i PYRAMIDING RESISTANCE GENES FOR APHANOMYCES ROOT ROT IN PEAS by Jenish Simon A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Plant Sciences MONTANA STATE UNIVERSITY Bozeman, Montana December 2024 ©COPYRIGHT by Jenish Simon 2024 All Rights Reserved ii DEDICATION First and foremost, I dedicate this work to God, whose guidance and grace have carried me through every challenge and triumph along this journey. To my family, especially my mom, Elizabeth Jaya, and my dad, Simon, I am forever grateful for your endless patience, strength, and the values you instilled in me. My greatest motivation is to thank my uncle, James Rajan, for your encouragement and belief in my abilities. To my best buddies, Janis Pohl and Sam Nicolas, for supporting me throughout this journey Finally, to my advisor, Dr. Kevin McPhee, whose mentorship and guidance were invaluable throughout this academic journey. iii ACKNOWLEDGEMENTS I would like to express my gratitude to my advisor, Dr. Kevin McPhee, for his continuous guidance and support throughout my Master’s project. I would also like to acknowledge my committee members, Dr. Alan Dyer and Dr. Jason Cook, for their valuable guidance and suggestions. I’m grateful for Dr. Norm Weeden’s guidance in molecular techniques. Thank you to Dr. Carmen Murphy for teaching me isolation and maintenance protocol for Aphanomyces root rot. Thanks to Jacob Tracy and Stacy Dreis for helping me with lab experiments. I would like to thank everyone who has worked in my lab: Amrit Poudel, Shreejana KC, Jacob Rasmussen, Shreya Gautam, Aoran Diao, and Jessica Ayers. This project was funded by the USA Dry Pea & Lentil Council. iv TABLE OF CONTENTS 1. PYRAMIDING RESISTANT GENES FOR APHANOMYCES ROOT ROT IN PEAS ............................................................................................................... 1 Introduction ................................................................................................................... 1 Dry Pea.......................................................................................................................... 2 Dry Pea Production ................................................................................................. 3 Origin and Domestication ....................................................................................... 5 Botany of Pea .......................................................................................................... 6 Market Class and End Use ...................................................................................... 8 Dry Pea in Montana ...................................................................................................... 8 Abiotic Stress .......................................................................................................... 9 Biotic Stress .......................................................................................................... 10 Bacterial Diseases ..................................................................................................11 Viral Diseases........................................................................................................ 12 Parasitic Nematodes .............................................................................................. 12 History of Oomycetes ................................................................................................. 12 Stages of Oomycetes ............................................................................................. 13 Aphanomyces Genus ............................................................................................ 13 Aphanomyces Root Rot in Peas ............................................................................ 14 Geographical Distribution ..................................................................................... 15 Environmental Characteristics .............................................................................. 16 Lifecycle of Aphanomyces euteiches .................................................................... 16 Symptoms and Pathotypes of Aphanomyces Root Rot......................................... 19 Detection of Aphanomyces euteiches.................................................................... 19 Soil Extraction Methods ....................................................................................... 19 Molecular Analysis ............................................................................................... 20 Management of Aphanomyces Root Rot in Peas .................................................. 21 Pioneer Breeding Efforts for Aphanomyces Root Rot in Peas ................................... 22 Pathogenicity of Aphanomyces Root Rot ............................................................. 23 Quantitative Trait Loci (QTL) Associated with ARR Resistance ............................... 25 Aphanomyces Root Rot QTL Meta-Analysis ....................................................... 28 Genome-Wide Association Mapping for ARR in Peas ............................................... 29 Gene Pyramiding ........................................................................................................ 31 2. MATERIALS AND METHODS ................................................................................ 33 Plant Materials ............................................................................................................ 33 Bi-parental Populations ............................................................................................... 33 Disease Inoculum ...................................................................................... 35 Inoculum Preparation ................................................................................ 35 Inoculation Method ................................................................................... 36 v TABLE OF CONTENTS CONTINUED Inoculation Method 2: ................................................................... 36 Experimental Setup ................................................................................... 37 Scoring Index ............................................................................................ 38 3. RESULTS.................................................................................................................... 40 Pathogenicity Test ....................................................................................................... 40 Pure-Culture Evaluation of Aphanomyces Root Rot Resistant Pea Parents ................................................................................ 41 Scoring Bi-Parental Populations ............................................................... 42 4. DISCUSSION ............................................................................................................. 48 5. CONCLUSION ........................................................................................................... 50 REFERENCES CITED ..................................................................................................... 51 vi LIST OF TABLES Table Page 1. Table 1. Aphanomyces root rot-resistant germplasm pea lines ......................................... 23 2. Table 2. Aphanomyces euteiches pathogenic variability and race identification globally. ...................................................................................................... 24 3. Table 3. Meta-QTL and QTLs were reported for partial resistance to Aphanomyces root rot in peas........................................................................................... 29 4. Table 4. Ten consistent regions for partial resistance to Aphanomyces root rot in peas (Leprévost et al., 2023). .................................................................................. 31 5. Table 5. Partially resistant and susceptible pea parent germplasm screened for resistance to Aphanomyces root rot ‘+’ denotes the partially resistant pea lines and ‘-’ denotes the susceptible pea lines................................................................... 33 6. Table 6. List of Pea Populations used in this experiment, along with their pedigree. ............................................................................................................................ 34 7. Table 7. Isolates of Aphanomyces euteiches collected from fields in Montana. ........................................................................................................................... 40 vii LIST OF FIGURES Figure Page 1. Figure 1. World production of dry pea and the production share of leading nations from 2012 to 2022 (FAO 2023). ............................................................................. 4 2. Figure 2. US Dry pea production data from 2014 to 2023 in million hundredweight (USDA 2024). ............................................................................................ 4 3. Figure 3. Acreage of dry peas in Montana. Source from USDA-NASS (2019). ................................................................................................................................. 9 4. Figure 4. Aphanomyces root rot in peas symptoms (a) chlorosis in lower leaves, (b) infected plant (right) vs. not infected plant (left), (c) honey brown discoloration of the roots. Source Wu et al., (2018). ........................................................ 15 5. Figure 5. Different structures of Aphanomyces euteiches: a. aseptate coenocytic hyphae, b. zoospore, c. oogonium, d. sexual stage (antheridium and oogonium), e. oospore. Source Wu et al., (2018). ...................................................... 18 6. Figure 6. 23 Additive-effect QTLs and 13 epistatic-effect QTLs partially resistant to ARR in pea (Ae-Ps: Aphanomyces euteiches-Pisum sativum, Ae- PsE: Aphanomyces euteiches-Pisum sativum epistasis) (Hamon et al., 2011). ................................................................................................................................ 27 7. Figure 7. The schematic diagram for incorporating ARR-resistant QTLs. ....................... 32 8. Figure 8. Photographic representation of each step of the screening protocol for plant reaction to Aphanomyces euteiches. ................................................................... 36 9. Figure 9. The disease severity score for reaction to Aphanomyces euteiches in peas was evaluated visually based on the presence of root discoloration. Photo: Carmen Murphy. .................................................................................................... 38 10. Figure 10. Disease severity scoring reaction to Aphanomyces euteiches by comparing individuals from a segregating population and the parents, PI 652444 and CDC BRONCO. ............................................................................................ 39 11. Figure 11. Disease reaction of five Aphanomyces euteiches isolates on two partially resistant and four susceptible pea cultivars. ....................................................... 41 viii TABLE OF FIGURES CONTINUED Figure Page 12. Figure 12. Disease Severity Score of 15 pea parental lines x-axis denotes the list of 15 pea parental lines and the y-axis denotes the disease rating scores ranges 0-5. .............................................................................................................. 42 13. Figure 13. Phenotypic scores for Set 1 populations and their parents. The partially resistant and susceptible parents were plotted as green and blue, respectively, in the graph. ................................................................................................. 43 14. Figure 14. Phenotypic scores for Set 2 populations and their parents. The partially resistant and susceptible parents were plotted as green and blue, respectively, in the graph. ................................................................................................. 44 15. Figure 15. Phenotypic scores for Set 3 populations and their parents. The partially resistant and susceptible parents were plotted as green and blue, respectively, in the graph. ................................................................................................. 45 16. Figure 16. Phenotypic scores for Set 4 populations and their parents. The partially resistant and susceptible parents were plotted as green and blue, respectively, in the graph. ................................................................................................. 46 17. Figure 17. Phenotypic scores for Set 5 populations and their parents. The partially resistant and susceptible parents were plotted as green and blue, respectively, in the graph. ................................................................................................. 47 ix ABSTRACT Dry pea is an important legume crop due to its protein content, nitrogen fixation ability, and value to crop rotation practices. These aspects contribute to both human and animal consumption. Pea cultivation is challenged from both abiotic and biotic stress. Soil-borne root rot pathogens lead to significant yield losses, up to 70%. Aphanomyces root rot (ARR) (caused by Aphanomyces euteiches Dreches) is one of the most destructive pea oomycete pathogens capable of attacking pea plants at any stage of development. Common symptoms include root decay, chlorosis, and wilting, particularly in wet soil conditions. Oospores are sexual spores produced by pathogens that can remain in the soil for many years, contributing to pathogen persistence. Standard practices such as seed treatment and other cultural practices have proven ineffective against this pathogen. Two major quantitative trait loci (QTL) Ae.Ps4.5 and Ae.Ps7.6 have been reported in previous studies. This research aims to pyramid quantitative trait loci Ae.Ps4.5 and Ae.Ps7.6 to increase ARR resistance in pea genotypes through marker-assisted selection. The derived lines with multiple QTL were evaluated using artificial inoculation methods in controlled environments. The first approach was to characterize reportedly resistant germplasm for reaction to ARR through rigorous pure culture screening. Subsequently generating molecular markers to facilitate introgression of ARR resistance QTLs into adapted genetic backgrounds as germplasm or cultivars suitable for cultivation in Montana. This study illustrates integration of multiple resistance genes minimizes the risk of pathogen adaptation and enables the development of cultivars with broad-spectrum resistance against ARR. Development of resistant varieties is crucial for ensuring sustainable pea production. This study demonstrates the application of marker assisted selection for pyramiding genes for ARR resistance. 1 PYRAMIDING RESISTANT GENES FOR APHANOMYCES ROOT ROT IN PEAS Introduction Pea (Pisum sativum L.) is a vital legume crop for protein, nitrogen fixation, and crop rotation. Its cultivation faces challenges from both biotic and abiotic stress. Soil-borne root rot pathogens cause significant yield losses, up to 70%. Aphanomyces root rot, caused by Aphanomyces euteiches, is one of the most destructive oomycete pathogens capable of attacking pea plants at any stage of development. Common symptoms include root decay, chlorosis, and wilting, especially in waterlogged conditions. Oospores are sexual spores produced by pathogens that can persist in the soil for many years, contributing to disease longevity. Efforts to control this pathogen, including commonly used methods like seed treatment and other cultural practices, have been found to be ineffective in combating its spread. In 2023, Montana's dry edible pea production covered an estimated 580,000 planted acres, with approximately 570,000 acres harvested. This represented an increase from 2022, where 550,000 acres were planted, and 510,000 acres were harvested. The yield for 2023 was also higher, averaging 1,740 pounds per acre, contributing to a total production of 9.92 million hundredweight, a 34% increase from the previous year and production was valued at $93,917,000 (USDA-NASS, 2024). Montana is the second-largest producing state for dry peas in the US, contributing 7,128,000 CWT in 2022, which accounts for one-third of US production (USDA-NASS, 2024). This study aims to screen available Aphanomyces root rot (ARR) resistant pea lines through greenhouse screening followed by validation of SSR markers linked to ARR resistance 2 QTLs in parents and the populations of pea genotypes that exhibit partial resistance to ARR. Several inoculation methods are available for greenhouse and nursery inoculation (Zitnick- Anderson et al., 2021). Zoospore suspension and dried inoculum methods are prevalent in controlled environments like greenhouses (Desgroux et al., 2016; Hamon et al., 2011; Pilet- Nayel et al., 2002; Wicker & Rouxel, 2001; Wu et al., 2018). Specifically, this research uses pure culture screening to characterize resistant pea germplasm for Aphanomyces root rot (ARR). Additionally, commercially available susceptible pea parents were selected to generate segregating populations in combination with the resistant germplasm to further integrate resistance QTLs Ae.Ps4.5 and Ae.Ps7.6 through marker-assisted selection to develop ARR- resistant pea varieties for sustainable cultivation. Dry Pea The worldwide need for peas or processed pea products is on the rise because of their versatile use as a source of protein for human consumption and as animal feed. Dry pea is a proteinaceous legume crop grown globally and is used as food, animal feed, and crop rotation practices (McPhee, 2003). North America is a leader in dry pea production and has been a major exporting continent for the past few decades (Janzen et al., 2014). Legumes, especially pea, act as a source of nitrogen through symbiotic nitrogen fixation. Most cereal crops use up to 20 – 40 kg N/ha over 3 to 5 months of pulses in crop rotation to fix atmospheric nitrogen via biological nitrogen fixation (BNF). Pulses are an important source of nutrients. In addition to food staples like wheat and rice, peas are a major source of protein (Joshi & Rao, 2017). Most peas produced are used as animal feed and exported to developing nations worldwide. Peas produced in North America are 3 used domestically as an important food source for the vegetarian community due to their nutritional properties (Joshi & Rao, 2017). Peas are rich in protein (20-25g/100g), amylose 32.2- 41.1% (Simsek et al., 2009), carbohydrates (12-15 g/100 g), essential amino acids and complex micronutrients such as iron (Fe:4.6-5.4 mg/100 g), zinc (Zn: 3.9-6.3 mg/100 g), and magnesium (Mg: 135-143 mg/100 g) (Thavarajah et al., 2022). In addition, most pulse breeding programs worldwide work on biofortification to increase iron, zinc, carotenoids, and folates to overcome micronutrient malnutrition (Jha & Warkentin, 2020). Dry Pea Production Globally, 14.1 MMT peas were produced in 2022 (Figure 1); in the US, 1,052,001 acres are pea-cultivated and contribute 7.1% of world production (Figure 2) (Thavarajah et al., 2022). Canada is the leading producer and exporter of dry peas globally, having produced 3.6 million metric tons and exported 3.2 million metric tons from 2018 to 2019. The following year, production increased to approximately 4.3 million metric tons, while exports decreased to 3.1 million metric tons (Mondor, 2020). Canada produces approximately 40% of the world's peas, followed by the Russian Federation at 14%, Australia at 10%, the United States at 8%, and Ukraine at 4%. (USDA, 2022). 4 Figure 1. World production of dry pea and the production share of leading nations from 2012 to 2022 (FAO 2023). Figure 2. US Dry pea production data from 2014 to 2023 in million hundredweight (USDA 2024). 5 Origin and Domestication Archaeological evidence suggests peas originated from the Middle East (Trněný et al., 2018). More than 171 germplasm collection centers possess 99,000 accessions worldwide (Trněný et al., 2018). Pea (Pisum sativum L.) belongs to the Leguminosae family, with 800 genera and more than 18,000 species (Kenicer, 2005). The Pisum genus belongs to the tribe Vicieae, subfamily Papilionoideae (Maxted & Ambrose, 2001). This genus has large leafy stipules and are semi-amplexical around the terete stem. Generally, pea is classified into two types, garden pea and field pea, based on commercial use (Amurrio et al., 1991). Numerical taxonomy was utilized to classify various pea landraces and four commercial pea varieties based on their quantitative traits. This classification resulted in six distinct groups. Among these, three groups are particularly noteworthy: Group 1 includes varieties intended for livestock, Group 2 consists of varieties meant for fresh consumption, and Group 4 encompasses varieties with tender green pods (Amurrio et al., 1991). The United States Department of Agriculture describes pea as Pisum sativum ssp. sativum L. (Duke, 2012)(Wiersema et al., 1990). Morphologically, peas are short with basal branching, small flowers of light yellow to dark orange color, and dehiscent pods with small dark brown round seeds (Makasheva, 1984). Many subspecies of Pisum sativum are categorized as different species or synonyms based on their geographical location and genetic traits. Pisum sativum ssp. abyssinicum A. Br. cultivated in Ethiopia and Yemen has a waxy stem coat, a bluish tinge on the juvenile stems and leaves, and irregularly rounded greenish-grey to dark violet seeds cultivated along with Pisum sativum L. (Makasheva, 1984; Weeden, 2018). Pisum elatius grows in the wild in the Caucasus and Mediterranean mountain valleys (Makasheva, 1984). Pisum pumilio grows as a type of weed distributed from Greece to Iran (Makasheva, 1984). Pisum arvense is the common fodder pea 6 type, and Pisum sativum var. macrocarpon is the edible pod type including sugar, snap, and snow peas (Gaskell, 1997). Pisum transcaucasicum has violet mottling seeds that are brown and marbled in color. Pisum formosum, or Vavilovia formosa, is distributed in southwestern Asia and is an ornamental type with carmine flowers (Duke, 2012). Pisum asiaticum is the primitive form of garden pea distributed in central and western Asia (Makasheva, 1984). These species within Pisum represent a wide range of genetic diversity which is vital for developing improved cultivars for yield, environment stress response, and response to other abiotic and biotic factors (Brhane & Hammenhag, 2024). Botany of Pea Pea belongs to the Leguminosae family, subfamily Fabaceae, and is a self-pollinated diploid (2n=2x=14) with seven chromosomes. It was the plant model for Gregor Mendel's discoveries (Smýkal et al., 2012). Peas are dicotyledonous with epigeal germination (Kenicer, 2005). A hilum is present on the seed near the micropyle portion where the radicle emerges (Makasheva, 1984). The root system is a taproot with many lateral and sub-lateral branches where nodules containing symbiotic nitrogen fixing bacteria can form (Kenicer, 2005). Based on leaf morphology, peas are divided into three types afila, stipulata, and tendrilled acacia three genes control this leaf morphology: af, st, and tl (Knott, 1987). The af gene plays a crucial role in determining whether a pea plant develops tendrils or leaflets. When a pea plant carries the dominant Af allele, it produces leaflets. However, if the plant possesses the recessive af allele, known as the afila mutation, the leaflets transform into tendrils (Knott, 1987). The st gene regulates the development of stipules, which are the leaf-like structures found at the base of the leaves. The dominant St allele produces normal stipules, whereas the recessive st allele leads 7 to reduced or modified stipules. Plants with modified stipules often have fewer surfaces for photosynthesis and may exhibit decreased overall vigor (Knott, 1987). The tl gene affects both the branching and size of leaflets in plants. When the dominant Tl allele is present, the leaflets develop normally and are fully formed. In contrast, the expression of the recessive tl allele results in smaller and more segmented leaflets, similar to those of the acacia tree. This leads to a divided or feathered appearance in the leaflets, which can reduce the plant's photosynthesis efficiency (Knott, 1987). Based on the leaf types, peas are divided into two categories: normal leaf and semi-leafless type. Peas with white to purple reddish flowers produce four to nine seeds per pod (McKay et al., 2003). Field peas are classified as determinate or indeterminate based on growth habit. Determinate types are compact with a bushy growth form that stops growing once the apical meristem terminates in a florescence. Determinate types often have synchronized harvest and indeterminate varieties produce new vines and branches throughout the growing season until terminal drought is prolonged harvest mature later (90 to 100 days) (McKay et al., 2003). Peas are self-pollinated and flowers are white, pink, purple, or bicolored. The number of seeds per pod ranges from 3 to 9, which can differ depending on the cultivar and environment. The seeds vary in size, ranging from 90 mg/seed to 400 mg/seed, and the number of pods per node or the plant varies depending upon the cultivar and environment (Knott, 1987). Vining peas generally produce three to five pods per fertile node (Knott, 1987). The seeds are wrinkled or spherical- shaped depending upon the presence of starch, and the dry seed color varies from blue-green, creamy white, olive, brown, or reddish brown. Some seed may also be speckled or have marbled markings (Knott, 1987). Combining pea varieties with leaf morphology traits, such as afila and 8 tendrilled acacia types, has improved crop performance by enhancing canopy structure and lowering disease susceptibility (Alemu et al., 2022). Market Class and End Use Pea are an essential source of protein for both human nutrition and livestock globally (Singh & Jauhar, 2005). It also has starch and fiber as ingredients in the food industry (Singh & Jauhar, 2005). Pea is classified under two classes based on their physiological maturity: green or vegetable pea and field or dry pea. Green pea is harvested in the immature stage and further divided into garden or vining types, which are shelled peas used for frozen or canned products and edible-podded peas, otherwise known as snow, snap, and Chinese peas (Singh & Jauhar, 2005). Dry peas are further divided into two classes: a. whole grain, used as a snack, ingredient in cooking, canning, and animal feed. Split peas are an ingredient for soups and Indian dhal cuisine (Singh & Jauhar, 2005). Dry Pea in Montana In Montana, the value of dry pea production was $94,090,000 in 2022 over 1,122,200 acres of pulse crop cultivation (Figure 3) (USDA-NASS, 2022). Dry pea production is agronomically viable in the Northern Great Plains in the dryland cropping system since it can be used for multiple purposes: grain, forage, or green manure (Miller et al., 2005). Dry peas are cultivated in a wide range of soils, from sandy to clay, and are not productive in saline and waterlogged soils (Miller et al., 2005). Symbiotic nitrogen fixation by pea reduces the need for nitrogen fertilizer in the subsequent crop. Research in Montana suggests that nitrogen contribution after a pea crop is 10 lb/N ac. The general row spacing is 6 to 12 inches, and the 9 planting date is between late March and early May (Miller et al., 2005). Flowering initiates at the 10- to 14-leaf stage, about 50 to 60 days after planting and matures in 90 days in Montana (Miller et al., 2005). Figure 3. Acreage of dry peas in Montana. Source from USDA-NASS (2019). Abiotic Stress Depending on the environment and soil properties, drought, temperature, soil pH, nutrient deficiency, salinity, and toxicity are the common factors affecting field pea production (Stoddard et al., 2006). Among all factors, heat and drought are the most common abiotic stressors that directly influence the physiological pathways and plant water relations (Stoddard et al., 2006). 10 Temperature above 25°C affects the reproductive stage, causing embryo abortion and failed seed set that reduces yield. Temperature above 45°C affects pollen viability, pollen tube formation, and pollen germination (Jiang et al., 2015). Heat stress is unpredictably correlated with weather parameters such as rainfall, evapotranspiration, and soil moisture (Jiang et al., 2015). Drought stress causes an electron transport chain imbalance and down-regulates response to stomatal closure (Stoddard et al., 2006). Cold and frost damage is most common in winter pea and affects reproductive stages (Stoddard et al., 2006). Developing cultivars with improved tolerance is an important cultural practice for frost and cold stress. Field peas require neutral to slightly alkaline soil pH. Acidic soil pH causes nutrient unavailability like phosphorous and causes mineral toxicity (Rice et al., 2000). A pH imbalance below 5.5 reduces symbiotic atmospheric nitrogen fixation via Rhizobium leguminosarum cv. viciae (Rice et al., 2000). Saline soils are also a threat to field pea production in Montana. Biotic Stress Field peas are influenced by a series of pests and diseases that attack all the stages of the crop and cause yield reduction. This creates the necessity to develop pest and disease resistant cultivars for field pea production. Most diseases are caused by fungi, bacteria, viruses, and nematodes affecting seeds, seedlings, and the reproductive phase of crop development (Grünwald et al., 2004). Fungal diseases are classified under seedling and soil-borne disease- causing fungi and foliar disease-causing fungi (McVay et al., 2013). The most common soil- borne diseases include Aphanomyces root rot, caused by Aphanomyces euteiches Drechs., in which plants show a stunted or wilted appearance, the infected root shows caramel-brown color with sloughing off root tissue exposing the vascular tissue (Grünwald et al., 2004; Markell et al., 11 2022); fusarium root rot (Fusarium solani f. sp. pisi) where affected plants show red to brown- black colored roots with yellowing and necrosis of leaves, other soil-borne fungal pathogens are Pythium seed and seedling rot (Pythium ultimum), and Rhizoctonia seed, seedling, and root rot (Rhizoctonia solani). Management practices of soil-borne fungal pathogens include seed treatments, crop rotation, and resistant cultivars (Grünwald et al., 2004). Foliar fungal diseases include Ascochyta blight (Ascochyta pisi, A. pinodes, Phoma medicaginis var. pinodella), rust (Uromyces viciae-fabea), powdery mildew (Erysiphe pisi and E. trifolii), Septoria blight (Septoria pisi), Botrytis gray mold (Botrytis cinerea), white mold (Sclerotinia sclerotiorum), Fusarium wilt (Fusarium oxysporum f. sp. pisi), and downy mildew (Peronospora viciae f. sp. pisi) (Markell et al., 2022). Each foliar fungal disease has distinctive symptoms, including leaf, pod, and stem necrotic lesions, discoloration of leaves (chlorosis), and stunted plant growth (wilting). Management practices include foliar fungicides, resistant cultivars, and cultural practices, including avoiding susceptible pea cultivars in disease-prone zones (Grünwald et al., 2004). Bacterial Diseases Bacterial blight (Pseudomonas syringae pv. pisi) and brown spot (Pseudomonas syringae pv. syringae) are bacterial diseases that cause water-soaked necrotic lesions in angular shapes on the above-ground portion of the plant's surface (Markell et al., 2022). Warm temperatures and high humidity favor bacterial infection in pea plants; bacterial ooze can be noticed at the site of infection. Management practices, including field sanitation and resistant pea cultivars (Grünwald et al., 2004; Markell et al., 2022). 12 Viral Diseases Field peas are susceptible to viral diseases, including alfalfa mosaic virus (AMV), bean leaf roll virus (BLRV), pea enation mosaic virus (PEMV), pea streak virus (PeSV), red clover vein mosaic virus (RCVMV), and pea seedborne mosaic virus (PSbMV) (Grünwald et al., 2004). Most of the viral diseases are transmitted by the pea aphid (Miller et al., 2005). Planting close to alfalfa or clover plantations increases the severity of viral diseases (Grünwald et al., 2004). Control measures involve resistant or tolerant pea cultivars and insect traps (Grünwald et al., 2004). Parasitic Nematodes The most important nematodes that attack pea is the pea cyst nematode (Heterodera goettingiana Liebs), the root-knot nematode (Meloidogyne incognita), and the root-lesion nematode (Pratylenchus penetrans) (Grünwald et al., 2004). Plant parasitic nematodes favor secondary infection to other fungal pathogens as they degrade the plant tissue. (Grünwald et al., 2004) Management of nematode infection is limited to crop rotation, which is effective in Meloidogyne and Heterodera but ineffective in Pratylenchus (Grünwald et al., 2004). History of Oomycetes Plant pathology entered a new era after the Irish famine caused by the potato blight (Oomycete) epidemic in the 1840s (Agrios, 1988). The second important threat caused by oomycetes was downy mildew in grapes in 1878 by the pathogen Plasmopara viticola, which drastically decreased grape production in Europe (Gobbin et al., 2006; Heyman, 2008). For the past two decades, the silviculture industry has experienced epidemics ‘sudden oak death’ and 13 other timber plants in North America by oomycete pathogens, Phytophthora ramorum, Phytophthora cinnamomi (Hardham, 2005; Ivors et al., 2004). Morphologically, oomycetes resemble true fungi with similar disease cycles but differ in physiological traits. The cell wall components of oomycetes are composed of cellulose, whereas in true fungi, they are made up of chitin. Previous phylogenetic studies revealed that oomycetes belong to the kingdom Stramenopila (Baldauf et al., 2000; Heyman, 2008). Stages of Oomycetes The oomycete pathogen mainly belongs to the aquatic ecosystem and exhibits saprotrophic and parasitic behavior (Heyman, 2008). The vegetative stages of their lifecycle are diploid, with both heterothallic and homothallic modes of reproduction (Heyman, 2008). The female reproductive system is called oogonium, which is fertilized by a sexual spore called an oospore produced by antheridium; oospores are unicellular desiccation resistant and capable of surviving extreme conditions (Heyman, 2008). The asexual spore is called a zoospore, which is motile with two flagella used for locomotion in the soil or water ecosystem to find a favorable host; once it is attached to the host, it sheds the flagella and forms a spherical walled cyst to form mycelium or secondary zoospore (Hardham & Hyde, 1997). Some groups of oomycetes have special structures or spores called sporangia and chlamydospores (Heyman, 2008). Aphanomyces Genus Most of the pathogens in the Aphanomyces genus have a broad host range in plants and aquatic species. Genus Aphanomyces belongs to the order Saprolegniales. Other plant pathogens causing downy mildew and pythium belong to the Peronosporales and Pythiales order of 14 oomycetes. Other than plants, crayfish plague is caused by Aphanomyces astaci, and it acts as a parasite (Aphanomyces invadans), causing ulcerative syndrome in fish. Aphanomyces Root Rot in Peas The term Aphanomyces is derived from the Greek word aphanes+myces= obscure fungus (Papavizas & Ayers, 1974). Aphanomyces root rot was first reported in 1920 in Wisconsin, United States, and first described by Drechsler in 1925. He identified the pathogen in the root cortex and the basal stem of the peas with thick-walled oospores at the site of infection in the major pea-growing area in the United States, causing rotting of the roots and the epicotyl region of the seedling, resulting in chlorosis and stunted growth or death of plants in severe disease conditions (Figure. 4) (Jones & Drechsler, 1925; Kraft & Pfleger, 2001). There are no effective control measures against Aphanomyces root rot except prolonged crop rotation in disease-prone zones with non-host crops, avoiding legume crops in moderate to high disease-infected fields based on the soil inoculum level (Kraft et al., 1990). Soil-borne root rot pathogens are a threat to field pea production. They attack all life stages of the crop causing significant yield loss. Aphanomyces euteiches Dreches is the most destructive, causing up to 80% yield loss (Jacobsen & Hopen, 1981). 15 Figure 4. Aphanomyces root rot in peas symptoms (a) chlorosis in lower leaves, (b) infected plant (right) vs. not infected plant (left), (c) honey brown discoloration of the roots. Source Wu et al., (2018). Geographical Distribution Aphanomyces root rot is a global threat in many continents, including North America and countries like Europe, Australia, New Zealand, and Japan (Papavizas & Ayers, 1974). In the United States, a severe form of Aphanomyces root rot was found in Wisconsin, Minnesota, Michigan, New Jersey, Delaware, Maryland, Virginia, Connecticut, New York, Utah, Idaho, and Montana. It is also found in the Pacific Northwest and Southeastern United States (Papavizas & Ayers, 1974). Disease occurrence peaks in the fields using river water sources, increasing soil moisture during the crop season (Papavizas & Ayers, 1974). Aphanomyces root rot is observed in France, Norway, southern Sweden, and Denmark in Europe. It caused drastic crop loss in France's Seine et Oise district in 1932-1933 (Papavizas & Ayers, 1974). It also occurs in England and Wales, the nonchernozem zone of the USSR, 16 Tasmania (pea yield loss 18.4 bushels per acre to 10 bushels per acre in 1933-1934), Jamaica, and Australia (Papavizas & Ayers, 1974). Environmental Characteristics Aphanomyces root rot is abundant in heavy clay soil (Smith & Walker, 1941). Walker suggested there is a relationship between Aphanomyces root rot infection and soil properties (Smith & Walker, 1941). Disease spread is increased in soils with poor drainage and high moisture at 75 percent of water holding capacity has resulted in infection of more than 70 percent of the plants (Smith & Walker, 1941). The optimum soil temperature for infection is 16°C, and the initial stage of disease development occurs at 28°C (Papavizas & Ayers, 1974). Aphanomyces root rot incidence is dominant in heavy, poorly drained, compacted soil, and soil with high water holding capacity favors oospore germination and zoospore release (Papavizas & Ayers, 1974). Lifecycle of Aphanomyces euteiches Asexual reproduction of the Aphanomyces genus produces two types of zoospores in filamentous zoosporangia with a single row of slender zoospores. The primary zoospores are slower and weaker than the secondary zoospores (Papavizas & Ayers, 1974; Walker & van West, 2007). In some species of Aphanomyces, there is a unique structure called evacuation hyphae that changes the thallus into zoosporangia (Papavizas & Ayers, 1974). The first stage of asexual reproduction is the formation of vigorous mycelium by transferring the thallus to fresh water for water replacement; this process is called mycelial washing, and the initial stage of asexual reproduction occurs after 5 – 6 hours (Papavizas & Ayers, 1974). The zoosporangia of Aphanomyces euteiches contain axial filaments that are 1 - 2 mm long and develop 6 – 10 branches (Papavizas & Ayers, 1974). The overall asexual reproduction is explained in three 17 phases, starting from the development of thallus, differentiation of primary zoospores within the zoosporangium, and release of zoospores from the zoosporangium (Papavizas & Ayers, 1974). The secondary zoospores are released from the primary zoospore cyst. The size of the primary zoospore is 8 – 11 µm in diameter, and the secondary zoospore is 13 µm long and 7 – 8 µm in diameter (Papavizas & Ayers, 1974). The zoospore produces cysts germinating into a tube through continuous discharge and assimilation of calcium ions on the root surface (Deacon & Saxena, 1998). The motile zoospore consists of two whiplash/tinsel-type flagella 24 µm long. The right side flagella possesses many tinsels compared to the left one, and the whip is present in the end portion of both flagella, helping in their swarming motion (Papavizas & Ayers, 1974). Oospores are the sexual spores released from the thallus of Aphanomyces cuticles exposed to adverse environmental conditions and are able to overwinter because of their thick-walled lipid sac (Papavizas & Ayers, 1974; Shang et al., 2000). The sexual reproductive organs are antheridium and oogonium, present on the vegetative mycelium. The female structure before mating appears thin-walled, subglobose to spherical bodies with densely granular vacuolate matter about 25 – 35 µm in diameter (Papavizas & Ayers, 1974). The single oosphere or unfertilized egg develops an oospore through mating. Antheridia measure 8 -10 µm in diameter and 15 - 18 µm in length, curved clavate, borne on the oogonial stalk (Papavizas & Ayers, 1974). The oospores are hyaline, subspherical, or ellipsoidal 10 -25 µm in diameter (Papavizas & Ayers, 1974). Mating occurs when fertilization hyphae transfer male nuclei from antheridia to the oogonium, resulting in diploid oospores (Scott, 1961). 18 Figure 5. Different structures of Aphanomyces euteiches: a. aseptate coenocytic hyphae, b. zoospore, c. oogonium, d. sexual stage (antheridium and oogonium), e. oospore. Source Wu et al., (2018). Both asexual (hyphae, zoospores, and zoosporangia) and sexual (oogonia, antheridia, and oospore) stages occur in subterranean regions (Scott, 1961). The outer membrane of the oospore acts as a protective covering against harsh environmental conditions and remains dormant on the soil surface for up to 10 years (Gaulin et al., 2007; Pfender & Hagedorn, 1983; Sherwood & Hagedorn, 1962). The moist soil conditions that favor the germination of oospores produce germ tubes entering the root tissue, producing hyphae and oospores (direct germination). On the other hand, the oospore produces sporangium under favorable soil conditions. It produces a zoospore with biflagellate flagella able to swim to locate the host plant through chemotaxis encyst on the host and begin a secondary infection (Cannesan et al., 2011; Scott, 1961; Sekizaki & Yokosawa, 1988). 19 Symptoms and Pathotypes of Aphanomyces Root Rot The typical symptoms of Aphanomyces root rot infected pea plants include water-soaked lesions, caramel brown straw color, and soft rot on the root (Hagedorn, 1984). Overall root damage restricts the uptake of nutrients to the plant, causing chlorosis or yellowing of lower leaves, wilting, and stunted growth (Gangneux et al., 2014). Aphanomyces euteiches isolates collected from the United States, and France differ in their genetic structure, and a wide range of genetic diversity occurs within France (Le May et al., 2018). France has 11 pathotypes or virulence types across different locations; in the U.S.A., there are four significant pathotypes. Pathotype 1 originated in France and is more virulent, and Pathotype 3 is found in Wisconsin, U.S.A. (Sivachandra Kumar et al., 2021; Wicker & Rouxel, 2001). Pathotypes 3, 4, and 5 are not found in French isolates (Wicker & Rouxel, 2001). Detection of Aphanomyces euteiches The severity of the infection depends upon the inoculum density of the pathogen in the soil (Bouhot, 1979). To quantify the severity of the disease, the oospore concentration is measured in the soil through alternate extraction methods (Boosalis & Scharen, 1959; Burke et al., 1969; Mitchell et al., 1969; Pfender & DI, 1981; Sherwood & Hagedorn, 1958). Boosalis and Scharen (1959) developed microscopic detection of oospore concentration from the soil . The total number of oospores in 100g of soil = X/10 * Y, where X is the number of oospores counted in 5 microscopic slides and Y is the volume of the final suspension. Soil Extraction Methods Pfender et al. (1981) developed the Most Probable Method of soil inoculum quantification using the ten-fold dilution series made from infected soil. The solution was 20 thoroughly mixed with sterilized vermiculite media, and the pea plants were grown 14 days later; the roots were observed for the honey-brown discoloration used for the MPN calculation (Chan & Close, 1987; Pfender & DI, 1981). Malvick et al. (1994) estimate soil inoculum potential causing Aphanomyces root rot. The inoculum potential measures soil factors, propagule infectivity, and propagule density (Malvick et al., 1994). Isolating Aphanomyces euteiches from the soil is performed using semi-selective media that contains amphotericin B, rifampicin, benomyl, vancomycin, and metalaxyl to avoid other bacterial and fungal contamination (Pfender et al., 1984). The well-grown Aphanomyces euteiches culture from the semi-selective media is reinoculated into the corn meal agar or potato dextrose agar (Zitnick-Anderson et al., 2021). The Aphanomyces euteiches cultures in the corn meal agar mainly produce oospores, and the cultures in the potato dextrose agar produce zoospores (Yokosawa et al., 1995; Zitnick-Anderson et al., 2021). Molecular Analysis Molecular detection of Aphanomyces root rot is effective compared to other detection methods. Aphanomyces euteiches detection using wet sieving or microscopic techniques to analyze the inoculum potential or the oospore concentration was time-consuming and labor- intensive (Vandemark et al., 2000). Polymerase chain reaction assays were used to detect the presence of Aphanomyces euteiches in soil and plant tissue. The SCAR marker OPC71332 has primers OPC7-FS-30 (5′-GTCCCGACGACAACACCAAGAAAGACAACG-3′) and OPC7-RS- 25 (5′-GTCCCGACGAGGTTGGTGGCAAGTG-3′) amplify the specific PCR product that detects the presence of Aphanomyces euteiches infected pea root samples (Vandemark et al., 2000). 21 The pathogen was amplified by 0.9-kb product using primers specific to the actin gene, and three different restriction enzymes were used to differentiate the related species (Vandemark et al., 2000). The SCAR primers can be amplified from the root tissue at the early flowering stage of peas (Vandemark et al., 2000). The presence of Aphanomyces euteiches was detected in the DNA isolated from the soil's organic debris or the mineral fraction of soil samples taken from a 20 cm depth in the rhizosphere zone (Almquist et al., 2016; Moussart et al., 2009). Some defects that influence pathogenic DNA extraction from soil inhibitors, such as tannins and humic acid, expose pathogenic DNA from thick-walled oospores without degradation; detection in high clay soils is more difficult than in sandy soils (Vandemark et al., 2000). The real-time PCR assay was used to compare the presence of Aphanomyces euteiches incidence in resistant and susceptible pea genotypes over different time periods (Vandemark & Ariss, 2007; Willsey et al., 2018). Management of Aphanomyces Root Rot in Peas The most strategic way to manage Aphanomyces root rot is using resistant varieties. Prophylactic measures, fungicides, and biocontrol agents are also important control measures (Bodah, 2017). There are no effective fungicides to control Aphanomyces root rot. The most recommended cultural practice is avoiding pea cultivation in disease-prone zones (Pilet-Nayel et al., 2002). Seed treatment with biocontrol agents Pseudomonas cepacia or Pseudomonas fluorescens (Parke et al., 1991). Long crop rotation can break the disease cycle; planting Brassicaceae family crops suppresses the growth of Aphanomyces euteiches. Brassicaceae family crops contain glucosinolates that produce volatile isothiocyanates that control Aphanomyces root rot through hydrolysis (Bodah, 2017). The common management practices are draining wet soil, 22 rotation, tillage, fertilization, and weed control before planting (Bodah, 2017). Hymexazol, azoxystrobin, and propamocarb are the common fungicides applied in soil drenches to control Aphanomyces root rot (Watson et al., 2013). Pre-planting soil assessment is considered the best control measure in disease-infected fields; the oospore concentration in the field soil is considered the primary source of infection (Watson et al., 2013). Pioneer Breeding Efforts for Aphanomyces Root Rot in Peas Genetic resistance is considered a key factor in controlling Aphanomyces root rot. From 1925 to 1950, no pea cultivars were identified as resistant to Aphanomyces root rot (Shehata et al., 1976). The first breeding program of peas for Aphanomyces euteiches resistance started in 1957, introducing a few PI lines with phenotypic characteristics of dwarf, intermediate or tall, green, wrinkled seeds (Lockwood, 1960). Breeding efforts have been carried out there since the 1950s, and they have been difficult because of polygenic inheritance of resistance with low heritability (Pilet-Nayel et al., 2002). Several P.I. lines were identified with Aphanomyces disease resistance, and the initial breeding program in 1951-1963 focused on incorporating available resistance to commercial types at the New York Agricultural Experiment Station, Geneva (Marx et al., 1972). The selection was made by testing 800 P.I. accessions and 1680 breeding lines (Marx et al., 1972). There were no great attempts made to transfer disease- resistance traits from PI lines to the commercial pea cultivars before the 1970s; resistance to Aphanomyces root rot was linked with dominant wild-type alleles at three unlinked marker loci Le (tall), A (colored flowers), Pl (black hilum), and the recessive allele controlling other physiological traits (Shehata et al., 1976). Pea lines with improved partial resistance to 23 Aphanomyces root rot were developed by one cycle of phenotypic recurrent selection examining the F2 generation in disease-prone zones (Table. 1) (Lewis & Gritton, 1992; Marx et al., 1972). Aphanomyces Root Rot Resistant Pea Lines Reference 86-638, 86-2197, 86-2231, 86-2236 (Kraft, 1989) PI538355, PI538356, PI538357, PI538358, PI538359 (Gritton, 1990) 90-2079, 90-2131, 90-2322 (Kraft, 1992) MN 144, MN 313, MN 314 (Davis et al., 1995) 96-2052, 96-2158, 96-2068, 96-2198, 96-2222 (Kraft & Coffman, 2000a) 97-261, 97-2154 (Kraft & Coffman, 2000b) 97-363, 97-2170, 97-2162 (Kraft & Coffman, 2000c) Table 1. Aphanomyces root rot-resistant germplasm pea lines Pathogenicity of Aphanomyces Root Rot Different races or isolates of Aphanomyces euteiches show pathogenic variability on the host has been identified over many regions (Table. 2). The main categorization of Aphanomyces euteiches isolates is based on the size of the sexual and asexual spores, sporulation ability, synthesis of pectinolytic and cellulolytic enzymes, and the growth rate on media (Papavizas & Ayers, 1974). King and Bissonnette 1954 first identified different isolates of Aphanomyces euteiches which varied in pathogenicity (Bissonnette, 1958). Later studies confirmed the presence of genetic variation in Aphanomyces euteiches isolates identified in different pea- growing areas (Beute & Lockwood, 1967; Carlson, 1965; Malvick et al., 1994; Manning & Menzies, 1984; Schren, 1960; Sundheim & Wiggen, 1972; Wicker & Rouxel, 2001) 24 Country Race/virulence type Method Reference United States Races 1 and 2 Race identification (Beute & Lockwood, 1967) Norway Races 1-4 Race identification (Sundheim & Wiggen, 1972) New Zealand Race 5 Race identification (Manning & Menzies, 1984) United States Virulence groups I-IV Pathogenic Variability (Malvick et al., 1994) Europe, North America, and New Zealand Virulence types I-XI Pathogenic Variability (Wicker & Rouxel, 2001) Table 2. Aphanomyces euteiches pathogenic variability and race identification globally. Variation of pathogenicity and genotype among single zoospore strains of Aphanomyces euteiches was studied using Random Amplified Polymorphic DNA (RAPD) markers to detect the pathogenic variation and suggested the genetic changes in Aphanomyces euteiches may occur during asexual reproduction. Genotypic variation of 114 stains Aphanomyces euteiches was evaluated from Central and Western United States on two resistant pea lines MN313 AND MN314 (Malvick & Percich, 1998a, 1998b). The Pisum sativum Plant Identification (PI) germplasm collection has 2500 accessions and was tested for Aphanomyces root rot resistance. One hundred twenty-three accessions were identified with variable resistance levels. The 123 accessions were further evaluated with multiple strains of Aphanomyces euteiches from different regions of the United States (Malvick & Percich, 1999). Wicker and Rouxel categorized 109 25 Aphanomyces euteiches isolates from different geographical locations as virulence types (I-IX) based on their virulence potential (Wicker et al., 2003; Wicker & Rouxel, 2001). Quantitative Trait Loci (QTL) Associated with Aphanomyces Root Rot Resistance Genetic resistance is considered a better solution than other management practices safeguarding the environment by eliminating the need for chemical control (Leprévost et al., 2023; Pilet-Nayel et al., 2002). Previous research findings identified several QTLs throughout the pea genome that provide different levels of partial resistance to Aphanomyces root rot (Pilet- Nayel et al., 2002). Partial resistance or quantitative resistance is minimum resistance controlled by many loci that are effective against all pathogenic variability, and it is highly stable and durable. Introgression of multiple available QTLs is a promising strategy for long-term control against all the pathotypes of Aphanomyces root rot in peas (Leprévost et al., 2023; Pilet-Nayel et al., 2002). Linkage mapping studies were performed using partially resistant parents 90-2079, 90- 2131, 552, and PI180693. SSR markers applied to mapping populations resulted in 27 meta- QTLs related to partial resistance to Aphanomyces root rot in peas comprising seven significant QTL regions. The most effective, consistent QTL Ae-Ps4.5 and Ae-Ps7.6 located in linkage groups 4 and 7, result in a higher level of partial resistance to Aphanomyces root rot (Hamon et al., 2011; Leprévost et al., 2023; Pilet-Nayel et al., 2002) The initial attempt to find QTLs for partial resistance to Aphanomyces root rot in peas and its consistency over different locations in the United States was performed by Pilet-Nayel et al. (2002). They used a mapping population of 127 recombinant inbred lines (RILs) derived from 26 the cross of ‘90-2079’ (Kraft, 1992) with partial resistance to Aphanomyces root rot in the United States and ‘Puget’, an Aphanomyces root rot susceptible line (Pilet-Nayel et al., 2002). QTL mapping identified a major QTL, Aph1, on Linkage Group Ⅳb that showed an additive effect of 47% variation over multiple years and locations. Similarly, Aph2 (32% and 8% variation over two locations Pullman and LeSueur) and Aph3 (14% and 11% variation over two locations Pullman and LeSueur) QTLs were identified on linkage groups Ⅴ and Ⅰa, respectively, in which the resistant alleles segregated from the susceptible parent (Pilet-Nayel et al., 2002). The identified QTLs for Aphanomyces root rot partial resistance were further evaluated for consistency of resistance in the greenhouse and field, with two isolates, SP7 and Ae106, of Aphanomyces euteiches (Pilet-Nayel et al., 2005). Isolate SP7 was extracted from a pea field in northern Idaho, United States, and isolate Ae106 was extracted from Basin, France (Wicker & Rouxel, 2001). QTLs Aph1 and Aph3 were detected when the population was challenged with both isolates, and QTL Aph2 was detected when challenged with the French isolate Ae106 (Pilet- Nayel et al., 2005). Hamon et al. (2011) worked with two pea lines, PI 180693 and 552, partially resistant to both French and United States isolates of Aphanomyces euteiches. They developed 178 recombinant inbred lines by crossing the PI 180693 with Baccara, a susceptible line (Hamon et al., 2011). As a result, 135 additive-effect QTL were identified over 23 additive-effect genomic regions and 13 epistatic interactions favoring partial resistance to Aphanomyces root rot were detected (Figure. 6) (Hamon et al., 2011). 27 Figure 6. 23 Additive-effect QTLs and 13 epistatic-effect QTLs partially resistant to ARR in pea (Ae-Ps: Aphanomyces euteiches-Pisum sativum, Ae-PsE: Aphanomyces euteiches-Pisum sativum epistasis) (Hamon et al., 2011). Ae109 and RB84 strains of Aphanomyces euteiches used for the experiment Ae109 were isolated from Wisconsin, United States, and RB84 was isolated from Riec-sur-Belon, France (Hamon et al., 2011; Moussart et al., 2007); they are known as pathotypes I and III. (Hamon et al., 2011; Wicker & Rouxel, 2001) Hamon et al. (2011) classified the additive-effect QTLs into three categories: stable ( Ae-Ps1.2, Ae-Ps2.2, Ae-Ps3.1, Ae-Ps4.1 and Ae-Ps7.6 a, and Ae- 28 Ps7.6 b), moderately stable (Ae-Ps2.1, Ae-Ps2.3, Ae-Ps3.2, Ae-Ps4.3, Ae-Ps5.1, Ae-Ps6.1, Ae- Ps6.2, Ae-Ps7.2, Ae-Ps7.3, Ae-Ps7.4 and Ae-Ps7.5) , and poorly stable (Ae-Ps1.1, Ae-Ps4.2, Ae- Ps4.4, Ae-Ps4.5, Ae-Ps5.2, Ae-Ps5.3 and Ae-Ps7.1) (Hamon et al., 2011). Overall, 13 epistatic- effect QTLs were identified in the mapping population in that QTLs Ae-PsE5, Ae-PsE7, Ae- PsE8, Ae-PsE10, Ae-PsE11, Ae-PsE12 and Ae-PsE13 (Hamon et al., 2011). Aphanomyces Root Rot QTL Meta-Analysis Meta-QTL-Analysis integrates multiple reported QTLs for a particular trait from different studies or mapping populations based on their confidence interval (Table. 3) (Goffinet & Gerber, 2000). Meta-analysis was conducted for the previously identified Aphanomyces root rot resistance QTL in peas four mapping population Puget x 90-2079, Baccara x PI180693, Baccara x 552, and DSP x 90-2131 (Hamon et al., 2013). More than 244 QTLs were identified in the four mapping populations (Hamon et al., 2013). The outcome of the meta-analysis found 27 meta- QTL that have partial resistance to Aphanomyces root rot in that 11 consistent meta-QTL were considered as consistent with high confidence intervals in seven genomic locations that act as the key source of marker-assisted selection for Aphanomyces root rot in peas (Hamon et al., 2013). 29 Meta-QTL QTL Linkage Group Reference MQTL-Ae3 Ae-Ps1.2, Aph3 I (Hamon et al., 2013) MQTL-Ae5 Ae-Ps2.2 II MQTL-Ae6 Ae-Ps2.2 II MQTL-Ae8 Ae-Ps3.1 III MQTL-Ae9 Ae-Ps3.1 III MQTL-Ae12 Ae-Ps4.1 IV MQTL-Ae15 Ae-Ps4.4, Ae-Ps4.5, Aph1 IV MQTL-Ae16 Ae-Ps5.1, Aph2 V MQTL-Ae17 Ae-Ps5.1, Aph2 V MQTL-Ae25 Ae-Ps7.6a VII MQTL-Ae26 Ae-Ps7.6a VII Table 3. Meta-QTL and QTLs were reported for partial resistance to Aphanomyces root rot in peas. Genome-Wide Association Mapping for Aphanomyces Root Rot in Peas Higher-density Single Nucleotide Polymorphisms (SNPs) are considered a valuable source of genetic variation for multiple breeding programs (Duarte et al., 2014). The whole genome of the pea (size 4.3 Gb) was sequenced and 1538 SNPs were validated with a genetic map containing 2070 markers (Duarte et al., 2014). The previously developed SNP assays and associated genetic linkage maps were applied in marker-assisted selection for different biotic and abiotic stress factors (Leonforte et al., 2013; Tayeh et al., 2015). Genome-wide association mapping studies refined the confidence interval of previously available QTLs that are partially resistant to Aphanomyces root rot (Table. 4) (Desgroux et al., 2016). The outcome of GWA identified numerous SNPs and linkage disequilibrium blocks for Aphanomyces root rot resistance (Desgroux et al., 2016). Association mapping was performed in 175 pea lines collectively known as the “Pea-Aphanomyces collection,” which includes most Aphanomyces-resistant pea lines 90-2131, 552, and PI180693. The cultivars were phenotypically 30 evaluated for Aphanomyces root rot in field and greenhouse conditions with two isolates (RB84 strain, a French isolate belonging to pathotype I, and Ae109 strain, an American isolate belonging to pathotype III) (Desgroux et al., 2016). The cultivars were genotyped using a wide range of available markers Af, R, A, SSR, SNP, and two known PCR primers for functional genes, and the results of GWA mapping studies revealed 79 markers in the pea genome comprising 33 variables for Aphanomyces disease-resistance (Desgroux et al., 2016). Three linkage disequilibrium (LD) blocks were found based on the significance and consistency of the markers 14 LD blocks and 3 to 26 haplotypes were identified as a consistent region for two to six disease-resistant trait markers, and the comparison of linkage analysis and mapping studies confirmed the presence of previously available QTLs and Meta-QTL which can be further used for breeding program (Desgroux et al., 2016). Leprévost et al. (2023) conducted QTL analysis, and GWAS in two pea populations, Eden x E11 and Eden x LISA. Eden is an Aphanomyces root rot susceptible pea line, E11 and LISA have partial resistance. Both populations were genotyped by SNP markers and phenotypically scored for Aphanomyces root rot resistance in a diseased field and controlled environment. A total of twenty-nine SNPs and 171 QTL were associated with partial resistance to Aphanomyces root rot found across the pea genome using the MLMM model with phenotypic variation ranging from 0% to 68% for the analyzed variables. The consistent genetic regions showing partial resistance to Aphanomyces root rot were categorized into groups from 1 to 10 based on the colocalization results of RIL/AB linkage analysis. (Leprévost et al., 2023). Leprévost et al. (2023) pooled the results of AB QTL analysis, RIL analysis, and GWAS studies 31 and identified ten consistent genetic regions that act as an important source for pyramiding resistant QTL and haplotypes against Aphanomyces root rot in peas (Leprévost et al., 2023). Linkage Group Consistent Genetic Region Ae-Psxx QTL repostitioning Total number of QTL I 1 Ae-Ps1.2 22 II 2 Ae-Ps2.2a 10 Ae-Ps2.2b 21 III 3 Ae-Ps3.1a 25 Ae-Ps3.1b 19 4 Ae-Ps3.2 17 IV 5 Ae-Ps4.1 12 6 Ae-Ps4.3 8 7 Ae-Ps4.5 8 V 8 Ae-Ps5.1 12 9 Ae-Ps5.2 6 VII 10 Ae-Ps7.6a 42 Ae-Ps7.6b 46 Table 4. Ten consistent regions for partial resistance to Aphanomyces root rot in peas (Leprévost et al., 2023). Gene Pyramiding The resistance provided by a single gene did not last long or endure compared to quantitative resistance. Consequently, plants can achieve a wide range of resistance by combining multiple genes through marker-assisted selection (Figure. 7) (Fang et al., 2022; Gautam et al., 2020; Hu et al., 2023; Jiang et al., 2012; Li et al., 2024; Li et al., 2022; Sharma et al., 2021). This strategy is successful in significant crops worldwide. In India, rice blast caused by Magnaporthe oryzae resistance genes Pi54 and Pi54rh were integrated using the co- transformation method in the TP309 rice variety, suggesting rice lines with two resistant genes 32 performed better than single resistance genes in the disease pressure (Kumari et al., 2017). The polygenic resistance mechanism has been proven to perform better than the monogenic resistant lines in the rice blast resistance (Chen et al., 2021; Peng et al., 2023). Using marker-assisted backcross selection in China, Fusarium head blight in major wheat growing areas was controlled by integrating three QTLs, Fhb1, Fhb4, and Fhb5 (Zhang et al., 2021). Figure 7. The schematic diagram for incorporating ARR-resistant QTLs. 33 MATERIALS AND METHODS Plant Materials Sixteen pea lines were selected to evaluate the resistance of pea cultivars against Aphanomyces euteiches, the causal agent of Aphanomyces root rot (Table. 5). Eleven lines were reported as partially resistant (McGee et al., 2012) (Kraft, 1989, 1992; Kraft & Coffman, 2000a, 2000b, 2000c), and the remaining five were commonly grown susceptible varieties (Table 5). No. Parental Lines Pedigree Resistant/ Susceptible 1. 96-2058 (79-2022 / 74-SN3)// (`Recette' / PD 606-8) + 2. 96-2068 75-786 / Dark Skin Perfection Tac + 3. 96-2198 Charo/79-2022 + 4. 97-2154 86-2197/VR-410-2 + 5. 97-2162 (244219-B /74 SN3)//PI 180693 + 6. 97-261 86-2197/VR-410-2 + 7. PI 652444 Dark Skin Perfection/90-2131 + 8. PI 652445 Dark Skin Perfection/90-2131 + 9. PI 652446 Dark Skin Perfection/90-2131 + 10. PI 180693 Landrace from Germany + 11. PS0877MT457 Stirling/PS0010946 - 12. MTP190144 Lifter/CDC Golden - 13. ND VICTORY Carneval/Stirling - 14. MTP191417 Hampton/NDP100144 - 15. HAMPTON PS810090/PS510718 - 16. LIFTER PS810102/Alaska81//IPS810106/3/PS010838 - Table 5. Partially resistant and susceptible pea parent germplasm screened for resistance to Aphanomyces root rot ‘+’ denotes the partially resistant pea lines and ‘-’ denotes the susceptible pea lines. Bi-Parental Populations Bi-parental crosses were made between partially resistant lines and susceptible pea varieties (Table 6). The populations screened were in the F5 generation, with seeds randomly 34 sampled from the harvested bulk of each population. Disease ratings were performed per pot, providing a composite severity measure rather than individual plant evaluations. Due to space constraints, the populations were divided into five sets for screening. Susceptible Line Resistant Line Name Pedigree CDC BRONCO PI 652444 M18P337 PI 652444/CDC BRONCO PI 652445 M19P090 PI 652445/CDC BRONCO PI 652446 M18P174 CDC BRONCO/PI 652446 97-2162 M18P132 97-2162/CDC BRONCO 97-261 M18P164 CDC BRONCO/97-261 96-2068 M18P161 CDC BRONCO/96-2068 CDC AMARILLO PI 652444 M18P336 PI 652444/CDC AMARILLO 97-2162 M18P131 97-2162/CDC AMARILLO 97-2154 M18P141 CDC AMARILLO/97-2154 97-261 M18P143 CDC AMARILLO/97-261 CDC GOLDEN PI 652445 M18P195 CDC GOLDEN/PI 652445 PI 652446 M18P196 CDC GOLDEN/PI 652446 96-2198 M18P177 CDC GOLDEN/96-2198 96-2068 M18P176 CDC GOLDEN/96-2068 CDC SAGE 97-2162 M18P133 97-2162/CDC SAGE 97-2154 M18P240 CDC SAGE/97-2154 97-261 M18P136 97-261/CDC SAGE 97-2198 M18P239 CDC SAGE/96-2198 CDC MEADOW 97-2154 M18P198 CDC MEADOW/97-2154 HAMPTON PI 652445 M18P341 PI 652445/HAMPTON 96-2198 M19P127 96-2198/HAMPTON 96-2068 M18P299 HAMPTON/96-2068 LIFTER 97-2154 M19P112 97-2154/LIFTER 96-2058 M19P120 96-2058/LIFTER MTP190144 PI 652446 M19P205 PI 652446/MTP190144 97-2162 M19P203 97-2162/MTP190144 PS0877MT457 96-2198 M19P143 PS0877MT457/96-2198 STIRLING 97-261 M19P106 97-261/STIRLING Table 6. List of Pea Populations used in this experiment, along with their pedigree. 35 Disease Inoculum Aphanomyces euteiches isolates used in this experiment were received from the Extension Plant Pathology Laboratory at Montana State University and cultured on corn meal agar plates at 23°C in an incubator. Agar was prepared by mixing 9 grams of corn meal agar powder in 500 mL of distilled water in a sterilized glass flask. 0.25 mg each Rifampicin and Penicillin G antibiotics was added in 500 mL of sterile corn meal agar. Cultures for each Aphanomyces euteiches isolate were allowed to grow for 12 to 14 days. Long-term storage of the Aphanomyces euteiches isolates was accomplished in sterilized corn kernel extract solution in the dark at room temperature. Inoculum Preparation Aphanomyces euteiches cultures were blended with 100 mL distilled water per petri plate . A Ninja Blender 1000W was used for blending operations, and the blades were thoroughly wiped with 75% ETOH before scooping out the Aphanomyces euteiches culture from the petri plates. For each cycle, cultures from 5 plates were blended with 500 mL of distilled water for 45 seconds. The liquid suspension was filtered using a single layer of cheesecloth to trap the agar. The spore suspension was further analyzed for oospore concentration with a hemocytometer. The target spore concentration was 500 spores per gram of soil. 36 Inoculation Method Zoospore suspensions were prepared from two-week old Aphanomyces euteiches cultures. Fifty mL of liquid culture was poured into a small pit in the middle of the pot. The plants were not watered for 12 hours to prevent the washing out the spores. The pea plants were allowed to grow for three weeks before destructive sampling. Inoculated pea plants were watered daily to keep them moist and to favor spore germination. Figure 8. Photographic representation of each step of the screening protocol for plant reaction to Aphanomyces euteiches. Inoculation Method 2: Eight to ten whole yellow corn kernels were placed into 250 ml Erlenmeyer flasks containing 75 ml of distilled water. The flasks were covered with aluminum foil and autoclaved for sterilization. After cooling to room temperature, five 6 mm² agar sections from a two-week- 37 old Aphanomyces euteiches culture were transferred into each flask. The flasks were then incubated at 22°C in the dark for two weeks. A visible mycelial mat developed within one week. After two weeks, the liquid was decanted, and 100 ml of sterilized water was added to the flasks, which were then incubated for 3 hours at 22°C. A pea exudate solution was prepared by soaking 10 surface-sterilized pea seeds (treated with 0.8% sodium hypochlorite) in 5 ml of sterile distilled water in a 150 ml Erlenmeyer flask. This solution was incubated for two weeks at 22°C. After the incubation period, 5 ml of a 1:1 mixture of distilled water and sterilized tap water was added to the solution. The mixture was incubated at room temperature for 1 hour and then filtered using a single layer of cheesecloth. The concentration of zoospores was verified before inoculating the pea plants 500 spores per gram of soil were aimed in each inoculation. Experimental Setup The experiment was conducted in a greenhouse with 64 pots arranged in a randomized complete block design, with four replications. Three blocks were treated, and one was untreated as a control. Pots were filled with MSU-steamed soil to remove other potential microbes in the soil. Tissue paper was used at the bottom of each pot to prevent soil loss. Four seeds were planted in each pot and allowed to grow for two weeks prior to inoculation. A 3 cm well was created in the middle of the pot, and 50ml of Aphanomyces euteiches inoculum was poured into the well. The spore concentration was maintained at 500 spores per gram of soil. No fertilizers were applied during the experiment. Plants were watered daily to keep them moist (Figure. 8). 38 Scoring Index Pea plants were grown in the greenhouse for 21 days after inoculation. Yellowing (chlorosis) and wilting symptoms were observed, indicating host susceptibility. Plants were carefully uprooted, washed, and scored for root rot (honey brown discoloration) using a disease severity scale ranging from 0 to 5, where 0 represented healthy roots, and 5 indicated severe root browning and tissue sloughing (Figure. 9). Figure 9. The disease severity score for reaction to Aphanomyces euteiches in peas was evaluated visually based on the presence of root discoloration. Photo: Carmen Murphy. 39 Figure 10. Disease severity scoring reaction to Aphanomyces euteiches by comparing individuals from a segregating population and the parents, PI 652444 and CDC BRONCO. 40 RESULTS Pathogenicity Test Five Aphanomyces euteiches isolates were received from the Plant Pathology Laboratory were collected from disease-prone zones in Montana (Table. 7). Isolate Place of Collection AE4.1MT18 Daniels County AE1.1MT17 NDSU Pulse Lab, Roosevelt County AE2.1MT18 Sheridan County AE8.1MT18 Valley County AE5.1MT18 Daniels County Table 6. Isolates of Aphanomyces euteiches collected from fields in Montana. A greenhouse experiment was conducted to identify the disease severity level of different isolates on pea parents PS0877MT457, MTP191417, PI 652446, PI 652445, and LIFTER. All the Aphanomyces euteiches isolates were inoculated using the zoospore suspension (Figure. 8) and the controls were inoculated with blank corn meal agar media. As a result, Aphanomyces euteiches isolates AE4.1MT18 and AE1.1MT17 were identified as the most virulent isolates compared to others (Figure 11.). 41 Figure 11. Disease reaction of five Aphanomyces euteiches isolates on two partially resistant and four susceptible pea cultivars. Pure-Culture Evaluation of Aphanomyces Root Rot Resistant Pea Parents Results of screening the pea parent lines are summarized in Figure 12 and demonstrate a range of reactions from highly resistant to moderately susceptible. PI 652444, PI 652445, and PI 652446 have lower disease incidence than other pea lines (mean 0.1, 0.1, 0). Similarly, ND VICTORY and MTP191417 have high disease incidence (Figure 12) (mean 2.3, 2.8, respectively). 42 Figure 12. Disease Severity Score of 15 pea parental lines x-axis denotes the list of 15 pea parental lines and the y-axis denotes the disease rating scores ranges 0-5. Scoring Bi-Parental Populations The segregating populations from crosses of partially resistant and susceptible parents were divided into five sets. The specific parents were included in the tests as positive and negative controls (Figure 10). The data were analyzed using R and plots were created using the visually observed disease rating scores for the population (Figure 13-17). 43 Figure 13. Phenotypic scores for Set 1 populations and their parents. The partially resistant and susceptible parents were plotted as green and blue, respectively, in the graph. 44 Figure 14. Phenotypic scores for Set 2 populations and their parents. The partially resistant and susceptible parents were plotted as green and blue, respectively, in the graph. 45 Figure 15. Phenotypic scores for Set 3 populations and their parents. The partially resistant and susceptible parents were plotted as green and blue, respectively, in the graph. 46 Figure 16. Phenotypic scores for Set 4 populations and their parents. The partially resistant and susceptible parents were plotted as green and blue, respectively, in the graph. 47 Figure 17. Phenotypic scores for Set 5 populations and their parents. The partially resistant and susceptible parents were plotted as green and blue, respectively, in the graph. 48 DISCUSSION Aphanomyces root rot (ARR), caused by Aphanomyces euteiches, represents a significant challenge to global pea production, with yield losses as high as 86% (Pfender & Hagedorn, 1983). This issue is particularly relevant in Montana, the leading state in pea acreage and production, where environmental conditions often favor the disease. Effective management strategies are essential for maintaining pea productivity in these high-risk areas. One traditional approach to controlling ARR involves extended crop rotations with non-host cereals, which can reduce disease severity by disrupting the pathogen's life cycle. This is ineffective primarily due to the long-term survival of oospores in the soil. However, given the pathogen's persistence and the growing demand for sustainable production practices, there is a critical need for more direct methods of control, such as developing genetically resistant pea cultivars. Historically, genetic resistance has been viewed as a vital component of disease management, particularly in the early 20th century when initial breeding efforts began (Davis et al., 1995; Gritton, 1990; Kraft, 1989, 1992). Despite these efforts, complete resistance to ARR has remained elusive, with only partial resistance identified in certain pea lines. In this study, nine pea lines previously reported to possess partial resistance were subjected to screening under controlled greenhouse conditions using inoculum cultured in corn meal agar. The greenhouse screening provided valuable insights into the performance of these lines under artificial infection, allowing us to evaluate their relative resistance levels effectively. Bi-parental crosses between these nine partially resistant lines and susceptible pea varieties were carried out to explore the potential for improved resistance through breeding. By advancing to the F5 generation, we aimed to identify progeny that exhibited enhanced resistance 49 to ARR. The results from these F5 populations indicate variability in resistance levels, suggesting that combining resistance genes from different sources may be a promising approach. The next step involves further genetic characterization, particularly using microsatellite markers. Microsatellite markers can play a crucial role in mapping quantitative trait loci (QTLs) associated with partial resistance. The previously identified major effect QTLs, Ae.Ps.4.5 and Ae.Ps.7.6, located in linkage groups IV and VII (Hamon et al., 2013), respectively, have been shown to contribute to ARR partial resistance. By designing markers specific to these QTL regions, we can track their inheritance in breeding populations. This molecular approach enables more precise selection of resistant individuals, which is particularly important given the complexity of partial resistance traits. Integration of multiple QTLs, known as pyramiding, offers a strategic advantage in breeding for disease resistance. Pyramiding QTLs minimizes the risk of pathogen adaptation by providing broad-spectrum resistance, which is less likely to be overcome by a single genetic change in the pathogen. This approach not only enhances the durability of resistance but also supports the development of pea cultivars that are resilient under diverse environmental conditions. The combination of multiple resistance sources can lead to a cumulative effect, providing stronger and more consistent protection against ARR. 50 CONCLUSION The development of resistant pea varieties is critical for achieving sustainable production, especially in regions like Montana, where the economic impact of ARR can be severe. Resistant cultivars can reduce the reliance on chemical controls and extended crop rotations, contributing to more environmentally friendly and economically viable production systems. The findings from this study highlight the potential for improving ARR resistance through targeted breeding strategies that combine traditional phenotypic selection with modern molecular techniques. Future research should focus on the validation and fine mapping of the identified QTLs to confirm their effects on resistance across different environments. Field trials are necessary to assess the stability of resistance under natural disease pressure and varying climatic conditions. Additionally, exploring the genetic basis of resistance in other pea germplasm may provide new sources of resistance that can be integrated into breeding programs. Overall, this study represents an important step towards developing ARR-resistant pea cultivars that can withstand the challenges of modern agriculture and contribute to global food security. 51 REFERENCES CITED Agrios, G. (1988). Genetics of plant disease. Plant Pathology, 116-146. Alemu, A., Brantestam, A. K., & Chawade, A. (2022). Unraveling the genetic basis of key agronomic traits of wrinkled vining pea (Pisum sativum L.) for sustainable production. Frontiers in Plant Science, 13, 844450. Almquist, C., Persson, L., Olsson, Å., Sundström, J., & Jonsson, A. (2016). Disease risk assessment of sugar beet root rot using quantitative real-time PCR analysis of Aphanomyces cochlioides in naturally infested soil samples. European journal of plant pathology, 145, 731-742. Amurrio, J. M., Ron Pedreira, A. M. d., & Casquero Luelmo, P. A. (1991). Practical importance of numerical taxonomy as a useful tool in the classification of pea landraces for their different uses. Baldauf, S. L., Roger, A. J., Wenk-Siefert, I., & Doolittle, W. F. (2000). A kingdom-level phylogeny of eukaryotes based on combined protein data. Science, 290(5493), 972-977. Beute, M., & Lockwood, J. (1967). Pathogenic variability in Aphanomyces euteiches. Phytopathology, 57(1), 57-+. Bissonnette, H. L. (1958). Physiologic specialization in Aphanomyces euteiches. University of Minnesota. Bodah, E. T. (2017). Root rot diseases in plants: a review of common causal agents and management strategies. Agric. Res. Technol. Open Access J, 5, 555661. Boosalis, M., & Scharen, A. L. (1959). Methods for microscopic detection of Aphanomyces eutiches and Rhizoctonia solani and for isolation of Rhizoctonia solani associated with plant debris. Phytopathology, 49(4). Bouhot, D. (1979). Estimation of inoculum density and inoculum potential: techniques and their value for diseases prediction. Brhane, H., & Hammenhag, C. (2024). Genetic diversity and population structure analysis of a diverse panel of pea (Pisum sativum). Frontiers in Genetics, 15, 1396888. Burke, D., Mitchell, J., & Hagedorn, D. (1969). Selective conditions for infection of Pea seedlings by Aphanomyces euteiches in soil. Phytopathology, 59(11), 1670-1674. Cannesan, M. A., Gangneux, C., Lanoue, A., Giron, D., Laval, K., Hawes, M., Driouich, A., & Vicré-Gibouin, M. (2011). Association between border cell responses and localized root infection by pathogenic Aphanomyces euteiches. Annuals of Botany, 108(3), 459-469. 52 Carlson, L. E. (1965). Studies on the root rot of peas caused by Aphanomyces euteiches Drechs. University of Minnesota. Chan, M., & Close, R. (1987). Aphanomyces root rot of peas 1. Evaluation of methods for assessing inoculum density of Aphanomyces euteiches in soil. New Zealand journal of agricultural research, 30(2), 213-217. Chen, Y.-C., Hu, C.-C., Chang, F.-Y., Chen, C.-Y., Chen, W.-L., Tung, C.-W., Shen, W.-C., Wu, C.-W., Cheng, A.-H., & Liao, D.-J. (2021). Marker-assisted development and evaluation of monogenic lines of rice cv. Kaohsiung 145 carrying blast resistance genes. Plant disease, 105(12), 3858-3868. Davis, D. W., Fritz, V. A., Pfleger, F. L., Percich, J. A., & Malvick, D. K. (1995). MN 144, MN 313, and MN 314: garden pea lines resistant to root rot caused by Aphanomyces euteiches Drechs. HortScience, 30(3), 639-640. Deacon, J., & Saxena, G. (1998). Germination triggers of zoospore cysts of Aphanomyces euteiches and Phytophthora parasitica. Mycological Research, 102(1), 33-41. Desgroux, A., L’anthoëne, V., Roux-Duparque, M., Rivière, J.-P., Aubert, G., Tayeh, N., Moussart, A., Mangin, P., Vetel, P., & Piriou, C. (2016). Genome-wide association mapping of partial resistance to Aphanomyces euteiches in pea. BMC genomics, 17, 1-21. Duarte, J., Rivière, N., Baranger, A., Aubert, G., Burstin, J., Cornet, L., Lavaud, C., Lejeune- Hénaut, I., Martinant, J.-P., & Pichon, J.-P. (2014). Transcriptome sequencing for high throughput SNP development and genetic mapping in Pea. BMC genomics, 15, 1-15. Duke, J. (2012). Handbook of legumes of world economic importance. Springer Science & Business Media. Fang, C., Wang, Z., Wang, P., Song, Y., Ahmad, A., Dong, F., Hong, D., & Yang, G. (2022). Heterosis derived from nonadditive effects of the BnFLC homologs coordinates early flowering and high yield in rapeseed (Brassica napus L.). Frontiers in Plant Science, 12, 798371. Gangneux, C., Cannesan, M.-A., Bressan, M., Castel, L., Moussart, A., Vicré-Gibouin, M., Driouich, A., Trinsoutrot-Gattin, I., & Laval, K. (2014). A sensitive assay for rapid detection and quantification of Aphanomyces euteiches in soil. Phytopathology, 104(10), 1138-1147. Gaskell, M. (1997). Edible-pod pea production in California. Gaulin, E., Jacquet, C., Bottin, A., & Dumas, B. (2007). Root rot disease of legumes caused by Aphanomyces euteiches. Molecular Plant Pathology, 8(5), 539-548. 53 Gautam, T., Dhillon, G. S., Saripalli, G., Rakhi, Singh, V. P., Prasad, P., Kaur, S., Chhuneja, P., Sharma, P., & Balyan, H. (2020). Marker-assisted pyramiding of genes/QTL for grain quality and rust resistance in wheat (Triticum aestivum L.). Molecular breeding, 40, 1-14. Gobbin, D., Rumbou, A., Linde, C. C., & Gessler, C. (2006). Population genetic structure of Plasmopara viticola after 125 years of colonization in European vineyards. Molecular plant pathology, 7(6), 519-531. Goffinet, B., & Gerber, S. (2000). Quantitative trait loci: a meta-analysis. Genetics, 155(1), 463- 473. Gritton, E. (1990). Registration of five root rot resistant germplasm lines of processing pea. Crop Science, 30(5), 1166-1167. Grünwald, N., Chen, W., & Larsen, R. (2004). Pea diseases and their management. In Diseases of Fruits and Vegetables: Volume II: Diagnosis and Management (pp. 301-331). Springer. Hagedorn, D. J. (1984). Compendium of pea diseases. (No Title). Hamon, C., Baranger, A., Coyne, C. J., McGee, R. J., Le Goff, I., L’Anthoëne, V., Esnault, R., Riviere, J.-P., Klein, A., & Mangin, P. (2011). New consistent QTL in pea associated with partial resistance to Aphanomyces euteiches in multiple French and American environments. Theoretical and Applied Genetics, 123, 261-281. Hamon, C., Coyne, C. J., McGee, R. J., Lesné, A., Esnault, R., Mangin, P., Hervé, M., Le Goff, I., Deniot, G., Roux-Duparque, M., Morin, G., McPhee, K. E., Delourme, R., Baranger, A., & Pilet-Nayel, M.-L. (2013). QTL meta-analysis provides a comprehensive view of loci controlling partial resistance to Aphanomyces euteiches in four sources of resistance in pea. BMC Plant Biology, 13(1), 45. https://doi.org/10.1186/1471-2229-13-45 Hardham, A., & Hyde, G. (1997). Asexual sporulation in the oomycetes. In Advances in Botanical Research (Vol. 24, pp. 353-398). Elsevier. Hardham, A. R. (2005). Phytophthora cinnamomi. Molecular plant pathology, 6(6), 589-604. Heyman, F. (2008). Root rot of pea caused by Aphanomyces euteiches. Calcium Dependent Soil Suppressiveness, Molecular Detection and Population Structure. Hu, W.-J., Fu, L.-P., Gao, D.-R., Li, D.-S., Sen, L., & Lu, C.-B. (2023). Marker-assisted selection to pyramid Fusarium head blight resistance loci Fhb1 and Fhb2 in the high-quality soft wheat cultivar Yangmai 15. Journal of Integrative Agriculture, 22(2), 360-370. Ivors, K. L., Hayden, K. J., Bonants, P. J., Rizzo, D. M., & Garbelotto, M. (2004). AFLP and phylogenetic analyses of North American and European populations of Phytophthora ramorum. Mycological research, 108(4), 378-392. 54 Jacobsen, B., & Hopen, H. (1981). Aphanomyces Root Rot of Peas. Plant disease, 11. Janzen, J. P., Brester, G. W., Smith, V. H., & Hall, L. (2014). Dry peas: trends in production, trade, and price. Agricultural Marketing Policy Center, briefing, 57. Jha, A. B., & Warkentin, T. D. (2020). Biofortification of pulse crops: Status and future perspectives. Plants, 9(1), 73. Jiang, H., Feng, Y., Bao, L., Li, X., Gao, G., Zhang, Q., Xiao, J., Xu, C., & He, Y. (2012). Improving blast resistance of Jin 23B and its hybrid rice by marker-assisted gene pyramiding. Molecular breeding, 30, 1679-1688. Jiang, Y., Lahlali, R., Karunakaran, C., Kumar, S., Davis, A. R., & Bueckert, R. A. (2015). Seed set, pollen morphology and pollen surface composition response to heat stress in field pea. Plant, cell & environment, 38(11), 2387-2397. Jones, F. R., & Drechsler, C. (1925). Root rot of peas in the United States caused by Aphanomyces euteiches (n. sp.). Joshi, P., & Rao, P. P. (2017). Global pulses scenario: status and outlook. Annals of the New York Academy of Sciences, 1392(1), 6-17. Kenicer, G. (2005). Legumes of the World. Edited by G. Lewis, B. Schrire, B. MacKinder & M. Lock. Royal Botanic Gardens, Kew. 2005. xiv+ 577pp., colour photographs & line drawings. ISBN 1 900 34780 6.£ 55.00 (hardback). Edinburgh journal of botany, 62(3), 195-196. Knott, C. (1987). A key for stages of development of the pea (Pisum sativum). Annals of applied Biology, 111(1), 233-245. Kraft, J. (1989). Registration of 86-638, 86-2197, 86-2231, and 86-2236 pea germplasms. Crop Science, 29(2), 494-495. Kraft, J. (1992). Registration of 90-2079, 90-2131, and 90-2322 pea germplasms. Crop Science, 32(4). Kraft, J., & Coffman, V. (2000a). Registration of 96-2052, 96-2058, 96-2068, 96-2198, and 96- 2222 Pea Germplasms. Crop Science, 40(1), 301-301. Kraft, J., & Coffman, V. (2000b). Registration of 97-261 and 97-2154 pea germplasms. Crop Science, 40(1), 302-302. Kraft, J., & Coffman, V. (2000c). Registration of 97-363, 97-2170, and 97-2162 Pea Germplasms. Crop Science, 40(1), 303-303. 55 Kraft, J., Marcinkowska, J., & Muehlbauer, F. (1990). Detection of Aphanomyces euteiches in field soil from northern Idaho by a wet-sieving/baiting technique. Plant disease, 74(9), 716-718. Kraft, J. M., & Pfleger, F. L. (2001). Compendium of pea diseases and pests. American Phytopathological Society (APS Press). Kumari, M., Rai, A. K., Devanna, B., Singh, P. K., Kapoor, R., Rajashekara, H., Prakash, G., Sharma, V., & Sharma, T. R. (2017). Co-transformation mediated stacking of blast resistance genes Pi54 and Pi54rh in rice provides broad spectrum resistance against Magnaporthe oryzae. Plant cell reports, 36, 1747-1755. Le May, C., Onfroy, C., Moussart, A., Andrivon, D., Baranger, A., Pilet-Nayel, M.-L., & Vandemark, G. (2018). Genetic structure of Aphanomyces euteiches populations sampled from United States and France pea nurseries. European journal of plant pathology, 150, 275-286. Leonforte, A., Sudheesh, S., Cogan, N. O., Salisbury, P. A., Nicolas, M. E., Materne, M., Forster, J. W., & Kaur, S. (2013). SNP marker discovery, linkage map construction and identification of QTLs for enhanced salinity tolerance in field pea (Pisum sativum L.). BMC Plant Biology, 13(1), 1-14. Leprévost, T., Boutet, G., Lesné, A., Rivière, J.-P., Vetel, P., Glory, I., Miteul, H., Le Rat, A., Dufour, P., & Regnault-Kraut, C. (2023). Advanced backcross QTL analysis and comparative mapping with RIL QTL studies and GWAS provide an overview of QTL and marker haplotype diversity for resistance to Aphanomyces root rot in pea (Pisum sativum). Frontiers in Plant Science, 14. Lewis, M. E., & Gritton, E. T. (1992). Use of one cycle of recurrent selection per year for increasing resistance to Aphanomyces root rot in peas. Journal of the American Society for Horticultural Science, 117(4), 638-642. Li, G., Xu, Z., Wang, J., Mu, C., Zhou, Z., Li, M., Hao, Z., Zhang, D., Yong, H., & Han, J. (2024). Gene pyramiding of ZmGLK36 and ZmGDIα-hel for rough dwarf disease resistance in maize. Molecular breeding, 44(4), 25. Li, M., Pan, X., & Li, H. (2022). Pyramiding of gn1a, gs3, and ipa1 Exhibits Complementary and Additive Effects on Rice Yield. International Journal of Molecular Sciences, 23(20), 12478. Lockwood, J. (1960). Progress and problems in breeding peas resistant to root rots. Quarterly Bulletin. Michigan State University Agricultural Experiment Station, 43, 358-366. Makasheva, R. K. (1984). The pea. 56 Malvick, D., & Percich, J. (1998a). Genotypic and pathogenic diversity among pea-infecting strains of Aphanomyces euteiches from the central and western United States. Phytopathology, 88(9), 915-921. Malvick, D., & Percich, J. (1998b). Variation in pathogenicity and genotype among single- zoospore strains of Aphanomyces euteiches. Phytopathology, 88(1), 52-57. Malvick, D., & Percich, J. (1999). Identification of Pisum sativum germ plasm with resistance to root rot caused by multiple strains of Aphanomyces euteiches. Plant disease, 83(1), 51-54. Malvick, D., Percich, J., Pfleger, F., Givens, J., & Williams, J. (1994). Evaluation of methods for estimating inoculum potential of Aphanomyces euteiches in soil. Plant disease, 78(4), 361-365. Manning, M., & Menzies, S. (1984). Pathogenic variability in isolates of Aphanomyces euteiches from New Zealand soils. New Zealand journal of agricultural research, 27(4), 569-574. Markell, S., Pasche, J., & Porter, L. (2022). Pea Disease Diagnostic Series. North Dakota State Extension. In. Marx, G., Schroeder, W., Provvidenti, R., & Mishanec, W. (1972). A Genetic Study of Tolerance in Pea (Pisum sativum L.) to Aphanomyces Root Rot. Journal of the American Society for Horticultural Science, 97(5), 619-621. Maxted, N., & Ambrose, M. (2001). Peas (Pisum L.). In Plant genetic resources of legumes in the Mediterranean (pp. 181-190). Springer. McGee, R. J., Coyne, C. J., Pilet-Nayel, M.-L., Moussart, A., Tivoli, B., Baranger, A., Hamon, C., Vandemark, G., & McPhee, K. (2012). Registration of pea germplasm lines partially resistant to aphanomyces root rot for breeding fresh or freezer pea and dry pea types. Journal of Plant Registrations, 6(2), 203-207. McKay, K., Schatz, B. G., & Endres, G. (2003). Field pea production. NDSU Extension Service USA. McPhee, K. (2003). Dry pea production and breeding: A minireview. Journal of Food Agriculture and Environment, 1, 64-69. McVay, K., Burrows, M., Menalled, F., Jones, C., Wanner, K., & O’Neill, R. (2013). Montana cool-season pulse. Production Guide, 1-28. Miller, P., McKay, K., Jones, C., Blodgett, S., Menalled, F., Riesselman, J., Cheng, C., & Wichman, D. (2005). Growing dry pea in Montana. Mont. State Univ. Ext. Serv. MT- 2500502-AG. 57 Mitchell, J., Bhalla, H., & Yang, G. (1969). An approach to study of population dynamics of Aphanomyces euteiches in soil. Phytopathology, 59(2), 206-+. Mondor, M. (2020). Pea. Pulses: Processing and Product Development, 245-273. Moussart, A., Onfroy, C., Lesne, A., Esquibet, M., Grenier, E., & Tivoli, B. (2007). Host status and reaction of Medicago truncatula accessions to infection by three major pathogens of pea (Pisum sativum) and alfalfa (Medicago sativa). European journal of plant pathology, 117, 57-69. Moussart, A., Wicker, E., Le Delliou, B., Abelard, J.-M., Esnault, R., Lemarchand, E., Rouault, F., Le Guennou, F., Pilet-Nayel, M.-L., & Baranger, A. (2009). Spatial distribution of Aphanomyces euteiches inoculum in a naturally infested pea field. European journal of plant pathology, 123, 153-158. Papavizas, G. C., & Ayers, W. A. (1974). Aphanomyces species and their root diseases in pea and sugarbeet. Parke, J., Rand, R., Joy, A., & King, E. (1991). Biological control of Pythium damping-off and Aphanomyces root rot of peas by application of Pseudomonas cepacia or P. fluorescens to seed. Plant disease, 75(10), 987-992. Peng, P., Jiang, H., Luo, L., Ye, C., & Xiao, Y. (2023). Pyramiding of multiple genes to improve rice blast resistance of photo-thermo sensitive male sterile line, without yield penalty in hybrid rice production. Plants, 12(6), 1389. Pfender, W., Delwiche, P., Grau, C., & Hagedorn, D. (1984). A medium to enhance recovery of Aphanomyces from infected plant tissue. Plant disease, 68(10), 845-847. Pfender, W., & DI, R. (1981). A" most probable number" method for estimating inoculum density of Aphanomyces euteiches in naturally infested soil. Pfender, W., & Hagedorn, D. (1983). Disease progress and yield loss in Aphanomyces root rot of peas. Phytopathology, 73(8), 1109-1113. Pilet-Nayel, M., Muehlbauer, F., McGee, R., Kraft, J., Baranger, A., & Coyne, C. (2002). Quantitative trait loci for partial resistance to Aphanomyces root rot in pea. Theoretical and Applied Genetics, 106, 28-39. Pilet-Nayel, M.-L., Muehlbauer, F., McGee, R., Kraft, J., Baranger, A., & Coyne, C. (2005). Consistent quantitative trait loci in pea for partial resistance to Aphanomyces euteiches isolates from the United States and France. Phytopathology, 95(11), 1287-1293. Rice, W., Clayton, G., Olsen, P., & Lupwayi, N. (2000). Rhizobial inoculant formulations and soil pH influence field pea nodulation and nitrogen fixation. Canadian Journal of Soil Science, 80(3), 395-400. 58 Schren, A. (1960). Germination of oospores of Aphanomyces euteiches embedded in plant debris. Phytopathology, 50(4). Scott, W. W. (1961). A monograph of the genus Aphanomyces. Technical Bulletin. Virginia Agricultural Experiment Station, 151. Sekizaki, H., & Yokosawa, R. (1988). Studies on zoospore-attracting activity. I. Synthesis of isoflavones and their attracting activity to Aphanomyces euteiches zoospore. Chemical and pharmaceutical bulletin, 36(12), 4876-4880. Shang, H., Grau, C., & Peters, R. (2000). Oospore germination of Aphanomyces euteiches in root exudates and on the rhizoplanes of crop plants. Plant disease, 84(9), 994-998. Sharma, A., Srivastava, P., Mavi, G., Kaur, S., Kaur, J., Bala, R., Singh, T. P., Sohu, V., Chhuneja, P., & Bains, N. S. (2021). Resurrection of wheat cultivar PBW343 using marker-assisted gene pyramiding for rust resistance. Frontiers in Plant Science, 12, 570408. Shehata, M., Davis, D., & Bissonnette, H. (1976). A New Testing Approach for Breeding Peas Resistant to Common Root Rot Caused by Aphanomyces euteiches Drechs. Journal of the American Society for Horticultural Science, 101(3), 257-261. Sherwood, R., & Hagedorn, D. (1962). Studies on biology of Aphanomyces euteiches. Phytopathology, 52(2), 150. Sherwood, R., & Hagedorn, D. J. (1958). Determining common root rot potential of pea fields. Simsek, S., Tulbek, M. C., Yao, Y., & Schatz, B. (2009). Starch characteristics of dry peas (Pisum sativum L.) grown in the USA. Food chemistry, 115(3), 832-838. Singh, R. J., & Jauhar, P. P. (2005). Genetic Resources, Chromosome Engineering, and Crop Improvement: Grain Legumes, Volume I. CRC Press. https://books.google.com/books?id=33qmDwAAQBAJ Sivachandra Kumar, N. T., Caudillo-Ruiz, K. B., Chatterton, S., & Banniza, S. (2021). Characterization of Aphanomyces euteiches pathotypes infecting peas in Western Canada. Plant disease, 105(12), 4025-4030. Smith, P. G., & Walker, J. (1941). Vol. 63 Washington, DC, July 1, 1941 Certain environmental and nutritional factors affecting Aphanomyces root rot of garden pea¹. Journal of Agricultural Research, 63, 1. Smýkal, P., Aubert, G., Burstin, J., Coyne, C. J., Ellis, N. T. H., Flavell, A. J., Ford, R., Hýbl, M., Macas, J., Neumann, P., McPhee, K. E., Redden, R. J., Rubiales, D., Weller, J. L., & Warkentin, T. D. (2012). Pea (Pisum sativum L.) in the Genomic Era. Agronomy, 2(2), 74-115. https://www.mdpi.com/2073-4395/2/2/74 59 Stoddard, F. L., Balko, C., Erskine, W., Khan, H., Link, W., & Sarker, A. (2006). Screening techniques and sources of resistance to abiotic stresses in cool-season food legumes. Euphytica, 147, 167-186. Sundheim, L., & Wiggen, K. (1972). Aphanomyces euteiches on peas in Norway. Isolation technique, physiological races, and soil indexing. Aphanomyces euteiches on peas in Norway. Isolation technique, physiological races, and soil indexing., 51(35). Tayeh, N., Aluome, C., Falque, M., Jacquin, F., Klein, A., Chauveau, A., Bérard, A., Houtin, H., Rond, C., & Kreplak, J. (2015). Development of two major resources for pea genomics: the GenoPea 13.2 K SNP Array and a high‐density, high‐resolution consensus genetic map. The Plant Journal, 84(6), 1257-1273. Thavarajah, D., Lawrence, T. J., Powers, S. E., Kay, J., Thavarajah, P., Shipe, E., McGee, R., Kumar, S., & Boyles, R. (2022). Organic dry pea (Pisum sativum L.) biofortification for better human health. PloS one, 17(1), e0261109. Trněný, O., Brus, J., Hradilová, I., Rathore, A., Das, R. R., Kopecký, P., Coyne, C. J., Reeves, P., Richards, C., & Smýkal, P. (2018). Molecular evidence for two domestication events in the pea crop. Genes, 9(11), 535. USDA-NASS. (2022). USDA National Agricultural Statistics Service Vandemark, G., & Ariss, J. (2007). Examining interactions between legumes and Aphanomyces euteiches with real-time PCR. Australasian Plant Pathology, 36, 102-108. Vandemark, G., Kraft, J., Larsen, R., Gritsenko, M., & Boge, W. (2000). A PCR-based assay by sequence-characterized DNA markers for the identification and detection of Aphanomyces euteiches. Phytopathology, 90(10), 1137-1144. Walker, C. A., & van West, P. (2007). Zoospore development in the oomycetes. Fungal biology reviews, 21(1), 10-18. Watson, A., Browne, S., Snudden, M., & Mudford, E. (2013). Aphanomyces root rot of beans and control options. Australasian Plant Pathology, 42, 321-327. Weeden, N. F. (2018). Domestication of pea (Pisum sativum L.): The case of the Abyssinian pea. Frontiers in Plant Science, 9, 515. Wicker, E., Moussart, A., Duparque, M., & Rouxel, F. (2003). Further contributions to the development of a differential set of pea cultivars (Pisum sativum) to investigate the virulence of isolates of Aphanomyces euteiches. European journal of plant pathology, 109, 47-60. 60 Wicker, E., & Rouxel, F. (2001). Specific behaviour of French Aphanomyces euteiches Drechs. populations for virulence and aggressiveness on pea, related to isolates from Europe, America and New Zealand. European journal of plant pathology, 107(9), 919-929. Wiersema, J. H., Kirkbride, J. H., & Gunn, C. R. (1990). Legume (Fabaceae) nomenclature in the USDA germplasm system. US Department of Agriculture, Agricultural Research Service. Willsey, T., Chatterton, S., Heynen, M., & Erickson, A. (2018). Detection of interactions between the pea root rot pathogens Aphanomyces euteiches and Fusarium spp. using a multiplex qPCR assay. Plant Pathology, 67(9), 1912-1923. Wu, L., Chang, K.-F., Conner, R. L., Strelkov, S., Fredua-Agyeman, R., Hwang, S.-F., & Feindel, D. (2018). Aphanomyces euteiches: A threat to Canadian field pea production. Engineering, 4(4), 542-551. Yokosawa, R., Kuninaga, S., Sakushima, A., & Sekizaki, H. (1995). Induction of oospore formation of Aphanomyces euteiches Drechsler by calcium ion. Japanese Journal of Phytopathology, 61(5), 434-438. Zhang, Y., Yang, Z., Ma, H., Huang, L., Ding, F., Du, Y., Jia, H., Li, G., Kong, Z., & Ran, C. (2021). Pyramiding of Fusarium head blight resistance quantitative trait loci, Fhb1, Fhb4, and Fhb5, in modern Chinese wheat cultivars. Frontiers in Plant Science, 12, 694023. Zitnick-Anderson, K., Porter, L. D., Hanson, L. E., & Pasche, J. S. (2021). Identification, laboratory, greenhouse, and field handling of Aphanomyces euteiches on pea (Pisum sativum). Plant Health Progress, 22(3), 392-403. PYRAMIDING RESISTANCE GENES FOR APHANOMYCES ROOT ROT IN PEAS ©COPYRIGHT DEDICATION ACKNOWLEDGEMENTS TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES ABSTRACT PYRAMIDING RESISTANT GENES FOR APHANOMYCES ROOT ROT IN PEAS Introduction Dry Pea Dry Pea Production Origin and Domestication Botany of Pea Market Class and End Use Dry Pea in Montana Abiotic Stress Biotic Stress Bacterial Diseases Viral Diseases Parasitic Nematodes History of Oomycetes Stages of Oomycetes Aphanomyces Genus Aphanomyces Root Rot in Peas Geographical Distribution Environmental Characteristics Lifecycle of Aphanomyces euteiches Symptoms and Pathotypes of Aphanomyces Root Rot Detection of Aphanomyces euteiches Soil Extraction Methods Molecular Analysis Management of Aphanomyces Root Rot in Peas Pioneer Breeding Efforts for Aphanomyces Root Rot in Peas Pathogenicity of Aphanomyces Root Rot Quantitative Trait Loci (QTL) Associated with Aphanomyces Root Rot Resistance Aphanomyces Root Rot QTL Meta-Analysis Genome-Wide Association Mapping for Aphanomyces Root Rot in Peas Gene Pyramiding MATERIALS AND METHODS Plant Materials Bi-Parental Populations Disease Inoculum Inoculum Preparation Inoculation Method Inoculation Method 2: Experimental Setup Scoring Index RESULTS Pathogenicity Test Pure-Culture Evaluation of Aphanomyces Root Rot Resistant Pea Parents Scoring Bi-Parental Populations DISCUSSION CONCLUSION REFERENCES CITED