Evaluation of Soybean Germplasm for Additional Sources of Resistance and. Characterization of Resistance towards Fusarium graminearum.

Transcription

1 Evaluation of Soybean Germplasm for Additional Sources of Resistance and Characterization of Resistance towards Fusarium graminearum. THESIS Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Bhupendra Acharya Graduate Program in Plant Pathology The Ohio State University 2014 Master's Examination Committee: Dr. Anne E. Dorrance. Advisor Dr. Leah McHale Dr. Pierce A. Paul Dr. Thomas Mitchell

2 Copyrighted by Bhupendra Acharya 2014

3 Abstract Fusarium graminearum was recently established as a primary pathogen of soybean causing seed rot and seedling root rot. Initial symptoms start as water soaked lesions which turn light brown or have pink discoloration at the later stages of disease development. The objective of this study was to evaluate soybean genotypes for resistance towards F. graminearum and to characterize resistance using recombinant inbred lines (RILs) of two soybean populations: Wyandot x PI567301B and Conrad x Sloan. All of the soybean germplasm, the parents and the RILs of the mapping populations were evaluated for resistance using the roll towel assay in either a randomized complete block design or augmented randomized incomplete block design. In this study, two hundred soybean genotypes were evaluated for resistance of which thirty had disease severity index ratings of less than 50% to F. graminearum indicating moderate to to high levels of resistance. Two of these genotypes, PI B and Conrad were identified with high and moderate levels of resistance to F. graminearum respectively. The genotypes were available in two mapping populations of 184 and 330 recombinant inbred lines (RILs) in a Wyandot x PI B and Conrad x Sloan, respectively. Both populations were phenotyped as described above and were genotyped using Infinium BARCSoySNP6K BeadChip array. Linkage maps and QTL analysis were constructed using JoinMap4 and MapQTL5. Using composite interval mapping, one major QTL on chromosome 8 and one minor QTL on chromosome 6 were identified in the Wyandot x PI B population which explained 38.5% and 8.1% of the ii

4 phenotypic variance, respectively. In the Conrad x Sloan population, three QTL were identified on chromosomes 10, 14, and 19 which explained 6.2, 4.8, and 8.9% the phenotypic variance, respectively. The QTL on chromosome 19 mapped to the region with QTL conferring resistance to Phytophthora sojae and Pythium irregulare. The QTL identified in the study will be useful in developing cultivars with resistance to F. graminearum. iii

5 Acknowledgments I would like to express my deepest gratitude to my advisor, Dr. Anne E. Dorrance for her excellent support, guidance, encouragement and providing an invaluable learning opportunity to develop my research and professional skills, in an excellent work environment. I would also like to thank my advisory committee: Dr. Leah McHale, Dr. Pierce A. Paul and Dr. Thomas Mitchell, who provided critical comments, advice and suggestions on my research work. I would like to thank Andika Gunadi, who as a good friend was always willing to help, teach and give his best suggestions. Many thanks to all the present and past members of Dorrance lab: Maggie Ellis, Sungwoo Lee, Damitha Wickramasinghe, Deloris Venney, Andreas Cruz, Anna Dixon, Charlotte Smith, Clifton Martin, Anna Stasko, Christine Balk, Jaqueline Huzar Novakowiski, Austin Pelyak and Thomas Fitz Gibbon for their technical assistance throughout my graduate studies. My research would not have been possible without their help. I would also like to thank my family: my parents Madhav Pd. Acharya and Manju Acharya, and brother Upendra Acharya for their continuous love, support and encouragement with their best wishes. Finally, I would like to thank my wife, Anita K.C. Acharya, who also helped me with my lab work, cheered me up and always stood by me through the good times and bad. iv

6 Vita December 14, Born - Chidipani-5, Palpa, Nepal B.Sc. Agriculture, Tribhuvan University Graduate Research Associate, Department of Plant Pathology, The Ohio State University Fields of Study Major Field: Plant Pathology v

7 Table of Contents Abstract... ii Acknowledgments... iv Vita... v Fields of Study... v Table of Contents... vi List of Figures... xiii Chapter 1: Introduction... 1 Introduction to Fusarium graminearum... 1 Fusarium seed and seedling rot of soybean... 6 Symptoms and Signs of Fusarium graminearum in soybean... 6 Literature Review... 7 References Chapter 2: Evaluation of Soybean Genotypes to Identify Sources of Resistance Towards Fusarium graminearum Introduction Materials Parental Lines of Soybean Nested Association Mapping (SoyNAM) vi

8 Soybean Genetic gain /Decade study OSU/OARDC Germplasm Inoculum production Phenotypic assay Statistical Analysis Results Parental Lines of Soybean Nested Association Mapping (SoyNAM) Soybean Genetic Gain /Decade study Maturity Group II (MG II) Maturity Group III (MG III) OSU/OARDC Germplasm OSU/OARDC Germplasm I OSU/OARDC Germplasm II Discussion References Chapter 3: Identification and Mapping of Quantitative Trait Loci (QTL) Conferring Resistance to Fusarium graminearum in Two Populations of Soybean Introduction vii

9 Materials and Methods Plant materials and DNA extraction Inoculum production Phenotypic evaluation Genotypic assay Results Phenotypic evaluation Wyandot x PI B population Conrad x Sloan population QTL identification Wyandot x PI B Conrad x Sloan Discussion References BIBLIOGRAPHY APPENDIX A: ANOVA tables of the evaluation of germplasm APPENDIX B: ANOVA tables for the checks of two populations APPENDIX C: List of genes within and close to the identified QTL viii

10 APPENDIX D: Protocols Mung Bean Agar (MBA) DNA extraction from soybean Lyophilized and ground tissues Preparation of inoculum and inoculation ix

11 List of Tables Table 1: Mean Disease Severity Index (DSI), mean score and non-inoculated percentage germination of parental lines, after excluding poor germinating genotypes, from the Soybean Nested Association Mapping (SoyNAM) population following inoculation of seeds with 100 µl of 2.5 x 10 4 macroconidia ml -1 of Fusarium graminearum in a rolled towel assay Table 2: Mean Disease Severity (DSI), mean score and non-inoculated germination percentage of Maturity Group II public cultivars, after excluding poor germinating cultivars, included in Genetic Gain study after inoculation of seeds with 100 µl of 2.5 x 10 4 macroconidia ml -1 of Fusarium graminearum in a rolled towel assay Table 3: Mean Disease Severity (DSI), mean score and non-inoculated germination percentage of Maturity Group III public cultivars, after excluding poor germinating cultivars, included in Genetic Gain study, after inoculation of seeds with 100 µl of 2.5 x 10 4 macroconidia ml -1 of Fusarium graminearum in a rolled towel assay Table 4: Mean Disease Severity (DSI), mean score and non-inoculated germination percentage of soybean genotypes, after excluding poor germinating genotypes, from OSU/OARDC Germplasm I, after inoculation of seeds with 100 µl of 2.5 x 10 4 macroconidia ml -1 of Fusarium graminearum in a rolled towel assay Table 5: Mean Disease Severity (DSI), mean score and non-inoculated germination percentage of 32 soybean genotypes from OSU/OARDC Germplasm II, after inoculation x

12 of seeds with 100µl of 2.5 x 10 4 macroconidia ml -1 of Fusarium graminearum in a rolled towel assay Table 6: Mean Disease Severity (DSI) and mean score of checks used in the Wyandot x PI B population study after inoculation of seeds with 100µl of 2.5 x 10 4 macroconidia ml -1 of F. graminearum in a rolled towel assay Table 7: Mean Disease Severity (DSI) and mean score of checks used in the Conrad x Sloan population study after inoculation of seeds with 100µl of 2.5 x 10 4 macroconidia ml -1 of F. graminearum in a rolled towel assay Table 8: Quantitative Trait Loci conferring resistance to F. graminearum identified via composite interval mapping (CIM) in the Wyandot x PI B RILs population Table 9: Quantitative Trait Loci conferring resistance to F. graminearum identified via composite interval mapping (CIM) based on version 1.0 SNP position using Conrad x Sloan RILs population Table 10: Genes within the QTL on chromosome 8 mapped from 'Wyandot'x PI B RILs population Table 11: Published genes close to the QTL in Chromosome 6 mapped from Wyandot x PI B RILs population (Soybase, 2014) Table 12: Published genes within or close to the QTL in Chromosome 10 mapped from Conrad x Sloan RILs population (Soybase, 2014) Table 13: Published genes within or close to the QTL in Chromosome 14 mapped from Conrad x Sloan RILs population (Soybase, 2014) xi

13 Table 14: Published genes within or close to the QTL in Chromosome 19 mapped from Conrad x Sloan RILs population (Soybase, 2014) xii

14 List of Figures Figure 1: Distribution of parental lines, after excluding poor germinating genotypes, of Soybean Nested Association Mapping (SoyNAM) population based on the disease severity index (DSI) Figure 2: Distribution of parental lines, after excluding poor germinating genotypes, of Soybean Nested Association Mapping (SoyNAM) population based on disease score Figure 3: Distribution of Maturity Group II public cultivars, after excluding poor germinating cultivars, included in Genetic Gain study based on the disease severity index (DSI) Figure 4: Distribution of Maturity Group II public cultivars, after excluding poor germinating cultivars, included in Genetic Gain study based on the disease score Figure 5: Distribution of Maturity Group III public cultivars after excluding poor germinating cultivars, included in Genetic Gain study based on the disease severity index (DSI) Figure 6: Distribution of Maturity Group III public cultivars, after excluding poor germinating cultivars, included in Genetic Gain study based on the disease score Figure 7: Distribution of soybean genotypes, after excluding poor germinating genotypes, from OSU/OARDC Germplasm I collection based on the disease severity index (DSI). 53 Figure 8: Distribution of soybean genotypes, after excluding poor germinating genotypes, from OSU/OARDC Germplasm I collection based on the disease score xiii

15 Figure 9: Distribution of soybean genotypes, after excluding poor germinating genotypes, from OSU/OARDC Germplasm II collection based on the disease severity index (DSI) Figure 10: Distribution of soybean genotypes, after excluding poor germinating genotypes, from OSU/OARDC Germplasm II collection based on the disease score Figure 11: Frequency distribution of BLUP values for DSI * of F 7: 10 recombinant inbred lines (RILs) population derived from the cross of Wyandot x PI B. Estimates of two parents and checks are indicated by arrows. A lower BLUP value means higher level of resistance to F. graminearum Figure 12: Frequency distribution of BLUP values for DSI * of F 9:11 recombinant inbred lines (RILs) population derived from the cross of Conrad x Sloan. Estimates of two parents and checks are indicated by arrows. A lower BLUP value means higher level of resistance to F. graminearum Figure 13: Graphical presentation of quantitative trait loci (QTL) for resistance to Fusarium graminearum identified in the Wyandot x PI B population by genome-wide LOD threshold 3.4 (a hatched line in LOD plots). The 1- and 2- LOD intervals are presented by a black bar and solid lines between the chromosome and the LOD plot for each QTL Figure 14: Graphical presentation of quantitative trait loci (QTL) for resistance to Fusarium graminearum identified in the Conrad x Sloan population by genome-wide LOD threshold 1.9 (a hatched line in LOD plots) xiv

16 Chapter 1: Introduction Introduction to Fusarium graminearum The study of the genus Fusarium dates back more than two centuries when H. F. Link in 1809, first introduced the genus (Leslie and Summerell, 2006; Snyder and Hansen, 1954). The publication, Grundlagen, published in Germany during 1910, by Appel and Wollenweber is believed to lay the foundation for all the later studies of Fusarium (Snyder and Hansen, 1954). Today this genus is reported around the globe in various climatic conditions and on many different species of plants as endophytes, epiphytes, parasites, or pathogens (Leslie and Summerell, 2006; Park et al., 1999). It is believed that even though plants look green, many contain at least one Fusarium-associated disease (Leslie and Summerell, 2006). Snyder and Hansen (1954) claimed that Fusarium is responsible for most of the economic damage to agriculture in the world in comparison to any other fungal genera. It was proposed that these fungi have modified their biosynthetic metabolism and other characteristics for better adaptation and colonization in different ecological systems (Mule et al., 2005). They have evolved to produce a diverse number of bioactive secondary metabolites, some of which are toxic to other organisms and also play an important role in pathogenesis and antagonism (Mule et al., 2005). Many Fusarium species produce mycotoxins, which are very detrimental to both animals and humans. Trichothecenes, which are 1

17 divided into two types: Type A and Type B. Type A trichothecene include T-2 toxins and its derivatives while Type B trichothecene include vomitoxins. Fusarium belongs to the kingdom Fungi and class Ascomycota, in which currently there are more than 80 different species recognized (Leslie and Summerell, 2006), of which some are pathogenic to plants, animals, and humans. This fungal genus produces three types of asexual spores: macroconidia, microconidia, and chlamydospores. There are certain morphological characters useful in identifying and differentiating species within the genus Fusarium (Leslie and Summerell, 2006) such as the presence or absence of microconidia or chlamydospores and the shape of macroconidia in sporodochia (Nelson et al., 1994). Fusarium graminearum Schwabe, (telemorph: Gibberella zeae (Schwein) Petch) is one of the major species in genus Fusarium that causes billions of dollars loss each year. This genus was the major causal agent of Fusarium Head Blight of wheat and related cereals and Gibberella ear and stalk rot of corn, but later additional closely related species were also found to cause the disease and hence this species is now considered as Fusarium graminearum species complex (FGSC). However, F. graminerum sensu lato is the predominant species in the United States (Horevaj et al., 2011). Recently, two novel species of Fusarium were discovered from the global surveillance of 2100 isolates, namely F. gerlachii and F. vorosii that fall within the FGSC (Starkey et al., 2007). In the United States, head scab alone caused an estimated loss of $ 3 billion on wheat and barley in 1990s (Windels, 2000). Fusarium graminearum is homothallic and sexual reproduction involves the development of perithecia that produce ascospores. The macroconidia are slender, elongated 2

18 containing 3-5 cells separated by septa and they play an important role in dissemination of the disease (Harris, 2005; Leslie and Summerell, 2006). Harris et al. (2005) explained that the apical cells are gradually tapering towards a rounded end and are the preferred site of germination, while bipolar germination is also commonly observed for the exploration of the local environment. The differences in morphology of the macroconidia and the culture can help in differentiating Fusarium graminearum from other closely related species. For instance: Fusarium graminearum isolates can be distinguished from isolates of F. sporotrichioides based on the absence of microconidia in the former even though both form similar colonies on PDA (Leslie and Summerell, 2006). Cultures of F. graminearum and F. culmorum (W. G. Smith) Saccardo, form red pigments in agar and lack microconidia, but F. culmorum forms rings of spore masses under alternating conditions of light and temperature (Leslie and Summerell, 2006), while F. graminearum does not. In addition, F. graminearum have slender, sickle-shaped, thick walled macroconidia with a distinct foot-shaped basal cell and tapered apical cell, while F. culmorum have short and stout macroconidia with a poorly developed foot cell and rounded apical cell (Leslie and Summerell, 2006). Initially, seven distinct phylogenetic lineages of F. graminearum species complex were found to exist throughout the world (O Donnell et al., 2000). Later, O Donnell et al. (2004) recognized nine phylogenetically distinct species within the FGSC based on genealogical concordance of genes. Recently, using a high resolution SNP-based multilocus genotyping (MLGT) assay combined with genealogical concordance phylogenetic species recognition (GCPSR) and molecular markers, the diversity within the F. graminearum complex led to the identification of several novel species 3

19 adding six distinct phylogenetic lineages to the previously described seven (Wang et al., 2011). Multilocus genotyping (MLGT) is a technique used as a guide to identify and characterize isolates of a microbial species by using the DNA sequences of multiple housekeeping genes internal fragments, and in general approximately 500bp internal fragments of each gene are used. Genealogical Concordance Phylogenetic Species Recognition (GCPSR) is a concept used to recognize and identify species based on comparison and concordance of multiple gene genealogies (Taylor et al, 2000). As underscored by Starkey et al. (2007), there are now 13 distinct phylogenetic lineages of F. graminearum and 16 phylogenetically divergent isolates and the number of distinct lineages is expected to increase with additional analysis. In addition to being pathogenic and causing yield loss, these species produce secondary metabolites such as mycotoxins: deoxynivalenol (DON), nivalenol (NIV) and zearalenone (ZEA). These are three of the most important mycotoxins that fall under type B trichothecenes produced by Fusarium species and are toxic to humans, animals, plants, insects and other microorganisms. These mycotoxins are also important in fungal pathogenesis and antagonism (Mule et al., 2005). In plants, Type B trichothecenes are produced by every species in the Fusarium graminearum complex (O Donnell et. al, 2008). The European and American isolates of F. graminearum produce DON whereas those isolates from Asia produce NIV and this mycotoxin is considered to be more toxic to humans and animals compared to DON (Mule et al., 2005). DON types can be further divided into 3-acetyl deoxynevalenol (3ADON) chemotype and 15- acetyl deoxynevalenol (15ADON) chemotype. But, F. graminearum isolates containing NIV or 3ADON chemotypes were found in the United States (Gale et al., 2011; Starkey et al., 2007). F. asiaticum producing NIV was also found for the first time in the United States (Gale et al., 2011). Furthermore, 4

20 a greenhouse experiment demonstrated NIV-type F. asiaticum was more aggressive than NIV-type F. graminearum when tested on wheat. A more recent study by Foroud et al. (2012) found that the aggressiveness of the isolates from wheat increased from NIV to 15ADONα to 15ADONβ to 3ADON chemotypes when point inoculated 10 µl of the 40,000 macroconidia ml -1 suspension of each chemotype at anthesis. The different chemotypes within the Fusarium graminearum complex with the appearance of NIV type in North America and the absence of standard NIV screening might pose a potential threat as NIV is more toxic in mammalian system. The study by Gale et al. (2011) included approximately 324 isolates from six Midwestern states: Illinois, Indiana, Kansas, Missouri, Nebraska and Ohio. There were 40 isolates of F. graminearum from six different counties in Ohio (Drake, Defiance, Mercer, Putnam, Van Wert and Wood) collected in three different years (2002, 2004, and 2006) included in the study. Their results suggested that F. graminearum in the mid western United States belongs predominantly to the MW15ADON population containing 15ADON trichothecene type. Since, this study included isolates only from six counties, the chemotype might not be the same in all the other parts of Ohio. Gale et al. (2012) further categorized Fusarium graminearum in United States into 3 different populations: MW15ADON population that contained predominantly 15ADON chemotype, Gulf Coast population that contains all 3 chemotypes (3ADON, 15ADON and NIV) and new Southern Louisiana population that predominantly contained NIV chemotype. 5

21 Fusarium seed and seedling rot of soybean Prior to 2004, F. graminearum was considered to be important pathogen of cereals only, especially wheat, oat and barley, causing head blight, popularly known as wheat head scab, and Gibberella ear and stalk rot of corn. This pathogen was also reported to reduce seedling emergence and cause root rot in several other crops that include rye, triticale, canary seed, flax, bean, lentil, and chickpea (Chongo et al., 2001). More recently, F. graminearum as a pathogen of soybean was reported from Argentina (Pioli et al., 2004) and subsequently from Brazil (Martinelli et al., 2004) and North America (Broders et al., 2007; Xue et al., 2007) in areas where soybean is rotated with wheat and/or corn. In addition, F. graminearum was also recently reported as a pathogen of potato (Ali et al., 2005), sugarbeet (Hanson, 2006) and dry beans (Bilgi et al., 2011). This indicates that F. graminearum is expanding its host range and causing disease to a wide range of crops and can become a threat to majority of crops in the near future. Recently, another species, F. proliferatum was reported to be highly pathogenic to soybean (Díaz Arias et al., 2011; Díaz Arias et al., 2013a), which was previously only considered to be a pathogen of corn. Symptoms and Signs of Fusarium graminearum in soybean In soybean, initial symptoms due to F. graminearum infection are observed as water soaked lesions followed by light brown or pink discoloration around the inoculation point at later stages. Pre- and post-emergence damping off, seed and seedling rot and root rot are other symptoms observed in North America (Broders et al., 2007; Ellis et al., 2012; Pioli et al., 2004; Xue et al., 2007). Additional symptoms observed in South America only include spreading of discoloration vertically on stem, interveinal 6

22 chlorosis of leaves leading to plant wilting, death, and pod blight (Martinelli et al., 2004; Pioli et al., 2004). Literature Review In a study by Martinelli et al. (2004), strains of F. graminearum complex obtained from soybean seeds, collected from the Southern Brazil, were pathogenic to soybean. They used macroconidia from six strains isolated from soybean and challenged seedlings and pods of adult plants. Ten ml of 10 3 macroconidia ml -1 suspension was added to pots with vermiculite containing 11-day old soybean plants and also to pots containing soil mixture prior to sowing seeds. All of the strains were pathogenic to soybean as primary pathogen, capable of invading the host tissue to initiate disease symptoms. They were also virulent to wheat causing Fusarium Head Blight. In addition, when soybean pods were inoculated at the beginning of the seed formation stage (R5 stage) with a macroconidial suspension of 10 3 spores ml-1, pod blight developed with brown, sunken necrotic spots, observed on the seeds along with the fluffy white mycelium on the heavily infected seeds. This study was the first to confirm F. graminearum as a primary pathogen of soybean and demonstrated that F. graminearum are capable of infecting seeds as the strains were first isolated from seeds. Detectable level of DON was produced in artificially inoculated pods and strains from soybean produced more NIV than DON. Furthermore, observation of pod blight in this study points to the potential threat of higher yield loss and mycotoxin contamination in the future. Similarly, Xue et al. (2007) compared the aggressiveness and pathogenicity of two closely related species, F. graminearum and F. pseudograminearum Aoki and 7

23 O Donnell, under controlled conditions and root rot severity symptoms were rated on a 0-4 rating scale. The results showed reduction in plant dry matter and shoot length. They concluded that F. graminearum was more pathogenic than F. pseudograminearum and there was variation in the aggressiveness among the isolates within the species evaluated for soybean root rot. Xue et al. (2007) suggested that the variation in the aggressiveness among the isolates might be due to variable adaptation of F. graminearum to the different hosts that are in the crop rotation. In North America, wheat and corn are grown in rotation with soybean. And, this pathogen now is capable of infecting all 3 crops, which are the major field crops grown in the world with a threat of declining the production and productivity of these crops. In an another study, Broders et al. (2007) isolated and identified several different Fusarium spp. associated with the seed and seedling damping-off and decline of both corn and soybean from different fields in Ohio. Broders et al. (2007) further tested the pathogenicity of isolates on corn and soybean. In addition, they also evaluated the sensitivity of F. graminearum to commonly-used commercial seed treatment fungicides. Pathogenicity was evaluated using seed-petri-plate and green house assays which showed that F. graminearum was both a seed and seedling pathogen of both corn and soybean. They confirmed that F. graminearum was more pathogenic to soybean compared to other species of Fusarium and there was variation in the levels of aggressiveness among the isolates. The studies by Martinelli et al (2004), Xue et al. (2007) and Broders et al. (2007) confirmed that F. graminearum is a primary pathogen of soybean compared to studies from 20 to 30 years prior that had concluded that F. graminearum was not a pathogen of soybean. Later, Ellis et al. (2011) studied the effect of inoculum density, 8

24 temperature and fungicide seed treatments on the disease development in the soybean seeds under the controlled conditions. They used isolates of F. graminearum collected from different symptomatic seedlings of corn, wheat and soybean from different fields in Ohio. They found that a minimum concentration of 2.5 x 10 4 macroconidia ml -1 was essential to obtain the disease severity of more than 50% on susceptible cultivars at the temperatures studied (18, 22 and 25 o C). F. graminearum isolate Fay 11 was one of the aggressive isolates obtained from soybean. They also concluded that new seed treatment fungicides (fludioxonil and strobilurins) were not as effective in managing F. graminearum as compared to the older ones, which supported the results of Broders et al. (2007). In 2012, Ellis et al., evaluated 24 soybean genotypes for resistance to F. graminearum. Five genotypes were found resistant: Prohio, Conrad, PI B, HC and PI Subsequently, four Quantitative Trait Loci (QTL) were identified on chromosomes 8, 13, 15, and 16 with the resistance alleles from Conrad and one QTL on chromosome 19 from Sloan, from a population of 262 F 6:8 recombinant inbred lines (RILs) derived from a cross of Conrad x Sloan. Since, F. graminearum was recently reported as a seedling pathogen to soybean, only 5 QTL have been identified for resistance (Ellis et al., 2012). In another study, nine commercial soybean cultivars of 57 were resistant to F. graminearum belonging to maturity group 000 to 0 (Zhang et al., 2010). In the past, many different methods were studied and suggested to effectively manage F. graminearum in fields such as crop rotation, host resistance, use of fungicides, and 9

25 use of fungal antagonists. A study by Chongo et al. (2001) showed that other cereals and pulse crops used in crop rotation might not help to reduce F. graminearum inoculum in fields where wheat and barley were also grown. The long term practice of no-till or reduced tillage in fields where there have been outbreaks of wheat head scab or Gibberella ear and stalk rot may have played a role in the development of F. graminearum as a pathogen to soybean in Ohio (Ellis et al., 2012). In addition, cooler temperature and high moisture levels during the planting season and changes in fungicide chemistries could be the other possible reasons (Broders et al., 2007; Ellis et al., 2011). No isolates that are fungicide insensitive have been identified in the field, but development of mutants that are resistant to the seed treatment fungicides, is a possibility (Broders et al., 2007). Therefore, the use of fungicide seed treatments alone as a measure to control F. graminearum might not be a long term strategy. Similarly, there was another study to manage F. graminearum in which several fungal antagonists were evaluated for the management of Fusarium graminearum by Luongo et al. (2005). In their bioassay, they used several saprophytic fungi obtained from the tissues of cereals and necrotic tissues of other crops to observe their ability to colonize wheat straw and corn stalk to suppress the sporulation of various pathogenic Fusarium spp. They concluded that the effect of many antagonists used in their study under controlled conditions were inconsistent except for isolates of Clonostachys rosea (Link: Fries) Schroers, Samuels, Seifert & W. Gams (syn. Gliocladium roseum) in demonstrating antagonism towards several Fusarium species that included F. graminearum. They did not find any significant correlation for the efficacy of the use of fungal antagonist, neither on different substrates used nor against the different Fusarium species. Isolates of Trichoderma harzianum Rifai, and Trichoderma viride 10

26 Pers.: Fries, included in this test only had a moderate effect on the reduction of sporulation of the Fusarium species. However, in a study by Zhang et al. (2009), novel Bacillus subtilis strains were found to reduce the mycelia growth and inhibit the spore germination in F. oxysporum and F. graminearum. Based on their greenhouse experiment, they suggested that the strains identified in their study may be effective alternatives for fungicides and soil treatment of these strains might provide better efficacy compared to seed treatment. Since the study was conducted under green house conditions, the result might not be replicated under field conditions due to high variations in the environmental conditions. More recently, Garcia et al. (2012) evaluated the growth and mycotoxin production by F. graminearum and F. verticillioides at different constant and cycling temperatures, in Argentina. Garcia et al. (2012) used milled soybean agar media and irradiated soybean seeds in petri dish using the isolates from Argentinean soybean. They found that F. graminearum had better growth when incubated at 15/20 o C and 15/25 o C (isothermal or cycling temperature). The higher level of DON and ZEA were recorded in soybean seeds at 15 days of incubation. DON production was found at higher level than to ZEA and temperature for higher DON production was 15 or 15/20 o C. Similarly, in a study by Barros et al. (2012) in Argentina, high frequency of Fusarium contamination was observed at the reproductive growth stages of soybean planted after wheat. Out of 389 Fusarium isolates recovered from the three reproductive stages (flowering, R6 and R8), 45% were isolated from pods, 38% from seeds and 17% from flowers. Fusarium graminearum species complex amounted to 11% of the total species that were present among the three stages, evaluated with the higher isolation in pods and seeds at R6 stage followed by pods and seeds in R8 stage and 11

27 flowers. In addition, seed samples from R6 and R8 stage were evaluated for DON and NIV contamination. Out of 40 samples evaluated, one sample from each stage had detectable DON contamination while NIV was not detected. Hence, they demonstrated the natural contamination of DON in soybean during the grain ripening stage, but the level of DON was less in soybean compared to wheat. In North America, F. graminearum is a seed, seedling and root pathogen, but in Brazil (Martinelli et. al., 2004) and in Argentina (Garcia et al., 2012; Barros et al., 2012), this is also a pathogen of seed and pod, which has not yet been reported in North America. In recent studies, F. graminearum was found to be highly aggressive in causing severe root rot in soybean compared to the other species (Broders et al., 2007; Díaz Arias et al., 2013 a; Ellis et al., 2011; Zhang et al., 2010). F. graminearum was not the most abundant species of Fusarium isolated at the field level, but was one of the fewer species frequently isolated from the soybean roots (Díaz Arias et al., 2013 b; Zhang et al., 2010). Therefore, seed, seedling and root rot caused by F. graminearum requires greater attention and warrants research for effective and long term management strategy before it becomes a major threat and causes major consequences towards yield, food quality and health. Identifying sources of host resistance may be the best management strategy for necrotrophic seedling pathogens affecting soybean (Ellis, 2011). The currently labeled fungicides and use of antagonists may not be as promising for management of this disease in field crops as the use of agrochemicals, and crop management strategies were only partly effective in controlling F. graminearum in another host crop, wheat (reviewed by Buerstmayr et al., 2009). 12

28 In crop plants, many traits such as yield, quality and disease resistance have been found to have quantitative variations resulting from the combined effect of the expression of several segregating genes and environmental factors (review by Asín, 2002). F. graminearum, as a head blight pathogen in wheat is more extensively studied than in any other crops. There are a plethora of studies showing that inheritance of resistance of wheat to FHB is quantitative and QTL for FHB resistance have been found on all the chromosomes except chromosome 7D (review by Buerstmayr et al., 2009). Fusarium strains/ isolates with wide differences in their aggressiveness have been detected but no biological races with specific host-pathogen interaction with wheat have been found to date (reviewed by Buerstmayr et al., 2009). Resistance to head blight in wheat, categorized into two types (Schroeder and Christensen, 1963) have been widely accepted compared to the five types. Type I resistance is resistance to initial infection and Type II resistance is resistance to spread of blight symptoms within the spike. Horevaj et al. (2011) found that the wheat lines having resistance to isolates of DON chemotype had more resistance to isolates of NIV chemotype of F. graminerum. Similarly, the study by Foroud et al. (2012) showed that aggressiveness increased from NIV to 15ADONα to 15ADONβ to 3ADON chemotypes. In an another study, Toth et al. (2008) found that the resistance in wheat cultivars to all the members of the F. graminearum species complex and lineages of F. culmorum is not specific and that one pathogenic isolate of any Fusarium species is enough for resistance breeding, even though isolates differing in aggressiveness and DON producing ability are found. Hence, resistant wheat genotypes can be cultivated successfully in the fields where different species of Fusarium are dominant or occur in a mix (Toth et. al., 2008). In addition to causing head blight, Fusarium graminearum has also been reported to cause Fusarium 13

29 seedling blight in wheat when it is seed borne and produce DON in the grain (Tamburic-Ilincic et. al., 2009). The authors identified a single QTL that controlled seedling blight, which was different from the QTL identified to control head blight. Similarly, Somers et al. (2003) reported that resistance to head blight and resistance to DON accumulation were controlled by different QTL. This signifies that the QTL conferring resistance to Fusarium seedling blight and head blight are different even though are caused by the same pathogen. Fusarium graminearum causes Gibberella ear and stalk rot of corn. F. graminearum infects corn ears through the silk channel or through wounds made by birds and insects damaging the husk tissue and grain (Chungu et al. 1996; Reid and Hamilton, 1996; Reid et al., 1992a and b). There are also two types of resistance to Gibberella ear rot (Chungu et al., 1996; Reid et al., 1992a and b). Silk resistance prevents the fungus from growing down the silk to the kernel. Kernel resistance in the kernel tissue prevents the fungus spreading from kernel to kernel when it overcomes the silk resistance. Ali et al. (2005) utilized recombinant inbred line population and detected 11 QTL for silk resistance and 18 QTL for kernel resistance and two of them were associated with both silk and kernel resistance. Similarly, Martin et al. (2011) using double haploid lines identified six major effect QTL that significantly enhanced ear rot resistance, four QTL that significantly lowered DON contamination and five QTL that lowered zearalenone contamination. This indicates that resistance to corn ear rot is quantitative as well. Mesterházy (1982) found a good correlation between the reaction of corn hybrids to F. graminearum and F. culmorum which showed possible common resistance mechanisms to these two pathogens. 14

30 Similarly, Gibberella stalk rot in corn is another devastating disease caused by F. graminearum. Both qualitative and quantitative resistances have been reported to stalk rot. Pè et al (1993) detected five resistance QTL to Gibberella stalk rot using inbred lines. Likewise, Yang et al. (2010) detected two QTL, one being a major QTL which was confirmed in a double haploid, F2 and back cross population. In addition, a single resistance gene Rfg1 has been detected to provide resistance to stalk rot (Yang et al., 2004). For F. verticilloides (Sacc.) Nirenberg, a closely relates species that causes Fusarium ear rot in corn, the resistance was quantitative and at least three QTL were detected (Ding et al., 2008). Fusarium virguliforme (ex. F. solani f. sp. glycines) is another species of Fusarium that causes sudden death syndrome (SDS) in soybean. Root rot and interveinal chlorosis (leaf scorch) from a toxin are key symptoms. Farias Neto et al. (2007) detected three resistance QTL in a Ripley x Spencer population and two QTL in PI x Omaha population. In the same study, one resistance QTL was mapped to the same location in both the populations which could be effectively utilized in SDS resistance breeding. Similarly, Yamanaka et al. (2006) detected four additional QTL different from those previously described for resistance to F. tucumaniae sp. nov., which also causes SDS in South America using RILs derived from Misuzudaizu and Moshidou Gong 503, a susceptible and resistant parent respectively. Similar to wheat and corn where two types of resistance were observed, separate loci were found to confer resistance to root infection and leaf scorch towards SDS in soybean (Kazi et al., 2008). To date, 14 QTL have been confirmed to be associated with SDS resistance (Luckew et al., 2013). 15

31 At Ohio State, Dorrance and Schmitthenner (2000) examined 1,015 soybean Plant Introductions (PI) obtained from the USDA Soybean Germplasm Collection, Urbana, IL., and identified 162 PIs which are resistant to three (7, 17 and 25) races of Phytophthora sojae, and 32 of which had resistance to additional 5 other races of P. sojae. In addition, more than 680 PIs, which were primarily from South Korea, had high level of partial resistance. Many of these PIs have been used in the development of new germplasm. Therefore, this soybean germplasm is a high priority to evaluate for resistance to F. graminerum to facilitate the identification of soybean germplasm which can serve as a source of host resistance to multiple seedling pathogens. In addition, many of these lines have been crossed with soybean genotypes that are susceptible to F. graminearum and large recombinant inbred line populations are available for mapping purposes. Fusarium graminearum and soybean genome both have been sequenced. The genome sequence of F. graminearum is available in the website of Broad Institute ( (Cuomo et al., 2007, Broad Institute, 2007). The assembly of soybean genome is about 85% complete and the remaining sequence is believed to be primarily consisting of the repetitive sequence with the expectation of the presence of nearly all the genes in the genome sequence (Cannon and Shoemaker, 2012). In addition, integrated physical, genetic and genome sequence map of F. graminearum was prepared by Chang et al. (2007) that can aide in studying the genes involved in pathogenicity and virulence of this fungus by utilizing the information on position of the genes in the genome. Similarly, the soybean genome is also now available in the website, Phytozome ( (Schmutz et al., 2010) and has 16

32 been a very useful tool in identifying molecular markers and gene associated with resistance. Therefore, the major objectives of this thesis were as follows: 1. Evaluate additional sources of resistance in soybean germplasm to F. graminearum 2. Characterize the resistance to F. graminearum using two populations of soybean 3. Identify molecular markers associated with loci that contribute to the expression of resistance so that this resistance can be incorporated into new disease resistant soybean cultivars 17

33 References Ali, M. L., Taylor, J. H., Jie, L., Sun, G., Williams, M., Kasha, K. J., Reid, L. M.,and Pauls, K. P Molecular mapping of QTLs for resistance to Gibberella ear rot, in corn, caused by Fusarium graminearum. Genome 48: Ali, S., Rivera, V. V., and Secor, G. A First report of Fusarium graminearum causing dry rot of potato in North Dakota. Plant Dis. 89:105. Asín, M. J Review: Present and future of quantitative trait locus analysis in plant breeding. Plant Breeding 121: Bai, G. -H., Plattner, R., Desjardins, A., and Kolb, F Resistance to Fusarium head blight and deoxynevalenol accumulation in wheat. Plant Breeding 120: 1-6. Barros, G., Alaniz Zanon, M. S., Abod, A., Oviedo, M.S., Ramirez, M.L., Reynoso, M. M., Torres, A.,and Chulze, S Natural deoxynivalenol occurrence and genotype and chemotype determination of a field population of the Fusarium graminearum complex associated with soybean in Argentina. Food Additives & Contaminants 29: Bilgi, V. N., Bradley, C. A., Mathew, F. M., Ali, S., and Rasmussen, J. B Root rot of dry edible beans caused by Fusarium graminearum. Online. Plant Health Progress doi: /php rs. Broders, K. D., Lipps, P. E., Paul P. A., and Dorrance, A. E Evaluation of Fusarium graminearum associated with corn and soybean seed and seedling in Ohio. Plant Dis. 91: Buerstmayr, H., Ban, T., and Anderson, J. A QTL mapping and markerassisted selection for Fusarium head blight resistance in wheat : a review. Plant Breeding 128: Cannon, S. B. and Shoemaker, R. C Evolutionary and comparative analyses of the soybean genome. Breeding Science 61: Chang, Y., Cho, S., Kistler, H. C., Hsieh, C., and Muehlbauer, G.J Bacterial artificial chromosome-based physical map of Gibberella zeae (Fusarium graminearum). Genome 50:

34 Chongo, G., Gossen, B. D., Kutcher, H. R., Gilbert, J., Turkington, T. K., Fernandez, M. R., and McLaren, D Reaction of seedling roots of 14 crop species to Fusarium graminearum from wheat heads. Can. J. Plant Pathol. 23: Chungu, C., Mather, D. E., Reid, L. M., and Hamilton, R. I Inheritance of kernel resistance to Fusarium graminearum in maize. J. Heredity 87: Cuomo, C. A., Gϋldener, U., Xu, J-R.,Trail, F., et al The Fusarium graminearum genome reveals a link between localized polymorphism and pathogen specialization. Science 317: Díaz Arias, M. M., Leandro, L. F., and Munkvold, G. P. 2013a. Aggressiveness of Fusarium species and impact of root infection on growth and yield of soybeans. Phytopathology 103: Díaz Arias, M. M., Munkvold, G. P., Ellis, and Leandro, L. F. S First report of Fusarium proliferatum causing root rot on soybean (Glycine max) in the United States. Online. Díaz Arias, M. M., Munkvold, G. P., Ellis, M. L., and Leandro, L. F. S. 2013b. Distribution and frequency of Fusarium species associated with soybean roots in Iowa. Plant Dis. 97: Dill-Macky, R., and Jones, R. K The effect of previous crop residues and tillage on Fusarium head blight of wheat. Plant Dis. 84: Ding, J-Q, Wang, X-M, Chander, S., Yan, J-B, and Li, J-S QTL mapping of resistance to Fusarium ear rot using a RIL population in maize. Mol. Breeding 22: Dorrance, A. E., and Schmitthenner, A. F New sources of resistance to Phytophthora sojae in the soybean plant introductions. Plant Dis. 84: Ellis, M. L., Broders, K. D., Paul, P. A., and Dorrance, A. E Infection of soybean seed by Fusarium graminearum and effect of seed treatments on disease under controlled conditions. Plant Dis. 95: Ellis, M. L., Wang, H., Paul, P. A., Martin, S. K. St., McHale, L. K. and Dorrance, A. E Identification of soybean genotypes resistant to Fusarium graminearum and genetic mapping of resistance Quantitative Trait Loci in the cultivar Conrad. Crop Science 52: Farias Neto, A. L., Hashmi, R., Schmidt, M., Carlson, S. R., Hartmen, G. L., Li, S., Nelson, R. L., and Diers, B. W Mapping and confirmation of a new sudden death syndrome resistance QTL on linkage group D2 from the soybean genotypes PI and Ripley. Mol. Breeding 20: Foroud, N. A., McCormick, S. P., MacMillan, T., Badea, A., Kendra, D. F., Ellis, B. E., and Eudes, F., Greenhouse studies reveal increased aggressiveness of 19

35 emergent Canadian Fusarium graminearum chemotypes in wheat. Plant Dis. 96: Gale, L. R., Harrison, S. A, Ward, T. J., O Donnell, K., Milus, E. A., Gale, S. W., and Kistler, H. C Nivalenol-type populations of Fusarium graminearum and F. asiaticum are prevalent on wheat in southern Louisiana. Phytopathology 101: Garcia, D., Barros, G., Chulze, S., Ramos, A. J., Sanchis, V., and Marín, S Impact of cycling temperatures on Fusarium verticilloides and Fusarium graminearum growth and mycotoxins production in soybean. J. Sci. Food Agric. 92: Hanson, L. E Fusarium yellowing of sugar beet caused by Fusarium graminearum from Minnesota and Wyoming. Plant Dis. 90:686. Harris, S. D Morphogenesis in germinating Fusarium graminearum macroconidia. Mycologia, 97: Horevaj, P., Gale, L. R., and Milus, E. A Resistance in winter wheat lines to initial infection and spread within spikes by deoxynivalenol and nivalenol chemotypes of Fusarium graminearum. Plant Dis. 95: Kazi, S., Shultz, J., Afzal, J., Johnson, J., Njiti, V., Lightfoot, D Separate loci underlie resistance to root infection and leaf scorch during soybean sudden death syndrome. Theor. Appl. Genet.116: Leslie, J. F., and Summerell, B. A The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA. Luckew, A. S., Leandro, L. F., Bhattacharyya, M. K., Nordman, D. J., Lightfoot, D. A., and Cianzio, S. R. Usefulness of 10 genomic regions in soybean associated with sudden death syndrome resistance. Theor. Appl. Genet. 126: Luongo, L., Galli, M., Corazza, L., Meekes, E., Haas. L. D., Plas, C. L. V., and Kohl, J Potential of fungal antagonists for biocontrol of Fusarium spp. in wheat and maize through competition in crop debris. Biocont. Sci. Tech. 15: Martin, M., Miedaner, T., Dhillon, B. S., Ufermann, U., Kessel, B., Ouzunova, M., Schipprack, W., and Melchinger, A. E Colocalization of QTL for Gibberella ear rot resistance and low mycotoxin contamination in early European maize. Crop Sci. 51: Martinelli, J.A., Bocchese, C.A.C., Xie, W., O Donnell, K. & Kistler, H.C Soybean pod blight and root rot caused by lineages of the Fusarium graminearum and the production of mycotoxins. Fitopatol. Bras. 29: Mesterházy, Á Resistance of corn to Fusarium ear rot and its relation to seedling resistance. Phytopathology 103:

36 Mule, G., González-Jaén, M. T., Hornok, L., Nicholson, P., and Waalwijk, C Advances in molecular diagnosis of toxigenic Fusarium species: A review. Food Additives and Contaminants 22: Nelson, P. E., Dignani, M. C., and Anaissie, E. J Taxonomy, biology, and clinical sspects of Fusarium species. Clinical Microbiol. Reviews 7: O Donnell, K., Ward, T. J., Geiser, D. M., Kistler, H. C., and Aoki, T Genealogical concordance between the mating type locus and seven other nuclear genes supports formal recognition of nine phylogenetically distinct species within the Fusarium graminearum clade. Fungal genetics and Biology 41: O Donnell, K., Kistler, H. C., Tacke, B. K., and Casper, H. H Gene genealogies reveal global phylogeographic structure and reproductive isolation among lineages of Fusarium graminearum, the fungus causing wheat scab. Proc. Natl. Acad. Sci. USA 97: O Donnell, K., Ward, T. J., Aberra, D., Kistler, H. C., Aoki, T., Orwig, N., Kimura, M., Bjornstad, A., Klemsdal, S. S Multilocus genotyping and molecular phylogenetics resolve a novel head blight pathogen within the Fusarium graminearum species complex from Ethiopia. Fungal Genet. Biol. 45: O Donnell, K., Ward, T.J., Geiser, D.M., Kistler, H.C. and Aoki, T Genealogical concordance between the mating type locus and seven other nuclear genes supports formal recognition of nine phylogenetically distinct species within the Fusarium graminearum clade. Fungal Genet. Biol. 41: Park, J. S., Lee, K. R., Kim, J. C., Lim, S. H., Seo, J. A. and Lee, Y. W A hemorrhagic factor (Apicidin) produced by toxic Fusarium isolates from soybean seeds. Appl. Environ. Microbiol. 65: Pè, M. E., Gianfranceschi, L., Taramino, G., Tarchini, R., Angelini, P., Dani, M., and Binelli, G Mapping quantative trait loci (QTLs) for resistance to Gibberella zeae infection in maize. Mol. Gen. Genet. 241: Pioli, R. N., Mozzoni, L., and Morandi, E. N First report of pathogenic association between Fusarium graminearum and soybean. Plant Dis. 88:220. Reid, L. M., Mather, D. E., Hamilton, R. I., and Bolton, A. T. 1992a. Diallel analysis of resistance in maize to Fusarium graminearum infection via the silk. Can. J. Plant Sci. 72: Reid, L. M., Mather, D. E., Hamilton, R. I., and Bolton, A. T. 1992b. Genotypic differences in the resistance of maize silk to Fusarium graminearum. Can. J. Plant Pathol. 14: Reid, L.M., and Hamilton, R. I Effects of inoculation position, timing, macroconidial concentration, and irrigration on resistance of maize to Fusarium graminearum infection through kernels. Can. J. Plant Pathol. 18:

37 Schmutz, J., Cannon, S. B., Schlueter, J., Ma, J. et al. Genome sequence of the palaeopolyploid soybean. Nature 463: Schroeder, H. W., and Christensen, J. J Factors affecting resistance of wheat to scab caused by Gibberella zeae. Phytopathology 53: Snyder, W. C. and Hansen, H. N Variation and speciation in the genus Fusarium. Annals of the New York Academy of Sciences, Oct 29; 60: Somers, D. J., Fedak, G., and Savard, M Molecular mapping of novel genes controlling Fusarium head blight resistance and deoxynivalenol accumulation in spring wheat. Genome 46: Starkey, D. E., Ward, T. J., Aoki, T., Gale, L. H., Kistler, H. C., Geiser, D.M., Suga, H., Tóth, B., Varga, J., and O Donnell, K Global molecular surveillance reveals novel Fusarium head blight species and trichothecene toxin diversity. Fungal Genet. Biol. 44: Tamburic-Ilincic, L., Somers, D., Fedak, G. and Schaafsma, A Different quantitative trait loci for Fusarium resistance in wheat seedlings and adult stage in the Wuhan/Nyubai wheat population. Euphytica 165: Taylor, J. W., Jacobson, D. J., Kroken, S., Kasuga, T., Geiser, D. M., Hibbett, D. S., and Fisher, M. C Phylogenetic species recognition and species concept in fungi. Fungal Genetics and Biology 31: Toth, B., Kaszonyi, G., Bartok, T., Varga, J. and Mesterhazy, A Common resistance of wheat to members of the Fusarium graminearum species complex and F. culmorum. Plant Breeding 127:1-8. Wang, J. H., Ndoye M., Zhang J. B., Li H. P., and Liao Y. C Population structure and genetic diversity of the Fusarium graminearum species complex. Toxins 3: Windels, C. E Economic and social impacts of Fusarium head blight: Changing farms and rural communities in the Northern Great Plains. Phytopathology 90: Xue, A. G., Cober, E., Voldeng, H. D., Babcook, C., and Clear, R. M Evaluation of the pathogenicity of Fusarium graminearum and Fusarium pseudograminearum on soybean seedlings under controlled conditions. Can. J. Plant Pathol. 29: Yamanaka, N., Fuentes, F. H., Gilli, J. R., Watanabe, S., Harada, K., Ban, T., Abdelnoor, R. V., Nepomuceno, A. L., and Homma, Y Identification of quantitative trait loci for resistance against soybean sudden death syndrome caused by Fusarium tucumaniae. Pesq. Agropec. Bras. 41:

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39 Chapter 2: Evaluation of Soybean Genotypes to Identify Sources of Resistance Towards Fusarium graminearum Introduction Soybean [Glycine max (L.) Merr.] is the second largest crop grown in the United States following corn. Soybean is produced both for protein meal and oil and is consumed worldwide. During 2013 in the United States, approximately 3.3 billion bushels of soybean were harvested from 76.5 million acres with a yield of approximately 43.3 bushels per acre (National Agricultural Statistics Service: More than 80% of the soybeans produced in the United States originates from the north central states, including Ohio which has 4.5 million acres harvested with approximately 49 bushels per acre in 2013 (National Agricultural Statistics Service: Soybean cultivars grown in United States are classified into 13 maturity groups (MG), 000 to X. MG 000 to MG IV are adapted to the northern region while MG V to MG X are adapted to the southern region. Cultivars belonging to MG 000 to MG IV exhibit indeterminate growth habit and cultivars in MG V to MG X exhibit determinate growth habit. Indeterminate growth habit is characterized by a continuation of vegetative growth after flowering begins, whereas determinate growth habit stops vegetative growth shortly after flowering (Pederson, 2004). Most of the soybean cultivars grown in Ohio belong to MG II and MG III (Pederson, 2004). 24

40 During the past decade, the incidence and severity of diseases of soybean reported in Ohio increased due to intensified production and adaptation towards resistance genes by some pathogens, particularly Phytophthora sojae and Soybean cyst nematode, used in cultivars (A. Dorrance and C. Taylor, personal communication). In addition, no-till and reduced tillage production systems are widely adapted to protect soil from erosion, which contributes to the buildup of soil borne pathogens that survive on crop residues (Dorrance and Lipps, 2002). There are a number of diseases that limit the production of soybean and seedling diseases are among the major limitations. The decrease in soybean yield due to seedling diseases ranked second to sixth among all diseases that suppressed yield, with the highest reported losses from 2005 to 2007 (Wrather and Koening, 2009). Fusarium graminearum Schwabe, (telemorph: Gibberella zeae (Schwein) Petch) is an economically important pathogen of a number of cereal crops and causes tremendous losses each year worldwide. Windels (2000) reported that head scab caused an estimated loss of $ 3 billion on wheat and barley during the 1990s, in the United States alone. F. graminearum is homothallic and sexual reproduction involves the development of perithecia that produce ascospores. This fungus produces macroconidia as asexual spores, which are slender, elongated containing 3-5 cells separated by septa and they play an important role in dissemination of the disease (Harris, 2005; Leslie and Summerell, 2006). In addition to being pathogenic to a number of crops and causing extensive yield losses, this fungus also produces secondary metabolites known as mycotoxins: Deoxynivalenol (DON), nivalenol (NIV) and zearalenone (Leslie and Summerell, 2006; Mule et al., 2005). These are three of the most important mycotoxins that fall under type B trichothecenes produced by 25

41 Fusarium species and are toxic to humans, animals, plants, insects, and other microorganisms, which are also important in fungal pathogenesis and antagonism (Mule et al., 2005). Prior to 2004, F. graminearum was considered to be an important pathogen of cereals only, especially wheat, oat, barley, causing head blight, popularly known as wheat head scab, and Gibberella ear and stalk rot of corn. This pathogen was also reported to reduce seedling emergence and cause root rot in several other crops that include rye, triticale, canary seed, flax, bean, lentil, and chickpea under controlled conditions (Chongo et al., 2001). More recently, F. graminearum was reported as a pathogen of soybean from Argentina (Pioli et al., 2004) and subsequently reported from Brazil (Martinelli et al., 2004) and North America in areas where soybean is rotated with wheat and/or corn (Broders et al., 2007; Xue et al., 2007). In addition, F. graminearum was also recently reported as a pathogen of potato (Ali et al., 2005), sugarbeet (Hanson, 2006) and dry beans (Bilgi et al., 2011). In soybean, initial symptoms start as water soaked lesions, which turn light brown or have pink discoloration at the later stages. Additional symptoms observed in North America include pre- and postemergence damping off, seed and seedling rot and root rot (Broders et al., 2007; Pioli et al., 2004; Xue et al., 2007). Other symptoms associated with F. graminearum in soybean include vertical spreading of discoloration on stem, interveinal chlorosis of leaves leading to plant wilting and death and pod blight in South America (Martinelli et al., 2004; Pioli et al., 2004). In North America, it is primarily a seed and seedling pathogen of soybean (Broders et al., 2007; Ellis et al., 2011). The long term practice of no-till or reduced tillage in fields where there have been outbreaks of wheat head scab or Gibberella ear and stalk rot of corn may have played a 26

42 role in the development of F. graminearum as a pathogen to soybean in Ohio (Broders et al., 2007; Ellis et al., 2011). Producers in Ohio use corn-soybean or corn-soybeanwheat rotation in their fields and F. graminearum can infect all the three crops, posing a high risk of loss in fields with the history of F. graminearum problems (Ellis et al., 2011). Furthermore, cooler temperatures and high moisture levels during the planting season and changes in fungicide chemistries may contribute to disease development (Broders et al., 2007; Ellis et al., 2011). Cooler temperatures and high moisture might have delayed seed germination and some of the newer fungicide chemistries are reportedly not as effective as some older chemistries, which were more broad spectrum (Broders et al., 2007; Ellis et al., 2011). Seed, seedling and root rot caused by F. graminearum requires greater attention and warrants research for an effective and long term management strategy towards improved yield, food quality and health. Identifying and characterizing sources of host resistance and incorporating resistance into cultivars may be the best management strategy for necrotrophic seedling pathogens affecting soybean (Ellis et al., 2011). The currently labeled fungicides and use of antagonists may not be as promising for management of this disease in field crops as isolates resistant to the most common fungicides have already been identified (Broders et al., 2007). Likewise, the use of agrochemicals and cultural management strategies were only partly effective in controlling F. graminearum, especially in wheat (reviewed by Buerstmayr et al., 2009). Resistance to F. graminearum was identified readily in a small preliminary screen of 24 soybean genotypes in which five of them had moderate to high levels of resistance (Ellis et al., 2012). Five QTL were subsequently identified for resistance, 4 resistance 27

43 alleles were from Conrad and 1 from Sloan (Ellis et al., 2012). In another study, nine out of 57 commercial soybean cultivars were resistant to F. graminearum, which belonged to maturity group 000 to 0 (Zhang et al., 2010). The objective of this study was to screen soybean genotypes for additional sources of resistance towards F. graminearum. To fulfill this objective, there is a well defined strategy for the types of materials to first screen for resistance to plant pathogens. These include: currently grown commercial cultivars, obsolete commercial cultivars, breeding lines and stocks, landrace cultivars, germplasm collections, undomesticated forms of crop plants, and related species (Stoskopf et al., 1993). For soybean, there are several collections of lines that have been used to develop segregating populations (Nested Association Mapping) or represent a historical set of lines (Genetic Gain study) (Diers et al., 2011; Rowntree et al., 2013) that are also a high priority to screen for resistance. Parental lines of Soybean Nested Association Mapping (SoyNAM) population were selected specifically to map QTL contributing to seed yield and drought tolerance while the genetic gain study consisted of highest yielding cultivars from 1923 to Hence, these soybean genotypes were worth evaluating for the potential source of resistance to F. graminearum. Materials Parental Lines of Soybean Nested Association Mapping (SoyNAM) The soybean NAM population was developed by crossing 40 diverse soybean genotypes to a common parent IA3023 to generate multiple families of recombinant inbred lines (RILs). Each individual RIL represents a mosaic of chromosomal segments of the parental genotypes (Stich et al., 2010; Yu et al., 2008). The major goal for developing the Soy NAM population was to map QTL that contribute to seed yield, 28

44 draught tolerance, agronomic traits such as maturity, plant height, lodging and seed protein and oil content (Diers et al., 2011). The genotypes chosen to serve as parents included MG II-V cultivars, experimental lines and plant introductions in order to diversify the sources of various traits of interest. Of the 40 parents, 17 are high yielding parents from eight states, 15 are lines with diverse ancestry and 8 are plant introductions (Diers et al., 2011). Soybean Genetic gain /Decade study There were approximately 119 soybean cultivars included in a study (Rowntree et al., 2013) that represent high yielding cultivars released from 1923 to For the assays in this study only 39 and 36 public cultivars from MG II and MG III from the soybean genetic gain study were evaluated. The cultivars of the different maturity groups were evaluated separately. OSU/OARDC Germplasm This set of 82 soybean genotypes consists of parents of OARDC/OSU breeding lines, recently released cultivars and stocks. In addition, many of these genotypes have been used to develop populations segregating for resistance to aphids, Phytophthora sojae, powdery mildew, soybean cyst nematode, and other quality traits. These soybean genotypes were divided into two groups: 50 genotypes in one series of experiments and 32 genotypes in another. 29

45 Inoculum production Fusarium graminearum isolate Fay 11, originally collected from soybean was used as it was highly aggressive among the different isolates collected from soybean (Ellis et al., 2011). The isolate has been stored as a suspension of macroconidia in sterile 10% glycerol at -80 o C. Macroconidia were transferred directly from the fungal suspension to petri plates with Mung Bean Agar (MBA) and grown for 10 days under fluorescent light with 12hr light: dark cycle. Plates were then flooded with 5-10 ml sterile distilled water; macroconidia were dislodged with a glass rod and filtered through 4-6 layer cheese cloth. The final macroconidia suspension was determined using a hemacytometer (Bright-Line Hemacytometer; Hausser Scientific, Horsham, PA) and adding sterile distilled water for a final concentration of 2.5 x 10 4 macroconidia ml -1. This inoculum concentration was previously determined to be optimal for seed infection (Ellis et al., 2011). Phenotypic assay A rolled towel assay was used as described by Ellis et al. (2011) with slight modifications. Fifteen to twenty seeds from each genotype were placed on a germination paper moistened with deionized water and inoculated with a 100µl suspension of 2.5 x 10 4 macroconidia ml -1. A second moistened towel was placed over the inoculated seeds, rolled up and placed on the top of a wire mesh in the 25 L bucket. The buckets were covered and stored in the dark at room temperature. Ten seeds from each genotype in each group of materials were also tested for the non-inoculated germination percentage using the rolled towel assay. Ten days after inoculation (dai), lesion length and total plant length were measured for each seedling of each genotype. A Disease Severity Index (DSI) was calculated by dividing lesion length by total 30

46 length of the seedlings and multiplying by 100. Disease severity was also rated using a 1-5 scale, where, 1= germination and healthy seedlings with no visible signs of colonization; 2= germination and colonization of the root and 1 to 19% of the root with lesions; 3= germination and colonization of the seeds, and 20 to 74% of the root with lesions; 4= germination and colonization of the seed and 75% or more of the seedling rot with lesions; and 5= no germination and complete colonization of the seed. The experimental design was a Randomized Complete Block Design (RCBD) with each replicate completed at a different time. Each soybean genotype group was evaluated separately. The genotypes in SoyNAM were evaluated in three replications and the genotypes in genetic gain study and OSU/OARDC breeding lines and stocks were evaluated in two replications. Statistical Analysis DSI was expressed as a percentage values, since majority of the values were unevenly distributed (Gumpertz, 1995) were arc sine square root transformed (sine -1 x) prior to analysis. Mean disease severity index and mean score of each genotype was analyzed using Proc GLM of SAS program version 9.3 (SAS; SAS Institute Inc., Cary, NC). The mean transformed DSI for each entry within each assay was compared using Fisher s Protected LSD at P = Results Parental Lines of Soybean Nested Association Mapping (SoyNAM) Five genotypes had non-inoculated seed germination less than 70%, including HS6-3976, LD , LG , LG and LG and were not included in final analysis. 31

47 Disease developed on all the parental lines of SoyNAM that were inoculated with F. graminearum isolate Fay 11. The overall mean lesion length for all of the genotypes was 35.6 mm, mean total length of plant was 60.5 mm, mean DSI was about 82.2% and mean score was 4.2. The genotypes were significantly different (P < ) for disease severity index at 10 DAI and ranged from 56.8% to 96.5% prior to transformation (Table 1, Figure 1) with the average disease score ranging from 3.5 to 4.7 (Table 1, Figure 2). The genotype LD had the lowest DSI (56.8%) and the least disease score (3.5) while LG had the highest DSI (96.5%) and the highest mean disease score (4.7). Thirteen of the genotypes had DSI values less than the common parent IA 3023 albeit only four of them were significantly different (Table 1). Soybean Genetic Gain /Decade study Maturity Group II (MG II) In a non-inoculated test, there was poor germination indicative of poor seed quality in 15 cultivars. The germination was less than 70% in these cultivars so were removed from the final analysis. Cultivars with poor germination include Korean, Beeson, Beeson 80, Wells II, Loda, Vickery, Harosoy, Preston, RCAT Angora, Hawkeye 63, Century, Century 84, Corsoy 79, Amsoy and IA Disease developed on all the soybean cultivars evaluated for resistance towards F. graminearum isolate Fay 11 in this study. The overall mean lesion length was 20.3 mm, mean total length of plant was 61.8 mm, mean DSI was about 75.9 mm and mean score was 4.1. Based on the arc sine transformed data for DSI, the cultivars were significantly different from each other (P < ). The DSI ranged from 34.0% to 93.6% (Table 2, Figure 3) and the mean disease score ranged from 2.7 to 4.7 (Table 2, 32

48 Figure 4). The cultivar Corsoy had the lowest DSI of 34.0% with the mean score of 2.7, while the cultivar Jack was the most susceptible with a DSI of 93.6% and cultivar IA 2021 had the highest mean score of 4.7. Four cultivars were moderately resistant to F. graminearum as indicated by DSI values of less than 50% including: Hawkeye, Corsoy, IA 2050 and IA2052 (Table 2). Maturity Group III (MG III) In the non-inoculated germination check, three cultivars in MG III had germination less than 70% including Pana, Dunfield and Adams, which were not included in the final analysis. All the soybean cultivars in MG III developed disease following inoculation with F. graminearum isolate Fay 11. The overall mean lesion length was 32.5 mm, mean total length of plant was 94.4 mm, mean DSI was about 61.6 mm and mean score was 3.7. The cultivars were significantly different from each other (P < ) based on the arc sine transformed DSI. The DSI ranged from 30.0% to 94.3% (Table 3, Figure 5) and the mean disease score ranged from 2.7 to 4.6 (Table 3, Figure 6). The genotype IA 3024 had the lowest DSI of 30.0% with the mean score of 2.7, while the cultivar Mingo was the most susceptible with a DSI of 94.3% with the mean score of 4.6. There were five cultivars that had DSI values of less than 50% including Maverick, Macon, Dunbar, Resnik, and IA 3024, indicating that these cultivars have some partial resistance to F. graminearum. 33

49 OSU/OARDC Germplasm OSU/OARDC Germplasm I All the soybean genotypes in this group developed disease after inoculation with F. graminearum isolate Fay 11. Two genotypes had non-inoculated germination percentage less than 70%: PI B and Cloud (PI ), were removed from the final analysis. The overall mean lesion length of the genotypes was 26.5 mm, mean total length of plant was mm, mean DSI was about 54.0% and mean score was 3.3. Based on the arc sine transformed data for DSI, the genotypes were significantly different from each other (P < ). The DSI ranged from 2.4% to 92.5% (Table 4, Figure 7) and the disease score ranged from 1.3 to 4.4 (Table 4, Figure 8). The genotype PI had the lowest DSI of 2.4% with the mean score of 1.3, while the genotype OX 20-8 was the most susceptible, with a DSI of 92.5% and the score of 4.4. Ninteen out of the 48 (39.5%) genotypes evaluated had DSI less than 50% (Table 4) which indicated moderate to high levels of resistance towards F. graminearum. OSU/OARDC Germplasm II In the non-inoculated checks, Minimax had only 30% germination so was not included in the final analysis. All the other soybean genotypes in this group developed disease after the inoculation of F. graminearum isolate Fay 11. The overall mean lesion length of the second group of genotypes was 30.8 mm, mean total length of plant was 71.9 mm, mean DSI was about 70.3% and mean score was 3.8. Based on the arc sine transformed data, all the soybean genotypes were significantly different from each other (P < ). The DSI ranged from 39.6% to 96.2% (Table 5, Figure 9) and the mean disease score ranged from 2.9 to 4.4 (Table 5, Figure 10). 34

50 The genotype PI had the lowest DSI of 39.6% with the mean score of 2.9, while the genotype Hutcheson was the most susceptible, with a DSI of 96.2% and the score of 4.4. Two of the genotypes out of thirty two evaluated had DSI less than 50% (Table 5) indicating moderate levels of resistance towards F. graminearum. Discussion Identifying and characterizing additional sources of resistance will provide breeders with a broader range of soybean genotypes to incorporate into their respective breeding program. There was a broad range of responses observed among the 200 soybean genotypes that were evaluated for resistance to F. graminearum following inoculation in a rolled towel assay (Table 1, 2, 3, 4 and 5). All the parents of Soy NAM population were considered to be moderate to highly susceptible to F. graminearum as DSI ratings greater than 50% was measured for all the genotypes. In the Genetic Gain, four cultivars in MG II (Table 2) and five in MG III (Table 3) had DSI of less than 50%. The cultivars in MG II include IA 2052, Hawkeye, IA 2050, and Corsoy and the cultivars in MG III include Maverick, Macon, Dunbar, Resnik, and IA Cultivars in MG II and MG III that had resistant reaction to F. graminearum were cultivars released in 1947, 1967, 1987, 1992, 1995, 2002 and The cultivar Maverick also has resistance to SCN (Sleper et al., 1998), while Corsoy, Dunbar and Macon were selected as high yielding cultivars (Graef et al., 1992; Nickell et al., 1996; Weber and Fehr, 1970) and cultivar Resnik had high yield and Rps1k for Phytophthora sojae (McBlain et al., 1990). There were 21 soybean genotypes in the OSU/OARDC germplasm collection with DSI rating of less than 50%. These included 14 plant introductions, 2 cultivars and 5 35

51 recombinant inbred lines from populations evaluated for resistance to other pathogens of soybean. The genotypes PI 88788, PI , PI , PI and Peking (PI ), are the major sources of resistance used in the development of Soybean Cyst Nematode (SCN) (Heterodera glycines Ichinohe) resistant cultivars and are used as differentials for characterizing Heterodera glycines (HG) type (Niblack, 2005). All of the SCN sources of resistance had low DSI values ranging from 16.4% in Peking (PI ) to 45.9% in PI In contrast, cultivar Lee74 which is a standard susceptible to all the HG types of SCN (Niblack, 2005) was highly susceptible to F. graminearum with the DSI of 74.4%. This raises the possibility that the resistance to SCN and to F. graminearum may be linked or there are some common genetic factors conferring resistance to these entirely different pathogens of soybean. Fusarium virguliforme (ex. F. solani f. sp. glycines), another related species of Fusarium, causes sudden death syndrome (SDS) in soybean. There are inconsistent reports concerning the interaction between SCN and SDS (Chang et al., 1997; Gao et al., 2006) although presence of both pathogens increases the severity of sudden death syndrome (Gao et al., 2006), which leads to greater yield loss. Gibson et al. (1994) reported that the sources of SCN and F. graminearum resistance namely, Peking, PI 88788, PI and PI were susceptible to SDS, which indicates that the resistance to SCN and F. graminearum compared to SDS may be different. However, genomic regions conferring resistance to both SDS and SCN have been reported in Essex X Forrest derived RILs (Chang et al., 1997; Prabhu et al., 1999). Similarly, there is soybean germplasm registered that confers resistance to both SCN and SDS such as LS-G96 and Saluki 4441 (Kantartzi et al., 2012; Schmidt et al., 1999). The cultivar Forrest is resistant to both SCN and SDS while Essex is susceptible to both (Chang et al., 1997). In the present study, the cultivar Forrest was more susceptible to F. 36

52 graminarum with mean DSI of 70. 7% compared to Essex that had mean DSI of 52.3%. Therefore, the presence of common resistance as reported to all the members of F. graminearum species complex and lineages of F. culmorum in wheat cultivars (Toth et al., 2008) as well as in corn hybrids (Mesterházy, 1982) does not appear to hold true in case of F. graminearum and F. virguliforme in soybean. This may be due to the fact that F. graminearum and F. culmorum cause the same disease in wheat and corn while F. graminearum is a seed and seedling pathogen and F. virguliforme causes root infection and leaf scorch with different disease symptoms in soybean. However, additional research is required to determine if resistance to F. graminearum and F. virguliforme are similar in soybean. In addition to PIs that are the common sources of resistance to SCN, several other PIs were identified with resistance to F. graminearum which were also the sources of resistance to other pathogens of soybean. PI and PI have Rps gene mediated resistance to P. sojae (Hegstad et al., 1998; Lee et al., 2014), while PI and PI C have resistance to SCN (Arelli et al., 2010; Yue et al., 2001). PI B and PI A are resistant to soybean aphid (Mian et al., 2008). PI B is resistant to soybean aphid, powdery mildew and P. sojae race 25 (Jun et al., 2012a, Jun et al 2012b, GRIN online database). PI has high level of resistance to Pythium irregulare (Ellis et al., 2013) and multiple Rps genes for P. sojae (Dorrance and Schmitthenner, 2000; Gordon et al., 2007). Interestingly, there are no additional reports linking PI , which had the lowest DSI of 2.44%, with resistance to other pathogens. One cultivar Ohio FG5 with high seed yield, large seeds, Rps3 gene and partial resistance to P. sojae and high seed protein content (St. Martin et al., 2006) had a low DSI to F. graminearum. 37

53 There were differences observed in the DSI of genotypes evaluated in this study compared to the previous evaluations (Ellis et al., 2012). Cultivars Resnik, Ohio FG5 and Prohio had DSI of 32.5%, 35.1% and 89.8% respectively in this study compared to 86.2%, 80.9% and 41.5% respectively in the study by Ellis et al. (2012). However, cultivars Kottman, Dennison and PI had similar DSI in both the studies, the DSI were 81.3%, 68.2% and 39.6% respectively in the present study compared to 81.7%, 74.4% and 24% respectively in the study by Ellis et al. (2012). The differences were observed within this study as well. For instance: cultivar Williams 82 included in the genetic gain had DSI of 49.8% while the same genotype had DSI of 70.2% in the OSU/OARDC germplasm. The rolled towel assay depends on the quality of the seeds. Hence, the differences observed between and/or within the studies might be due to the differences in seed quality, seed age, seed lots and sources of seeds used in these evaluations. Further analysis is required to determine what seed quality factors may influence this response to inoculation. In conclusion, a total of 30 out of 200 soybean genotypes were identified with resistance to F. graminearum indicating that resistance to F. graminearum is readily available in soybean. Eleven soybean cultivars were identified that can be recommended for fields with high inoculum levels and the RILs identified can be utilized in developing a cultivar with resistance to at least two different pathogens of soybean. Likewise, the plant introductions (PIs) identified in this study can be used as sources of resistance to characterize and map loci associated with F. graminearum resistance. Many of the genotypes identified represent adapted germplasm and can 38

54 immediately be incorporated into a breeding program for development of cultivars with high levels of resistance to F. graminearum. 39

55 Soy NAM Parental Lines Mean DSI * Grouping Mean Score Grouping Non inoc. % germination LG A 4.7 A 90.0 LG AB 4.5 ABC 79.2 PI B 94.7 ABC 4.6 AB 73.3 LD ABCD 4.4 ABCDE 93.3 PI ABCDE 4.5 ABCD 72.5 U ABCDEF 4.4 ABCDEF 82.5 LD ABCDEFG 4.4 BCDEFG 90.0 PI ABCDEFG 4.3 BCDEFGH 93.3 Prohio 89.8 ABCDEFG 4.3 BCDEFG 72.5 NE ABCDEFG 4.3 DEFGHI 73.3 PI ABCDEFGH 4.3 CDEFGHI M BCDEFGH 4.2 DEFGHIJ 96.7 LG BCDEFGH 4.3 CDEFGHI 96.7 LG CDEFGH 4.2 DEFGHIJ LG DEFGHI 4.2 FGHIJKL 95.8 CLOJO DEFGHI 4.3 DEFGHI 92.5 LG CDEFGHI 4.2 DEFGHIJK 86.7 LG EFGHIJ 4.3 DEFGHI 66.7 Skylla 82.2 EFGHIJ 4.2 EFGHIJK 86.7 PI EFGHIJ 4.2 FGHIJKL 78.3 LG EFGHIJK 4.2 FGHIJKL 96.7 IA GHIJK 4.1 GHIJKL 96.7 LG FGHIJK 4.2 FGHIJKL 93.3 PI404188A 80.5 GHIJK 4.2 FGHIJKL 78.3 LG GHIJK 4.1 HIJKLM 72.5 Continued Table 1: Mean Disease Severity Index (DSI), mean score and non-inoculated percentage germination of parental lines, after excluding poor germinating genotypes, from the Soybean Nested Association Mapping (SoyNAM) population following inoculation of seeds with 100 µl of 2.5 x 10 4 macroconidia ml -1 of Fusarium graminearum in a rolled towel assay. * Disease severity index equals lesion length divided by total length of the seedling multiplied by 100. Disease severity data were arcsine transformed. The actual mean DSI are reported in the table. The experimental design was randomized complete block design with three replications repeated over time. Values with the same uppercase letter were not significantly different according to Fisher s protected Least Significance Difference (LSD) at α = 0.05, based on the arcsine transformed data. 40

56 Table1 continued Soy NAM Parental Lines Mean DSI * Grouping Mean Score Grouping Non inoc. % germination PI GHIJK 4.1 HIJKLM 78.3 CLOJ GHIJK 4.0 IJKLM 93.3 Maverick 75.9 HIJKL 3.9 KLMNO 88.3 PI437169B 73.5 IJKLM 4.0 JKLMN J KLM 3.9 LMNO 85.0 LG JKLM 3.9 KLMNO 90.0 LG LM 3.9 MNO 82.5 TN MN 3.8 NO 96.7 Magellan 63.5 MN 3.7 OP 93.3 LD N 3.5 P 85.0 Mean

57 20 SoyNAM Parental Lines 18 No. of SoyNAM parental Lines Disease Deverity Index (DSI) Figure 1: Distribution of parental lines, after excluding poor germinating genotypes, of Soybean Nested Association Mapping (SoyNAM) population based on the disease severity index (DSI). 42

58 30 SoyNAM Parental Lines No. of SoyNAM Parental Lines Disease Score Figure 2: Distribution of parental lines, after excluding poor germinating genotypes, of Soybean Nested Association Mapping (SoyNAM) population based on disease score. 43

59 Genetic Gain MG II Mean DSI * Grouping Mean Score Grouping Non inoc.% germination Jack 93.6 A 4.6 AB 90.0 Mukden 91.9 AB 4.6 AB 85.0 IA AB 4.7 A 75.0 Harosoy AB 4.5 AB 75.0 Lindarin 90.2 AB 4.5 ABC 70.0 Kenwood 89.6 ABC 4.6 AB 80.0 Wells 88.8 ABC 4.5 ABC 85.0 Richland 88.7 ABC 4.5 AB 80.0 Amsoy ABC 4.5 ABC 70.0 Savoy 86.6 ABC 4.5 ABC 80.0 IA ABCD 4.3 ABCD 95.0 Dwight 84.6 ABCD 4.3 ABCD 90.0 IA ABCD 4.4 ABC 70.0 Burlison 79.9 ABCD 4.2 BCD 90.0 Harcor 76.2 BCD 4.2 BCD 75.0 Elgin CDE 4.0 CDE 90.0 Amcor 70.2 DEF 3.8 DEF IA EFG 3.7 EFG Elgin 55.3 FGH 3.4 GH 90.0 IA GHI 3.4 FGH Hawkeye 45.8 HIJ 3.1 HI 90.0 IA GHIJ 3.3 GH 85.0 IA IJ 2.8 I 95.0 Corsoy 34.0 J 2.7 I 90.0 Mean Table 2: Mean Disease Severity (DSI), mean score and non-inoculated germination percentage of Maturity Group II public cultivars, after excluding poor germinating cultivars, included in Genetic Gain study after inoculation of seeds with 100 µl of 2.5 x 10 4 macroconidia ml -1 of Fusarium graminearum in a rolled towel assay. * Disease severity index equals lesion length divided by total length of the seedling multiplied by 100. Disease severity data were arcsine transformed. The actual mean DSI are reported in the table. The experimental design was randomized complete block design with three replications repeated over time. Values with the same uppercase letter were not significantly different according to Fisher s protected Least Significance Difference (LSD) at α = 0.05, based on the arcsine transformed data. 44

60 9 Genetic Gain Maturity Group II 8 No. of soybean cultivars Disease Severity Index (DSI) Figure 3: Distribution of Maturity Group II public cultivars, after excluding poor germinating cultivars, included in Genetic Gain study based on the disease severity index (DSI). 45

61 16 Genetic Gain Maturity Group II 14 No. of soybean cultivars Disease Score Figure 4: Distribution of Maturity Group II public cultivars, after excluding poor germinating cultivars, included in Genetic Gain study based on the disease score. 46

62 Genetic Gain MG III Mean DSI * Grouping Mean Score Grouping Non inoc.% germination Mingo 94.3 A 4.6 A 95.0 Mandell 85.5 AB 4.3 ABC 90.0 Calland 85.2 AB 4.3 AB 85.0 Pella BC 4.2 BCD 95.0 Woodworth 74.5 BCD 4.0 BCDEF 95.0 Illini 74.3 BCD 4.0 BCDE Pella 74.1 BCD 4.0 BCDE 95.0 Lincoln 71.5 CDE 4.0 DEFGH 85.0 AK (Harrow) 70.7 CDE 4.0 BCDEF 85.0 Harper 70.3 CDE 3.9 DEFGHIJ 95.0 Oakland 70.0 CDEF 4.0 CDEFG 95.0 Williams 68.0 CDEFG 4.0 DEFGHI 90.0 Chamberlain 67.2 CDEFG 3.8 DEFGHIJK 90.0 Ford 66.0 CDEFG 3.7 EFGHIJKL 85.0 Ross 65.4 CDEFGH 3.8 EFGHIJKL 85.0 Wayne 64.0 CDEFGHI 3.7 EFGHIJKL Zane 63.5 CDEFGHI 3.7 EFGHIJKLM 90.0 Shelby 60.9 DEFGHIJ 3.6 EFGHIJKLMN IA EFGHIJ 3.6 GHIJKLMN NE EFGHIJ 3.5 IJKLMN 80.0 IA EFGHIJ 3.6 FGHIJKLMN U FGHIJK 3.6 GHIJKLMN 85.0 IA GHIJKL 3.5 KLMNO Adelph 53.3 GHIJKL 3.5 JKLMNO 80.0 Cumberland 52.8 GHIJKL 3.5 HIJKLMN 95.0 Continued Table 3: Mean Disease Severity (DSI), mean score and non-inoculated germination percentage of Maturity Group III public cultivars, after excluding poor germinating cultivars, included in Genetic Gain study, after inoculation of seeds with 100 µl of 2.5 x 10 4 macroconidia ml -1 of Fusarium graminearum in a rolled towel assay. * Disease severity index equals lesion length divided by total length of the seedling multiplied by 100. Disease severity data were arcsine transformed. The actual mean DSI are reported in the table. The experimental design was randomized complete block design with three replications repeated over time. Values with the same uppercase letter were not significantly different according to Fisher s protected Least Significance Difference (LSD) at α = 0.05, based on the arcsine transformed data. 47

63 Table 3 continued Genetic Gain MG III Mean DSI * Grouping Mean Score Grouping Non inoc.% germination IA HIJKL 3.4 LMNOP 95.0 Thorne 51.0 HIJKL 3.3 MNOP Williams IJKL 3.3 MNOP 80.0 Maverick 47.5 JKLM 3.3 NOP 90.0 Macon 42.5 KLMN 3.1 OPQ 95.0 Dunbar 40.4 LMN 3.1 PQR Resnik 32.5 MN 2.8 QR 95.0 IA N 2.7 R Mean

64 10 Genetic Gain Maturity Group III 9 No. of soybean cultivars Disease Severity Index (DSI) Figure 5: Distribution of Maturity Group III public cultivars after excluding poor germinating cultivars, included in Genetic Gain study based on the disease severity index (DSI). 49

65 30 Genetic Gain Maturity Group III 25 No. soybean cultivars Disease Score Figure 6: Distribution of Maturity Group III public cultivars, after excluding poor germinating cultivars, included in Genetic Gain study based on the disease score. 50

66 OSU/OARDC Germplasm I Mean DSI * Grouping Mean Score Grouping Non inoc.% germination OX A 4.4 A 95.0 PI AB 4.2 AB 90.0 PI A 82.8 ABC 4.1 ABC 90.0 PI ABCD 4.1 ABC PI 54618(27) 78.8 ABDE 4.0 ABCD 95.0 S ABCD 4.0 ABCD PI (299) 77.0 BCDEF 4.0 ABCD 90.0 R BCDEF 4.0 ABCDE PI B 74.8 BCDEFG 4.0 BCDEF PI A 74.6 BCDEF 4.0 BCDEF 95.0 Defiance 72.9 BCDEF 3.9 BCDEF 95.0 PI BCDEFGH 3.8 BCDEFG 85.0 Forrest 70.7 CDEFGH 3.9 BCDEFGH Williams CDEFGHI 3.8 BCDEFGH 95.0 PI A 69.4 CDEFGHI 3.8 BCDEFGHI 80.0 PI A 68.3 CDEFGHI 3.7 CDEFGHIJ 85.0 Dennison 68.2 CDEFGHI 3.8 BCDEFGH Magellan 67.3 DEFGHIJ 3.8 BCDEFGHI 95.0 PI A 63.7 DEFGHIJK 3.7 CDEFGHIJ 95.0 PI FGHIJKL 3.4 GHIJKL 75.0 S EFGHIJKL 3.6 DEFGHIJ PI FGHIJKL 3.6 EFGHIJ PI FGHIJKL 3.5 FGHIJK Dilworth 56.9 GHIJKLM 3.4 HIJKLM PI GHIJKLMN 3.4 HIJKLM 95.0 Continued Table 4: Mean Disease Severity (DSI), mean score and non-inoculated germination percentage of soybean genotypes, after excluding poor germinating genotypes, from OSU/OARDC Germplasm I, after inoculation of seeds with 100 µl of 2.5 x 10 4 macroconidia ml -1 of Fusarium graminearum in a rolled towel assay. * Disease severity index equals lesion length divided by total length of the seedling multiplied by 100. Disease severity data were arcsine transformed. The actual mean DSI are reported in the table. The experimental design was randomized complete block design with three replications repeated over time. Values with the same uppercase letter were not significantly different according to Fisher s protected Least Significance Difference (LSD) at α = 0.05, based on the arcsine transformed data. 51

67 Table 4 continued OSU/OARDC Germplasm I Mean DSI * Grouping Mean Score Grouping Non Inoc. % germination Jack 55.1 HIJKLMN 3.4 IJKLM HS IJKLMN 3.3 IJKLM Essex 52.3 JKLMNO 3.4 IJKLM Wyandot 51.6 JKLMNO 3.3 JKLMN 95.0 RIL KLMNO 3.1 LMNO 90.0 PI MNO 3.0 LMNO 85.0 RIL LMNO 3.0 LMNO 90.0 PI MNO 3.0 LMNO 85.0 PI MNO 2.9 MNO 80.0 PI MNO 3.1 KLMNO 90.0 RIL MNOP 2.9 MNO 75.0 PI NOPQ 2.8 OP 75.0 Ohio FG OPQR 2.9 NOP RIL PQRS 2.5 PQ 85.0 PI B 26.3 QRS 2.4 QR 75.0 RIL RST 2.2 QRS Wooster 21.3 RST 2.3 QR Peking (PI ) 16.4 STU 1.9 RST 95.0 PI TUV 1.8 ST 95.0 PI A 11.6 TUV 1.8 T 80.0 PI B 11.2 TUV 1.7 TU PI C 8.7 UV 1.6 TU 70.0 PI V 1.3 U 95.0 Mean

68 12 OSU/OARDC Germplasm I No. of soybean germplasm Disease Severity Index (DSI) Figure 7: Distribution of soybean genotypes, after excluding poor germinating genotypes, from OSU/OARDC Germplasm I collection based on the disease severity index (DSI). 53

69 30 OSU/OARDC Germplasm I No. of soybean germplasm Disease Score Figure 8: Distribution of soybean genotypes, after excluding poor germinating genotypes, from OSU/OARDC Germplasm I collection based on the disease score. 54

70 OSU/OARDC Germplasm II Mean DSI * Grouping Mean Score Grouping Non inoc.% germination Hutcheson 96.2 A 4.4 A 70.0 PI AB 4.3 AB 90.0 PI ABCD 4.3 ABC ALTB 90.0 ABC 4.3 ABC PI ABCDE 4.2 ABCD SLT 82.4 BCDEF 4.1 ABCDE Kottman 81.3 BCDEFG 4.1 ABCDEF ALTRR 77.1 CDEFGH 4.0 BCDEF ALTB 76.3 CDEFGHI 4.0 CDEFG PI DEFGHI 4.0 BCDEFG ALTA 75.3 EFGHIJ 3.9 DEFGHI Lee EFGHIJ 3.9 DEFGH PI EFGHIJ 3.9 CDEFGH ALTB 73.0 EFGHIJ 3.9 DEFGHI ALTA 71.5 EFGHIJK 3.9 DEFGHIJ ALTB 69.8 FGHIJK 3.8 EFGHIJ SLT 68.9 FGHIJK 3.8 EFGHIJ ALTB 68.1 FGHIJK 3.8 EFGHIJK Sloan 67.1 FGHIJK 3.7 FGHIJK ALTRR 66.8 GHIJKL 3.8 EFGHIJ ALTB 66.0 GHIJKL 3.8 EFGHIJK SLT 65.3 HIJKL 3.7 FGHIJK SLT 62.0 IJKL 3.6 GHIJK PI IJKL 3.6 HIJKL ALTA 60.9 JKL 3.6 HIJKL 90.0 Conrad 58.0 KLM 3.5 IJKL 80.0 PI KLM 3.4 KLM ALTRR 56.4 KLM 3.5 JKL Tiffin 51.1 LMN 3.3 LMN Continued Table 5: Mean Disease Severity (DSI), mean score and non-inoculated germination percentage of 32 soybean genotypes from OSU/OARDC Germplasm II, after inoculation of seeds with 100µl of 2.5 x 10 4 macroconidia ml -1 of Fusarium graminearum in a rolled towel assay. * Disease severity index equals lesion length divided by total length of the seedling multiplied by 100. Disease severity data were arcsine transformed. The actual mean DSI are reported in the table. The experimental design was randomized complete block design with three replications repeated over time. Values with the same uppercase letter were not significantly different according to Fisher s protected Least Significance Difference (LSD) at α = 0.05, based on the arcsine transformed data. 55

71 Table 5 continued OSU/OARDC Breeding Lines and Stocks II Mean DSI * Mean Score Non inoc.% germination Grouping Grouping PI MN 3.0 MN 90.0 PI N 2.9 N 90.0 Mean

72 12 OSU/OARDC Germplasm II No. of soybean germplasm Disease Severity Index (DSI) Figure 9: Distribution of soybean genotypes, after excluding poor germinating genotypes, from OSU/OARDC Germplasm II collection based on the disease severity index (DSI). 57

73 25 OSU/OARDC Germplasm II No. of soybean germplasm Disease Score Figure 10: Distribution of soybean genotypes, after excluding poor germinating genotypes, from OSU/OARDC Germplasm II collection based on the disease score. 58

74 References Ali, S., Rivera, V. V., and Secor, G. A First report of Fusarium graminearum causing dry rot of potato in North Dakota. Plant Dis. 89:105. Arelli, P. R., Concibido, V. C., and Young, L. D QTL associated with resistance in soybean PI567516C to synthetic nematode population infecting cv. Hartwig. J. Crop Sci. Biotech. 13: Bilgi, V. N., Bradley, C. A., Mathew, F. M., Ali, S., and Rasmussen, J. B Root rot of dry edible beans caused by Fusarium graminearum. Online. Plant Health Progress doi: /php rs. Broders, K. D., Lipps, P. E., Paul P. A., and Dorrance, A. E Evaluation of Fusarium graminearum associated with corn and soybean seed and seedling in Ohio. Plant Dis. 91: Buerstmayr, H., Ban, T., and Anderson, J. A QTL mapping and markerassisted selection for Fusarium head blight resistance in wheat : A review. Plant Breeding 128:1-26. Chang, S. J. C., Doubler, T. W., Kilo, V. Y., Abu-Thredeih, J., Prabhu, R., Freire, V., Suttner, R., Klein, J., Schmidt, M. E., Gibson, P. T., and Lightfoot, D. A Association of loci underlying field resistance to Soybean Sudden Death Syndrome (SDS) and Cyst Nematode (SCN) Race 3. Crop Sci. 37: Chongo, G., Gossen, B. D., Kutcher, H. R., Gilbert, J., Turkington, T. K., Fernandez, M. R., and McLaren, D Reaction of seedling roots of 14 crop species to Fusarium graminearum from wheat heads. Can. J. Plant Pathol. 23: Diers, B., Specht, J., Hyten, D., Cregan, P., Nelson, R., and Beavis, B Soybean Nested Association Mapping. ers.pdf Dorrance, A. E. and Lipps, P. E Profitable soybean disease management in Ohio. p Ohio State University Extension Bulletin 895. Dorrance, A. E., and Schmitthenner, A. F New sources of resistance to Phytophthora sojae in the soybean plant introductions. Plant Dis. 84:

75 Ellis, M. L., Broders, K. D., Paul, P. A., and Dorrance, A. E Infection of soybean seed by Fusarium graminearum and effect of seed treatments on disease under controlled conditions. Plant Dis. 95: Ellis, M. L., McHale, L. H., Paul, P. A., St. Martin, S. K., and Dorrance, A. E Soybean germplasm resistant to Pythium irregulare and molecular mapping of resistance quantitative trait loci derived from the soybean accession PI Crop Sci. 53: Ellis, M. L., Wang, H., Paul, P. A., Martin, S. K. St., McHale, L. K. and Dorrance, A. E Identification of soybean genotypes resistant to Fusarium graminearum and genetic mapping of resistance Quantitative Trait Loci in the cultivar Conrad. Crop Science 52: Foroud, N. A., McCormick, S. P., MacMillan, T., Badea, A., Kendra, D. F., Ellis, B. E., and Eudes, F., Greenhouse studies reveal increased aggressiveness of emergent Canadian Fusarium graminearum chemotypes in wheat. Plant Dis. 96: Gale, L. R., Harrison, S. A, Ward, T. J., O Donnell, K., Milus, E. A., Gale, S. W., and Kistler, H. C Nivalenol-type populations of Fusarium graminearum and F. asiaticum are prevalent on wheat in southern Louisiana. Phytopathology 101: Gao, X., Jackson, T. A., Hartman, G. L., and Niblack, T. L Interactions between the Soybean Cyst Nematode and Fusarium solani f. sp. glycines based on greenhouse factorial experiment. Phytopathology 96: Gibson, P. T., Shenaut, M. A., Njiti, V. N., Suttner, R. J., Myers, O. Jr Soybean varietal response to sudden death syndrome. p In D. Wilkinson (ed.) Proc. Twenty-fourth Soybean Seed Res. Conf., Chicago, IL. 6-7 Dec. Am. Seed Trade Assoc., Washington, DC. Gordon, S. G., Berry, S. A., St. Martin, S. K., and Dorrance, A. E Genetic analysis of soybean plant introductions with resistance to Phytophthora sojae. Phytopathology 97: Graef, G. L., Specht, J. E., Korte, L. L., and White, D. M Registration of Dunbar soybean. Crop Sci. 32:497. Gumpertz, M. L Special report: Data transformation. Biological and Cultural tests 10:1-5. Hanson, L. E Fusarium yellowing of sugar beet caused by Fusarium graminearum from Minnesota and Wyoming. Plant Dis. 90:686. Harris, S. D Morphogenesis in germinating Fusarium graminearum macroconidia. Mycologia, 97:

76 Hegstad, J. M., Nickell, C. D., and Vodkin, L. O Identifying resistance to Phytophthora sojae in selected soybean accessions using RFLP techniques. Crop Sci. 38: Horevaj, P., Gale, L. R., and Milus, E. A Resistance in winter wheat lines to initial infection and spread within spikes by deoxynivalenol and nivalenol chemotypes of Fusarium graminearum. Plant Dis. 95: Jun, T-H, Mian, M. A., and Michel, A. P. 2012a. Genetic mapping revealed two loci for soybean aphid resistance in PI B. Theor. Appl. Genet. 124: Jun, T-H, Mian, M. A., Kang, S. T., and Michel, A. P. 2012b. Genetic mapping of the powdery mildew resistance gene in soybean PI B. Theor. Appl. Genet. 125: Kantartzi. S. K., Klein III, J., and Schmidt, M Registration of Saluki 4411 soybean with resistance to Sudden Death Syndrome and HG Type 0 (Race 3) Soybean Cyst Nematode. Journal of Plant Registrations 6: Lee, S., Rouf Mian, M. A., Sneller, C. H., Wang, H., Dorrance, A. E., and McHale, L. K Joint linkage QTL analyses for partial resistance to Phytophthora sojae in soybean using six nested inbred populations with heterogenous conditions. Theor. Appl. Genet. 127: Leslie, J. F., and Summerell, B. A The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA. Martinelli, J.A., Bocchese, C.A.C., Xie, W., O Donnell, K. & Kistler, H.C Soybean pod blight and root rot caused by lineages of the Fusarium graminearum and the production of mycotoxins. Fitopatol. Bras. 29: McBlain, B. A., Fioritto, R. J., St. Martin, S. K., Calip-DuBios, A., Schmitthenner, A. F., Cooper, R. L., and Martin, R. J Registration of Resnik soybean. Crop Sci. 30: Mesterházy, Á Resistance of corn to Fusarium ear rot and its relation to seedling resistance. Phytopathology 103: Mian, M. A. R., Hammond, R. B., and St. Martin, S. K New plant introductions with resistance to the soybean aphid. Crop Sci. 48: Mule, G., González-Jaén, M. T., Hornok, L., Nicholson, P., and Waalwijk, C Advances in molecular diagnosis of toxigenic Fusarium species: A review. Food Additives and Contaminants 22: Niblack, T. L Soybean cyst nematode management reconsidered. Plant Dis. 89: Nickell, C. D., Thomas, D. J., Cary, T. R., and Heavner, D Registration of Macon soybean. Crop Sci. 36:

77 Pederson, P Soybean growth and development. p Iowa State University, University Extension, Ames, IA. Pioli, R. N., Mozzoni, L., and Morandi, E. N First report of pathogenic association between Fusarium graminearum and soybean. Plant Dis. 88:220. Prabhu, R.R., Njiti, V.N., Bell-Johnson, B., Johnson, J.E., Schmidt, M.E., Klein, J.H., Lightfoot, D.A Selecting soybean cultivars for dual resistance to soybean cyst nematode and sudden death syndrome using two DNA markers. Crop Sci. 39: Rouf Mian, M. A., Hammond, R. B., and St. Martin, S. K. New plant introcuctions with resistance to the soybean aphid Crop Sci. 48: Rowntree, S. C., Suhre, J. J., Weidenbenner, N. H., Wilson, E. W., Davis, V. M., Naeve, S. L., Casteel, S. N., Diers, B. W., Esker, P. D., Specht, J. E., and Conley, S. P Genetic gain x management interactions in soybean: I. Planting date. Crop Sci. 53: Schmidt, M. E., Suttner, R. J., Klein, J. H., Gibson, P. T., Lightfoot, D. A., and Myers, O. Jr Registration of LS-G96 soybean germplasm resistant to Soybean Sudden Death Syndrome and Soybean Cyst Nematode Race 3. Crop Sci. 39: 598. Sleper, D. A., Nickell, C. D., Noel, G. R., Cary, T. R., Thomas, D. J., Clark, K. M., and Rao Arelli, A. P Registration of Maverick soybean. Crop Sci. 38: St. Martin, S. K., Mills, G. R., Fioritto, R. J., McIntyre, S. A., Dorrance, A. E., and Berry, S. A Registration of Ohio FG5 soybean. Crop Sci. 46:2709. Starkey, D. E., Ward, T. J., Aoki, T., Gale, L. H., Kistler, H. C., Geiser, D.M., Suga, H., Tóth, B., Varga, J., and O Donnell, K Global molecular surveillance reveals novel Fusarium head blight species and trichothecene toxin diversity. Fungal Genet. Biol. 44: Stich, B., Friedrich Utz, H., Piepho, H-P., Maurer, H. P., Melchinger, A. E Optimum allocation of resources for QTL detection using a nested association mapping strategy in maize. Theor. Appl. Genet. 120: Stoskopf, N. C., Tomes, D. T., and Christie, B. R Plant Breeding Theory and Practice.Westview Press, Boulder, Co. p. 91. Toth, B., Kaszonyi, G., Bartok, T., Varga, J. and Mesterhazy, A Common resistance of wheat to members of the Fusarium graminearum species complex and F. culmorum. Plant Breeding 127:1-8. Weber, C. R. and Fehr, W. R Registration of Corsoy soybeans. Crop Sci. 10:

78 Windels, C. E Economic and social impacts of Fusarium head blight: Changing farms and rural communities in the Northern Great Plains. Phytopathology 90: Wrather, J. A., and Koening, S. R Effets of diseases on soybean yields in the United States 1996 to Online. Plant Health Progress doi: /PHP RS. Xue, A. G., Cober, E., Voldeng, H. D., Babcook, C., and Clear, R. M Evaluation of the pathogenicity of Fusarium graminearum and Fusarium pseudograminearum on soybean seedlings under controlled conditions. Can. J. Plant Pathol. 29: Yu, J., Holland, J. B., McMullen. M. D., and Buckler, E. S Genetic design and statistical power of nested association mapping in maize. Genetics 178: Yue, P., Sleper, D. A., and Arelli, P. R Mapping resistance to multiple races of Heterodera glycines in soybean PI Crop Sci. 41: Zhang, J. Z., Xue, A. G., Zhang, H. J., Nagasawa, A. E. and Tambong, J. T Response of soybean cultivars to root rot caused by Fusarium species. Can. J. Plant Sci. 90:

79 Chapter 3: Identification and Mapping of Quantitative Trait Loci (QTL) Conferring Resistance to Fusarium graminearum in Two Populations of Soybean Introduction Soybean (Glycine max (L.) Merr.) is one of the major crops grown in the world as a food, oilseed and animal feed crop. Soybean seed has close to 40% protein content and is considered as an inexpensive source of protein. Soybean produces twice as much protein per acre as produced by any other vegetable or grain crop and has fifteen times as much protein per acre as the land set aside for meat production (National Soybean Research Laboratory: For 2011, USDA estimated that 45% of the soybean would be exported and also estimated that the world soybean production increased by about 500% (National Soybean Research Laboratory: In 2013, the United States harvested approximately 3.3 billion bushels of soybean from 76.5 million acres with approximately 43.3 bushels per acre (National Agricultural Statistics Service: In the United States, more than 80% of the soybeans are produced in the north central states and Ohio is one of them, where 4.5 million acres produced approximately 49 bushels of soybean per acre in 2013 (National Agricultural Statistics Service: In addition to the 64

80 success and importance of soybean in the region, there are also a number of pathogens that can negatively impact yield. In Ohio, the incidence of the soybean seedling diseases has increased substantially since the previous decade with the increase in acreage and continuous production (Ellis et al., 2011). A greater number of pathogens are soil or residue borne, which are of major concern to the soybean farmers. Resistance genes in soybean cultivars are no longer effective towards many of the populations of Phytophthora sojae as well as new reports of pathogens that infect soybean (Broders et al., 2007; Dorrance and Lipps, 2002) are documented. P. sojae and Pythium spp. have long been known in Ohio for association with seed and seedling diseases causing pre and post-emergence damping off in soybean and are responsible for severe production losses in the state (Schmitthenner, 1985). More recently, SCN incidence has been found to be increasing in the fields in Ohio and has been identified in fields in 68 counties (Dorrance et al., 2012). In addition to these, another residue borne pathogen, Fusarium graminearum has also been identified as a primary pathogen of soybean in South America (Martinelli et al., 2004, Pioli et al., 2004) and is a seed and seedling pathogen of soybean in North America (Broders et al., 2007; Xue et al., 2007). More recently, F. proliferatum was also reported to be highly pathogenic to soybean (Díaz Arias et al., 2011; Díaz Arias et al., 2013a), which was previously only considered to be a pathogen of corn. In soybean, symptoms due to F. graminearum infection are observed on seedlings first as water soaked lesions followed by light brown or pink discoloration around the inoculation point. Pre- and post-emergence damping off, seed and seedling rot and 65

81 root rot are other symptoms observed in North America (Broders et al., 2007; Ellis et al., 2012; Pioli et al., 2004; Xue et al., 2007). Additional symptoms observed only in South America at later growth stages include spreading of discoloration vertically on stem, interveinal chlorosis of leaves leading to plant wilting, death, and pod blight (Martinelli et al., 2004; Pioli et al., 2004). Fusarium graminearum is a homothallic, ascomycete fungus and its sexual reproduction involves the development of perithecia that produce ascospores. This fungus produces macroconidia as asexual spores but lacks microconidia (Leslie and Summerell, 2006). The macroconidia are slender and elongated containing 3-5 cells separated by septa and they play an important role in the dissemination of the disease (Harris, 2005; Leslie and Summerell, 2006). In addition to F. graminearum, many closely related species were also found to cause the head scab disease in wheat and releated cereal crops, and Gibberella stalk and ear rot in corn, hence, now is a Fusarium graminearum species complex (FGSC) (O Donnell et al., 2004; Starkey et al. 2007; Taylor et al, 2000; Wang et al., 2011). In the United States, F. graminerum sensu lato is the predominant species (Horevaj et al., 2011), which resulted in an estimated loss of $3 billion in 1990s causing head scab in wheat and barley (Windels, 2000). In addition to being pathogenic and causing yield loss, secondary metabolites as mycotoxins are also produced: Deoxynivalenol (DON), Nivalenol (NIV) and Zearalenone (ZEN). These are the three of the most important mycotoxins that fall under type B trichothecenes which are toxic to humans, animals, plants, insects and other microorganisms and are also important in fungal pathogenesis and antagonism (Mule et al., 2005). In plants, Type B trichothecenes are produced by every species in the Fusarium graminearum species complex (O Donnell et. al, 2008). 66

82 In crop plants, many traits such as yield, quality and disease resistance have quantitative variations, which result from the combined effect of the expression of several segregating genes and environmental factors (review by Asín, 2002). F. graminearum is more extensively studied as a head blight pathogen of wheat than in any other crops. There are a plethora of studies showing that inheritance of resistance of wheat to FHB is quantitative and QTL for FHB resistance have been found on all the chromosomes except chromosome 7D (review by Buerstmayr et al., 2009). Fusarium isolates with wide differences in aggressiveness have been detected, but no biological races with specific host-pathogen interaction with wheat have been found to date (reviewed by Buerstmayr et al., 2009). Resistance to head blight in wheat, categorized into two types (Schroeder and Christensen, 1963) have been widely accepted compared to the five types. Type I resistance is resistance to initial infection and Type II resistance is resistance to spread of blight symptoms within the spike. Horevaj et al. (2011) found that the wheat lines having resistance to isolates of DON chemotype had more resistance to isolates of NIV chemotype of F. graminerum. Similarly, the study by Foroud et al. (2012) showed that aggressiveness increased from NIV to 15ADONα to 15ADONβ to 3ADON chemotypes. In an another study, Toth et al. (2008) found that the resistance in wheat cultivars to all the members of the F. graminearum species complex and lineages of F. culmorum is not specific and that one pathogenic isolate of any Fusarium species is enough for resistance breeding, even though isolates differ in aggressiveness and DON producing ability. Hence, resistant wheat genotypes can be cultivated successfully in the fields where different species of Fusarium are dominant or occur in a mix (Toth et. al., 2008). In addition to causing head blight, Fusarium graminearum has also been reported to cause Fusarium 67

83 seedling blight when it is seed borne and produce DON in the grain (Tamburic-Ilincic et. al., 2009). The authors identified a single QTL that controlled seedling blight, which was different from the QTLs identified to control head blight. Similarly, Somers et al. (2003) reported that resistance to head blight and resistance to DON accumulation were controlled by different loci. This signifies that the QTL conferring resistance to Fusarium seedling blight and head blight in wheat might be different even though caused by the same pathogen. Fusarium graminearum also causes Gibberella ear and stalk rot of corn. F. graminearum infects corn ears through the silk channel or through wounds made by birds and insects damaging the husk tissue and grain (Chungu et al. 1996; Reid and Hamilton, 1996; Reid et al., 1992a and b). There are also two types of resistance to Gibberella ear rot (Chungu et al., 1996; Reid et al., 1992a and b). Silk resistance prevents the fungus from growing down the silk to the kernel. Kernel resistance, in kernel tissue prevents the fungus from spreading kernel to kernel when it overcomes the silk resistance. Ali et al. (2005) utilized a recombinant inbred line population and detected 11 QTL for silk resistance and 18 QTL for kernel resistance and two of them were associated with both silk and kernel resistance. Similarly, Martin et al. (2011), using double haploid lines, identified six major effect QTL that significantly enhanced ear rot resistance, four QTL that significantly lowered DON contamination and five QTL that lowered zearalenone contamination. This indicates that resistance to corn ear rot is quantitative as well. Mesterházy (1982) found a good correlation between the reaction of corn hybrids to F. graminearum and F. culmorum, which showed a possible common resistance mechanism to these two pathogens. 68

84 Gibberella stalk rot in corn is another devastating disease caused by F. graminearum. Both qualitative and quantitative resistances have been reported to stalk rot. Pè et. al (1993) detected five resistance QTL to Gibberella stalk rot using inbred lines. Likewise, Yang et. al (2010) detected two QTL, one being a major QTL, which was confirmed in a double haploid, F2 and back cross population. In addition, a single resistance gene Rfg1 has been detected to provide resistance to stalk rot (Yang et al., 2004). For F. verticilloides (Sacc.) Nirenberg, a closely relates species that causes Fusarium ear rot in corn, at least three resistance QTL were detected (Ding et al., 2008) indicating quantitative nature of resistance. Fusarium virguliforme (ex. F. solani f. sp. glycines) is another species of Fusarium that causes sudden death syndrome (SDS) in soybean. Root rot and intervenal chlorosis (leaf sorch) from a toxin are key symptoms. Farias Neto et al. (2007) detected three resistance QTL in a Ripley x Spencer population and two QTL in PI x Omaha population. In the same study, one resistance QTL was mapped to the same location in both the populations, which could be effectively utilized in SDS resistance breeding. Similarly, Yamanaka et al. (2006) detected four additional QTL different from those previously described for resistance to F. tucumaniae sp. nov., which also causes SDS in South America, using RILs derived from Misuzudaizu and Moshidou Gong 503, a susceptible and resistant parent respectively. Similar to wheat and corn where two types of resistance were observed, separate loci were found to confer resistance to root infection and leaf scorch of SDS in soybean (Kazi et al., 2008). To date, 14 QTL have been confirmed to be associated with SDS resistance (Luckew et al., 2013). 69

85 In recent studies, F. graminearum was found to be highly aggressive in causing severe root rot in soybean compared to the other species of Fusarium (Arias et al., 2013 a; Broders et al., 2007; Ellis et al., 2011; Zhang et al., 2010). F. graminearum was not the most abundant species of Fusarium in the field, but it was one of the few species frequently isolated from the soybean roots (Arias et al., 2013 b; Zhang et al., 2010). In a study, nine out of 57 commercial soybean cultivars evaluated, belonging to maturity group 000 to 0, were resistant to F. graminearum (Zhang et al., 2010) and in another small preliminary screening of 24 soybean genotypes, five had moderate to high levels of resistance (Ellis et al., 2012). Hence, very few sources of resistance have been identified to date and only five QTL conferring resistance to F. graminearum were detected, 4 resistance alleles were from Conrad and 1 from Sloan (Ellis et al., 2012). Previously, 200 soybean genotypes were evaluated for resistance to F. graminearum and 30 genotypes had a resistant reaction and one of them was PI B that had high level of resistance while Wyandot had moderate level of resistance (Chapter 2). PI B has resistance to soybean aphid (Jun et al., 2012a) and to Phytophthora sojae race 25 (GRIN online database). Jun et al. (2012b) also reported that this PI is resistant to powdery mildew of soybean caused by Microsphaera diffusa (Cooke & Peck) Jacz. Wyandot has the Rps3 gene for resistance to Phytophthora root and stem rot, high protein content and high yield (St. Martin et al., 2006). Conrad was identified as moderately resistant to F. graminearum while Sloan was susceptible in an earlier study (Ellis et al., 2012). Conrad is also moderately resistant to purple stain and P. sojae but susceptible to brown stem rot, bacterial tan spot and soybean mosaic virus (Fehr et al., 1989). Sloan is moderately resistant to pod and 70

86 stem blight and purple stain but moderately susceptible to brown stem rot and bacterial blight and susceptible to P. sojae (Bahrenfus and Fehr, 1980). Both the cultivars are also resistant to powdery mildew but susceptible to Soybean Cyst Nematode (SCN) and soybean aphid (A. E. Dorrance, personal communication). Therefore, the objectives of this study were to identify and fine map QTL for resistance to F. graminearum using recombinant inbred lines (RILs) from two populations derived from the crosses of Wyandot x PI B and Conrad x Sloan. Materials and Methods Plant materials and DNA extraction A population of 184 F 7:10 recombinant inbred lines (RILs) derived from the cross of the cultivar Wyandot by PI B, from the Mian Lab (USDA-ARS, Department of Horticulture and Crop Science, OSU) was used to map resistance QTL towards F. graminearum. The cross was made in 2006 at Ohio Agricultural Research and Development Center (OARDC), Wooster and was advanced by single seed decent to develop RILs used in this study. Wyandot is a maturity group (MGII) soybean cultivar released by OARDC Crop Variety Release and Distribution Committee in 2006 (St. Martin et al., 2006). The plant introduction, PI B, is a MG IV accession originally collected from Gansu, China. Using F 7 derived RILs from the same parents, a minor QTL for aphid resistance near the Rag2 gene on chromosome 8 and a single dominant powdery mildew resistance gene on chromosome 16 was reported (Jun et al., 2012a; Jun et al., 2012b). 71

87 Young leaf samples collected from each RILwere lyophilized for two days and ground using Mixer Mill (Model MM301, Retch, Hannover, Germany). DNA was extracted from the ground samples using a CTAB protocol with minor modification (Mian et al. 2008). A second population used to fine map resistance QTL towards F. graminearum consisted of 330 F 9:11 RILs derived from the cross of moderately resistant cultivar Conrad and a susceptible cultivar Sloan. The cross was made in 2002 at Waterman Farm, West Campus, Columbus, OH. The F 1 plants obtained from the cross were grown in a greenhouse at the OARDC during the winter The seeds were advanced by single seed decent each year in the field and greenhouse to develop RILs used in this study. Both Sloan and Conrad are MG II cultivars released in 1977 and 1988 respectively due to their higher yield compared to the other cultivars in the same MG at the time (Bahrenfus and Fehr, 1980; Fehr et al., 1989). Young leaf samples collected from each RIL and stored at -80 o C until used for DNA extraction. The samples were ground using liquid nitrogen in mortar and pestle. DNA was extracted from the ground samples using a CTAB protocol with minor modification (Gunadi, 2012) Inoculum production Fusarium graminearum isolate Fay 11, originally collected from soybean (Ellis et al., 2011) was used in this study. Macroconidia were stored in cryovails (Nalgene cryogenic tubes, Fisher Scientific Inc.) in 10% glycerol at -80 o C. This isolate was highly aggressive on soybean among the different isolates initially evaluated (Ellis et al., 2011). Macroconidia were transferred to petri plates with Mung Bean Agar 72

88 (MBA) and grown for 10 day under fluorescent light with 12hr light: dark cycle. Plates were then flooded with 5-10 ml sterile distilled water; macroconidia were dislodged with a glass rod and filtered through 4-6 layers of cheese cloth. The final macroconidia suspension was determined using a hemacytometer (Bright-Line Hemacytometer; Hausser Scientific, Horsham, PA) and sterile distilled water was added to achieve a final concentration of 2.5 x 10 4 macroconidia ml -1. Phenotypic evaluation A rolled towel assay was used to assess resistance to F. graminearum in these RILs as previously described by Ellis et al. (2011) with slight modifications. Fifteen seeds from each RIL and checks were placed on a germination paper moistened with deionized water and inoculated with a 100 µl suspension of 2.5 x 10 4 macroconidia ml -1. A second moistened towel was placed over the inoculated seeds, rolled up and placed on the top of a wire mesh in a 25 L bucket. The buckets were covered with a dark plastic bag and stored in the dark at room temperature which was approximately o C. Ten days after inoculation (dai), lesion length and total plant length were measured for each seedling of each genotype. A Disease Severity Index (DSI) was calculated by dividing the lesion length by total length of the seedlings and multiplying by 100. Disease was also rated using a 1-5 scale, where, 1= germination and healthy seedlings with no visible signs of colonization; 2= germination and colonization of the root and 1 to 19% of the root with lesions; 3= germination and colonization of the seeds, and 20 to 74% of the root with lesions; 4= germination and colonization of the seed and 75% or more of the seedling rot with lesions; and 5= no germination and complete colonization of the seed. 73

89 In the Wyandot x PI B population, a total of 184 RILs were evaluated by dividing into two groups. Grouping was done to only divide the RILs into two halves. Each group consisted of 92 RILs and each RIL within a group was evaluated in three replications. Each replication consisted of multiple sets and each set had 48 RILs arranged in incomplete blocks with 2-4 buckets containing 12 RILs, two parents and Conrad, Williams and Sloan as checks in each bucket. Bucket was not included in the model as the bucket effect was not significant in the previous analysis (Ellis et al., 2012). In Conrad x Sloan population, a total of 330 RILs were evaluated in three replications. RILs were not divided into groups in this population. Each replication consisted of multiple sets and each set consisted of 60 RILs arranged in incomplete blocks with 2-4 buckets containing 15 RILs, two parents and PI B, Wyandot and Williams as checks. At least 45 individuals were evaluated for each RIL. The mean DSI and root rot score of 15 seedlings of each RIL was calculated from each rolled towel in each rep of each experiment. The DSI data were not transformed and analyzed using BLUP analysis. This data from each experiment within each population was combined and analyzed to obtain the best linear unbiased predictor (BLUP) (Stoup, 1989) by using PROC MIXED procedure of SAS version 9.3 (SAS Institute Inc., Cary, NC, USA). The model used for Wyandot x PI567301B was Y ijklm = µ + G i + R(G) ij + S(RG) ijk + C l + L(C) lm + ε ijklm where, µ is overall mean, G i is the effect of i th group, R(G) ij is effect of j th replication in i th group, S(R) ijk is effect of k th set in j th replication in i th group, C l is effect of l th class of entry, L(C) lm is effect of m th genotype within class for 74

90 recombinant inbred lines only (Genotypic variance, σ g 2 ) and ε ijklm is experimental error (σ 2 ). The heritability was calculated as: σ g / (σ g + σ 2 /r), where r is the number of replications per RIL. The model used for Conrad x Sloan was Y ijklm = µ + R i + S(R) ij + B(RS) ijk + C l + G(C) lm + ε ijklm where, µ is overall mean, R i is the effect of i th replication, S(R) ij is effect of j th set in i th replication, B(RS) ijk is effect of k th bucket in j th set in i th replication, C l is effect of l th class of entry, G(C) lm is effect of m th genotype within class for recombinant inbred lines only (Genotypic variance, σ 2 g ) and ε ijklm is experimental error (σ 2 ). The heritability was calculated as: σ g / (σ g + σ 2 /r), where r is the number of replications per RIL. Genotypic assay DNA was shipped on dry ice in 96-well plates to University of California, Davis for genotyping of both the RIL populations and their parents. Single nucleotide polymorphism (SNP) genotyping was performed by Illumina Infinium BARCSoySNP6k BeadChip with a total of 5,403 SNPs. Three hundred and sixty eight RILs along with Wyandot and PI B were genotyped previously (Lee et al., in press) and 2,563 SNPs were found polymorphic between the parents. Finally, 2,545 polymorphic SNPs and 184 RILs were integrated using Kosambi mapping function of JoinMap 4 (Van Ooijen, 2006) to develop a genetic map. There were 20 linkage groups and total length of the genetic linkage map was 2, 342 cm. The average marker interval ranged from cm by chromosome with the overall average marker distance of 1.2 cm. The map covered from % of each chromosome, based on the physical positions of the SNPs integrated. 75

91 Two hundred and fifty eight RILs including Conrad and Sloan were genotyped with 5403 SNPs and 2,085 SNPs were found polymorphic between the parents. Prior to developing a genetic map all the markers that had more than 10% missing data or that had more than 10% heterozygotes were removed. In addition, markers in the same position in the genetic map were removed. Hence, a total of 1012 polymorphic SNPs and 258 of the 330 RILs were integrated using Kosambi mapping function of JoinMap 4 (Van Ooijen, 2006) to develop a genetic map. Selected 10 SSR and 1 SNP markers were used to fill the large gaps in the preliminary SNP genetic map of Conrad x Sloan population to have better marker coverage. Based on this preliminary genetic map, there were 20 linkage groups and a total length of genetic linkage map was 1697 cm. The average marker interval among the 669 markers used to make the map ranged from cm by chromosome while the overall marker distance was 2.5 cm. For QTL identification, interval mapping (IM) and composite interval mapping (CIM) were conducted using MapQTL 5 (Van Ooijen, 2004). For IM and CIM, the significance threshold level was determined by a 1000-permutation test at α = 0.05 (Churchill and Doerge 1994). For the identification of the genes in the QTL region mapped from these two populations, was accessed on July, Results Phenotypic evaluation Wyandot x PI B population The mean DSI was significantly different among the two parents and the checks indicating that PI B had high level of resistance and Wyandot had moderate 76

92 level of resistance towards F. graminearum (Table 6). The mean DSI for the checks and parents of the population averaged over all the buckets from both the groups was 87.4%, 66.4%, 43.1%, 35.7%, and 8.6% respectively for Sloan, Williams, Conrad, Wyandot and PI B. Similarly, the mean score were 4.3, 3.7, 3.0, 2.8 and 1.6 for Sloan, Williams, Conrad, Wyandot and PI B respectively. The means of parents and checks were separated by Fisher s Protected LSD using PROC GLM procedure (P < ) (Table 6). The mean DSI of individual RILs ranged from 0.75% to 63.7%, and the overall mean DSI of all the RILs was 32.6%. The BLUP values were calculated using the DSI and the frequency of BLUP values were normally distributed in this population (Figure 11). Lower BLUP values means higher level of resistance to F. graminearum. Using the mixed model analysis, the BLUP values were estimated and 9.6 for PI B and Wyandot respectively. The BLUP values of checks: Conrad, Williams and Sloan were 17.0, 40.4 and 59.9 respectively. Six RILs had BLUP values lower than PI B and 38 RILs had BLUP values higher than Wyandot. The broad-sense heritability for the mean DSI was Conrad x Sloan population The mean DSI was significantly different among the two parents and the checks indicating that Conrad had moderate level of resistance and Sloan had susceptible reaction towards F. graminearum (Table 7). The mean DSI averaged over all the buckets from all the sets were 80.7%, 60.6%, 43.2%, 40.6%, and 8.1% respectively for Sloan, Williams, Conrad, Wyandot and PI B. Similarly, mean scores 77

93 were 4.1, 3.5, 3.0, 3.0 and 1.6 for Sloan, Williams, Conrad, Wyandot and PI B respectively. The means of parents and checks were separated by Fisher s Protected LSD using PROC GLM procedure (P < ) (Table 7). The mean DSI of individual RIL ranged from 11.2% to 89.9%, and the overall mean DSI of all the RILs was 49.7%. The BLUP values were calculated using the DSI and the frequency of BLUP values were normally distributed in this population (Figure 12). Lower BLUP values means higher level of resistance towards F. graminearum. Using the mixed model analysis, the BLUP values were estimated -8.5 and 29.1 for Conrad and Sloan respectively. The BLUP values of checks: PI B, Wyandot and Williams were -43.5, and 9.0 respectively. Sixty one RILs had lower BLUP values than Conrad. The broad-sense heritability for the mean DSI was QTL identification Wyandot x PI B Two QTL conferring resistance to F. graminearum were identified one major on chromosome 8 and one minor on chromosome 6 by composite interval mapping (CIM) (Table 8). The genome-wide significance threshold calculated by a permutation test was 3.4 for Wyandot x PI B population. The major QTL on chromosome 8 was closely linked to marker BARC_2.0_Gm08_ and accounted for 38.5% of the phenotypic variance (Table 8). A minor QTL, which explained 8.1% of the phenotypic variance, was closely linked to marker BARC_2.0_Gm06_ on chromosome 6 (Table 8). The resistance allele for QTL mapped on both the chromosomes were contributed by PI B (Table 8). 78

94 There were a total of 39 genes within the QTL region on chromosome 8 (Soybase, accessed on July, 2014). Conrad x Sloan Three QTL conferring resistance to F. graminearum were identified on chromosomes 10, 14, and 19 by CIM (Table 9). The QTL on chromosome 10 was closely linked to marker BARC_1.0_Gm10_ _A_G, QTL on chromosome 14 was closely linked to marker BARC_1.0_Gm14_ _G_A, and QTL on chromosome 19 was closely linked to marker BARC_Gm19_ _T_C which explained 6.2, 4.8, and 8.9% of the phenotypic variance respectively (Table 9). The genome-wide LOD threshold calculated by 1000 permutation test was 1.9 for Conrad x Sloan population. The resistance allele for QTL on chromosomes 14 and 19 were contributed by Sloan and on chromosomes 10 was contributed by Conrad (Table 9). Discussion In this study, two large RIL populations derived from a cross of Wyandot x PI B and a cross of Conrad x Sloan were evaluated for resistance to F. graminearum using a rolled towel assay. Wyandot and PI B had BLUP values less than Conrad, a moderately resistant cultivar (Figure 11) indicating both the parents are resistant to F. graminearum and that it was a resistant by moderately resistant cross. Both the resistance alleles associated with resistance to F. graminearum were contributed by the resistant parent (PI B). The QTL on chromosome 8, which overlaps the 79

95 Rhg4 gene, contributed a major effect (38.5% of phenotypic variance) for resistance (Table 8, Figure 13). The major QTL detected on chromosome 8 is a novel QTL and is 14Mb distant from F. graminearum resistance QTL previously identified (Ellis et al., 2012). This QTL mapped close to QTL conferring resistance to P. sojae (Wang et al., 2012) and SCN (Ariagada et al., 2012; Ferreira, 2011; Guo et al., 2006; Mahalingam et al., 1995; Meksem et al., 2001c; Vuong et al., 2011; Webb et al., 1995; Wu et al., 2009). This locus is at a different location from a minor QTL reported by Jun et al. (2012a) for aphid resistance near the Rag2 gene on chromosome 8 using the F 7:9 RILs derived from the same parents. Hence, PI B contains resistance genes and resistance alleles conferring resistance to at least three different pathogens and pests of soybean. Based on the current annotation in Soybase [ accessed June 2014], there were 39 genes within or close to the QTL on chromosome 8 (Table 10) which included the Rhg4 for SCN resistance. Based on Williams 82 sequence, this QTL also contains four serine hydromethyltransferases (SHMT), which were reported to function in photorespiratory pathway influencing resistance to biotic and abiotic stresses (Moreno, 2005). A study by Liu et al. (2012) confirmed that SHMT is the Rhg4 gene by using mutation analysis, gene silencing and transgenic complementation techniques and also cloned the gene from cultivar Forrest. However, in a large evaluation of soybean genotypes for resistance to F. graminearum, Forrest was found to be susceptible with the disease severity index of 70.7% and mean disease score of 3.9 (Chapter 2). Interestingly, all the sources of resistance and differentials used to identify Heterodera glycines (HG) types were resistant to F. graminearum (Chapter 2). This indicates that the resistance to F. graminearum is potentially linked to Rhg4 genes identified in the QTL region and SHMT is less likely to be involved in 80

96 the resistance response potentially due to the susceptibility of the cultivar Forrest. However, further investigation is required to address this hypothesis. This QTL region also includes chalcone synthase, which is a key regulatory enzyme in flavonoid pathway and contains eight-member gene family and some of them are involved in tissue specific gene silencing, such as seed coat pigmentation (Tuteja et al., 2004). Additionally, two subtilisin-like proteases were located within the region, which has a predicted function in recognition of cell wall components during the plant development and in the plant defense responses (Nelsen et al., 2004). Another protein, agamous-like 3 protein was located upstream of QTL which has sequence specific DNA binding transcription factor activity ( and a bzip transcription factor bzip110 was located downstream. Other genes of interest that may be candidates for a defense response are Brassinosteroid-regulated protein, that regulates the gene expression and promotes elongation in soybean (Zurek and Clouse, 1994); DNAJ-like protein, that play role in protein translation, folding, unfolding, translocation and degradation ( lectins, which are carbohydrate-binding proteins found mostly in seeds and also in roots of soybean (Vodkin and Raikhel, 1986); and aspartokinase-homoserine dehydrogenase, that catalyze the pathway for the synthesis of lysine, threonine, and methionine from aspartate (Weisemann and Matthews, 1993). The QTL on chromosome 6 mapped close to the resistant QTL for SDS (Kazi et al., 2008), flood tolerance (Githiri et al., 2006), Sclerotinia sclerotiorum (Huynh et al., 2010) and also mapped close to a minor QTL for soybean aphid resistance (Jun et al., 2013) (Soybase, accessed on July, 2014). On chromosome 6, four genes mapped close to the QTL conferring resistance to F. graminearum (Table 11). 81

97 Using the RILs derived from a cross of Conrad x Sloan, three QTL were detected on chromosomes 10, 14, and 19 (Figure 14). The resistance QTL on chromosome 10 was mapped close to QTL conferring resistance to SCN (Chang et al., 2011), and P. sojae (Wu et al., 2010) and the resistance QTL on chromosomes 14 mapped close to QTL conferting resistance to Sclerotinia sclerotiorum (Vuong et al., 2008). The QTL on chromosome 19 in this study overlapped the region previously reported by Ellis et al. (2012). The QTL on chromosome 19 also mapped to the region with resistance QTL for P. sojae (Stasko et al., personal communication; Wang et al., 2012) and Pythium irregulare (Nauth et al., in press) in this same population. This QTL region has been associated with resistance for SDS (Njiti and Lightfoot, 2006), SCN (Guo et al., 2006), and P. sojae (Wang et al., 2012). Numerous genes which have been previously shown to contribute to the defense response in other host pathogen system were in the QTL regions. On chromosome 10, four genes including two phenylalanine ammonia-lyase 2 genes, Nodulin 6I mrna and a putative phytosulfokine peptide precursor (Table 12) are close to the QTL. On chromosome 14, six genes were close to the QTL which included rfls6 protein, MAPK mrna, glutathione S-transferase GST-24 and three other genes (Table 13). Glutathione S-transferases are transcription factors involved in cell protection from oxidative damages to support nitrogen fixation (Dalton et al., 2009; McGonigle et al., 2000). The resistance QTL on chromosome 19 contained nine genes within or close to the region. The region included bzip transcription factors and MYB transcription factors in addition to other genes (Table 14). 82

98 Most of the QTL detected from the two populations in this study mapped close to the QTL region for SCN and P. sojae with the exception on chromosomes 6 and 14. The QTL on both chromosomes 6 and 14, mapped close to QTL conferring resistance to Sclerotinia sclerotiorum. Similarly, chromosomes 8, 14 and 19 have defense related genes and transcription factors in the QTL region or in close proximity indicating their potential role in contributing to the expression of resistance to F. graminearum. Genes, particularly in the bzip family, MYB family and glutathione S-transferase family might be the candidates for further studies as they were found close to resistant QTL in several chromosomes. Future studies will need to focus on the expression and functional analysis of these genes. In a study by Ellis et al. (2012), four resistance alleles for resistance QTL were contributed by Conrad on chromosomes 8, 13, 15 and 16 and one resistant allele from Sloan for QTL on chromosome 19. In the present study, one resistant allele was contributed by Conrad for resistance QTL on chromosomes 10, not detected in the study by Ellis et al. (2012) while no QTL were detected on chromosomes 8, 13, 15 and 16 in this study compared to the earlier. The resistance allele for QTL on chromosomes 14 was also contributed by Sloan in this study which was not detected in the previous study by Ellis et al. (2012). However, the resistance allele for QTL on chromosome 19 was from Sloan that mapped to the same region as reported in the previous study by Ellis et al. (2012). The differences in the QTL locations in this study compared to the study by Ellis et al. (2012) might be due to the use of more advanced population with higher number of RILs and different experimenter as the same isolate was used. 83

99 More recently, a study by Garcia et al. (2012) in Argentina, observed higher level of DON and ZEA on milled soybean agar media and in irradiated soybean seeds in petri dish assay. The suitable temperature for higher DON production was found to be 15 or 15/20 o C cycling temperature. Similarly, in a study by Barros et al. (2012) in Argentina, high frequency of natural Fusarium contamination was observed in the reproductive growth stages of soybean, planted after wheat. Fusarium graminearum species complex amounted to 11% of the total species that were present among the three stages (flowering, R6 and R8) evaluated, with the higher isolation in pods and seeds at R6 stage followed by pods and seeds in R8 stage and in flowers. In addition, seed samples from R6 and R8 stage were evaluated for DON and NIV contamination. Out of 40 samples evaluated, one sample from each stage had detectable DON contamination while NIV was not detected. Hence, the natural contamination of DON in soybean during the grain ripening stage was observed, but the level of DON was less in soybean compared to wheat. The observation of pod and seed infection in addition to seed and seedling rot indicates the potential threat of higher yield loss and DON contamination. In addition, there might be two types of resistance to Fusarium graminearum in soybean as reported in head blight in wheat, Gibberella ear rot in corn and SDS in soybean as it causes seed and seedling rot as well as pod and seed infection. Recently, F. proliferatum was also reported to be highly pathogenic to soybean (Díaz Arias, 2011). Therefore, it is worth investigating to know if soybean has common resistance to pathogenic species of Fusarium (especially, F. graminearum, F. virguliforme and F. proliferatum) as observed in wheat (Toth et al., 2008) and corn 84

100 (Mesterházy, 1982). Similarly, F. graminearum isolates containing 3 ADON (more aggressive compared to 15 ADON) and NIV chemotypes were reported recently in North America (Foroud et al., 2012; Gale et al., 2011; Starkey et al., 2007). Hence, if a change in the chemotype of F. graminearum is causing this fungus to be pathogenic to soybean and other crop species would be another question worth of investigation. To date, F. graminearum has not been reported as a seed and pod pathogen in North America, however, the observation of pod blight in Brazil (Martinelli et. al., 2004) and seed and pod infection in Argentina (Garcia et al., 2012; Barros et al., 2012) signals the possibility in the areas where wheat, corn and soybean are grown in rotation and in the fields with high inoculum levels and favorable environmental conditions. However, QTL detected in this study should be introgressed into the breeding program to develop cultivars with resistance to F. graminearum for seed and seedling phase. To help prevent the threat of mycotoxin contamination of pods and seeds of soybean, more studies are needed to determine if the same QTL will confer resistance to these later infections. 85

101 No. of RILs Wyandot x PI567301B F 7:10 population Wyandot PI567301B Conrad Williams Sloan BLUP value Figure 11: Frequency distribution of BLUP values for DSI * of F 7: 10 recombinant inbred lines (RILs) population derived from the cross of Wyandot x PI B. Estimates of two parents and checks are indicated by arrows. A lower BLUP value means higher level of resistance to F. graminearum. * Disease severity index (DSI) equals lesion length divided by total length of the seedling multiplied by 100. The experimental design was augmented incomplete block design with three replications repeated over time. Fifteen seeds of each RIL were used; each seed was inoculated with 100 µl of 2.5 x 10 4 macroconidia ml -1 suspension of F. graminearum isolate Fay

102 No. of RILs Conrad x Sloan F 9:11 RILs Population Conrad Wyandot PI567301B Williams Sloan More BLUP value Figure 12: Frequency distribution of BLUP values for DSI * of F 9:11 recombinant inbred lines (RILs) population derived from the cross of Conrad x Sloan. Estimates of two parents and checks are indicated by arrows. A lower BLUP value means higher level of resistance to F. graminearum. * Disease severity index (DSI) equals lesion length divided by total length of the seedling multiplied by 100. The experimental design was augmented incomplete block design with three replications repeated over time. Fifteen seeds of each RIL were used; each seed was inoculated with 100 µl of 2.5 x 10 4 macroconidia ml -1 suspension of F. graminearum isolate Fay

103 Checks DSI Mean Grouping Score Mean Grouping Sloan 87.4 A 4.3 A Williams 66.4 B 3.7 B Conrad 43.1 C 3.0 C Wyandot 35.7 D 2.8 D PI B 8.6 E 1.6 E Table 6: Mean Disease Severity (DSI) and mean score of checks used in the Wyandot x PI B population study after inoculation of seeds with 100µl of 2.5 x 10 4 macroconidia ml -1 of F. graminearum in a rolled towel assay. Checks DSI Mean Grouping Score Mean Grouping Sloan 80.7 A 4.1 A Williams 60.6 B 3.5 B Conrad 43.2 C 3.0 C Wyandot 40.6 C 3.0 C PI B 8.1 E 1.6 D Table 7: Mean Disease Severity (DSI) and mean score of checks used in the Conrad x Sloan population study after inoculation of seeds with 100µl of 2.5 x 10 4 macroconidia ml -1 of F. graminearum in a rolled towel assay. 88

104 Chr. a Peak marker b LOD c PVE (%) d R-allele from e 6 BARC_2.0_Gm06_ PI B 8 BARC_2.0_Gm08_ PI567301B Table 8: Quantitative Trait Loci conferring resistance to F. graminearum identified via composite interval mapping (CIM) in the Wyandot x PI B RILs population. a Chromosome b Marker with the highest LOD score at each locus. A SNP name consisted of BARC, genome assembly ver. 2.0, chromosome, and physical position. c Log of Odd d Percentage of phenotypic variance explained by a QTL e Resistance allele contribution 89

105 Chr. a Peak marker b LOD c PVE (%) d R-allele from 10 BARC_1.0_Gm10_ _A_G Conrad 14 BARC_1.0_Gm14_ _G_A Sloan 19 BARC_1.0_Gm19_ _T_C Sloan Table 9: Quantitative Trait Loci conferring resistance to F. graminearum identified via composite interval mapping (CIM) based on version 1.0 SNP position using Conrad x Sloan RILs population. a Chromosome b Marker with the highest LOD score at each locus. A SNP name consisted of BARC, genome assembly ver. 2.0, chromosome, and physical position. c Log of Odd d Percentage of phenotypic variance explained by a QTL e Resistance allele contribution 90

106 Figure 13: Graphical presentation of quantitative trait loci (QTL) for resistance to Fusarium graminearum identified in the Wyandot x PI B population by genome-wide LOD threshold 3.4 (a hatched line in LOD plots). The 1- and 2- LOD intervals are presented by a black bar and solid lines between the chromosome and the LOD plot for each QTL. 91

107 * Continued Figure 14: Graphical presentation of quantitative trait loci (QTL) for resistance to Fusarium graminearum identified in the Conrad x Sloan population by genomewide LOD threshold 1.9 (a hatched line in LOD plots). 92

108 Figure 14 continued * Continued 93

109 Figure 14 continued * * Nearest marker to the mapped quantitative trait loci (QTL) on the respective chromosomes 94