, et al., 2018).

Triticale has been regarded as resistant to diseases and pests, mainly rust, smut, bunt, wheat streak mosaic, brome mosaic, barley stripe mosaic and barley yellow dwarf, cereal cyst and Hessian fly (Mergoum, et al., 2004). However, expansion of triticale cultivation has led to an increase in disease susceptibility, with many wheat and rye diseases now affecting triticale (Zillinsky, 1985; Singh and Saari, 1991; Mergoum, 1994).

The causative agent of tan spot of wheat, Pyrenophora tritici-repentis, a global pathogen, also affects triticale (De Wolf et al. 1998; Krupinsky, 1992; Wakulinski, et, al., 2005).

Diseases such as Fusarium head blight (Fusarium graminearum), Septoria leaf blotch

(Zymoseptoria tritici) and powdery mildew (Blumeria graminis) have also reported in triticale (Parry, et al., 1995; Oettler and Schmid 2000; Oettler, et al., 2004; Haesaert, et al.,

2006; Miedaner, et al., 2006). This plant species to be more susceptible to spot blotch

(Bipolaris sorokiniana), ergot (Claviceps purpurea) and scab (Fusarium sp.) than wheat

(Mergoum, et al., 2004). It is also affected by bacterial diseases caused by Pseudomonas sp. (Skovmand, et al., 1984), as well as pathogens like Gaeumannomyces graminis and

Pseudocercosporella herpotrichoides (Arseniuk, 1996). Stem rust was the first epidemic reported in triticale in Australia (Arseniuk, 1996). Leaf and stripe rusts (Puccinia recondita and P. striformis) are also becoming economically significant (Arseniuk, 1996; Schinkel,

2002; Sodkiewicz and Strzembicka, 2004). 15

Tan spot

Importance Pyrenophora tritici-repentis is a necrotrophic fungus and belongs to the phylum

Ascomycota, class Dothideomycetes, subclass Pleosporomycetidae, order Pleosporales, and family Pleosporaceae. It is primarily a foliage pathogen that causes tan spot in wheat

(Ali and Francl 2003; DeWolf et al. 1998). Furthermore, it uses a wide variety of host plants such as barley, rye, triticale, and oat, in addition to several non-cereal grasses

(Sprague, 1950; Krupinsky, 1992; Ali and Francl, 2003; Wakulinski, et, al., 2005).

Tan spot is an important disease of wheat with severe consequences to global food security, as it can cause a yield reduction of up to 50% and sometimes, even as far as 70%

(Sharp, et al., 1976; Shabeer and Bockus, 1988; Moreno and Perelló, 2010). For this reason, tan spot and its causative agent, Pyrenophora tritici-repentis, is the subject of extensive research around the world. In South Dakota, 5% grain yield loss has been reported.

However, in some fields, grain yield loss may reach higher than 30% (Buchneau et al.

1983). Tan spot is still considered to be the most common and yield-limiting disease in

South Dakota (Byamukama, 2013).

History The pathogen P. tritici-repentis was first isolated from a persistent weed grass

Agropyron repens in Germany and entitled Pleospora trichostoma (DeWolf et al. 1998;

Moreno and Perelló, 2010). It was recovered two decades later from wheat and wrongly given another name, Helminthosporium tritici-repentis (Drechsler, 1923). It reproduces by both sexually and asexually, yet the structures included; namely, the pseudothecia

(telopmorph), and the conidium (anamorph) are morphologically diverse. 16

Of particular importance in its history, its isolation from wheat in Japan (Nisikado,

1928b), associated with yellow spot and called Helminthosporium tritici-vulgaris

(Nisikado, 1928a). Drechslera tritici-repentis and Helminthosporium tritici-vulgaris were identified as the same, shortly after that (cited in DeWolf et al. 1998).

Based on the similarity in morphology and other characteristics, this Pleospora was later classified with other fungal pathogens of Ascomycota that affect cereal grains, called

Pyrenophora (Shoemaker, 1961; 1962). Following that, its pathogenicity in wheat causing tan spot was established (Hosford, 1971; Hosford and Busch, 1974).

Distribution P. tritici-repentis is observed in most wheat growing countries worldwide (Fig. 1)

(DeWolf et al. 1998). The distribution of P. tritici-repentis can infer from the trail of tan spot occurrences it generates. Following the report of tan spot in Japan in 1928, the disease was recorded in India (Mitra, 1934). Shortly after that it had dispersed into the North

American continent, commencing with Canada (Conners, 1937; 1939) then, three years later, to the USA (Barrus and Johnson, 1942). It spread to Australia (Shaw and Valder,

1953) and Kenya (Duff, 1954) in the following decade.

Nevertheless, it rose into a threat in the 1970s, owing to changes in agricultural practices, and expanded worldwide. It is presently found all wheat growing countries/continents of the South American continent, Europe and Russia, Asia, Africa, and Australia and New Zealand (Moreno and Perelló, 2010; Moreno, et al., 2012; Ciuffetti, et al., 2014; Weith, 2015; Abdullah, et al., 2017a).

The performance of contemporary agricultural practices including minimal tillage, in an effort at soil conservation, have enabled the persistence of this organism in the post- harvest stubble (Rees, et al., 1989; Ali and Francl, 2001; Moreno and Perelló, 2010). The 17 threat posed by this pathogen has more been amplified via agricultural practices such as very short, or no crop rotation, and the cultivation of susceptible wheat varieties (Moreno, et al., 2012).

Host range Wheat is the main host of P. tritici-repentis. However, the fungus attacks rye and triticale (Hosford, 1971; Krupinsky, 1992; Ali and Francl, 2003; Wakulinski, et, al.,2005) and, to a limited extent, some genotypes of barley (Aboukhaddour and Strelkov, 2016).

Other than these cereals, P. tritici-repentis strikes a variety of non-cereal grasses too

(Sprague, 1950).

Numerous of these grasses (Krupinsky, 1992; Ali and Francl, 2003) utilized in pastures, such as the perennial rye-grass (Lolium perenne), turkey foot (Andropogon gerardi), orchard grass (Dactylis glomerata) and reed canary grass (Phalaris arundinacea) and serve as alternative hosts. It is possible for livestock that wanders these pastures, to transmit these spores to crop plants in the field (Krupinsky, 1992; Ali and Francl, 2003).

Likewise, wild oat (Avena fatua), as well as various species of Agropyron, are persistent weeds in the field, and may help to spread the pathogen inoculum elsewhere.

Also, they may grow near the fields, in such a way that they might escape weed control yet spread the fungal spores to the fields nearby (Ali and Francl 2003).

Such alternate hosts can serve as reservoirs of the infectious pathogen during seasons of crop rotation when an unsuitable host, such as soybean, mustard or flax, is grown. Moreover, it is possible that such frequent transfers between various host species might contribute to the genetic variability in P. tritici-repentis (De Wolf, et al., 1998), by shuffling existing genes or uptake of new genes through horizontal gene transfer. 18

However, not all lines of wheat are susceptible to P. tritici-repentis, and lines resistant to tan spot exist (Hosford, 1971), such as BH 1146 (Larez, et al., 1986) Gernaro

8 1, Vicam 7 1 (Rees and Platz, 1989) 4B1149, 4B242; Erik, and Salamouni (Gamba and

Lamari, 1998) and ND 735 (Singh, et al., 2010). Most of oats genotypes are also resistant to tan spot, like the Lodi strain (Larez, et al., 1986).

Symptoms and significance The fungus produces lens-shaped tan necrotic spots surrounded by a chlorotic halo with a pin head size black spot in the center in tan spot susceptible wheat genotypes (Fig

2). However, on leaves of wheat plants that are resistant to tan spot, a small dark brown or blackish spot corresponding to the initial site of infection may be seen, but the other symptoms of chlorosis and necrosis are absent (Hosford, 1971; De Wolf, et al., 1998;

Moreno and Perelló, 2010).

Being a necrotrophic fungus, P. tritici-repentis causes necrosis or toxic cell death to the leaf cells, and then feed on them. These tan necrosis in susceptible wheat leaves observed is the primary sites of infection. Surrounding this necrotic lesion is a yellow halo, where degradation of chlorophyll (chlorosis) occurs on the leaf (Moreno and Perelló,

2010). Often, young leaves are heavily infected, and regions of chlorosis can expand and merge together, covering major portions of the leaf.

Since leaves are the primary sites of photosynthesis, energy and starch production in plants, the necrosis and chlorosis caused by P. tritici-repentis significantly reduced the photosynthetic efficiency. This reduction in photosynthetic yield corresponds to stunting, delay in maturation and reduction in biomass (Kremer and Hoffman, 1992), calorific value, weight of the kernel (Shabeer and Bockus, 1988), size of the grain head, the quantity of 19 grains presents in each wheat grain head (Schilder and Bergstrom 1994), and their quality

(Fernandez, et al., 1994).

The fungus can infect plants at any growth stage during the season (Moreno and

Perelló, 2010). When infection occurs during the latter stage (flowering and the early grain- filling), it infects grain and produces symptom called “red smudge,” with the name derived from the reddish coloration exhibited by the infected seeds (Valder, 1954; Fernandez, et al., 1994; Schilder and Bergstorm, 1994).

Virulence variation in the pathogen The major hurdle in understanding the genetic basis of the pathogenicity of P. tritici-repentis was that it is difficult genetically generating amenable crosses and mutants.

So, one of the methods possible was to study the natural variation in the pathogen.

Accordingly, various studies were conducted to identify possible differences, which ranged from documenting variation in the pathogen isolates based on necrotic spot size, number of leaf spot, and incubation period (Luz and Hosford, 1980; Gilchrist, et al., 1984;

Krupinsky, 1987; Schilder and Bergstrom, 1994), but these features did not correlate with difference in virulence.

A significant breakthrough was achieved in the study of when it was observed that the two symptoms of extensive chlorosis and tan necrosis produced by the fungal isolates

(Lamari and Bernier, 1989. They were not related to one another, but rather two distinct and separable symptoms, each exhibited by one variant of the fungus (Lamari and Bernier,

1989). This led to the conclusion that the pathogenicity of P. tritici-repentis was the result of at least two separate gene products, and that some of the variants possessed only one of these genes or in combination. 20

Consequently, a race classification system was put forwarded and the detection of the variation in virulence in the pathogen isolates based on these two distinct symptoms on a set of bread wheat differential: Glenlea, Salamouni, 6B365 and 6B662 (Lamri and

Bernier 1989; Lamari et al. 1995). The resulting variants were classified into four races

(Lamari and Bernier, 1989) initially, which later expanded on to a total of 8 races (Table

1). With the discovery of their gene sequences, the races were genotyped accurately using

PCR to minimize errors in phenotypic characterization (Andrie, et al., 2007).

Of these eight races, race 1 is the most predominant worldwide, with its central dominion being Eurasia, Africa and the two American continents (Abdullah, et al.,

2017a). It is closely followed by race 2 (Ali and Francl, 2003; Gamba, et al., 2012), with race 3 and 4 being uncommon (Lamari and Bernier, 1989; Ali and Francl 2003). The distribution of race 5 is rather diverse but discontinuous, occupying regions of Africa,

Central Asia, USA and Canada (Strelkov, et al., 2002; Lamari, et al., 2003; Abdullah et al., 2017a). While race 6 was found in Africa (Strelkov, et al., 2002). The races 7 and 8 were observed to be prevalent in the Caucasus and Fertile Crescent regions (Lamari and

Strelkov, 2010).

Role of Host-Selective toxins in Disease development P. tritici-repentis is known to produce three host-selective effectors (toxins), Ptr

ToxA, Ptr ToxB, and Ptr ToxC which are associated with necrosis and chlorotic symptoms, and play an essential role in the disease development, depending on the wheat susceptible wheat genotype (Lamari et al. 2005; Ali et al. 2010). Ptr ToxA gene was not initially present in P. tritici-repentis It was postulated (Rosewich and Kistler, 2000) and then demonstrated to have been acquired through horizontal gene transfer from Phaeosphaeria nodorum, sometime just before 1941 (Friesen, et al., 2006). 21

The high degree of similarity between the toxin of the two species, the requirement of the same host gene for resistance, and the fact that both these pathogens affect the same host, strengthen the probability that such a horizontal gene transfer had indeed taken place.

It is this gene transfer that has most likely contributed to the dominance of P. tritici-repentis as a global pathogen of wheat from the latter half of the 20th century (Friesen, et al., 2006).

The three characterized toxins produced by P. tritici-repentis are designated Ptr

ToxA, Ptr ToxB and Ptr ToxC respectively (Martinez, et al., 2001; Effertz, et al., 2002).

Of the three, ToxA is known to be a secreted protein with a molecular mass of 13.3 kDa and ToxB, a protein 6.5 kDa in size (Ballance, et al., 1989; Tomas, et al., 1990; Strelkov and Lamari, 2003; Martinez et al., 2004; Manning and Ciuffetti, 2005; Kim et al., 2010;

Andrie and Ciuffetti, 2011). Ptr ToxC characterized as a non-ionic, but small polar molecule (Effertz, et al., 2002).

Studies on PtrToxA indicate that this secreted protein which causes necrosis must be internalized into the host cells for it to exert its toxic effects and cause cell death

(Manning and Ciuffetti, 2005). It was also demonstrated that the resistant wheat plants did not internalize this protein, which could be because of the absence of the necessary import machinery, or modifications to the mechanism in such a way that the toxin is no longer compatible (Manning and Ciuffetti, 2005).

While Ptr ToxA is encoded by a single-copy gene, Ptr ToxB is encoded by multiple copies (Orolaza, et al., 1995; Strelkov, et al., 1999; Martinez, et al., 2001), with the virulence and the extent of chlorosis it causes, being impacted by the copy number. A homolog very similar in sequence (Martinez, et al., 2004) and structure (Nyarko, et al.,

2014) to Ptr ToxB has been found to occur in non-pathogenic P. tritici-repentis. 22

Ptr ToxC has not been purified, and the gene that is responsible for its production is not known. It is known that this toxin induces chlorosis in sensitive wheat genotypes.

The only method presently available for detecting the presence of Ptr ToxC is by inoculating Ptr ToxC sensitive wheat line, 6B365 based on chlorosis induction (Effertz, et al., 1998; Strelkov and Lamari, 2003). It has been partially purified using ion exchange and reverses phase chromatography (Effertz, et al., 2002).

Disease cycle P. tritici-repentis is a homothallic fungus that undergoes self-fertilization. It is capable of dispersal and infection in a moisture-laden atmosphere, in a range (18-22oC) of temperatures (Hosford, 1971). However, disease progression depends on the wheat genotype susceptibility level, the pathogen virulence, and conducive environment for disease development (De Wolf, et al., 1988). Following the life cycle of the organism leads to the routes of infection and transmission of the disease. The seed is the primary mode of spread of the pathogen over large distances. The fungus lives in the pericarp of wheat in the form of mycelium and gets transmitted to new fields (Schilder and Bergstorn, 1994).

Other sources of P. tritici-repentis are the post-harvest stubble, or wheat straw, and the alternate host plants. During winter, the ascospores of the fungus reside as a saprotroph on this substratum to tide over the unfavorable cold. This process is called overwintering

(Ciuffetti, et al., 2014). Following the return of favorable conditions, rain/splash dispersal transmits the ascospores for short distances, and they encounter the host plant, where they initially penetrate the leaf and induce tan spot and begin the asexual cycle under favor weather condition (Schilder and Bergstorn, 1994; Ciuffetti, et al., 2014).

This process involves spores multiplying many times, spreading over the leaf, and sometimes, even the plant stem. While on the leaf, they cause tan necrotic spots and release 23 another cycle of mature asexual spores in the form of conidia during the same growing season. These spores are capable of long-distance travel by wind to spread the pathogen to more regions (Ciuffetti, et al., 2014).

Disease management P. tritici-repentis is the dominant pathogen of wheat globally. It requires proper management to keep the disease pressure minimal and prevent it from resulting in substantial yield loss. Tan spot can also occur along with other foliage diseases, which may complicate the situation (Moreno and Perelló, 2010). The disease can be managed by adopting suitable agricultural practices, cultivating resistant cultivars, and using biocontrol strategies. Fungicides need to be used sparingly for acute and efficient control, because, P. tritici-repentis may become fungicide resistant (Reimann and Deising, 2005; Patel et al,

2012).

Use of certain agricultural practices like tillage and pretreatment of seeds may help in minimizing the chances of infection. Crop rotation using plant species that do not support

P. tritici-repentis growth, such as soybean, flax, and mustard (Bockus and Claassen, 1992) limits the pathogen viability of the sexual stage with reduction of suitable substratum over time (Moreno and Perelló, 2010). Cultivation of tan spot resistant varieties of wheat is the best and most sustainable approach. Biocontrol agents can be used to manage the tan spot in a variety of ways. The possibility of using antagonistic microbes is actively explored, to keep a check on P. tritici-repentis (Moreno and Perelló, 2010).

Table 1: Races of Pyrenophra tritici-repentis and their symptoms on tan spot wheat differentials set (Lamari et al., 2003) 24

Race Toxin Wheat differential lines Salamouni Glanlea 6B365 6B662 (Ptr ToxA)* (Ptr ToxC)* (Ptr ToxB)* 1 ToxA_ToxC Avirulent Necrosis Chlorosis Avirulent 2 ToxA Avirulent Necrosis Avirulent Avirulent 3 ToxC Avirulent Avirulent Chlorosis Avirulent 4 No toxin Avirulent Avirulent Avirulent Avirulent 5 ToxB Avirulent Avirulent Avirulent Chlorosis 6 ToxB_ToxC Avirulent Avirulent Chlorosis Chlorosis 7 ToxA_ToxB Avirulent Necrosis Avirulent Chlorosis 8 ToxA_ToxB_ToxC Avirulent Necrosis Chlorosis Chlorosis *Toxin sensitivity in parentheses

Figure 1.4: World map depicting the global occurrence of tan spot in the wheat growing countries (image credit: Ciuffetti, et al., 2014). Yellow dots and circles indicate countries and regions of tan spot occurrence.

Figure 1.5: Tan necrotic leaf spots on a susceptible triticale genotype inoculated with race 1

25

Figure 1.6: Schematic representation of the life cycle of tan spot (image credit: Ciuffetti, et al., 2014).

The fungus overwinters saprotrophically on wheat straw in the form of pseudothecia, the sexual fruiting body containing ascospores, serve as a source of primary inoculum. Mature ascospores are released in spring and infect young plants and serve as primary inoculum.

Later, conidia from mature are produced and dispersed over long distances by the wind to infect new plants or the same plants upward if suitable conditions for sporulation, infection, and disease development persist.

26

Rational, Significance and Objectives There is an increase in the general demand for a nutritious, safe, and reliable supply of food and nutrients across the world. Agriculture is of significant importance to developing nations in obtaining food security, and agricultural products are a significant source of national revenue. Some challenges such as climate change, desertification may reduce the productivity of the crops, decrease in the availability of food and raise food costs, which might push millions of people deeper into hunger and poverty.

Among highest cereal production oat is also considered as an important crop

(Hoffman, 1995) although it is a secondary crop considering the wheat and rice as major cereal crops. (Zhou, et al., 1999). Oats provide calories in the form of carbohydrates and fats, and about 100g of oats gives 389 calories (Ahuja, et al., 2018). Oats has been well known for reducing the risk of heart diseases, and it has soluble fiber named as beta-glucan which lowers the cholesterol level (Whitehead, et al., 2014). Along with rich in soluble fiber, oat also has B-complex vitamins and minerals like manganese which are beneficial to human health (Ahuja, et al., 2018). Its beneficial effects were demonstrated which are higher than any other cereal crop, and that causes an increase in its production and demand

(Whitehead, et al., 2014).

Drechslera avenae has been reported in southern USA, Western Europe, Japan and

India and caused enormous damage to oats (Simmons, 1985). The loss was reported around

30-40%, and epidemics of leaf spot was reported from Germany and the southern USA and considered as the second most severe disease of oats after the crown rust (Gough and

McDaniel, 1974). The typical Drechslera avenae leaf spot incidence was recorded about

14% (Kunovski and Breshkov, 1981), but the fungal seed-borne infection ranging from 40-

70% could cause yield loss of 3-5 % (Olofsson, 1976). 27

Triticale was developed by the hybridization of wheat and diploid rye. In the process of hybridization, where wheat was used as a female parent (Wilson, 1875). It is considered as a minor crop and is used for fodder, but due to its better biomass yield and performance increased its value. It has a large canopy cover which helps it to utilize more sunlight and to survive in suboptimal conditions (Mergoumet al.,2009; Ayalew, et al.,

2018). It has been known to be generally resistant to diseases and pests (Mergoum, et al.,

2004). Currently, it has been used in wheat breeding programs as a bridge for the transfer of useful genes from rye (Saulescu, et al., 2011). This gene transfer includes but not limited to the genes for resistance to biotic and abiotic stress (Jessop, 1996; Blum, 2014; Ayalew, et al., 2018). Increasing trend in triticale production due to its use as a cover crop for soil health, forage, and sources of resistance for multiple diseases including tan spot warrants its germplasm evaluation against most prevalent races ( 1 and 5) of P. tritici-repentis worldwide including the US northern Great Plains (Abdullah et al. 2016; Ali and Francl

2003; Lamari et al. 2003).

There is lack of information available on the reaction oat germplasms including commercial cultivars grown in South Dakota and the reaction of triticale genotypes to tan spot against race 1 and race 5. Also, a little on information is available on the prevalence of leaf spot pathogens of oats in South Dakota. The availability of information on the reaction of oats germplasm to Drechslera avenae leaf spot and the reaction of triticale germplasm to tan spot will help in the development diseases management strategies in oats, triticale, and wheat by identifying sources of resistance to oat leaf spot and tan in wheat and triticale. 28

The objectives of this study were to 1) recover and characterize the causal agent(s) of leaf spot of oats in South Dakota. 2) evaluate oat germplasms for its reaction to

Drechslera avenae leaf spot, and 3) characterize triticale genotypes for their reaction to P. tritici-repentis races 1 and 5 and sensitivity to Ptr ToxA.

29

Literature cited Abdullah S, Sehgal SK and Ali S (2017a) “Race diversity of Pyrenophora tritici-repentis

in South Dakota and response of predominant wheat cultivars to tan spot” J. Plant

Pathol Microbiol. 8, 5, 409.

Abdullah S, Sehgal SK, Ali S, Liatukas Z, Ittu M and Kaur N (2017b) "Characterization of

Pyrenophora tritici-repentis (tan spot of wheat) races in Baltic states and Romania"

Plant Pathol J. 33, 2, 133-139.

Aboukhaddour R and Strelkov SE (2016) “Exploring de novo specificity: the Pyrenophora

tritici-repentis–barley interaction” Plant Path. 65, 1347–1357.

Ahuja J, Haytowitz DB, Pehrsson PR, Roseland JM, Wu X, et al., (2018) “USDA national

nutrient database for standard reference” US Department of Agriculture,

Agricultural Research Service, Nutrient Data Laboratory.

Ali S and Francl LJ (2001) “Recovery of Pyrenophora tritici-repentis from barley and

reaction of 12 cultivars to five races and two host-specific toxins” Plant Dis. 85,

580-584.

Ali S and Francl LJ (2003) “Population race structure of Pyrenophora tritici-repentis

prevalent on wheat and non-cereal grasses” Plant Dis. 87, 418-422.

Ali, S., Gurung, S., & Adhikari, T. B. (2010). “Identification and characterization of novel

isolates of Pyrenophora tritici-repentis from Arkansas”. Plant disease, 94(2), 229-

235.

Amelung D (1990) “Die Bedeutung pilzlicher Schaderreger des Hafers (The importance

of fungal pathogens of oats)” Nachricht fur Pflanzenschutz. (News for crop

protection) 44, 10, 235–238. 30

Andrie RM, and Ciuffetti LM (2011) “Pyrenophora bromi, causal agent of brown spot of

brome grass, expresses a gene encoding a protein with homology and similar

activity to Ptr ToxB, a host-selective toxin of wheat” Mol Plant Microbe Interact.

24, 359–367.

Andrie RM, Pandelova I and Ciuffetti LM (2007) “A combination of phenotipic and

genotypic characterization Pyrenophora tritici-repentis race identification”

Phytopathol. 97: 694-701.

Ariyawansa HA, Kang JC, Alias SA, Chukeatirote E, Hyde KD (2014) –“Pyrenophora”.

Mycosphere 5(2), 351–362, Doi 10.5943/mycosphere/5/2/9

Arseniuk, E (1996) “Triticale diseases-a review. In Triticale: today and tomorrow (pp. 499-

525). Springer, Dordrecht.

Ayalew H, Kumssa TT, Butler TJ and Ma X-F (2018) “Triticale improvement for forage

and cover crop uses in the southern great plains of the United States” Front Plant

Sci. 9, 1130.

Ballance GM, Lamari L and Bernier CC (1989) “Purification of a host-selective necrosis

toxin from Pyrenophora tritici-repentis” Physiol Mol Plant Pathol. 35, 203-213.

Barrus MF and Johnson AG (1942) “Yellow-spot disease of wheat in the New York state.

Helminthosporium tritici-vulgaris on wheat in Maryland” Plant Dis. Rep. 26, 246.

Blum A (2014) “The abiotic stress response and adaptation of triticale—a review” Cereal

Res Commun. 42, 359–375.

Blum MMC (1997) “Pyrenophora avenae: ocorrência, inóculo, patogenicidade e

sobrevivência (Pyrenophora avenae: occurrence, inoculum, pathogenicity and 31

survival)” Masters Dissertation in Phytopathology, Federal University of Rio

Grande do Sul: Porto Alegre.

Bocchese CAC, Martinelli JA, Federizzi LC and Rosa CRE (2006) “Processo de infecção

e formação de mancha em grãos de aveia branca com níveis diferenciados de

resistência para Pyrenophora chaetomioides (Infection process and spot

development on kernels of white oats with differentiated levels of resistance against

Pyrenophora chaetomioides)” Fitopatologia Brasileira (Brazilian Phytopathology)

31, 284–290.

Bocchese CAC, Martinelli JA, Matsumura ATS, Federizzi LC and Prestes AM (2003)

“Virulência, atividade enzimática e padrões de isoesterases de isolados de

Pyrenophora chaetomioides, agente etiológico da mancha de grãos e folhas de

aveia (Virulence, enzymatic activity and isoesterase patterns of isolates of

Pyrenophora chaetomioides, causal agent of the oat leaf and kernel spot)”

Fitopatologia Brasileira (Brazilian Phytopathology) 28, 1, 11–16.

Bockus WW and Claassen MM (1992) “Effects of crop rotation and residue management

practices on severity of tan spot of winter wheat” Plant Dis. 76, 633-636.

Borojevic K and Borojevic K (2005) “The transfer and history of “reduced height genes” (Rht) in wheat from Japan to Europe” J Hered. 96, 4, 455–459. Braithwaite M, Alexander BJR and Adams RLM (1998) “Nationwide survey of pests and

diseases of cereal and grass seed crops in New Zealand. 2. Fungi and Bacteria”

Proceedings of the 51st New Zealand Plant Protection Conference 51, 51–59.

Braun U (2012) “The impacts of the discontinuation of dual nomenclature of pleomorphic

fungi: the trivial facts, problems, and strategies” IMA Fungus 3, 1, 81–6. 32

Brigham AP (1910) "The development of wheat culture in North America" Geogr J. 35,

1, 42–56.

Brigham AP (1910) "The development of wheat culture in North America" Geogr J. 35, 1, 42–56. Briosi G and Cavara F (1889) I Fungi Parassiti della Piante Coltivate od utili essicati,

delineati e descritti (The fungal parasites of cultivated cereals or fodder, outlined

and described), Pavia Vol. II–IV, 26–50.

Buchenau, GW., Smolik, JD., Ferguson, MW., and Cholick, FA. 1983. “Wheat diseases in

South Dakota”. Ann. Wheat Newsletter, 29:147.

Butler EJ and Jones SG (1956) Plant Pathology, MacMillan & Co. Ltd: New York.

Byamukama, E. 2013. Leaf spot diseases beginning to develop in winter wheat. igrow.

SDSU Extension 6/6/2013 Retrieved 09/13/2018

http://igrow.org/agronomy/wheat/leafspot-diseases-beginning-to-develop-in-

winter-wheat/

CABI (2018) “Pyrenophora chaetomioides” In: Crop Protection Compendium,

Wallingford: CAB International.

Carmona M, Zweegman J and Reis EM (2004) “Detección y transmisión de Drechslera

avenae en semillas de avena (Detection and transmission of Drechslera avenae

from oat seed)” Fitopatologia Brasileira (Brazilian Phytopathology) 29, 3, 319–

321.

Cegiełko M (2008) “Pathogenicity of Drechslera avenae (Eidam) Scharif to selected oat

genotypes (Avena sativa L.)” Elec J Polish Agri Univ. 11, 2, 5. 33

Cegiełko M, Kiecana I, Kachlicki P and Wakuliski (2011) “Pathogenicity of Drechslera

avenae for leaves of selected oat genotypes and 1st ability to produce anthraquinone

compounds” Acta Sci Pol Hortorum Cultus 10, 2, 11–22.

Chełkowski J (1995) Helminthosporia: metabolites, biology, plant diseases. Bipolaris,

Drechslera, Exserohilum” Institute of Plant Genetics, Polish Academy of Sciences:

Poznań.

Ciacci C, Ciclitira P, Hadjivassiliou M, Kaukinen K, Ludvigsson JF, McGough N, et al.,

(2015) "The gluten-free diet and its current application in coeliac disease and

dermatitis herpetiformis" United European Gastroenterol J. 3, 2, 121–35.

Ciuffetti LM, Manning VA, Pandelova I, Faris JD, Friesen TL, Strelkov S, Weber GL,

Goodwin SB, Wolpert TJ and Figueroa, M (2014) "Chapter 1: Pyrenophora tritici-

repentis: a plant pathogenic fungus with global impact" Genomics of plant-

associated fungi: monocot pathogens” Springer-Verlag Berlin: Heidelberg, 1-39.

Clark W, Bryson R, Tonguç L, Kelly C and Jellis G (2008) The encyclopedia of cereal

diseases, HGCA: Kenilworth.

Clay J (2004) World agriculture and the environment: a commodity-by-commodity guide to impacts and practices, Island Press: Washington, DC. Coffman FA (1977) “Oat history, identification and classification” USDA-ARS Tech Bull.

1516, US Gov. Print Office: Washington, DC.

Colledge S and Connolly J (2007) The origins and spread of domestic plants in southwest Asia and Europe, Left Coast Press: Walnut Creek. Conners IL (1937) “17th Annual report” Can. Plant Dis. Surv. 17, 5.

Conners IL (1939) “Nineteenth annual report” Can. Plant Dis. Surv. 19, 12-14. 34

Contreras-Govea FE and Albrecht KA (2006) "Forage production and nutritive value of

oat in autumn and early summer" Crop Sci. 46, 2382–2386.

Cunfer, B. M. (2000). Stagonospora and Septoria diseases of barley, oat, and rye. Canadian

journal of plant pathology, 22(4), 332-348.

Curtis BC, Rajaram S and MacPherson HG (2002) "Bread Wheat" FAO Plant Production and Protection Series, FAO: Rome. De Tempe J (1964) “Helminthosporium spp. in seeds of wheat, barley, oats and rye” Proc

Int Seed Test Ass. 29, 117–140.

De Wolf ED, Effertz RJ, Ali S and Francl LJ (1998) “Vistas of tan spot research” Can J

Plant Pathol. 20, 349-370.

Dennis WG (1933) “Studies on the morphology and biology of Helminthosporium avenae”

Trans Br Mycol Soc. 18, 223–238.

Derzhavin A (1938) “Results of work on breeding perennial varieties of wheat and rye” Izv

Acad Nauk USSR Ser Biol. 3, 663–665.

Drechsler C (1923) “Some graminicolous species of Helminthosporium. I” J Agric Res. 24,

641-740 &. 24, 641–739.

Duff ADS (1954) “A new disease of wheat in Kenya caused by a specie of Pyrenophora”

The East African Agri. J. 225-229.

Dvorak J, di Terlizzi P, Zhang H-B and Resta P (1993) “The evolution of polyploid wheats: identification of the A genome donor species” Genome 36, 1, 21–31. Earhart RW and Shands HL (1952) “Oat varietals responses to infection by

Helminthosporium avenae” Bid Agronom J. 44, 234–238. 35

Effertz RJ, Anderson JA and Francl LJ (1998) “QTLs associated with resistance to

chlorosis induction by Pyrenophora tritici-repentis in adult wheat” Can J Plant

Pathol. 20, 438–439.

Effertz RJ, Meinhardt SW, Anderson JA, Jordahl JG and Francl LJ (2002) “Identification

of a chlorosis-inducing toxin from Pyrenophora tritici-repentis and the

chromosomal location of an insensitivity locus in wheat” Phytopathology 92, 527–

533.

Eidam E (1891) “Das Vorkommen der Fleckenkrankheit auf Gersten und auf Haferblättern

(The occurrence of spot disease on barley and on oat leaves)” Der Landw. (The

farmer’s newspaper), Breslau 27, 509.

Ellis MB (1971) “Dematiaceous hyphomycetes” Mycological Papers 125, 1–30.

Engström K, Brishammar S, Svensson C, Bengtsson M and Andersson R (1993)

“Anthraquinones from some Drechslera species and Bipolaris sorokiniana” Mycol

Res. 97, 3, 381–384.

Faris JD (2014) “Wheat domestication: key to agricultural revolutions past and future” In: Tuberosa R, et al., (eds.) Genomics of plant genetic resources, Springer: Dordrecht. Feldman M (2001) “Origin of cultivated wheat” In: Bonjean AP and Angus WJ (ed.) The world wheat book: a history of wheat breeding, Lavoisier Publishing: Paris. Feldman M and Kislev ME (2007) “Domestication of emmer wheat and evolution of free- threshing tetraploid wheat” Isr J Plant Sci. 55, 3–4, 207–21. Fernandez MR, Clarke JM and de Pauw RM (1994) “Response of durum wheat kernels

and leaves at different growth stages to Pyrenophora tritici-repentis” Plant Dis. 78:

597-600.

Figueroa, M., Hammond-Kosack, K. E., and Solomon, P. S. 2018. “A review of wheat

diseases—a field perspective. Mol. Plant Path”. 19:1523–1536. 36

Filipas, A., Oulianenko, L., Alexakhin, R., Pimenov, E., Rudakov, O., & Mikhaleva, S.

(1997). “Phytopathological state of cereal crop stands on agricultural lands

contaminated by radioactive substances following the accident at the Chernobyl

NPP”. Archives of Phytopathology & Plant Protection, 31(2), 133-140.

Food and Agriculture Organization (FAO) of the United Nations (2012) “Second global

conference on Agricultural research for development” Breakout session P1.1

National Food Security – The Wheat Initiative – an International Research

Initiative for Wheat Improvement Retrieved Feb 9, 2019, from

http://www.fao.org/docs/eims/upload/306175/Briefing%20Paper%20(3)Wheat%2

0Initative%20-%20H%C3%A9l%C3%A8ne%20Lucas.pdf

Frank JA and Christ BJ (1988) “Rate limiting resistance to Pyrenophora leaf blotch in

spring oats” Phytopathol. 78, 7, 957–960.

Friesen TL, Stukenbrock EH, Liu Z, Meinhardt S, Ling H, Faris JD, Rasmussen JB,

Solomon PS, McDonald BA and Oliver RP (2006) “Emergence of a new disease as

a result of interspecific virulence gene transfer" Nat Genet. 38, 8, 953-6.

Gair R, Jenkins JEE and Lester E (1978) Cereal pests and diseases, Farming Press Ltd:

London.

Gamba FM and Lamari L (1988) “Mendelian inheritance of resistance to tan spot

(Pyrenophora tritici-repentis) in selected genotypes of durum wheat (Triticum turgidum)”

Can J Plant Pathol. 20, 408-414.

Gamba FM, Strelkov SE and Lamari L (2012) "Virulence of Pyrenophora tritici-repentis

in the southern cone region of South America" Can J Plant Pathol. 34, 4, 545–550. 37

Gilchrist L, Fuentes SF and de Bauer IML (1984) “Determinación de fuentes de Resistencia

contra Helminthosporium tritici-repentis bajo condiciones de campo y de

invernáculo” Agrociencia 56, 95–106.

Gough FJ and McDaniel ME (1974) “Occurrence of oat leaf blotch in Texas in 1973” Plant

Disease Reporter, 58, 80–81.

Graniti A and Pugila PA (1984) “Osservazioni sulla sensibilità di cultivar di avena alle

tossine di Pyrenophora avenae Ito et Kurib (Observations on the sensitivity of

cultivar of oats to the toxins of Pyrenophora avenae Ito et Kurib)” Phytopathol

Medit. 23, 1, 39–42.

Gupta PK and Priyadarshan PM (1982) “Triticale: present status and future prospects” Adv

Genet. 21, 255–345.

Haesaert G, Derycke V, Latre J and Hermans B (2006) “Evolution of disease pressure on

triticale under Belgian growing conditions: overview of the last 20 years” In: Botes

W (ed.) Proceedings of the 6th International Triticale Symposium, International

Triticale Association: Stellenbosch.

Harder DE and Haber S (1992) “Oat diseases and pathologic techniques” In: Marshall HG

and Sorrells ME (eds) Oat science and technology, Crop Science Society of

America: Madison.

Hetherington, SD, Smith, HE, Scanes, MG, & Auld, B. A. (2002). “Effects of some

environmental conditions on the effectiveness of Drechslera avenacea” (Curtis ex

Cooke) Shoem.: a potential bioherbicidal organism for Avena fatua L. Biological

Control, 24(2), 103-109. 38

Hoffman LA (1995) “World production and use of oats” In: Welch RW (eds) The Oat

Crop, World Crop Series, Springer: Dordrecht.

Hosford RM (1971) “A form of Pyrenophora trichostoma pathogenic to wheat and other

grasses” Phytopathol. 61, 28–32.

Hosford RM and Busch RH (1974) “Losses in wheat caused by Pyrenophora trichostoma

and Leptosphaeria avenaria f. sp. triticea” Phytopathol. 64, 184–187. https://downloads.usda.library.cornell.edu/usdaesmis/files/5t34sj573/gq67jv49w/1j92gc0

6p/smgr0918.pdf

Internationals Wheat Genome Sequencing Consortium (IWGSC) (2018) “Shifting the limits in wheat research and breeding using a fully annotated reference genome” Science 361, 6403, 7191–7203. Ito S (1930) “On some new ascigerous stages on the species of Helminthosporium parasitic

on cereals” Proc Imp Acad Tokyo 6, 352–355.

Jalli M (2004) “First studies on the virulence spectrum of Finnish Drechslera avenae

isolates” Pirjo Peltonen-Sainio and Mari Topi-Hulmi (Proceedings 7th

International Oat Conference) 51, 185.

Jessop, RS (1996). Stress tolerance in newer triticale compared to other cereals. In

Triticale: today and tomorrow (pp. 419-427). Springer, Dordrecht.

Johnson EC (1914) “Some Imperfecti fungi from wheat, oat and barley” J Agric Res. 1, 6,

475–87.

Jones DG and Clifford DBC (1983) Cereal diseases, their pathology and control, John

Wiley: Chichester. 39

Kastanias MA and Chrysayi-Tokousbalides M (1999) “Comparative phytotoxicity of

pyrenophorin and pyrenophorol isolated from a Drechslera avenae pathotype”

Proceedings of the 1999 Brighton Conference: Weeds, 2, 581–582.

Kastanias MA and Chrysayi-Tokousbalides M (2000) “Herbicidal potential of

pyrenophorol isolated from a Drechslera avenae pathotype” Pest Manag Sci. 56,

3, 227–232.

Kim S, Hatori M, Ojika M, Sakagami Y and Marumo S (1998) “KM-01 a brassinolide

inhibitor, its production, isolation and structure from two fungi Drechslera avenae

and Pycnoporus coccineus” Bioorg Med Chem. 6, 11, 1975–1982.

Kim SK, Asano T and Marumo S (1995) “Biological activity of brassinosteroid inhibitor

KM-01 produced by a fungus Drechslera avenae” Biosci Biotech Biochem. 59, 8,

1394–1397.

Kim YM, Bouras N, Kav NN and Strelkov SE (2010) “Inhibition of photosynthesis and

modification of the wheat leaf proteome by Ptr ToxB: a host-specific toxin from

the fungal pathogen Pyrenophora tritici-repentis” Proteomics 10, 2911–2926.

Kostoff D (1938) “Colchicine and acenaphthene as polyploidizing agents” Nature 142,

753.

Kremer M and Hoffmann GM (1992) “Effect to Drechslera tritici-repentis as the cause of

wheat yellow leaf spot disease on kernel yield and dry matter production” J

Phytopathol. 99: 509-605.

Krolow KD (1973) “4× Triticale, production and use in triticale breeding” Proc Int Wheat

Genet Symp. 4, 237–243. 40

Krupinsky JM (1987) “Pathogenicity on wheat of Pyrenophora tritici-repentis isolated

from Bromus inermis” Phytopathol. 77, 760–765.

Krupinsky JM (1992) “Grass hosts of Pyrenophora tritici repentis” Plant Dis. 76, 92-95.

Kunovski J and Breshkov T (1981) “Field resistance of oat cultivars from a world

collection to stem rust (Puccinia graminis Pers. f. sp. avenae Eriks et Henn.) and

helminthosporiosis (Pyrenophora avenae Ito)” Plant Sci. 18, 3, 118–123.

La Vieille S, Pulido OM, Abbott M, Koerner TB and Godefroy S (2016) "Celiac disease

and gluten-free oats: a Canadian position based on a literature review" Can J

Gastroenterol Hepatol. 2016, 1–10.

Ladizinsky G (1988) “The domestication and history of oats” In: Mattsson B and Lyhagen

R (ed.) Proceedings of the 3rd International Oat Conference, Svalof AB: Lund.

Lamari L and Bernier CC (1989) “Virulence of isolates of Pyrenophora tritici-repentis on

11 wheat cultivars and cytology of the differential host reactions” Can J Plant

Pathol. 11, 284-290.

Lamari L, Strelkov SE, Yahyaoui A, Orabi J and Smith RB (2003) “The identification of

two new races of Pyrenophora tritici-repentis from the host center of diversity

confirms a one-to-one relationship in tan spot of wheat” Phytopathol. 93, 391-396.

Lamari L., and Strelkov, SE (2010). Minireview/Minisynthèse The wheat/Pyrenophora

tritici-repentis interaction: progress towards an understanding of tan spot disease.

Canadian Journal of Plant Pathology, 32(1), 4-10.

Lamari, L., Sayoud, R., Boulif, M., and Bernier, CC (1995). “Identification of a new race

in Pyrenophora tritici-repentis: implications for the current pathotype classification

system”. Canadian Journal of Plant Pathology, 17(4), 312-318. 41

Larez CR, Hosford RM and Freeman TP (1986) “Infection of wheat and oats by

Pyrenophora tritici-repentis and initial characterization of resistance” Phytopathol.

76, 4, 931-938.

Lasztity R (1999) The chemistry of cereal proteins (2nd ed.) CRC Press: Boca Raton.

Lawrie J, Greaves MP and Down M (2010) “Drechslera sp. (Pyrenophora chaetomioides

(Speg.)), a potential biocontrol agent for Bromus sterilis and other Bromus spp.”

Biocontrol Sci Technol. 8, 4, 479–484.

Leonard KJ (2003). “Regional frequencies of virulence in oat crown rust in the United

States from 1990 through 2000”. Plant Disease, 87(11), 1301-1310.

Lindschau MU and Oehler E (1935) “Untersuchungen am konstant intermediaren

additiven Rimpau’schen Weizen-Roggen-Bastard” Zurchter 7, 228–233.

Luke HH, Wallace, AT and Chapman, WH (1957) “A new symptom incited by the oat leaf

blotch pathogen Heterosporium avenae” Plant Disease Reporter 2, 109–110.

Luz WC and Hosford RM (1980) “Twelve Pyrenophora trichostoma races for virulence to

wheat in the central plains of North America” Phytopathol. 70, 1193–1196.

Manning VA and Ciuffetti LM (2005) “Localization of Ptr ToxA Produced by

Pyrenophora tritici-repentis Reveals Protein Import into Wheat Mesophyll Cells”

The Plant Cell 17, 3203-3212.

Margot P, Huggenberger F, Amrein J and Weiss B (1998) "CGA 279202: a new broad-

spectrum strobilurin fungicide" Brighton Crop Protection Conference, 375–382.

Martens, JW (1985). Oat stem rust. In Diseases, Distribution, Epidemiology, and Control

(pp. 103-129). 42

Martinez JP, Oesch NW and Ciuffetti LM (2004) “Characterization of the multiple-copy

host-selective toxin gene, ToxB, in pathogenic and nonpathogenic isolates of

Pyrenophora tritici-repentis” Mol Plant-Microbe Interact. 17, 467-474.

Martinez JP, Ottum SA, Ali S, Francl LJ and Ciuffetti LM (2001) “Characterization of the

ToxB gene from Pyrenophora tritici-repentis” Mol Plant-Microbe Interact. 14,

675-677.

Maude R (1998) “Progressos recentes no tratamento de sementes (Recent progress in seed

treatment)” In: Seminário Panamericano de Semillas (Pan American Seed

Seminar), CESML: Passo Fundo.

McGinnis MR, Rinaldi MG and Winn RE (1986) “Emerging agents of

phaeohyphomycosis: pathogenic species of Bipolaris and Exserohilum” J Clin

Microbiol. 24, 2, 250–9.

Meister GK (1930) “The present purposes of the study of interspecific hybrids” Proc USSR

Congr Genet Plant-Anim Breed. 2, 27–43.

Mergoum M (1994) “Performance and adaptation of triticale to Morrocan environments”

In: Proceedings of the 3rd International Triticale Symposium, International Triticale

Association: Lisbon.

Mergoum M, Pfeiffer WH, Peña RJ, Ammar K and Rajaram S (2004) “Triticale crop

improvement: the CIMMYT programme” In: Mergoum M (ed.) Triticale

improvement and production, FAO: Rome.

Mergoum M, Singh PK, Peña RJ, Río L, et al., (2009) “Triticale: a “new” crop with old

challenges” In: Carena, MJ (ed.) Cereals, Springer: Fargo. 43

Mergoum, M., Singh, P. K., Pena, R. J., Lozano-del Río, A. J., Cooper, K. V., Salmon, D.

F., & Macpherson, H. G. (2009). Triticale: a “new” crop with old challenges. In

Cereals (pp. 267-287). Springer, New York, NY.

Meyer RO and Barnett RD (2011) “Triticale grain in swine diets” Institute of Food and

Agricultural Sciences, University of Florida: Gainesville.

Miedaner T, Schneider B and Oettler G (2006) “Means and variances for Fusarium head

blight resistance of F2-derived lines from winter triticale and winter wheat crosses”

Euphytica 152, 405–411.

Mitra MA (1934) “Leaf spot disease of wheat caused by Helminthosporium tritici-

repentis” Indian J Agr Sci 4, 692–700.

Moreno MV and Perelló AE (2010) “Chapter 21: Occurrence of Pyrenophora tritici-

repentis causing tan spot in Argentina” Management of Fungal Plant Pathogens,

CAB International: Wallingford.

Moreno MV, Stenglein SA and Perelló AE (2012) “Chapter 14: Pyrenophora tritici-

repentis, causal agent of tan spot: a review of intraspecific genetic diversity” The

Molecular Basis of Plant Genetic Diversity, Intech Open: Rijeka.

Morrissey, J. P., Wubben, J. P., & Osbourn, A. E. (2000). Stagonospora avenae secretes

multiple enzymes that hydrolyze oat leaf saponins. Molecular plant-microbe

interactions, 13(10), 1041-1052.

Müller HJ (1963) “Untersuchungen uber Blattflecken-krankheiten des Hafers. II. Pilzliche

Blattfleckenerreger des Hafers: Helminthosporium avenae Eidam, Septoria avenae

Frank, Helminthosporium sativum, Fusarium spec., Epicoccum spec. und

Heterosporium avenae Oud (Studies on leaf spot diseases of oats. II. Fungal leaf- 44

spot pathogens of the oat: Helminthosporium avenae Eidam, Septoria avenae

Frank, Helminthosporium sativum, Fusarium spec., Epicoccum spec. and

Heterosporium avenae Oud)” Phytopathol Zeitschr. (Journal of Phytopathology)

49, 266–290.

Mundt CC and Browning JA (1985). “Development of crown rust epidemics in genetically

diverse oat populations: effect of genotype unit area”. Phytopathology, 75(5), 607-

610.

Murphy JP and Hoffman LA (1992) “The origin, history and production of oat” In:

Marshall HG and Sorrells ME (ed.) Oat science and technology, Agron Monogr.

33, ASA and CSSA: Madison.

Muskett AE (1937) “A study of the epidemiology and control of Helminthosporium disease

of oats” Ann Bot N S. (London) 1, 763–784.

Nakurte, I., Klavins, K., Kirhnere, I., Namniece, J., Adlere, L., Matvejevs, J., ... &

Muceniece, R. (2012). Discovery of lunasin peptide in triticale (X Triticosecale

Wittmack). Journal of cereal science, 56(2), 510-514

Nisikado Y (1928a) “Preliminary notes on yellow leaf spot disease of wheat caused by

Helminthosporium tritici-vulgaris” Inst Agric Biol. 4, 103–109.

Nisikado Y (1928b) “Studies on the Helminthosporium diseases of Gramineae in Japan”

Ann Phytopath Soc Jpn. 2, 96.

Nyarko A, Singarapu KK, Figueroa M, Manning VA, Pandelova I, Wolpert TJ, Ciuffetti

LM and Barbar E (2014) “Solution NMR Structures of Pyrenophora tritici-repentis

ToxB and Its Inactive Homolog Reveal Potential Determinants of Toxin Activity”

J Biol Chem. 37, 25946-25956. 45

O'Brien DG and M'Naughton EI (1933) The Helminthosporium disease of oats” Res Bull

W Scotl Coll Agric. 3, 1–74.

Oettler G and Schmid T (2000) “Genotypic variation for resistance to Septoria nodorum in

triticale” Plant Breed. 199, 487–490.

Oettler G, Heinrich N and Miedaner T (2004) “Estimates of additive and dominance effects

for Fusarium head blight resistance of winter triticale” Plant Breed. 123, 525–530.

Olofsson B (1976) “Undersökningar rörande Drechslera arter hos korn och havre (Studies

on Drechslera species in barley and oats)” Meddn St VäxtskAnnst. (Announcement

by the State Plant Protection Agency) 16, 172, 352–416.

Orolaza NP, Lamari L, and Balance GM (1995) “Evidence of a host-specific chlorosis

toxin from Pyrenophora tritici-repentis, the causal agent of tan spot of wheat”

Phytopathology 85, 1282-1287.

Parry DW, Jenkinson P and McLeod L (1995) "Fusarium ear blight (scab) in small grain

cereals—a review" Plant Pathol. 44, 2, 207–238.

Patel JS, Gudmestad NC, Meinhardt, S., and Adhikari TB (2012). “Pyraclostrobin

sensitivity of baseline and fungicide exposed isolates of Pyrenophora tritici-

repentis”. Crop Protection, 34, 37-41.

Paveley ND, Rennie WJ, Reeves JC, Wray MW, Slawson DD, Clark WS, Cockerell V and

Mitchell AG (1996) “Cereal seed health strategies in the UK” Home Grown Cereals

Authority (HGCA) Research review, HGCA: London.

Petrova OS, Afanasenko OS and Loskutov IG (2006) “Oat resistance to Pyrenophora

avenae Ito et Kurib” Oat Newsletter, Vol. 50. 46

Pinhasi R, Fort, J and Ammerman, AJ (2005). “Tracing the origin and spread of agriculture

in Europe”. PLoS biology, 3(12), e410.

Rees RG and Platz GJ (1989) “Effectiveness of incomplete resistance to Pyrenophora

tritici-repentis in wheat” Aust J Agric Res. 40, 1, 43-48.

Reimann S and Deising HB (2005) “Inhibition of efflux transporter-mediated fungicide

resistance in Pyrenophora tritici-repentis by a derivative of 4'-hydroxyflavone and

enhancement of fungicide activity” Am Soc Microbiol. 71, 6, 3269-3275.

Rekola O, Ruokola A-L and Kurtto J (1970) “Damage caused by Helminthosporium

avenae Eidam on the crop yield of oats in Finland” Acta Agric Scand. 20, 4, 225–

229.

Rimpau W (1891) “Kreuzungsprodukte landwirtschaftlicher Kulturpflanzen”

Landwirtschaftl. Jahrb. 20, 335–371.

Ronquist E (1994) “Seed treatment with rhizosphere bacteria for control of fungal diseases”

Växtskyddsnotiser (Plant protection notes) Stockholm 58, 2, 42–44.

Rosentrater KA and Evers AD (2018) “Introduction to cereals and pseudocereals and their

production” In: Kent's technology of cereals (5th ed.) Elsevier: Cambridge.

Rosewich UL and Kistler HC (2000) “Role of horizontal gene transfer in the evolution of

fungi” Annu Rev Phytopathol. 38, 325–363.

Saulescu NN, Ittu G, Ciuca M, Ittu M, et al., (2011) “Transferring useful rye genes to

wheat, using triticale as a bridge” Czech J Genet Plant Breed. 47, S56–S62.

Schilder AMC and Bergstrom GC (1994) “Infection of wheat seed by Pyrenophora tritici-

repentis” Can J Bot. 72, 510–519. 47

Schinkel B (2002) “Triticale – still a healthy crop?” In: Arseniuk E (ed.) Proceedings of

the 5th International Triticale Symposium, Plant Breeding and Acclimatization

Institute: Radzików.

Sebesta J, Zwatz B, Roderick HW, Corazza L, Starzyk MH, Reitan L and Loskutov IG

(2001) “Incidence of Pyrenophora avenae Ito et Kurib. in Europe between 1994–

1998, and the varietal reaction of oats to it” Plant Protect Sci. 37, 3, 91–95.

Šebesta J, Zwatz B, and Corazza, L (1995). “Incidence of Pyrenophora avenae ito et kurib.

In Europe and the varietal reaction of oat to it”. Archives of Phytopathology & Plant

Protection, 29(6), 485-490.

Shabeer A and Bockus WA (1988) “Tan spot effects on yield and yield components relative

to growth stage in winter wheat” Plant Dis. 72, 599-602.

Shaner G (1981) “Effect of environment on fungal leaf blights of small grains” Ann Rev

Phytopathol. 19, 273–296.

Sharp EL, Sally BK and McNeal FH (1976) “Effect of Pyrenophora wheat leaf blight on

the thousand-kernel weight of 30 spring wheat cultivars” Plant Dis. 60, 135-138.

Shaw DE and Valder PG (1953) “A study of microflora of wheat grains in New South

Wales” Proc. Linn. Soc. NSW 77:307-322.

Sheridan JE and Tan PET (1973) “Incidence and survival of Pyrenophora avenae in New

Zealand seed oats” New Zeal J Agr Res. 16, 251–253.

Shewry PR (2009) “Wheat” J Exp Bot. 60, 6, 1537–1553. Shoemaker RA (1961) “Pyrenophora phaecomes (Reb. Ex. Fr.)” Fr. Can J Bot. 39, 901–

908.

Shoemaker RA (1962) “Drechslera Ito.” Can J Bot. 40, 809–908. 48

Simmons MD (1985) “Diseases of oats” In: Lawes DA and Thomas H (eds) World crops:

production, utilization, description, Proceedings of the Second International Oats

Conference, Kluwer Academic Publishers: Lancaster.

Singh PK, Mergoum M, Adhikari TB, Shah T, Ghavami F and Kianian SF (2010) “Genetic

and molecular analysis of wheat tan spot resistance effective against Pyrenophora

tritici-repentis races 2 and 5” Mol Breed. 25, 3, 369–379.

Singh RP and Saari EE (1991) “Biotic stresses in triticale” In: Proceedings of the 2nd

International Triticale Symposium, CIMMYT: Passo Fundo.

Sisterna M and Carranza M (2004) “Occurrence of Drechslera avenae on durum wheat

(Triticum durum) seed in Argentina” Australas Plant Pathol. 33, 459–460.

Sivanesan A (1987) “Graminicolous species of Bipolaris, Curvularia, Drechslera,

Exserohilum and their teleomorphs” Mycological Papers 158, 1–261.

Skovmand B, Fox PN and Villareal RL (1984) “Triticale in commercial agriculture:

progress and promise” Adv Agron. 37, 1–45.

Sodkiewicz W and Strzembicka A (2004) “Application of Triticum monococcum for the

improvement of triticale resistance to leaf rust (Puccinia triticina)” Plant Breed.

123, 1, 39–42.

Sprague R (1950) Diseases of cereals and grasses in North America, New York: Ronald

Press Co.

Stace CA (1987) “Triticale: a case of nomenclatural mistreatment” Taxon 36, 2, 445–452.

Strelkov SE and Lamari L (2003) “Host-parasite interaction in tan spot (Pyrenophora

triticirepentis) of wheat” Can J Plant Path. 25, 339-349. 49

Strelkov SE, Kowatsch, RF, Ballance GM, and Lamari L (2005). Characterization of the

ToxB gene from North African and Canadian isolates of Pyrenophora tritici-

repentis. Physiological and Molecular Plant Pathology, 67(3-5), 164-170.

Sugawara F and Strobel GA (1986) “(−)-Dihydropyrenophorin, a novel and selective

phytotoxin produced by Drechslera avenae” Plant Sci. 43, 1, 1–5.

Tanno K and Willcox G (2006) "How fast was wild wheat domesticated?" Science 311, 5769, 1886. Tomas A, Feng GH, Bockus WW and Leach JE (1990) “Purification of a cultivar specific

toxin from Pyrenophora tritici-repentis, causal agent of tan spot of wheat” Mol

Plant-Microbe Interact. 3, 221-224.

Turner DM and Millard WA (1931) “Leaf-spot of oats, Helminthosporium avenae (Bri.

& Cav,) Eid” Ann Appl Biol. 18, 535–559.

United States Department of Agriculture (2018, September) “Small grains 2018

summary” National Agricultural Statistics Service 1949-162X Retrieved Feb 9,

2019, from

https://.usda.library.cornell.edu/usdaesmis/files/5t34sj573/gq67jv49w/1j92gc06p/

smgr0918.pdf

United States Department of Agriculture (2019) “World Agricultural Production”

Circular Series WAP 3-19 Retrieved Feb 9, 2019, from

https://usda.library.cornell.edu/concern/publications/5q47rn72z?locale=en

Valder PG (1954) “Yellow leaf spot and pink grain in wheat” Agric. Gaz. NSW 65, 36–37.

Wakulinski, W., Zamorski, C., Schollenberger, M., Nowicki, B., & Kachlicki, P. (2005).

Tan spot epiphytosis in winter wheat and triticale crop-comparative studies. Acta

Agrobotanica, 58(1) 50

Weiss E and Zohary D (2011) "The Neolithic southwest Asian founder crops: their biology and archaeobotany" Curr Anthropol. 52, S4, S239–S240. Weith, S (2015) “Pyrenophora tritici-repentis the causal agent of tan spot: characterisation

of New Zealand populations” Master’s Thesis, Lincoln University.

Whitehead, A., Beck, E. J., Tosh, S., & Wolever, T. M. (2014). Cholesterol-lowering

effects of oat β-glucan: a meta-analysis of randomized controlled trials. The

American journal of clinical nutrition, 100(6), 1413-1421.

Wilson AS (1875) “Wheat and rye hybrids” Trans Proc Bot Soc Edinb. 12, 286–288.

Zhou X, Jellen EN and Murphy JP (1999) "Progenitor germplasm of domesticated

hexaploid oat" Crop Science 39, 4, 1208–1214.

Zillinsky, F. J. (1985). Triticale—An Update on Yield, Adaptation, and World

Production 1. Triticale, (triticale), 1-7. 51

CHAPTER 2

Survey of oat leaf spot pathogens in South Dakota and evaluation of a worldwide collection of oats germplasm against Drechslera avenae leaf spot

ABSTRACT

Oat is one of the most important crops in cereals, and it is highly used crop in cereal production. It is relatively less prone to pests and diseases as compared to other grains like wheat and rice. Leaf spot diseases like Drechslera avenae leaf spot and Stagonospora avenae leaf blotch have been reported on oats in various oat-producing countries to cause significant yield losses under conducive environment for disease development. Information on the prevalence of leaf spot pathogens and reaction of commercial cultivars grown in

South Dakota is not available. The objectives of this study were to conduct a survey of oat leaf spot pathogens in the state and evaluate an extensive collection of oat genotypes including commercial cultivars grown in South Dakota for their reaction to Drechslera avenae leaf spot. Oat diseased leaves, exhibiting leaf spots, samples were collected from various locations in 2017 and 2018 growing seasons in South Dakota. Leaves were analyzed for leaf spot pathogens using standard protocols. Also, 155 oat genotypes representative of 33 countries were characterized for their reaction to D. avenae leaf spot at seedlings stage under controlled conditions in the greenhouse. Four leaf spot pathogens that include D. avenae, Stagonospora avenae, Bipolaris victoriae, and Colletotrichum graminicola were observed on oats in the state during the two years survey. However, D. avenae and Stagonospora avenae were more common than the other two pathogens. The pathogens prevalence percentage varied during the two growing seasons due to the weather, and a higher percentage of diseases incidence found in 2017. The evaluated 155 52 oat genotypes differed in their reaction to D. avenae leaf spot in ranging from resistant

(14.8%), moderately resistant (15.5%), moderately susceptible (28.4%), and susceptible

(41.3%). The five commercial cultivars in the screening were found susceptible to D. avenae leaf spot. The results of this study warrants monitoring leaf spot pathogens in the state at regular basis and incorporation of resistance to Drechslera avenae leaf spot in future to avoid any potential disease epidemic in the region. Further, oat genotypes exhibited resistance to the disease in this study can serve as a source of resistance in oat breeding program.

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Introduction Oat is one of the important cereal crops, and it is considered to be among the highest cereal productions (Zhou et al. 1999; Hoffman, 1995). Oat is among the healthiest grains; thus, its production and demand have considerably increased in recent years (Whitehead, et al., 2014). Oat can be a reason to decrease the risk of heart disease due because it is a rich source of the soluble fiber, β-glucan that lowers cholesterol (Whitehead, et al., 2014).

Additionally, it is an excellent source of some vitamins of the B-complex and various minerals (Ahuja, et al., 2018). Globally, the 2018/2019 projection of oat to be produced is

22.27 million metric tons (USDA, 2019). The USA is among the vital oat producers

(USDA, 2018). In 2016, the production of oats in the United States of America was about

64.7 million bushels. In 2017, this production was decreased to 49.3 million bushels due to some abiotic stresses, but in 2018 it was again raised to 56.1 million bushels. In 2016, highest oat production, 9 million bushels, in the USA produced in South Dakota. However, this production reduced to 4.2 million bushels in 2017. In 2018, through increasing its production from 4.2 to 7.7 million bushels, South Dakota was ranked second oats producer in the country (USDA, 2018).

Oat is relatively less affected by pests and diseases except for foliar diseases

(Rosentrater and Evers, 2018). Drechslera avenae (teleomorph: Pyrenophora chaetomioides) is a necrotrophic fungal pathogen is the causal agent of Drechslera avenae leaf spot. The fungus produces reddish-brown with purple margins leaf spots in susceptible oats genotypes (Clark, et al., 2008). These leaf spots cause a decrease in green leaf area hence impact the photosynthesis process. With reduced photosynthesis, there is a decrease in energy and starch production that affect plant growth in the form of stunting and decrease in tillering (Dennis, 1933). The fungus can spread to grain sheath which causes the 54 dropping of spikelet, under a favorable environment (Ivanoff, 1963). The kernels become darkened with the reduction in their weights (Blum, 1997). This is because of the fungal mycelium that makes the grain quality poor (Bocchese, et al., 2006). Drechslera avenae is a worldwide pathogen of oats and has been documented to cause significant harm in southern USA, Western Europe, Japan, and India by 1984 (Simmons, 1985). Its damages were estimated to be around 30–40% under disease epidemics reported in Germany and the southern USA (Gough and McDaniel, 1974). Typical incidences of seed-borne infection around 14% have been documented (Kunovski and Breshkov, 1981). The seed borne infection with 40–70% could cause 3–5% yield losses (Olofsson, 1976). Cool temperatures and high humidity are optimal for the growth of Drechslera avenae (Turner and Millard, 1931). The disease has been reported in South Dakota; nevertheless, no information is available on the cultivars’ reactions to Drechslera avenae leaf spot in the state. The disease can be managed through cultural practices, fungicide application, and deployment of leaf spot resistant cultivars. However, disease management through resistant cultivars is a more promising strategy due to their durability and environment-friendly.

Therefore, the objectives of this study were 1) conduct a disease survey to document leaf spot pathogens of oats across the state; 2) evaluate oat germplasms for their reaction to

Drechslera avenae leaf spot.

Materials and methods Recovery and characterization of Drechslera avenae isolates from oat. Sixty-nine symptomatic oat leaf samples were randomly collected from research plots at Volga, Aurora, North East Farm, Selby and Southeast Research Farms. Of these

69 samples, 11 were collected in 2017, and the rest were obtained in 2018. The samples were placed individually in paper bags and stored in a refrigerator until isolations were 55 done. To recover fungal isolates from the sampled leaves, each leaf sample was cut into 1 to 2 cm pieces with leaf spots. 40-50 randomly selected leaf pieces were placed in Petri dishes contained water agar medium. The dishes were incubated for 72 hours at 220 C under the 12 hours light and dark cycle. The incubated leaf pieces were examined for leaf spot causing pathogens. All potential leaf spot causing pathogens were transferred individually onto V8PDA (V8 juice: 150 ml; CaCO3: 3 grams; Potato Dextrose Agar: 10 grams; Agar

10 grams; distilled water 850 ml) plates. Single conidium (8-10 conidia/sample if present) of Drechslera avenae and Bipolaris victorie was collected with the help of a sterilized steel needle as described in Ali and Francl (2001). The fungal isolates Stagonspora avenae and

Colletotrichum graminicola were picked by touching an individual pycnidium/acrevuli if present with a sterile needle and then streaked them individually onto V8PDA plates. All pathogens identity was confirmed based on their morphological characteristics. The isolates were stored as dry plugs at -20oC in the freezer until tested.

Plant Materials One hundred fifty oat genotypes seed from 33 countries belong to six continents:

Africa, Asia, Australia, Europe, North America, and South America (33 countries) was obtained from USDA National Plant Germplasm Bank, Aberdeen, ID (Figure 2.1). Also, the seed of five commercial cultivars grown in South Dakota was obtained from SDSU oat breeding program and included in this study. Greenhouse experiments were conducted in the Young Brothers Seed Technology Building greenhouse, South Dakota State University,

Brookings. Two weeks old seedlings of all 155 oats genotypes were raised by planting the seed in plastic containers (5 x 23 cm) (Stuewe & Sons, Inc. 31933 Rolland Drive, Tangent,

Oregon 97389 USA) filled with Sunshine Mix 1 (770 Silver Street, Agawam, MA, USA).

Nine seedlings (three seedlings/cone) of each genotype were evaluated for reaction to D. 56 avenae leaf spot. Three seedlings in each cone served as an experimental unit and replication, respectively. The seedlings were watered, were kept in a greenhouse at 21-

22oC with 16 hours’ photoperiod till the experiment was completed. The seedlings were watered and fertilized as needed.

Figure 2.1: Countries of origin of oat genotypes screened in this study Inoculum preparation, plants inoculation, and disease rating The D. avenae isolate “SD1” that was stored at -20oC was used for producing the inoculum. The plugs were transferred to V8-PDA Petri dishes. Then, they were incubated for 10 days under the alternate 12 cycles of light and dark. The conidia were dislodged by adding 30 ml of distilled water with the help of with looped wire inoculating needle. The conidial suspension was adjusted to 3000 spores per ml as described in Ali and Francl,

2003). Before the inoculation, a drop of tween 20 was added into the suspension.

Nine seedlings of each genotype at the 2-leaf stage were inoculated with Drechslera avenae using its spore suspension (3000 spores/ml) with a hand-held sprayer (Power Sprayer,

Prevail, Chicago Aerosol, 1300 E. North Street, Coal City, IL60416). To promote infection, the seedlings were placed in the humidity chamber with about 100% humidity for 24 hours. 57

After that, they were transferred to the bench in a greenhouse for 10 days until rated for the disease reaction, based on 1-5 modified rating scale of Lamari and Bernier (1989) (Figure

2.2), where lesion type 1-2 is resistant to moderately resistant and 3-5 is moderately susceptible to susceptible.

Data analysis The greenhouse experiment was analyzed utilizing completely randomize design. Analysis of variance (ANOVA) was conducted to find out the descriptive statistics on the genotypes’ reactions. Then, Fisher’s Least Significant Difference (LSD) was used to compare the differences among the genotypes’ reactions.

Figure 2.2: Range of oat genotypes reaction (lesion types 1- 5) induced by Drechslera avenae. Results Recovery and characterization of Drechslera avenae All oat leaf samples collected from 5 South Dakota locations were examined for harboring leaf spot causing pathogens. The total number of leaf segments examined in 2017 and 2018 were 462 and 1672, respectively. In both years, the majority of samples were infected with Stagnospera avenae. Stagonospera avenae was observed in 168 segments 58 which mean the infection incidence was approximately 36% in 2017; whereas, in the following year, the infection incidence diminished to about 20%. Drechslera avenae came second for both years, and it has occurred in 60 (around 13%) and 121 (7%) segments, respectively. Bipolaris victoriae infection incidences were roughly 12% and 6%; while

Colletotrichum graminicola were detected in 2% and 3% of the total number of segments

(Table 2.1). All pathogens were characterized based on their morphological characters.

Drechslera avenae was characterized based on its morphology (Fig 2.3 and 2.4). It can be seen under the stereoscope that brown conidiophores arise separately or in groups of 2 to 4 carry olvicious green colored conidia. Under the microscope the conidia were straight, rod-shaped and sometimes a with a little pointed end (Ellis, 1971). Stagonospora avenae produces plentiful pycnidia that content oozing creamy conidial mass. Conidia are hyaline, smooth, thin-walled, tube-shaped, with a round tip and shorten base, and transversely septate (Quaedvlieg et, al., 2013; Cunfer, 2000). Conidiophores of Bipolaris victoriae occur individually or as groups of a handful conidia. Its conidia are simple, straight or flexuous, septate, extended near the midpoint, occasionally geniculate at the top portion, smooth, and light to mid brown (Manamgoda et, al. 2014). Colletotrichum graminicola produces erumpent acervuli comprising heavily melanized, sterile hairs named setae which is a structure that differentiates the group of Colletotrichum from the morphologically similar genus. Also, its conidia is curved like a sickle (falcate conidia)

(Crouch and Beirn, 2009). Other fungal species, such as Aspergillus, Penicillium,

Rhizopus, Fusarium were infrequently observed.

Reaction of oat genotypes to leaf spot All 155 oat genotypes screened against leaf spot varied significantly in their responses by exhibiting reactions that ranged from moderately susceptible (MS) to 59 susceptible (S) and moderately resistant (MR) to resistant (R) (Fig 2.5 and Table 2.2).

14.8% (n=23) oat genotypes showed resistant (lesion type 1) reactions to leaf spot while

41.3% (n=64) lines exhibited susceptible (lesion type 4-5) including the five commercial cultivars, Shelby, Streaker, Deon, Horsepower, and Hayden (Table 2.3). 15.5% (n= 24) of the genotypes were moderately resistant (lesion type 2) to Drechslera avenae; whereas the rest 28.4% (n= 44) demonstrated moderately susceptible (lesion type 3). In Africa, there were only two genotypes exhibited moderately resistant to resistant reactions; while the rest three showed susceptible (n=1) and moderately susceptible (n=2) reactions. Two of the nine genotypes from Asia showed resistance whereas the majority (n=7) were susceptible.

Of the three genotypes from Australia continent tested against D. avenae leaf spot, two exhibited moderately resistant, and one developed a susceptible reaction. Eight percent

(n=2) of the genotypes from Europe were resistant. Four percent (1) and 24% (6) were moderately resistant and moderately susceptible, respectively; however, the majority (16) of the cultivars exhibited susceptible reaction. Of the 95 genotypes evaluated from North

America, 15.8% (n=15) and 13.7% (n=13) were resistant and moderately resistant, respectively. Whereas, 29.5% (n= 28) and 41.1% (n= 39) of the genotypes were moderately susceptible and susceptible, receptively. The majority of the genotypes from South

America were either resistant (30%) or moderately resistant (50%) to D. avenae leaf spot.

The rest 20% were grouped under moderately susceptible to susceptible. At the country level, the most resistant genotypes were collected from the USA (n=13) than Brazil (n=3) and Canada (n=2). Only one resistant cultivar was found in each of Finland, Belgium,

Turkey, China, and South Africa (Table 2.4).

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Table 2.1: Fungi associated with oat leaf samples collected from various locations in South Dakota in 2017 and 2018 Samples Pathogen % Sample Infected 2017 11 Stagnospera avenae 36.36% Drechslera avenae 12.98% Bipolaris victoriae 12.33% Colletotrichum graminicola 2.10% 2018 58 Stagnospera avenae 20.50% Drechslera avenae 7.06% Bipolaris victoriae 6.77% Colletotrichum graminicola 3.27%

Table 2.2: ANOVA of Drechslera avenae leaf spot Df Sum Sq Mean Sq F value Pr (> F) Genotypes 154 400.2 2.5984 15.79 2e-16 Residuals 155 25.5 0.1645

Table 2.3: Reaction of 155 oat genotypes from six continents to Drechslera avenae

Continent Countries Genotype R MR MS S Africa 4 5 1 1 2 1 Asia 5 18 2 4 5 7 Australia 2 3 0 0 2 1 Europe 16 26 2 1 6 16 North America 3 94 15 13 28 38 South America 3 10 3 5 1 1 Total 33 155 23 24 44 64 (P ≤ 0.05) Table 2.4. Reaction of oats genotypes to Drechslera avenae leaf spot from six continents (33 countries) Continent Country Genotype R MR MS S (number of genotpes) Africa (5) South Africa 1 1 0 0 0 Ethiopia 2 0 1 1 0 61

Table 2.4: continued from previous page Morocco 1 0 0 0 1 Zimbabwe 1 0 0 1 0 Asia (18) China 6 1 1 3 1 Japan 2 0 0 1 1 Mongolia 1 0 0 0 1 Turkey 7 1 3 1 2 India 2 0 0 0 2 Australia (3) Australia 2 0 0 1 1 New Zealand 1 0 0 1 0 Europe (24) Austria 1 0 0 0 1 Belgium 2 1 0 1 0 Czech 2 0 0 0 2 Denmark 1 0 0 0 1 England 2 0 0 0 2 Finland 1 1 0 0 0 Germany 2 0 0 0 2 Ukraine 3 0 0 1 2 Italy 1 0 0 0 1 Portugal 1 0 0 0 1 Romania 1 0 0 1 0 Russia 2 0 0 0 2 Scotland 2 0 1 1 0 Serbia 2 0 0 1 1 Sweden 1 0 0 1 0 Wales 1 0 0 0 1 N. America (95) Canada 6 2 2 1 1 USA 84 13 11 26 34 Mexico 4 0 0 1 3 S. America (10) Argentina 4 0 3 1 0 Brazil 5 3 1 0 1 Uruguay 1 0 1 0 0 Total 33 155 23 24 44 64

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Figure 2.3. Drechslera avenae conidia on an infected leaf segment

Figure 2.4: Drechslera avenae conidia under microscope

Figure 2.5. Tan necrotic leaf spots on two-week-old seedlings of a susceptible oat genotype (top) and small tan necrotic spot on a resistant genotype (bottom) inoculated with Drechslera avenae in greenhouse

63

Discussion In this study, four leaf spot causing pathogens were recovered from the collected leaves samples during 2017 and 2018 and diverse locations in South Dakota. D. avenae and S. avenae were found to be the most prevalent pathogens of oats in South Dakota.

However, the percent recovery of the pathogens varied among the growing seasons. This variation in the pathogens’ recovery could be due to weather conditions during the seasons and sampling locations. Our results are in agreement with Hetherington and Auld, (2001) and Kastanias & Chrysayi‐Tokousbalides, (2000). They recovered Drechslera avenae from

Avena sterilis and Avena fatua (wild oat) leaves samples, respectively. D. avenae is well known to be a seed-borne pathogen and play an important role in the disease occurrence

(Turner and Millard, 1931). Though we did not attempt to analyze oats seed samples from

South Dakota, we suspect diseased seed may be one of the factors playing a role in the disease occurrence in the state, as it has been reported in other studies (Tariq et al., 2004;

Sheridan and Tan, 1973). Tariq et al., (2004) recovered Drechslera avenae from 28 seed samples of oat cultivars collected from various locations in Pakistan. Similarly, Sheridan and Tan (1973) recovered from large numbers of seed samples from New Zealand and

British. In this study, we also recovered Stagonospora avenae, Colletotrichum graminicola, and Bipolaris victoriae from oat leaves. These pathogens have been reported to cause leaf blotch, anthracnose, and leaf spot in oats (Politis, 1976; Luttrell, 1955; Ueng,

1998). The recovery of these pathogens from the region warrants screening oats germplasm against these pathogens and monitor oats periodically for these pathogens to avoid any potential disease epidemic.

In this study, all four kinds of reactions that include susceptible, moderately susceptible, moderately resistant, and resistant, were observed in the evaluated oat 64 genotypes to Drechslera avenae leaf spot. Further, differential reactions of the evaluated oats genotypes exist across the continents, not only to one region or the country. Similar results were reported by (Mehta 2001). In his study that conducted on a small number of oat lines, showed differential response to 18 oat genotypes for their reaction to D. avenae leaf spots and observed differential response to the disease. However, he did not find any genotypes with complete resistance to the disease, and that may be a few numbers of genotypes tested. In another independent study, 63 oats genotypes (from Austria, the Czech

Republic, Finland, Germany, Italy, Poland, Russia, and Sweden) were screened against D. avenae leaf spot (Šebesta et al., 1995). They reported that all 63 oat accessions varied in their resistance level to the disease. They further reported variation in the pathogen virulence and could impact the outcome of their study. We used only one isolate for screening the germplasm. We did not find any virulence variation based on symptom development in the fungal isolates collected from South Dakota when they were tested on some oat cultivars grown in the state. Cegiełko et al., (2011) also reported variation in oats cultivars level of resistance to D. avenae leaf spot under both greenhouse and field condition. They performed a study on 12 selected oat cultivars and breeding lines under field and laboratory conditions. They concluded that there is variability in susceptibility of the oat cultivars and breeding lines to leaf spot in field and laboratory (Cegiełko et al.,

2011). The results of this study indicate that most of the oats commercial cultivars grown in South Dakota are susceptible to the D. avenae that may increase the chances of disease epidemics if conducive environment prevails for the disease development. It can be concluded from our results that there are variable levels of resistance to D. avenae exists in the evaluated germplasm, and the genotypes with resistance can be exploited as sources 65 resistance in the breeding for D. avenae resistant cultivars. Further, the evaluated germplasm should be screened against S. avenae blotch to find some genotypes resistance to both diseases.

66

Literature cited Ahuja J, Haytowitz DB, Pehrsson PR, Roseland JM, Wu X, et al., (2018) “USDA national

nutrient database for standard reference” US Department of Agriculture,

Agricultural Research Service, Nutrient Data Laboratory.

Blum MMC (1997) “Pyrenophora avenae: ocorrência, inóculo, patogenicidade e

sobrevivência (Pyrenophora avenae: occurrence, inoculum, pathogenicity and

survival)” Masters Dissertation in Phytopathology, Federal University of Rio

Grande do Sul: Porto Alegre.

Bocchese CAC, Martinelli JA, Federizzi LC and Rosa CRE (2006) “Processo de infecção

e formação de mancha em grãos de aveia branca com níveis diferenciados de

resistência para Pyrenophora chaetomioides (Infection process and spot

development on kernels of white oats with differentiated levels of resistance against

Pyrenophora chaetomioides)” Fitopatologia Brasileira (Brazilian

Phytopathology) 31, 284–290.

Clark W, Bryson R, Tonguç L, Kelly C and Jellis G (2008) The encyclopedia of cereal

diseases, HGCA: Kenilworth.

Crouch, J. A., & Beirn, L. A. (2009). Anthracnose of cereals and grasses. Fungal

Diversity, 39, 19 Crouch and Beirn

Cunfer, B. M. (2000). Stagonospora and Septoria diseases of barley, oat, and rye. Canadian

journal of plant pathology, 22(4), 332-348.

Ellis MB (1971) “Dematiaceus Hyphomycetes”. Commonwealth Mycological Institute

Kew, Surrey England: 608 p.

Gough FJ and McDaniel ME (1974) “Occurrence of oat leaf blotch in Texas in 1973”

Plant Disease Reporter, 58, 80–81. 67

Hetherington, S. D., & Auld, B. A. (2001). Host range of Drechslera avenacea, a fungus

with potential for use as a biological control agent of Avena fatua. Australasian

Plant Pathology, 30(3), 205-210.

Hoffman LA (1995) “World production and use of oats” In: Welch RW (eds) The Oat

Crop, World Crop Series, Springer: Dordrecht.

Kastanias, M. A., & Chrysayi‐Tokousbalides, M. (2000). Herbicidal potential of

pyrenophorol isolated from a Drechslera avenae pathotype. Pest Management

Science: formerly Pesticide Science, 56(3), 227-232.

Kunovski J and Breshkov T (1981) “Field resistance of oat cultivars from a world

collection to stem rust (Puccinia graminis Pers. f. sp. avenae Eriks et Henn.) and

helminthosporiosis (Pyrenophora avenae Ito)” Plant Sci. 18, 3, 118–123.

Lamari L and Bernier CC (1989) “Virulence of isolates of Pyrenophora tritici-repentis on

11 wheat cultivars and cytology of the differential host reactions” Can J Plant

Pathol. 11,

Luttrell, E. S. (1955). A taxonomic revision of Helminthosporium sativum and related

species. American Journal of Botany, 42(1), 57-68. 284-290.

Manamgoda, D. S., Rossman, A. Y., Castlebury, L. A., Crous, P. W., Madrid, H.,

Chukeatirote, E., & Hyde, K. D. (2014). The genus bipolaris. Studies in

mycology, 79, 221-288.

Mehta, Y. R. (2001). Molecular and pathogenic variability of Drechslera isolates from oats.

Fitopatologia Brasileira, 26(3), 590-596.

Politis, D. J. (1976). Ultrastructure of penetration by Colletotrichum graminicola of highly

resistant oat leaves. Physiological Plant Pathology, 8(2), 117-122. 68

Quaedvlieg, W., Verkley, G. J. M., Shin, H. D., Barreto, R. W., Alfenas, A. C., Swart, W.

J., ... & Crous, P. W. (2013). Sizing up septoria. Studies in mycology, 75, 307-

390.Dennis WG (1933) “Studies on the morphology and biology of

Helminthosporium avenae” Trans Br Mycol Soc. 18, 223–238.

Rosentrater KA and Evers AD (2018) “Introduction to cereals and pseudocereals and their

production” In: Kent's technology of cereals (5th ed.) Elsevier: Cambridge.

Šebesta, J., Zwatz, B., & Corazza, L. (1995). Incidence of pyrenophora avenae ito et kurib.

In Europe and the varietal reaction of oat to it. Archives of Phytopathology & Plant

Protection, 29(6), 485-490.

Sheridan, J. E., & Tan, P. E. T. (1973). Incidence and survival of Pyrenophora avenae in

New Zealand seed oats. New Zealand Journal of Agricultural Research, 16(2), 251-

253.

Simmons MD (1985) “Diseases of oats” In: Lawes DA and Thomas H (eds) World crops:

production, utilization, description, Proceedings of the Second International Oats

Conference, Kluwer Academic Publishers: Lancaster.

Tariq, A. H., Ahmad, S., Hussain, M. A., Akhtar, L. H., Arshad, M., & Siddiqui, S. Z.

(2004). Seed-borne mycoflora of oats in the Punjab. Pakistan Journal of Scientific

and Industrial Research, 47(1), 46-49.

Turner DM and Millard WA (1931) “Leaf-spot of oats, Helminthosporium avenae (Bri.

& Cav,) Eid” Ann Appl Biol. 18, 535–559.

Ueng, P. P., Subramaniam, K., Chen, W., Arseniuk, E., Wang, L., Cheung, A. M., ... &

Bergstrom, G. C. (1998). Intraspecific genetic variation of Stagonospora avenae

and its differentiation from S. nodorum. Mycological Research, 102(5), 607-614. 69

United States Department of Agriculture (2018) “Small grains 2018 summary” National

Agricultural Statistics Service 1949-162X Retrieved Feb 9, 2019, from

https://.usda.library.cornell.edu/usdaesmis/files/5t34sj573/gq67jv49w/1j92gc06p/

smgr0918.pdf

United States Department of Agriculture (2019) “World Agricultural Production”

Circular Series WAP 3-19 Retrieved Feb 9, 2019, from

https://usda.library.cornell.edu/concern/publications/5q47rn72z?locale=en

Whitehead, A., Beck, E. J., Tosh, S., & Wolever, T. M. (2014). Cholesterol-lowering

effects of oat β-glucan: a meta-analysis of randomized controlled trials. The

American journal of clinical nutrition, 100(6), 1413-1421.

Zhou X, Jellen EN and Murphy JP (1999) "Progenitor germplasm of domesticated

hexaploid oat" Crop Science 39, 4, 1208–1214. 70

CHAPTER 3

Characterization of a global collection of triticale genotypes to Pyrenophora tritici- repentis (tan spot) race 1, race 5, and Ptr ToxA

ABSTRACT Triticale was developed through hybridization between wheat and rye. It is used as a forage crop and a rotational crop in improving soil health. Tricale is used in wheat breeding to bring good agronomic traits like yield, winter hardiness and pest, and disease resistance traits to improve wheat quality. Triticale is susceptible to some foliar diseases including the tan spot that impact wheat. It can serve as a source of tan spot causing pathogen,

Pyrenophora tritici-repentis, inoculum/virulence variation when cultivated in the vicinity of wheat. So far, information on the reaction of triticale to commonly prevalent P. tritici- repentis races1 and 5 is unknown. In this study, we evaluated 366 triticale genotypes representing 21 countries of all six continents for their reaction to tan spot (P. tritici- repentis race 1 and race 5) and Ptr ToxA. Our results indicate that triticale genotypes differ in response to tan spot caused by P. tritici-repentis race 1 and race 5. One-hundred-seventy

(53%) of the evaluated genotypes were found either resistant or moderately resistant to race

1. Sixty-eight (18.55%) genotypes showed sensitivity to Ptr ToxA. Also, no correlation between the genotype susceptibility to P. tritici-repentis and Ptr ToxA sensitivity or resistance and Ptr ToxA insensitivity was observed in our study. The majority of the evaluated genotypes (64.75%) developed a resistant – moderately resistant reaction against race 5. The results of this study indicate that most of triticale germplasm/genotypes exhibit resistance to both race 1 and race 5. Also, Ptr ToxA was not necessary for disease development in every genotype as was observed in wheat and rye in previous studies. 71

Based on our results, it is necessary to evaluate triticale lines/varieties against P. tritici- repentis before their release for forage to minimize the risk of an additional source of inoculum for tan spot development in wheat if planted in close vicinity. Further, tan spot resistant triticale genotypes can be utilized as a source of resistance genes for wheat.

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Introduction Triticale was developed by the hybridization of wheat being the female parent with diploid rye (Wilson, 1875). This plant has been regarded as the minor crop which is used for forage, but it has biomass yield and better performance against abiotic and biotic stresses which is causing an increase in its popularity and making it a major crop (Mergoum et al.,2009; Ayalew, et al., 2018). It has large canopy cover, which helps it to utilize more sunlight and increase photosynthesis, even in the harsh conditions of the environment

(Long et al., 2013, Ayalew, et al., 2018). The production of triticale is averaging 17 million metric tons annually, and the area which is used for this production is 4 million hectares over the world. The production and area for triticale were increased to 17% in 2014 from

8% in the previous years. (Ayalew, et al., 2018). The greatest production was recorded in

Poland, following by Belarus, France, Germany, and Russia. (Ayalew, et al., 2018).

In wheat breeding programs, triticale has been used widely as a bridge which transfers some useful genes of many traits from rye to wheat. The rye genome has several resistant genes which help this crop to withstand biotic and abiotic stress as a result of which quality of wheat has improved (Saulescu, et al., 2011; Jessop, 1996; Blum, 2014;

Ayalew, et al., 2018). Triticale is known to be resistant to a number of diseases, which includes rust, smut, bunt, wheat streak mosaic, brome mosaic, barley stripe mosaic and barley yellow mosaic, cereal cyst and Hessian fly, and this resistance may be inherited from rye (Mergoum, et al., 2004; Saulescu, et al., 2011). Nonetheless, the increase in its production has enhanced the risk of certain diseases, and the diseases which are previously affecting rye and wheat are now impacting triticale (Zillinsky, 1985; Singh and Saari, 1991;

Mergoum, 1994). Pyrenophora tritici repentis, (Ptr) is a pathogen which causes the disease 73

“tan spot” in wheat, can also infect triticale (Krupinsky, 1992; Wakulinski et al.,2005,

GRDC, 2018).

Tan spot is an important disease of wheat with serious consequences to global food security. It can cause a yield reduction of up to 50% (Sharp, et al., 1976; Shabeer and

Bockus, 1988) and sometimes, even as far as 70% (Moreno and Perelló, 2010) depending on the level of the cultivar susceptibility. In South Dakota, it is the most common leaf spot disease (Byamukama, 2013). The fungus produces oval-shaped tan necrotic spots; and these spots have a chlorotic halo with a small dark brown spot in the center (DeWolf et al.

1998). Three host-selective effectors (toxins) Ptr ToxA, Ptr ToxB, and Ptr ToxC are produced by the fungus, which are associated in the development of necrosis and chlorosis symptoms depending on the wheat genotype (Lamari et al. 2003; Martinez, et al., 2001;

Effertz, et al., 2002). Eight races have been identified in P. tritici-repentis population based on the isolate’s ability to produce necrosis and/or chlorosis reactions on appropriate wheat differential genotype (Lamari and Bernier, 1989; Ali and Francl 2003). In North America, the most important races are 1 and 5 (Ali and Francl 2003; Abdullah et al. 2017b). Race 1 produces Ptr ToxA and Ptr ToxC is the predominant race across the world (Lamari et al.

2003; Ali and Francl, 2003). Race 5, produces Ptr ToxB, is reported from Africa, central

Asia, USA and Canada (Lamari et al. 2003; Ali and Francl 2003; Abdullah et al. 2017a;

Strelkov, et al., 2002). Triticale can be a good source of resistance of tan spot inherited from the rye. Additionally, due to the increase in triticale production and the lack of information available on its reaction to tan spot, it essential to evaluate triticale germplasm against most prevalent races 1 and 5 of P. tritici-repentis. The objectives of this study were 74

1) evaluate a worldwide collection of triticale genotypes for their reaction to P. tritici- repentis races 1 and 5, and 2) test their sensitivity to Ptr ToxA.

Materials and methods Plant material and seedlings production The seeds of 366 triticale genotypes belonging to 21 countries located in Africa,

Asia, Australia, Europe, North America, and South America continents were obtained from

USDA Small Grain Germplasm Bank, Aberdeen, ID (Figure 3.1). Two weeks old seedlings of all 366 triticale genotypes were raised in 5 x 23 cm plastic containers (Stuewe & Sons,

Inc. 31933 Rolland Drive, Tangent, Oregon 97389) by placing three seeds/cone. In total

18 seedlings of each genotype were maintained until tested for their reaction to tan spot.

The seedlings were watered and fertilized as needed until the experiment was terminated.

The experiments were conducted in the greenhouse (22oC and 18oC day and night temperature, respectively with 16 hours’ photoperiod) at the Young Brothers Seed

Technology Building, South Dakota State University, Brookings, SD. All four tan spot differentials genotype Glenlea, 6B365, 68662, and Salamouni were also included in the experiment as checks. Three seedlings/cone were considered as an experimental unit, and each cone served as one replication. The seedlings of all 366 genotypes were grouped into two sets before testing them against (tan spot) P. tritici-repentis race 1 and race 5. 75

Figure 3.1. Origin of 366 triticale genotypes evaluated in this study

Inoculum preparation, plants inoculation, and disease rating The race 1 and 5 isolates frozen plugs were taken from the freezer, placed in petri dishes (one plug/plate) contained V8-potato dextrose agar medium and inoculated for four days or until the growth of colony reaches to about 4 cm. Thereafter, distilled sterile water is poured into Petri dishes to knock down the mycelia with a flame sterilized glass test tube.

The excess water was removed from the dishes. To induce conidia, the plates were incubated in an alternate cycle of 24 light at room temperature (~22-23 oC and 24 hours dark at 16 oC). The conidia were dislodged by adding about 30 ml of distilled water/plate with the help of a looped wire inoculating needle. The suspension of conidia was adjusted to 3000 spores per ml by following the method of Jordhal and Francl 1992. A drop of tween

20 per 100 ml of the conidial suspension was added before inoculation. 76

Nine seedlings of each genotype at 2-leaf stage were inoculated with P. tritici- repentis race 1 and race 5 isolate using its spore suspension (3000 spores/ml) with a hand- held sprayer (Power Sprayer, Prevail, Chicago Aerosol, 1300 E. North Street, Coal City,

IL60416). Then the seedlings from the genotypes and 4 from differential genotypes were placed in 100% humidity in a humidity chamber for 24 hours, and this was done to increase the fungal infection chances. Then for symptom development, these seedlings were placed to a greenhouse bench. These seedlings were rated based on 1-5 disease rating scale where

1-2 = resistant to moderately resistant; 3-5 =moderately susceptible to susceptible (Lamari and Bernier, 1989). The wheat differential lines 6B365 (susceptible to race 1) and Glenlea

(susceptible to race 1), 6B662 (susceptible to race 5), and Salamouni (resistant to race 1 and race 5) were included in the experiment as checks.

Date analysis The greenhouse experiment was analyzed utilizing completely randomize design. Analysis of variance (ANOVA) was conducted to find out the descriptive statistics on the genotype’s reactions. Then, Fisher’s Least Significant Difference (LSD) was used to compare the differences among the genotypes’ reactions.

Results Reaction of triticale genotypes to tan spot (race 1) and Ptr ToxA All 366 triticale genotypes that were screened for their reaction to P. tritici-repentis race 1 varied significantly in their response ranging from moderately resistant (MR) to resistant (R) and moderately susceptible (MS) to susceptible (S) (Table 3.1). Of all the genotypes screened, 11.4 % (n=42) showed a moderately resistant reaction as they developed lesion type 2; whereas, 28.4% (104) of the genotypes exhibited a moderately susceptible reaction with lesion type 3 (Table 3.2 Fig 3.2). The resistant reaction was 77 observed in 153 (41.8%) lines while 67 (18.3%) genotypes exhibited susceptibility with lesion type 4-5. Overall, the ratio of resistant to susceptible accessions were 1 (195):1

(171). The ratio of resistant (R+MR) to susceptible (S+MS) accessions from South Africa to race 1 was estimated 4 (30):1 (7), the only African country from the triticale genotypes were tested in this study. Four and five of the 37 genotypes exhibited MR and MS reactions, respectively. Thirty-three genotypes from Asia, 30.3% (10) were resistant; 15 (45.5%) moderately resistant; 15 (45.45) moderately susceptible; and only three (9.1%) germplasms were susceptible. The three genotypes from Australia, one was resistant and two were susceptibility. Of the 157 genotypes screened from Europe, 86 (23.5%) were resistant and

71 (19.4%) were susceptible; whereas, within the resistant genotypes (R=71 and MR=15) and susceptible (S=28 and MS= 43). In general, more genotypes (57%) from North

America were grouped as susceptible as compared to grouped as resistant (43%). However, the genotypes exhibited all four types, R (30%), MR (12.5%), MS (31.5), and S (25.2) reactions. The majority (89%) of genotypes evaluated from South America developed a resistant reaction. Only one of the 9 genotypes tested shown susceptibility. The highest number of resistant genotypes (n=35) were from Russia followed by South Africa (26),

Canada (14), Mexico and Polnad (13), and the USA (12). Of the 366, only 18.55% (n= 68) showed sensitivity to Ptr ToxA as they developed necrosis in the toxin infiltrated leaves sites (Table 3.1and 3.2 and Fig 3.1, Fig 3.2, Fig 3.3). Further, the 171 genotypes showed susceptible-moderately susceptible reaction to race 1, 11.7% exhibited sensitivity (n= 43) and 35% insensitivity (128) to Ptr ToxA. Similar trend was observed in resistant genotypes

(sensitive = 25 and insensitive = 170) (Table 3.2 and 3.3 and Fig 3.1, Fig 3.2, Fig 3.3). The differential lines Glenlea and 6B365 exhibited necrotic and chlorotic symptoms as 78 expected to race 1, respectively; while 6B662 and Salamouni remained symptomless.

Glenlea (Ptr ToxA sensitive) developed necrosis to Ptr ToxA and Salamouni (Ptr ToxA insensitive) did not develop any symptom thus verified the successful inoculations; race 1 reaction; and Ptr ToxA viability.

Table 3.1: ANOVA of tan spot (race 1) Df Sum Sq Mean Sq F value Pr (> F) Genotypes 365 1144.2 3.1349 26.24 2e-16 Residuals 366 43.7 0.1195

Table 3.2: Reaction of 366 triticale genotypes from six continents to Pyrenophora tritici- repentis race 1 and Ptr ToxA

Continent Country Genotypes R MR MS S Ptr ToxA Africa 1 37 26 4 5 2 4 Asia 5 33 10 5 15 3 5 Australia 1 3 1 0 0 2 0 Europe 8 157 71 15 43 28 37 N. America 3 127 39 16 40 32 20 S. America 3 9 6 2 1 0 2 Total 21 366 153 42 104 67 68 (P ≤ 0.05)

Table 3.3: Reaction of 366 triticale genotypes from 21 countries to Ptr race 1 and Ptr ToxA Continent Country Genotype R MR MS S Ptr ToxA (n= genotypes) Africa (37) South Africa 37 26 4 5 2 4 Asia ( 33) China 4 2 0 2 0 0 India 16 6 2 6 2 2 Japan 10 2 1 7 0 3 Azerbaijan 2 0 1 0 1 0 Pakistan 1 0 1 0 0 0 Australia (3) Australia 3 1 0 0 4 0 79

Table 3.3: continued from previous page Europe (157) Russia 55 35 3 6 11 12 Ukraine 16 8 5 2 1 2 Poland 31 13 0 11 7 9 Spain 12 5 2 5 0 2 Hungary 13 2 1 6 4 6 Germany 4 1 1 2 0 0 France 6 3 1 2 0 0 Sweden 20 4 2 9 5 6 N. America Canada 32 14 2 9 7 5 (127) Mexico 40 13 7 8 12 8 USA 55 12 7 23 13 7 S. America Argentina 1 1 0 0 0 1 (9) Brazil 7 5 2 0 0 0 Ecuador 1 0 0 1 0 1 Total 366 153 42 104 69 68

Figure 3.2. Reaction of genotypes to Pyrenophora tritici-repentis race 1 and Ptr ToxA. R = resistant; MR= moderately resistant; MS= moderately susceptible S = susceptible; I= insensitive to Ptr ToxA; Sen = sensitive to Ptr ToxA.

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Figure 3.3: Two triticale genotypes inoculated with race 1 (susceptible-top leaf and resistant-bottom leaf).

Figure 3.4. Two triticale genotypes infiltrated with Ptr ToxA (sensitive-top leaf and insensitive bottom leaf).

Reaction of triticale genotypes to tan spot (race 5) Three hundred sixty-six triticale genotypes were evaluated for their reaction to Ptr race 5 and observed a range of reactions from moderately resistant to resistant and moderately susceptible to susceptible among the genotypes. Overall, the majority of the genotypes 47.2% (n=173) exhibited a resistant reaction as they developed lesion type 1 symptom. Only 9% (n=33) of the genotypes developed chlorosis with lesion type 4-5 and were grouped as susceptible. The moderately resistant reaction was observed in 17.5%

(n=64) and moderately susceptible in 26.2% (n=96) of the evaluated genotypes. Both tan spot susceptible and resistant triticale were observed in four continents (Africa, Asia,

Europe, North America). All genotypes except one from Australia and South America were grouped as resistant. Nonetheless, the percentage of susceptible to resistant genotypes was different from continent to continent. In Africa 46.9 % (n= 17) of the genotypes were found resistant while only 5.4% (n= 2) were susceptible. Whereas, the genotypes from Asia,

42.4% (n=14) was developed resistance reaction; whereas, 9% (n=3) lines were susceptible. Both moderately susceptible and moderately resistant lines were from Africa 81

were 29.7% and 18.9%, respectively. Also, 21.2% (n=7) of the genotypes grouped as moderately resistant, and 27.3% (n=9) were moderately susceptible from Asia. A similar trend was observed in genotypes from Europe where more number (74) of genotypes were resistant as compared to susceptible (14) genotypes. While 15.2% (n=24) showed a moderately resistant reaction and 28.7% (n=45) were moderately susceptible. One hundred-twenty-seven genotypes form North America 47.2% (n=60) were grouped as resistant a reaction and 11% (n=11) were susceptible. Moderately susceptible and moderately resistant responses were observed in 22.8% and 18.9% respectively. Majority of the genotypes from Australia (67%) and South America (67%) were found resistant to race 5. The similar trend was observed within the countries where the majority of the genotypes were either resistant or moderately resistant to race 5. The differential line

6B662 (race 5 susceptible) developed chlorosis and the rest three differential lines exhibited resistance as expected and validated the successful inoculation process and the race validity.

Table 3.4: ANOVA of tan spot (race 5) Df Sum Sq Mean Sq F value Pr (> F) Genotypes 365 870.4 2.3913 17.96 2e-16 Residuals 366 48.9 0.1331

Table 3.5: Reaction of 366 triticale genotypes from six continents to Ptr race 5 Continent Countries Genotypes R MR MS S Africa 1 37 17 7 11 2 Asia 5 33 14 7 9 3 Australia 1 3 2 0 1 0 Europe 8 157 74 24 45 14 N. America 3 127 60 24 29 14 S. America 3 9 6 2 1 0 Total 21 366 173 64 96 33 (P ≤ 0.05) 82

Table 3.6: Reaction of oats genotypes to Ptr race 5 from 21 countries. Continent Country Genotype R MR MS S (number of genotypes) Africa (37) South Africa 37 17 7 11 2 Asia (4) China 4 4 0 0 0 India 16 7 2 4 3 Japan 10 2 4 4 0 Azerbaijan 2 1 1 0 0 Pakistan 1 0 0 1 0 Australia (3) Australia 3 2 0 1 0 Europe (157) Russia 55 28 9 15 3 Ukraine 16 8 5 1 2 Poland 31 13 4 12 2 Spain 12 6 1 4 1 Hungary 13 3 3 7 0 Germany 4 3 0 0 1 France 6 4 0 1 1 Sweden 20 9 2 5 4 N. America Canada 32 14 5 9 4 (127) Mexico 40 20 4 8 8 USA 55 26 15 12 2 S. America Argentina 1 1 0 0 0 (9) Brazil 7 4 2 1 0 Ecuador 1 1 0 0 0 Total 366 173 64 96 33

Figure 3.5: Two triticale race 5 resistant genotypes exhibiting resistant lesion type 1 (black spots without chlorosis) reaction (top two leaves) and Two susceptible triticale genotypes inoculated with race 5 exhibiting chlorosis symptoms (bottom two leaves).

83

Discussion Resistance and susceptible responses in the evaluated 366 genotypes to P. tritici- repentis (tan spot) were observed. The outcomes of this study suggested that there is variability for resistance to tan spot (race 1 and 5). A study conducted by Wakulinski et al., (2005) on 327 wheat and 352 triticale genotypes demonstrated that wheat and triticale could be heavily infected by P. tritici-repentis. However, the pathogen was more aggressive on wheat and induced symptoms quicker than triticale. However, the researcher did not mention the race of P. tritici-repentis was used in his study (Wakulinski et al.,

2005). Abdullah et al. (2017a) studied the reaction of the global collection of rye to P. tritici-repentis (race1). Most of the 211 genotypes showed a resistant reaction, and we also observed a similar trend in triticale. This similarity may be the resistance trait came from rye into triticale. Our results also suggest that variability of resistant level against P. tritici- repentis race1 exists. In another study in which both hard red spring wheat (n=33) and hard red winter wheat (12) genotypes were characterized for their reaction to tan spot. They observed variation in the resistance level among the evaluated genotypes to both race 1 and race 5. (Abdullah et al. 2017b). These results further indicate that resistance to tan spot races may be shared by rye, triticale, and wheat. It is a fact that triticale is between rye and wheat (Wilson, 1875), and the outcomes of the previous studies that illustrated above shared the similar fact that the reaction of rye, wheat, and triticale to race 1 were variable from susceptible to resistant.

In this study, Ptr ToxA role in disease tan spot (necrosis) development in triticale was investigated by infiltrating Ptr ToxA into all 366 genotypes. The majority (79%) of the evaluated genotypes showed insensitivity to Ptr ToxA. Only 19% of the accessions tested developed necrosis and were grouped as sensitive to Ptr ToxA. Our toxin results showed 84 that the toxin sensitivity or insensitivity in the genotypes is genotype specific, may not be necessarily playing role in the pathogenicity or aggressiveness in all tan spot susceptible genotypes (Ali et al. 2010). Similar findings were reported by (Abdullah et al. 2017a;

Abdullah et al. 2017c) who observed four different possible combinations (susceptibility- sensitivity, susceptibility-insensitivity; resistant-sensitivity, and resistant insensitivity) of

Ptr ToxA and tan spot susceptibility in wheat and rye. Abdullah et al. (2017a) and Ali et al. (2010) characterized a large number of P. tritici-repentis isolates from different countries or the US states for their race structure. They found some isolates behaved like race 1 but lack in Ptr ToxA. Their results further support our findings that the toxin does not act as the sole factor in tan spot development.

All the 366 genotypes inoculated with P. tritici-repentis race 5 displayed different type of disease reaction. Race 5 was firstly observed in Algeria (Lamari et al., 1995), and it was tested in various plant species including triticale for its host range. The triticale genotype used in their study showed susceptibility to race 5. In 1998, Race 5 was reported in North America (North Dakota, USA (Ali et al. 1999) and later it was reported from

South Dakota in 2016 (Abdullah et al. 2016). On global collocation of rye genotypes, P. tritici-repentis race 5 showed both of susceptible and resistant responses. The resistant cultivars percentage was higher than the susceptible percentage which is matched with our results (Abdullah et al. 2017a). The differential wheat line 6B662 showed susceptibly and while Glenlea, 6B365, and Salamouni exhibited resistance to race 5. Further, the susceptibility and resistance exhibited in different wheat cultivars (Singh, et al., 2008).

They screened 17 genotypes against race 5. The majority of their accessions showed a resistant reaction, but the susceptible reaction was observed in a few genotypes too. Thus, 85 diverse chlorotic lesions were observed in rye, wheat, and triticale. Finally, tan spot resistant triticale genotypes found in our study can be used in the development of tan spot resistant cultivars in wheat breeding programs in the northern Great Plains and wherever the tan spot occurs in wheat and triticale. To our knowledge, this is the first study where a large (366) number of triticale genotypes were screened against tan spot using the pathogen race 1, and 5.

86

Literature cited Abdullah S, Sehgal SK, Jin Y, Turnipseed B, Ali S (2017c) “Insights into tan spot and stem

rust resistance and susceptibility by studying the pre-green revolution global

collection of wheat”. Plant Pathol J 33: 125-132.

Abdullah S, Ali S, Byamukama E and Langham MAC (2016). “First report of

Pyrenophora tritici-repentis race 5 from South Dakota”. Plant Disease, 100(11),

2322.

Abdullah S, Sehgal SK and Ali S (2017b). “Race diversity of Pyrenophora tritici-repentis

in South Dakota and response of predominant wheat cultivars to tan spot”. J. Plant

Pathol. Microbiol, 8, 409.

Abdullah S, Sehgal SK, Glover KD and Ali S (2017a). Reaction of Global Collection of

Rye (Secale cereale L.) to Tan Spot and Pyrenophora tritici-repentis Races in South

Dakota. The plant pathology journal, 33(3), 229.

Ali S and Francl LJ (2003) “Population race structure of Pyrenophora tritici-repentis

prevalent on wheat and non-cereal grasses” Plant Dis. 87, 418-422.

Ali S, Gurung S and Adhikari TB 2010. Identification and characterization of novel

isolates of Pyrenophora tritici-repentis from Arkansas. Plant Dis. 94:229-

235Blum A (2014) “The abiotic stress response and adaptation of triticale—a

review” Cereal Res Commun. 42, 359–375.

Ayalew H, Kumssa TT, Butler TJ and Ma X-F (2018) “Triticale improvement for forage

and cover crop uses in the southern great plains of the United States” Front Plant

Sci. 9, 1130.

Byamukama E 2013. Leaf spot diseases beginning to develop in winter wheat. igrow.

SDSU Extension 6/6/2013 Retrieved 11/13/2018 87

http://igrow.org/agronomy/wheat/leafspot-diseases-beginning-to-develop-in-

winter-wheat/

De Wolf ED, Effertz RJ, Ali S and Francl LJ (1998) “Vistas of tan spot research” Can J

Plant Pathol. 20, 349-370.

Effertz RJ, Meinhardt SW, Anderson JA, Jordahl JG and Francl LJ (2002) “Identification

of a chlorosis-inducing toxin from Pyrenophora tritici-repentis and the

chromosomal location of an insensitivity locus in wheat” Phytopathology 92, 527–

533.

Grains Research and Development Corporation (2018). Triticale Section 9 Diseases.

GRDC Grownotes Retrieved 3/21/2019

https://grdc.com.au/__data/assets/pdf_file/0029/370649/GrowNote-Triticale-

South-09-Diseases.pdf

Jessop RS (1996). “Stress tolerance in newer triticales compared to other cereals”. In

Triticale: today and tomorrow (pp. 419-427). Springer, Dordrecht.

Krupinsky JM (1992) “Grass hosts of Pyrenophora tritici repentis” Plant Dis. 76, 92-95

Lamari L and Bernier CC (1989) “Virulence of isolates of Pyrenophora tritici-repentis on

11 wheat cultivars and cytology of the differential host reactions” Can J Plant

Pathol. 11, 284-290.

Lamari L, Strelkov SE, Yahyaoui A, Orabi J and Smith RB (2003) “The identification of

two new races of Pyrenophora tritici-repentis from the host center of diversity

confirms a one-to-one relationship in tan spot of wheat” Phytopathol. 93, 391-396.

Long E, Van Slyke K, Ketterings QM, Goodwin G and Czymmek K (2013). “Triticale as

a cover and double crop on a New York dairy”. What’s Cropping Up, 23(1), 3-5. 88

Martinez JP, Ottum SA, Ali S, Francl LJ and Ciuffetti LM (2001) “Characterization of the

ToxB gene from Pyrenophora tritici-repentis” Mol Plant-Microbe Interact. 14,

675-677.

Mergoum M (1994) “Performance and adaptation of triticale to Morrocan environments”

In: Proceedings of the 3rd International Triticale Symposium, International Triticale

Association: Lisbon.

Mergoum M, Pfeiffer WH, Peña RJ, Ammar K and Rajaram S (2004) “Triticale crop

improvement: the CIMMYT programme” In: Mergoum M (ed.) Triticale

improvement and production, FAO: Rome.

Mergoum M, Singh, PK, Pena, RJ, Lozano-del Río AJ, Cooper KV, Salmon DF, and

Macpherson, HG (2009) “Triticale: a “new” crop with old challenges”. In Cereals

(pp. 267-287). Springer, New York, NY.

Moreno MV and Perelló AE (2010) “Chapter 21: Occurrence of Pyrenophora tritici-

repentis causing tan spot in Argentina” Management of Fungal Plant Pathogens,

CAB International: Wallingford.

Noriel AJ, Xiaochum S, Willimam B, Guihua B (2011). “Resistance to tan spot and

insensitivity to Ptr ToxA in wheat”. Crop Sci 51: 1059-1067

Orolaza NP, Lamari L, ane Balance G M (1995). “Evidence of a host-specific chlorosis

toxin from Pyrenophora tritici-repentis, the causal agent of tan spot of wheat”.

Phytopathology, 85(10), 1282-1287.

Saulescu NN, Ittu G, Ciuca M, Ittu M, et al., (2011) “Transferring useful rye genes to

wheat, using triticale as a bridge” Czech J Genet Plant Breed. 47, S56–S62. 89

Shabeer A and Bockus WA (1988) “Tan spot effects on yield and yield components relative

to growth stage in winter wheat” Plant Dis. 72, 599-602.

Sharp EL, Sally BK and McNeal FH (1976) “Effect of Pyrenophora wheat leaf blight on

the thousand-kernel weight of 30 spring wheat cultivars” Plant Dis. 60, 135-138.

Singh RP and Saari EE (1991) “Biotic stresses in triticale” In: Proceedings of the 2nd

International Triticale Symposium, CIMMYT: Passo Fundo.

Singh PK, Mergoum M, Ali S, Adhikari TB and Hughes G R (2008). Genetic analysis of

resistance to Pyrenophora tritici-repentis races 1 and 5 in tetraploid and hexaploid

wheat. Phytopathology, 98(6), 702-708.

Strelkov SE, Lamari L, Sayoud R and Smith RB (2002) “Comparative virulence of

chlorosis-inducing races of Pyrenophora tritici-repentis” Can J Plant Path. 24, 29-

35.

Wakulinski W, Zamorski C, Schollenberger M, Nowicki B and Kachlicki P (2005). “Tan

spot epiphytosis in winter wheat and triticale crop-comparative studies”. Acta

Agrobotanica, 58(1)

Wilson AS (1875) “Wheat and rye hybrids” Trans Proc Bot Soc Edinb. 12, 286–288.

Zillinsky FJ (1985). “Triticale—An Update on Yield, Adaptation, and World Production

1”. Triticale, (triticale), 1-7.

90

General Conclusion

The present study provides information on the prevalence of leaf spot pathogens of oats in South Dakota, the reaction of a worldwide collection of oat germplasm and commercial cultivars grown in South Dakota to Drechslera avenae leaf spot. Additionally, a global collection of 366 triticale genotypes were screened for their reaction to tan spot, caused by the fungus Pyrenophora tritici-repentis, race 1, race 5, and Ptr ToxA. A little or no information on all the three aspects was available. In previous studies, the P. tritici- rpentis population have been grouped into eight races based on the isolates availability to produce necrosis and chlorosis symptoms alone or in combination on appropriate tan spot differential lines. Race 1 and 5 are prevalent in most wheat growing countries including in the US northern Great Plains (North Dakota and South Dakota).

The results of this study reveal that Drechslera avenae, Stagonospor avenae,

Bipolaris victoriae, and Colletotrichum graminicola are the leaf spot pathogens prevalent on oats in South Dakota. However, D. avenae and S. avenae were the most common leaf spot pathogens during the both 2017 and 2018 season. Further, the global collection of oat germplasm including South Dakota commercial cultivars exhibited diverse reaction in ranging from resistant, moderately resistant, moderately susceptible, and susceptible reaction D. avenae leaf spot. All the commercial cultivars developed a susceptible reaction.

Nearly 15% of the evaluated genotypes exhibited complete resistance to D. avenae leaf spot.

Triticale is developed by the hybridization of wheat being the female parent with diploid rye. It is served as a connector between rye and wheat to bring some good traits like pest and disease resistance genes, cold hardiness, etc. to improve wheat quality. In general triticale is considered resistant to most of the pests and diseases; however, some 91 leaf spot diseases including tan spot, an important foliar disease of wheat, has been reported in triticale. In this study, the results of screening of a global collection of three hundred and sixty-six triticale genotypes against tan spot race 1 and 5 reveal that they varied in their reaction to race 1 and race 5. In general, most of the genotypes exhibited resistance reaction to race 1 and race 5. The majority (n= 298) of 366 genotypes showed insensitivity to Ptr

ToxA. Our results further suggest that there is no correlation between the susceptibility and the toxin sensitivity or resistance and the toxin insensitivity. Our results suggest monitoring oats crop on regular bases for leaf spot pathogens to avoid any disease epidemic and availability of resistant germplasm to D. avenae leaf spot and tan spot race 1 and race 5 can be utilized in the development of resistant cultivars and other disease management strategies. Moreover, oats germplasm should be screened against S. avenae leaf blotch to select resistant lines to both important oat leaf spot pathogens.

92

93

APPENDIX

Table A 2.1. Disease reaction of 155 oat genotypes to Drechslera avenae leaf spot No. Taxon Accession # Country Lesion Reaction type 1 Avena sativa Clav 732 USA 4.3 S 2 Avena sativa Clav 887 USA 3.2 MS 3 Avena sativa Clav 905 USA 4.3 S 4 Avena sativa Clav 949 USA 3.4 MS 5 Avena sativa Clav 951 USA 1.0 R 6 Avena sativa Clav 966 USA 3.2 MS 7 Avena sativa Clav 975 USA 2.9 MR 8 Avena sativa Clav 1020 USA 4.7 S 9 Avena sativa Clav 1027 USA 3.5 MS 10 Avena sativa Clav 1070 USA 4.8 S 11 Avena sativa Clav 1117 USA 5.0 S 12 Avena sativa Clav 1143 Germany 4.7 S 13 Avena sativa Clav 1160 USA 2.8 MR 14 Avena sativa Clav 2173 USA 4.8 S 15 Avena sativa Clav 2203 USA 4.5 S 16 Avena sativa Clav 2206 Denmark 4.8 S 17 Avena sativa Clav 2217 USA 3.4 MS 18 Avena sativa Clav 2218 USA 3.8 MS 19 Avena sativa Clav 2226 USA 4.5 S 20 Avena sativa Clav 2228 USA 4.6 S 21 Avena sativa Clav 2250 USA 3.0 MS 22 Avena sativa Clav 2344 USA 4.3 S 23 Avena sativa Clav 3033 Zimbabwe 3.8 MS 24 Avena sativa Clav 3065 USA 3.0 MS 25 Avena sativa Clav 3172 USA 1.8 R 26 Avena sativa Clav 3180 USA 3.9 MS 27 Avena sativa Clav 3215 USA 3.5 MS 28 Avena sativa Clav 3279 Romania 4.5 S 29 Avena sativa Clav 3332 USA 4.7 S 30 Avena sativa Clav 3343 USA 3.3 MS 31 Avena sativa Clav 3391 USA 1.4 R 32 Avena sativa Clav 3421 USA 3.5 MS 33 Avena sativa Clav 3424 USA 3.8 MS 34 Avena sativa Clav 4123 USA 3.7 MS 35 Avena sativa Clav 4135 Canada 1.9 R 36 Avena sativa Clav 4211 USA 3.2 MS 94

37 Avena sativa Clav 4213 USA 4.1 S 38 Avena sativa Clav 4215 USA 4.4 S 39 Avena sativa Clav 4223 USA 1.9 R 40 Avena sativa Clav 4258 USA 4.4 S 41 Avena sativa Clav 4263 USA 3.8 MS 42 Avena sativa Clav 4274 USA 3.5 MS 43 Avena sativa Clav 4278 USA 4.5 S 44 Avena sativa Clav 4287 USA 4.1 S 45 Avena sativa Clav 4294 USA 2.3 MR 46 Avena sativa Clav 4328 USA 2.0 MR 47 Avena sativa Clav 4371 USA 2.9 MR 48 Avena sativa Clav 4421 USA 1.3 R 49 Avena sativa Clav 4466 USA 1.6 R 50 Avena sativa Clav 4468 USA 1.3 R 51 Avena sativa Clav 4519 Argentina 3.3 MS 52 Avena sativa Clav 4542 USA 3.0 MS 53 Avena sativa Clav 4525 USA 1.8 R 54 Avena sativa Clav 4527 USA 2.7 MR 55 Avena sativa Clav 4538 Canada 1.8 R 56 Avena sativa Clav 4542 Sweden 2.3 MR 57 Avena sativa Clav 4606 USA 4.3 S 58 Avena sativa Clav 4629 USA 3.2 MS 59 Avena sativa Clav 4634 Brazil 4.7 S 60 Avena sativa Clav 4636 Brazil 4.9 S 61 Avena sativa Clav 4656 USA 4.5 S 62 Avena sativa Clav 4663 USA 3.5 MS 63 Avena sativa Clav 4683 USA 4.7 S 64 Avena sativa Clav 4684 USA 4.7 S 65 Avena sativa Clav 4687 USA 2.2 MR 66 Avena sativa Clav 4692 USA 1.3 R 67 Avena sativa Clav 4694 USA 1.6 R 68 Avena sativa Clav 4696 USA 3.2 MS 69 Avena sativa Clav 4711 USA 4.3 S 70 Avena sativa Clav 4721 USA 3.0 MS 71 Avena sativa Clav 6190 USA 4.3 S 72 Avena sativa Clav 6266 USA 3.0 MS 73 Avena sativa Clav 6270 USA 2.7 MR 74 Avena sativa Clav 6529 USA 3.3 MS 75 Avena sativa Clav 6532 USA 2.7 MR 76 Avena sativa Clav 7557 Canada 4.2 S 77 Avena sativa Clav 7569 USA 3.7 MS 95

78 Avena sativa Clav 7670 USA 2.0 MR 79 Avena sativa Clav 7671 USA 2.0 MR 80 Avena sativa Clav 7751 USA 1.0 R 81 Avena sativa Clav 7754 USA 1.5 R 82 Avena sativa Clav 7782 USA 2.5 MR 83 Avena sativa Clav 7969 USA 2.8 MR 84 Avena sativa Clav 8016 USA 2.3 MR 85 Avena sativa Clav 8067 USA 3.7 MS 86 Avena sativa Clav 8170 Canada 2.3 MR 87 Avena sativa Clav 8172 Canada 3.2 MS 88 Avena sativa Clav 8179 Canada 3.8 MS 89 Avena sativa Clav 9006 Scotland 1.9 R 90 Avena sativa Clav 9029 New Zealand 3.7 MS 91 Avena sativa Clav 9032 Ethiopia 1.9 R 92 Avena sativa Clav 9133 Ethiopia 2.0 MR 93 Avena sativa Clav 9179 USA 3.7 MS 94 Avena sativa Clav 9181 USA 3.3 MS 95 Avena sativa Clav 9218 USA 3.2 MS 96 Avena sativa Clav 9240 USA 3.0 MS 97 Avena sativa Clav 9247 USA 2.7 MR 98 Avena sativa Clav 48224 China 1.0 R 99 Avena sativa PI 66486 Scotland 4.3 S 100 Avena sativa PI 71055 Ukraine 3.3 MS 101 Avena sativa PI 73695 China 4.2 S 102 Avena sativa PI 73739 China 4.2 S 103 Avena sativa PI 73883 China 4.3 S 104 Avena sativa PI 73891 China 4.7 S 105 Avena sativa PI 73898 China 3.3 MS 106 Avena sativa PI 74032 Russia 4.3 S 107 Avena sativa PI 76805 Czech 3.5 MS 108 Avena sativa PI 76816 Czech 4.8 S 109 Avena sativa PI 78002 England 4.6 S 110 Avena sativa PI 80239 Australia 4.5 S 111 Avena sativa PI 80722 Australia 1.9 R 112 Avena sativa PI 81730 Japan 4.3 S 113 Avena sativa PI 110258 Italy 5.0 S 114 Avena sativa PI 110836 Ukraine 4.2 S 115 Avena sativa PI 125173 Japan 4.8 S 116 Avena sativa PI 132780 South Africa 4.2 S 117 Avena sativa PI 134604 England 4.8 S 118 Avena sativa PI 135731 Morocco 4.3 S 96

119 Avena sativa PI 137597 Portugal 3.5 MS 120 Avena sativa PI 158141 Russia 5.0 S 121 Avena sativa PI 158222 Ukraine 4.8 S 122 Avena sativa PI 158224 Mongolia 5.0 S 123 Avena sativa PI 159141 Mexico 5.0 S 124 Avena sativa PI 159148 Mexico 5.0 S 125 Avena sativa PI 159150 Mexico 4.7 S 126 Avena sativa PI 159173 Mexico 4.7 S 127 Avena sativa PI 159934 Wales 3.3 MS 128 Avena sativa PI 159779 Germany 5.0 S 129 Avena sativa PI 161959 Austria 2.7 MR 130 Avena sativa PI 166026 India 1.9 R 131 Avena sativa PI 166170 India 2.7 MR 132 Avena sativa PI 167378 Turkey 4.4 S 133 Avena sativa PI 177784 Turkey 2.5 MR 134 Avena sativa PI 177809 Turkey 3.2 MS 135 Avena sativa PI 177811 Turkey 2.1 MR 136 Avena sativa PI 177843 Turkey 3.0 MS 137 Avena sativa PI 177845 Turkey 2.8 MR 138 Avena sativa PI 178484 Turkey 2.7 MR 139 Avena sativa PI 184031 Serbia 4.5 S 140 Avena sativa PI 185658 Argentina 1.1 R 141 Avena sativa PI 185790 Argentina 1.0 R 142 Avena sativa PI 186271 Argentina 1.3 R 143 Avena sativa PI 186473 Uruguay 4.5 S 144 Avena sativa PI 186604 Brazil 1.8 R 145 Avena sativa PI 186611 Brazil 3.7 MS 146 Avena sativa PI 186618 Brazil 4.8 S 147 Avena sativa PI 221279 Finland 4.4 S 148 Avena sativa PI 221286 Serbia 4.0 S 149 Avena sativa PI 225027 Belgium 4.3 S 150 Avena sativa PI 225029 Belgium 3.6 MS 151 Avena sativa Deon USA 4.4 S 152 Avena sativa Hayden USA 4.4 S 153 Avena sativa Horspower USA 5.0 S 154 Avena sativa Shelby USA 4.9 S 155 Avena sativa Streaker USA 4.0 S CV = 5.8415; LSD =0.3959

97

Table A 3.1. Disease reaction of 366 triticale genotypes to Pyrenophora tritici-repentis (tan spot) races 1 and 5 and Ptr ToxA No Taxon Accession country Ptr Race1 Race5 . # Tox lesion Reaction lesion Reaction A type type 1 X Triticosecale Clxt 2 Canada - 4.2 S 4.0 S spp. 2 X Triticosecale Clxt 3 Canada + 3.2 MS 2.0 MR spp. 3 X Triticosecale Clxt 5 USA - 4.0 S 3.6 MS spp. 4 X Triticosecale Clxt 6 USA - 4.3 S 2.3 MR spp. 5 X Triticosecale Clxt 7 USA - 4.0 S 3.3 MS spp. 6 X Triticosecale Clxt 9 Hungary + 3.4 MS 3.5 MS spp. 7 X Triticosecale Clxt 10 USA - 4.3 S 4.2 S spp. 8 X Triticosecale Clxt 11 USA - 4.0 S 3.0 MS spp. 9 X Triticosecale Clxt 12 USA - 1.3 R 1.3 R spp. 10 X Triticosecale Clxt 13 USA - 1.3 R 1.0 R spp. 11 X Triticosecale Clxt 14 USA - 4.1 S 2.5 MR spp. 12 X Triticosecale Clxt 15 USA - 3.8 MS 2.7 MR spp. 13 X Triticosecale Clxt 16 USA - 1.0 R 1.0 R spp. 14 X Triticosecale Clxt 17 USA - 2.0 MR 1.0 R spp. 15 X Triticosecale Clxt 18 USA - 3.3 MS 1.0 R spp. 16 X Triticosecale Clxt 19 USA - 3.2 MS 1.6 R spp. 17 X Triticosecale Clxt 20 USA - 1.4 R 1.0 R spp. 18 X Triticosecale Clxt 21 USA - 4.2 S 1.0 R spp. 19 X Triticosecale Clxt 22 USA - 1.3 R 1.1 R spp. 98

20 X Triticosecale Clxt 23 Canada - 4.0 S 3.2 MS spp. 21 X Triticosecale Clxt 24 Canada - 3.5 MS 2.2 MR spp. 22 X Triticosecale Clxt 25 Canada - 4.0 S 3.3 MS spp. 23 X Triticosecale Clxt 26 USA - 3.7 MS 1.3 R spp. 24 X Triticosecale Clxt 28 China - 1.0 R 1.4 R spp. 25 X Triticosecale Clxt 30 China - 1.0 R 1.2 R spp. 26 X Triticosecale Clxt 31 USA + 3.3 MS 1.0 R spp. 27 X Triticosecale Clxt 35 USA - 3.8 MS 1.2 R spp. 28 X Triticosecale Clxt 40 USA - 2.0 MR 3.3 MS spp. 29 X Triticosecale Clxt 41 USA - 3.0 MS 3.0 MS spp. 30 X Triticosecale Clxt 43 USA - 3.4 MS 3.2 MS spp. 31 X Triticosecale Clxt 46 USA - 1.3 R 2.3 MR spp. 32 X Triticosecale Clxt 47 USA + 4.0 S 2.7 MR spp. 33 X Triticosecale Clxt 48 USA - 3.5 MS 1.0 R spp. 34 X Triticosecale Clxt 49 USA - 3.0 MS 1.9 R spp. 35 X Triticosecale Clxt 50 USA - 3.8 MS 3.0 MS spp. 36 X Triticosecale Clxt 51 USA - 3.8 MS 2.7 MR spp. 37 X Triticosecale Clxt 52 USA - 3.5 MS 2.1 MR spp. 38 X Triticosecale Clxt 53 USA - 2.0 MR 3.9 MS spp. 39 X Triticosecale Clxt 54 USA - 3.8 MS 2.3 MR spp. 40 X Triticosecale Clxt 55 USA - 3.7 MS 4.0 S spp. 41 X Triticosecale Clxt 56 USA + 2.8 MR 1.0 R spp. 42 X Triticosecale Clxt 57 USA - 1.0 R 1.0 R spp. 99

43 X Triticosecale Clxt 58 USA - 2.4 MR 2.7 MR spp. 44 X Triticosecale Clxt 59 USA - 3.5 MS 2.7 MR spp. 45 X Triticosecale Clxt 60 USA - 4.0 S 1.5 R spp. 46 X Triticosecale Clxt 64 USA + 2.7 MR 3.2 MS spp. 47 X Triticosecale Clxt 65 USA - 3.7 MS 3.2 MS spp. 48 X Triticosecale Clxt 68 USA - 3.5 MS 1.4 R spp. 49 X Triticosecale Clxt 70 USA + 3.8 MS 2.7 MR spp. 50 X Triticosecale Clxt 74 USA + 4.1 S 1.0 R spp. 51 X Triticosecale Clxt 76 USA + 3.8 MS 3.5 MS spp. 52 X Triticosecale Clxt 77 USA - 2.5 MR 1.0 R spp. 53 X Triticosecale Clxt 101 USA - 4.0 S 1.2 R spp. 54 X Triticosecale Clxt 104 USA - 3.5 MS 1.7 R spp. 55 X Triticosecale Clxt 105 USA - 1.0 R 2.2 MR spp. 56 X Triticosecale PI 94602 Russia - 1.0 R 1.2 R spp. 57 X Triticosecale PI 94603 Russia - 1.2 R 3.5 MS spp. 58 X Triticosecale PI 94605 Russia - 4.0 S 2.3 MR spp. 59 X Triticosecale PI 94606 Russia - 4.0 S 2.5 MR spp. 60 X Triticosecale PI 94609 Russia - 1.0 R 1.3 R spp. 61 X Triticosecale PI 197132 Japan - 3.7 MS 3.8 MS spp. 62 X Triticosecale PI 197133 Japan - 3.5 MS 3.3 MS spp. 63 X Triticosecale PI 218251 Japan - 3.0 MS 2.0 MR spp. 64 X Triticosecale PI 235640 Spain - 3.0 MS 1.4 R spp. 65 X Triticosecale PI 256030 Spain - 3.5 MS 3.5 MS spp. 100

66 X Triticosecale PI 256032 Spain - 2.0 MR 2.2 MR spp. 67 X Triticosecale PI 256033 Spain - 2.0 MR 1.0 R spp. 68 X Triticosecale PI 271075 Russia - 3.9 MS 2.5 MR spp. 69 X Triticosecale PI 280457 Russia - 1.0 R 1.0 R spp. 70 X Triticosecale PI 282899 Argentin + 1.0 R 1.0 R spp. a 71 X Triticosecale PI 285753 Poland - 1.2 R 1.0 R spp. 72 X Triticosecale PI 285754 Spain + 3.8 MS 1.3 R spp. 73 X Triticosecale PI 308880 Spain - 3.8 MS 3.2 MS spp. 74 X Triticosecale PI 308881 Spain - 1.2 R 1.0 R spp. 75 X Triticosecale PI 320251 Spain - 1.8 R 4.0 S spp. 76 X Triticosecale PI 340749 Germany - 3.0 MS 1.3 R spp. 77 X Triticosecale PI 355950 Russia + 1.0 R 1.5 R spp. 78 X Triticosecale PI 355951 Russia + 1.3 R 2.0 MR spp. 79 X Triticosecale PI 355953 Russia + 1.0 R 1.0 R spp. 80 X Triticosecale PI 381430 Hungary - 3.7 MS 3.1 MS spp. 81 X Triticosecale PI 381431 Hungary - 4.0 S 1.6 R spp. 82 X Triticosecale PI 381432 Hungary + 4.0 S 3.4 MS spp. 83 X Triticosecale PI 381433 Hungary + 3.5 MS 3.0 MS spp. 84 X Triticosecale PI 383408 Poland + 1.0 R 1.0 R spp. 85 X Triticosecale PI 383409 Poland + 3.8 MS 3.0 MS spp. 86 X Triticosecale PI 386001 Russia - 3.0 MS 3.0 MS spp. 87 X Triticosecale PI 386002 Russia + 4.1 S 1.0 R spp. 88 X Triticosecale PI 386003 Russia - 1.0 R 1.0 R spp. 101

89 X Triticosecale PI 386004 Russia - 1.6 R 1.5 R spp. 90 X Triticosecale PI 386005 Russia + 1.1 R 2.4 MR spp. 91 X Triticosecale PI 386113 Ukrine + 2.0 MR 1.0 R spp. 92 X Triticosecale PI 386114 Russia + 3.3 MS 1.0 R spp. 93 X Triticosecale PI 386116 Russia - 2.6 MR 3.7 MS spp. 94 X Triticosecale PI 386118 Ukrine - 2.3 MR 1.0 R spp. 95 X Triticosecale PI 386119 Russia - 4.0 S 3.8 MS spp. 96 X Triticosecale PI 386120 Russia - 4.0 S 3.8 MS spp. 97 X Triticosecale PI 386122 Ukrine - 2.0 MR 2.2 MR spp. 98 X Triticosecale PI 386123 Ukrine - 4.0 S 1.5 R spp. 99 X Triticosecale PI 386124 Ukrine - 2.3 MR 1.0 R spp. 10 X Triticosecale PI 386125 Russia - 1.0 R 1.0 R 0 spp. 10 X Triticosecale PI 386126 Russia - 3.2 MS 4.0 S 1 spp. 10 X Triticosecale PI 386127 Ukrine - 1.0 R 3.0 MS 2 spp. 10 X Triticosecale PI 386128 Ukrine - 1.3 R 4.0 S 3 spp. 10 X Triticosecale PI 386130 Russia + 1.9 R 1.0 R 4 spp. 10 X Triticosecale PI 386131 Russia - 1.0 R 2.8 MR 5 spp. 10 X Triticosecale PI 386132 Russia + 1.3 R 1.0 R 6 spp. 10 X Triticosecale PI 386134 Russia - 1.0 R 3.5 MS 7 spp. 10 X Triticosecale PI 386135 Russia - 1.0 R 1.8 R 8 spp. 10 X Triticosecale PI 386136 Russia - 1.2 R 3.0 MS 9 spp. 11 X Triticosecale PI 386137 Russia - 4.0 S 2.8 MR 0 spp. 11 X Triticosecale PI 386144 Ukraine - 2.8 MR 2.5 MR 1 spp. 102

11 X Triticosecale PI 386145 Ukraine - 1.0 R 1.3 R 2 spp. 11 X Triticosecale PI 386146 Ukraine - 3.0 MS 2.0 MR 3 spp. 11 X Triticosecale PI 386147 Azerbaij - 4.0 S 1.5 R 4 spp. an 11 X Triticosecale PI 386152 Ukraine - 3.0 MS 2.6 MR 5 spp. 11 X Triticosecale PI 386153 Azerbaij - 2.0 MR 2.0 MR 6 spp. an 11 X Triticosecale PI 386154 Russia - 1.0 R 1.1 R 7 spp. 11 X Triticosecale PI 386156 Russia - 1.0 R 3.6 MS 8 spp. 11 X Triticosecale PI 386157 Ukraine - 1.0 R 1.5 R 9 spp. 12 X Triticosecale PI 388656 South - 4.0 S 2.2 MR 0 spp. Africa 12 X Triticosecale PI 388657 South - 3.8 MS 2.7 MR 1 spp. Africa 12 X Triticosecale PI 388658 South - 1.8 R 2.9 MR 2 spp. Africa 12 X Triticosecale PI 388659 South - 1.1 R 2.5 MR 3 spp. Africa 12 X Triticosecale PI 388660 South - 4.0 S 1.6 R 4 spp. Africa 12 X Triticosecale PI 388664 South - 2.1 MR 1.4 R 5 spp. Africa 12 X Triticosecale PI 388667 South - 3.3 MS 3.2 MS 6 spp. Africa 12 X Triticosecale PI 388668 South - 3.0 MS 4.0 S 7 spp. Africa 12 X Triticosecale PI 388671 South - 1.2 R 3.0 MS 8 spp. Africa 12 X Triticosecale PI 388673 South - 1.0 R 1.4 R 9 spp. Africa 13 X Triticosecale PI 388674 South - 1.5 R 1.3 R 0 spp. Africa 13 X Triticosecale PI 388675 South - 1.0 R 3.8 MS 1 spp. Africa 13 X Triticosecale PI 388676 South - 1.0 R 1.0 R 2 spp. Africa 13 X Triticosecale PI 388677 South - 1.0 R 3.3 MS 3 spp. Africa 13 X Triticosecale PI 388678 South - 1.6 R 1.5 R 4 spp. Africa 103

13 X Triticosecale PI 388682 South - 1.0 R 3.5 MS 5 spp. Africa 13 X Triticosecale PI 388686 South + 1.2 R 1.0 R 6 spp. Africa 13 X Triticosecale PI 388688 South + 2.5 MR 1.0 R 7 spp. Africa 13 X Triticosecale PI 388689 South - 1.3 R 2.9 MR 8 spp. Africa 13 X Triticosecale PI 388690 South - 2.0 MR 3.0 MS 9 spp. Africa 14 X Triticosecale PI 388691 South + 1.0 R 1.0 R 0 spp. Africa 14 X Triticosecale PI 388692 South - 1.0 R 1.2 R 1 spp. Africa 14 X Triticosecale PI 388693 South - 1.0 R 1.7 R 2 spp. Africa 14 X Triticosecale PI 388696 South - 1.0 R 3.0 MS 3 spp. Africa 14 X Triticosecale PI 388699 South + 3.8 MS 3.0 MS 4 spp. Africa 14 X Triticosecale PI 405019 South - 1.0 R 3.0 MS 5 spp. Africa 14 X Triticosecale PI 405021 South - 2.1 MR 2.3 MR 6 spp. Africa 14 X Triticosecale PI 405023 South - 1.2 R 2.2 MR 7 spp. Africa 14 X Triticosecale PI 405024 South - 1.5 R 1.0 R 8 spp. Africa 14 X Triticosecale PI 405025 South - 3.0 MS 1.0 R 9 spp. Africa 15 X Triticosecale PI 405029 South - 1.3 R 3.0 MS 0 spp. Africa 15 X Triticosecale PI 405030 South - 1.0 R 1.0 R 1 spp. Africa 15 X Triticosecale PI 405032 South - 1.2 R 1.2 R 2 spp. Africa 15 X Triticosecale PI 405034 South - 1.0 R 3.4 MS 3 spp. Africa 15 X Triticosecale PI 410802 Poland - 3.7 MS 3.6 MS 4 spp. 15 X Triticosecale PI 410803 Poland - 1.0 R 3.0 MS 5 spp. 15 X Triticosecale PI 410804 Poland - 1.3 R 1.7 R 6 spp. 15 X Triticosecale PI 410806 Poland - 1.0 R 3.1 MS 7 spp. 104

15 X Triticosecale PI 410808 Poland - 4.0 S 3.3 MS 8 spp. 15 X Triticosecale PI 410809 Poland - 1.2 R 3.5 MS 9 spp. 16 X Triticosecale PI 410883 Ukraine - 1.3 R 2.1 MR 0 spp. 16 X Triticosecale PI 410904 Spain - 3.8 MS 3.7 MS 1 spp. 16 X Triticosecale PI 410906 Spain - 1.4 R 3.5 MS 2 spp. 16 X Triticosecale PI 413008 South - 1.0 R 1.0 R 3 spp. Africa 16 X Triticosecale PI 414627 China - 3.0 MS 1.0 R 4 spp. 16 X Triticosecale PI 414945 Ukraine - 1.0 R 4.0 S 5 spp. 16 X Triticosecale PI 414950 Russia - 1.0 R 1.2 R 6 spp. 16 X Triticosecale PI 414952 Russia - 1.0 R 1.2 R 7 spp. 16 X Triticosecale PI 414953 Russia - 1.4 R 1.3 R 8 spp. 16 X Triticosecale PI 414955 Russia - 2.2 MR 4.0 S 9 spp. 17 X Triticosecale PI 414962 Russia - 1.0 R 3.9 MS 0 spp. 17 X Triticosecale PI 414963 Russia - 1.0 R 3.5 MS 1 spp. 17 X Triticosecale PI 422258 Mexico - 3.8 MS 3.6 MS 2 spp. 17 X Triticosecale PI 422259 Mexico - 4.2 S 3.0 MS 3 spp. 17 X Triticosecale PI 422260 Mexico - 4.0 S 3.5 MS 4 spp. 17 X Triticosecale PI 422263 Mexico - 1.8 R 2.7 MR 5 spp. 17 X Triticosecale PI 422264 Mexico - 3.8 MS 4.0 S 6 spp. 17 X Triticosecale PI 422265 Mexico - 1.0 R 1.0 R 7 spp. 17 X Triticosecale PI 422266 Mexico - 2.8 MR 1.0 R 8 spp. 17 X Triticosecale PI 422268 Mexico + 1.3 R 1.5 R 9 spp. 18 X Triticosecale PI 422270 Mexico - 1.0 R 2.0 MR 0 spp. 105

18 X Triticosecale PI 428730 Japan - 1.3 R 1.5 R 1 spp. 18 X Triticosecale PI 428736 Canada - 4.5 S 3.5 MS 2 spp. 18 X Triticosecale PI 428738 Canada - 3.8 MS 1.0 R 3 spp. 18 X Triticosecale PI 428739 Sweden - 1.0 R 2.8 MR 4 spp. 18 X Triticosecale PI 428742 Japan + 3.5 MS 3.6 MS 5 spp. 18 X Triticosecale PI 428743 Japan - 1.5 R 2.8 MR 6 spp. 18 X Triticosecale PI 428744 Japan - 3.8 MS 3.0 MS 7 spp. 18 X Triticosecale PI 428745 Japan + 3.7 MS 2.5 MR 8 spp. 18 X Triticosecale PI 428747 Japan - 3.8 MS 1.0 R 9 spp. 19 X Triticosecale PI 428755 Canada - 1.0 R 1.5 R 0 spp. 19 X Triticosecale PI 428756 Japan + 2.2 MR 2.0 MR 1 spp. 19 X Triticosecale PI 428762 Canada + 1.0 R 1.0 R 2 spp. 19 X Triticosecale PI 428769 USA - 1.0 R 1.0 R 3 spp. 19 X Triticosecale PI 428770 USA - 1.0 R 1.3 R 4 spp. 19 X Triticosecale PI 428771 India - 3.6 MS 2.8 MR 5 spp. 19 X Triticosecale PI 428774 Sweden + 2.5 MR 1.7 R 6 spp. 19 X Triticosecale PI 428777 Sweden - 4.0 S 1.8 R 7 spp. 19 X Triticosecale PI 428778 Sweden - 3.4 MS 1.0 R 8 spp. 19 X Triticosecale PI 428780 Sweden - 4.0 S 1.8 R 9 spp. 20 X Triticosecale PI 428785 Sweden - 1.7 R 1.8 R 0 spp. 20 X Triticosecale PI 428787 Sweden - 4.3 S 4.0 S 1 spp. 20 X Triticosecale PI 428792 Sweden + 3.3 MS 1.0 R 2 spp. 20 X Triticosecale PI 428793 Sweden + 2.6 MR 2.9 MR 3 spp. 106

20 X Triticosecale PI 428794 Sweden + 3.7 MS 3.1 MS 4 spp. 20 X Triticosecale PI 428796 Canada - 1.0 R 1.0 R 5 spp. 20 X Triticosecale PI 428803 Canada - 1.0 R 3.0 MS 6 spp. 20 X Triticosecale PI 428804 Canada + 3.1 MS 2.0 MR 7 spp. 20 X Triticosecale PI 428805 Canada - 1.7 R 2.6 MR 8 spp. 20 X Triticosecale PI 428806 USA - 3.3 MS 1.3 R 9 spp. 21 X Triticosecale PI 428807 Canada - 1.9 R 1.3 R 0 spp. 21 X Triticosecale PI 428810 Germany - 1.3 R 1.7 R 1 spp. 21 X Triticosecale PI 428811 Germany - 3.2 MS 1.3 R 2 spp. 21 X Triticosecale PI 428812 Canada - 1.0 R 2.0 MR 3 spp. 21 X Triticosecale PI 428813 Russia - 1.0 R 3.3 MS 4 spp. 21 X Triticosecale PI 428814 Russia - 1.8 R 3.0 MS 5 spp. 21 X Triticosecale PI 428821 Spain - 1.1 R 1.0 R 6 spp. 21 X Triticosecale PI 428825 Spain + 1.0 R 1.0 R 7 spp. 21 X Triticosecale PI 428830 Russia + 3.0 MS 3.0 MS 8 spp. 21 X Triticosecale PI 428836 Canada - 4.3 S 3.2 MS 9 spp. 22 X Triticosecale PI 428839 Canada - 1.0 R 1.0 R 0 spp. 22 X Triticosecale PI 428842 Canada - 2.0 MR 3.0 MS 1 spp. 22 X Triticosecale PI 428844 Russia - 3.8 MS 3.1 MS 2 spp. 22 X Triticosecale PI 428845 Russia + 1.0 R 1.0 R 3 spp. 22 X Triticosecale PI 428846 Russia - 1.0 R 4.0 S 4 spp. 22 X Triticosecale PI 428853 Russia + 1.2 R 1.0 R 5 spp. 22 X Triticosecale PI 428855 Russia - 1.0 R 1.0 R 6 spp. 107

22 X Triticosecale PI 428860 Russia - 1.0 R 2.2 MR 7 spp. 22 X Triticosecale PI 428870 France - 1.0 R 1.0 R 8 spp. 22 X Triticosecale PI 428871 France - 2.5 MR 1.4 R 9 spp. 23 X Triticosecale PI 428875 France - 1.3 R 1.2 R 0 spp. 23 X Triticosecale PI 428878 France - 3.7 MS 4.0 S 1 spp. 23 X Triticosecale PI 428879 France - 3.0 MS 1.1 R 2 spp. 23 X Triticosecale PI 428887 Hungary + 3.8 MS 1.0 R 3 spp. 23 X Triticosecale PI 428889 Hungary + 3.6 MS 2.8 MR 4 spp. 23 X Triticosecale PI 428892 Hungary - 1.5 R 3.5 MS 5 spp. 23 X Triticosecale PI 428893 Hungary - 2.0 MR 1.0 R 6 spp. 23 X Triticosecale PI 428894 Hungary - 3.7 MS 3.3 MS 7 spp. 23 X Triticosecale PI 428896 USA - 4.0 S 1.0 R 8 spp. 23 X Triticosecale PI 428902 Canada - 3.8 MS 4.0 S 9 spp. 24 X Triticosecale PI 428903 Canada - 1.3 R 1.7 R 0 spp. 24 X Triticosecale PI 428936 Sweden + 3.4 MS 1.0 R 1 spp. 24 X Triticosecale PI 428937 Sweden - 1.5 R 1.0 R 2 spp. 24 X Triticosecale PI 428938 Sweden - 1.7 R 3.0 MS 3 spp. 24 X Triticosecale PI 428945 Canada - 1.8 R 1.0 R 4 spp. 24 X Triticosecale PI 428946 Canada - 3.7 MS 4.0 S 5 spp. 24 X Triticosecale PI 428953 Canada + 3.5 MS 3.8 MS 6 spp. 24 X Triticosecale PI 428955 Canada - 1.0 R 1.3 R 7 spp. 24 X Triticosecale PI 428979 Hungary + 4.0 S 2.5 MR 8 spp. 24 X Triticosecale PI 428980 Hungary - 4.0 S 3.5 MS 9 spp. 108

25 X Triticosecale PI 428981 Hungary - 1.0 R 2.0 MR 0 spp. 25 X Triticosecale PI 428987 Canada - 3.4 MS 1.0 R 1 spp. 25 X Triticosecale PI 428989 Canada - 1.0 R 1.0 R 2 spp. 25 X Triticosecale PI 428992 Canada - 1.5 R 1.0 R 3 spp. 25 X Triticosecale PI 429006 India - 2.5 MR 3.8 MS 4 spp. 25 X Triticosecale PI 429007 India - 4.0 S 3.8 MS 5 spp. 25 X Triticosecale PI 429010 India - 1.7 R 1.0 R 6 spp. 25 X Triticosecale PI 429011 India - 3.8 MS 3.3 MS 7 spp. 25 X Triticosecale PI 429013 France - 1.3 R 3.0 MS 8 spp. 25 X Triticosecale PI 429023 Mexico - 1.3 R 4.0 S 9 spp. 26 X Triticosecale PI 429025 Mexico + 3.8 MS 4.0 S 0 spp. 26 X Triticosecale PI 429028 Mexico + 4.0 S 3.0 MS 1 spp. 26 X Triticosecale PI 429038 Mexico + 4.0 S 1.1 R 2 spp. 26 X Triticosecale PI 429042 India - 3.1 MS 2.9 MR 3 spp. 26 X Triticosecale PI 429043 India - 1.8 R 1.2 R 4 spp. 26 X Triticosecale PI 429044 India - 3.3 MS 3.0 MS 5 spp. 26 X Triticosecale PI 429045 India - 4.2 S 1.0 R 6 spp. 26 X Triticosecale PI 429046 India - 1.0 R 4.0 S 7 spp. 26 X Triticosecale PI 429047 India - 1.2 R 1.0 R 8 spp. 26 X Triticosecale PI 429048 India - 1.0 R 1.6 R 9 spp. 27 X Triticosecale PI 429053 Canada - 2.9 MR 1.0 R 0 spp. 27 X Triticosecale PI 429060 Mexico + 4.6 S 4.0 S 1 spp. 27 X Triticosecale PI 429064 Sweden - 3.3 MS 3.8 MS 2 spp. 109

27 X Triticosecale PI 429070 Sweden + 3.0 MS 1.0 R 3 spp. 27 X Triticosecale PI 429071 Poland - 1.0 R 1.0 R 4 spp. 27 X Triticosecale PI 429072 Poland - 3.0 MS 1.0 R 5 spp. 27 X Triticosecale PI 429073 Poland - 3.2 MS 1.4 R 6 spp. 27 X Triticosecale PI 429074 Poland + 3.8 MS 1.4 R 7 spp. 27 X Triticosecale PI 429079 Poland - 1.0 R 1.0 R 8 spp. 27 X Triticosecale PI 429080 Poland - 1.0 R 1.8 R 9 spp. 28 X Triticosecale PI 429081 Poland - 1.0 R 1.1 R 0 spp. 28 X Triticosecale PI 429087 India - 1.1 R 1.0 R 1 spp. 28 X Triticosecale PI 429089 India + 3.0 MS 4.0 S 2 spp. 28 X Triticosecale PI 429092 India - 2.0 MR 1.0 R 3 spp. 28 X Triticosecale PI 429098 Germany - 2.2 MR 4.0 S 4 spp. 28 X Triticosecale PI 429103 Canada - 1.0 R 4.0 S 5 spp. 28 X Triticosecale PI 429113 Mexico - 1.3 R 3.4 MS 6 spp. 28 X Triticosecale PI 429114 Mexico - 1.2 R 2.0 MR 7 spp. 28 X Triticosecale PI 429121 Mexico - 4.0 S 1.0 R 8 spp. 28 X Triticosecale PI 429123 Mexico + 3.5 MS 1.0 R 9 spp. 29 X Triticosecale PI 429124 Mexico + 2.0 MR 1.3 R 0 spp. 29 X Triticosecale PI 429125 Mexico - 3.0 MS 1.7 R 1 spp. 29 X Triticosecale PI 429128 Mexico - 2.0 MR 1.0 R 2 spp. 29 X Triticosecale PI 429136 Poland - 1.0 R 2.3 MR 3 spp. 29 X Triticosecale PI 429137 Poland - 3.8 MS 3.0 MS 4 spp. 29 X Triticosecale PI 429138 Poland + 3.0 MS 1.0 R 5 spp. 110

29 X Triticosecale PI 429139 Poland - 5.0 S 4.0 S 6 spp. 29 X Triticosecale PI 429140 Poland + 4.8 S 3.6 MS 7 spp. 29 X Triticosecale PI 429142 Poland - 1.0 R 3.0 MS 8 spp. 29 X Triticosecale PI 429143 Poland + 4.3 S 2.8 MR 9 spp. 30 X Triticosecale PI 429145 Poland + 3.0 MS 3.0 MS 0 spp. 30 X Triticosecale PI 429144 Poland + 1.2 R 2.0 MR 1 spp. 30 X Triticosecale PI 429147 Poland - 3.2 MS 1.9 R 2 spp. 30 X Triticosecale PI 429148 Poland - 3.5 MS 1.0 R 3 spp. 30 X Triticosecale PI 429153 Sweden - 4.0 S 4.0 S 4 spp. 30 X Triticosecale PI 429155 Sweden - 3.3 MS 4.0 S 5 spp. 30 X Triticosecale PI 429157 Sweden - 3.6 MS 3.8 MS 6 spp. 30 X Triticosecale PI 429160 Sweden - 3.0 MS 4.0 S 7 spp. 30 X Triticosecale PI 429162 Sweden - 4.0 S 3.8 MS 8 spp. 30 X Triticosecale PI 429164 Canada - 4.2 S 3.0 MS 9 spp. 31 X Triticosecale PI 429171 Canada - 3.8 MS 3.5 MS 0 spp. 31 X Triticosecale PI 429189 USA - 4.0 S 2.0 MR 1 spp. 31 X Triticosecale PI 429192 Mexico - 1.8 R 3.0 MS 2 spp. 31 X Triticosecale PI 429193 Mexico + 3.5 MS 1.0 R 3 spp. 31 X Triticosecale PI 429196 Mexico - 5.0 S 1.0 R 4 spp. 31 X Triticosecale PI 429198 Mexico - 4.0 S 3.5 MS 5 spp. 31 X Triticosecale PI 429203 Mexico - 4.0 S 1.0 R 6 spp. 31 X Triticosecale PI 429205 Mexico - 4.0 S 4.0 S 7 spp. 31 X Triticosecale PI 429207 Mexico - 3.0 MS 4.0 S 8 spp. 111

31 X Triticosecale PI 429210 Mexico - 1.3 R 2.3 MR 9 spp. 32 X Triticosecale PI 429211 Mexico - 1.1 R 1.0 R 0 spp. 32 X Triticosecale PI 429213 Mexico - 3.3 MS 4.0 S 1 spp. 32 X Triticosecale PI 429223 Mexico - 4.5 S 3.6 MS 2 spp. 32 X Triticosecale PI 429224 Canada + 4.0 S 1.0 R 3 spp. 32 X Triticosecale PI 429253 Russia - 4.5 S 1.8 R 4 spp. 32 X Triticosecale PI 429255 Russia - 1.8 R 1.0 R 5 spp. 32 X Triticosecale PI 429260 Ukraine + 1.1 R 1.0 R 6 spp. 32 X Triticosecale PI 429261 Russia - 1.4 R 1.5 R 7 spp. 32 X Triticosecale PI 429264 Russia - 1.7 R 1.5 R 8 spp. 32 X Triticosecale PI 429270 Russia - 2.0 MR 2.5 MR 9 spp. 33 X Triticosecale PI 429271 Russia - 4.8 S 1.0 R 0 spp. 33 X Triticosecale PI 429280 Mexico - 2.3 MR 1.0 R 1 spp. 33 X Triticosecale PI 429281 Mexico - 4.5 S 1.7 R 2 spp. 33 X Triticosecale PI 429282 Mexico - 1.8 R 4.0 S 3 spp. 33 X Triticosecale PI 429287 Mexico - 1.0 R 1.1 R 4 spp. 33 X Triticosecale PI 429289 Mexico - 2.0 MR 1.0 R 5 spp. 33 X Triticosecale PI 429296 Poland + 4.0 S 3.8 MS 6 spp. 33 X Triticosecale PI 429298 Poland - 4.2 S 2.2 MR 7 spp. 33 X Triticosecale PI 429304 Poland - 3.0 MS 3.8 MS 8 spp. 33 X Triticosecale PI 429306 Poland - 4.0 S 4.0 S 9 spp. 34 X Triticosecale PI 434716 China - 3.8 MS 1.0 R 0 spp. 34 X Triticosecale PI 434889 South - 1.0 R 4.0 S 1 spp. Africa 112

34 X Triticosecale PI 434891 South - 1.4 R 1.0 R 2 spp. Africa 34 X Triticosecale PI 445677 Ukraine - 1.0 R 1.0 R 3 spp. 34 X Triticosecale PI 445678 Russia - 4.0 S 1.0 R 4 spp. 34 X Triticosecale PI 483066 Australia - 1.0 R 1.0 R 5 spp. 34 X Triticosecale PI 491409 USA - 1.0 R 2.0 MR 6 spp. 34 X Triticosecale PI 491549 Mexico - 2.0 MR 1.0 R 7 spp. 34 X Triticosecale PI 495821 Australia - 4.0 S 3.1 MS 8 spp. 34 X Triticosecale PI 519232 Pakistan - 2.8 MR 3.5 MS 9 spp. 35 X Triticosecale PI 519817 Mexico - 2.5 MR 1.0 R 0 spp. 35 X Triticosecale PI 519879 Mexico - 1.0 R 1.0 R 1 spp. 35 X Triticosecale PI 520433 Brazil - 2.0 MR 1.0 R 2 spp. 35 X Triticosecale PI 520434 Brazil - 1.0 R 1.8 R 3 spp. 35 X Triticosecale PI 520435 Brazil - 1.5 R 1.3 R 4 spp. 35 X Triticosecale PI 520436 Brazil - 2.0 MR 3.0 MS 5 spp. 35 X Triticosecale PI 520438 Brazil - 1.3 R 2.1 MR 6 spp. 35 X Triticosecale PI 520439 Brazil - 1.4 R 2.3 MR 7 spp. 35 X Triticosecale PI 520441 Brazil - 1.1 R 1.3 R 8 spp. 35 X Triticosecale PI 520460 Ecuador + 3.7 MS 1.0 R 9 spp. 36 X Triticosecale PI 525197 Australia - 4.3 S 1.0 R 0 spp. 36 X Triticosecale PI 591863 Russia - 1.0 R 1.0 R 1 spp. 36 X Triticosecale PI 591864 Russia - 5.0 S 1.0 R 2 spp. 36 X Triticosecale PI 591912 Russia + 4.7 S 3.0 MS 3 spp. 36 X Triticosecale PI 601077 USA - 3.5 MS 3.3 MS 4 spp. 113

36 X Triticosecale PI 601078 USA - 1.5 R 2.0 MR 5 spp. 36 X Triticosecale PI 648395 India + 3.8 MS 4.0 S 6 spp. Ptr ToxA reaction (+ = sensitive and - = insensitive) Race 1: CV = 8.0679; LSD = 0.3959; Race 5: CV = 9.1405; LSD = 0.3959