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Theses and Dissertations

7-2015 : Characterization of Resistance in and Population Diversity Keiddy Esperanza Urrea Romero University of Arkansas, Fayetteville

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This Dissertation is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of ScholarWorks@UARK. For more information, please contact [email protected], [email protected]. Pythium: Characterization of Resistance in Soybean and Population Diversity

A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Plant Science

by

Keiddy E. Urrea Romero

Universidad Nacional de Colombia Agronomic Engineering, 2003 University of Arkansas Master of Science in Plant Pathology, 2010

July 2015 University of Arkansas

This dissertation is approved for recommendation to the Graduate Council.

______Dr. John C. Rupe Dissertation Director

______Dr. Craig S. Rothrock Dr. Pengyin Chen Committee Member Committee Member

______Dr. Burton H. Bluhm Dr. Brad Murphy Committee Member Committee Member

Abstract

Pythium spp. are an important group of pathogens causing stand losses in Arkansas soybean production. New inoculation methods and advances in molecular techniques allow a better understanding of cultivar resistance and responses of Pythium communities to cultural practices. The objectives of this research were to i) characterize the resistance of soybean to P. aphanidermatum with two phenotyping assays that evaluated the seed rot phase of the disease; and ii) understand the effect of long term crop rotation on species diversity and iii) to determine the effect of location, temperature and continuous soybean and soybean-rice rotation on Pythium spp. diversity in several locations in Arkansas. For objective one, resistance to seedling disease caused by P. aphanidermatum, was characterized in 84 F2:6 soybean lines derived from a cross of

‘Archer’ and ‘Hutcheson’ cultivars using a seed plate assay and an infested vermiculite assay.

The lines were assayed with 5,403 SNP markers and genetic maps and QTL mapping was performed. With both inoculation methods, two quantitative trait loci (QTL) were identified on chromosomes 4 and 7. In objective two, the effect of crop rotation and location on species diversity was determined by using soybean seed to bait Pythium spp. from soil of plots following a ten year rotation. Of the 320 isolates, 12 species identified, P. spinosum, P. irregulare, P. pareocandrum, and P. sylvaticum were the most common. There were significant differences in number of Pythium isolates from the ten rotation systems with the rice ()-soybean (wheat) rotation having the highest recovery of these six Pythium spp. In objective three, soils from a soybean-rice and a soybean- soybean rotation were collected from three locations in 2012. A total of 275 isolates were identified representing 25 species. The most frequently recovered species were P. irregulare, P. pareocandrum, P. sylvaticum, P. corolatum and P. spinosum.

Location had a large effect on Pythium population composition and diversity. Distinctive species

prevailed in each of the three locations across two temperature and two rotations. Overall, populations of Pythium spp. varied the most among locations, but were not influenced by the previous crop or the isolation temperature.

Acknowledgments

I would like to thank my major advisor Dr. John Rupe for his direction, support and encouragement during these years and to my dissertation committee members: Dr. Craig

Rothrock, Dr. Burton Bluhm, Dr. Pengyin Chen and Dr. Brad Murphy for their guidance, constructive criticism and suggestions.

My deepest gratitude to my beloved parents Norberto and Flor and my bothers Edwin and

Alexis, for their love and encouragement to pursue my dreams. I want to thank my wonderful husband Ruben Morawicki for all his love, support, care, and encouragement all these years.

There are many people in the Plant Pathoplogy department that have been a big part of this project and I want to say thank you to all of them. My gratitude to Dr. Rick Bennett, professors, fellow students, and support personnel. In Dr. Rupe’s laboratory, without the help of

Adele Steger, Bob Holland, Sandy Goeke, and Kartlyn Jones this project would not have be possible. Also, I would like to thank John Guerber, Scot Winters, Dr. Chunda Fenn, Dr. Joannis

Tzanetakis and his laboratory, and Dr. Jim Correll and his laboratory. In the Crop Soil and

Environmental Science department, I would like to thanks Dr. Pengyin Chen and Dr. Esten

Mason and theirs laboratory, especially, Liliana Florez, Ailan Zeng, Cindy Lopez, Nythia,

Topher Addison, and Andrea Acuña for their help with the genotyping analysis. Dr. Edward Gbur for his invaluable help whit the statistical analysis. Dr. Martin Chilvers and Alejandro Rojas at

Michigan State University, for their help with the molecular identification of the Pythium isolates.

Lastly, I would like to thank my entire family in Colombia and my friends: Camila,

Natalia, Juliana, Andrea, Catalina, Valeria, Cristina, Sergio and Claudia that have been supporting me here and from the distance all these years.

Dedication

I dedicate this dissertation to my parents Flor Maria Romero and Norberto Urrea

Bejarano, to my brothers Edwin and Alexis Urrea Romero, and my husband Ruben Morawicki for all their love and support during these years.

Tables of Contents

Chapter I: Introduction 1

Seedling diseases in Soybean 1

Importance of Pythium spp. 1

Effect of crop rotation on Pythium spp. 4

Environmental factors affecting Pythium spp. 6

Management of Pythium spp. 7

Fungicide seed treatments 7

Soybean resistance to Pythium spp. 8

Research justification and objectives 11

References 13

Chapter II: Characterization seed rot the resistance in soybean to Pythium 19 aphanidermatum

Abstract 19

Introduction 20

Materials and Methods 24

Isolate 24

Plant material 24

Phenotypic data 25

Genotyping data 27

Data analysis 28

Results 29

Phenotypic data 30

Quantitative trait loci mapping 30

Discussion 31

Conclusion 36

References 37

Chapter III: Effect of crop rotation on Pythium spp. population composition in 61 Arkansas soybean fields Abstract 61

Introduction 62

Materials and Methods 66

Sample collection 66

Baiting and isolation 67

Molecular identification 67

Pathogenicity tests 68

Statistical analysis 69

Results 69

Isolation and identification 69

Crop rotation 70

Pathogenicity tests 70

Discussion 71

Conclusion 74

References 75

Chapter IV: Effect of crop rotation, location and isolation temperature on 88 Pythium spp. population composition in Arkansas

Abstract 88

Introduction 89

Materials and Methods 93

Soil collection 93

Soil sampling 94

Isolation 94

Molecular identification 95

Experimental design and statistical analysis 95

Results 96

Pythium species isolation and identification 96

Pythium species by location 96

Pythium species diversity 97

Discussion 97

Conclusion 100

References 101

Overall conclusion 113

Appendix 114

List of Tables

Chapter II

Table 1. Analysis of variance of seed plate and infested vermiculite assay with 41 of 84 F2:6 lines and the parents, Archer and Hutcheson.

Table 2. Percent of seed for the 84 F2:6 lines and parents evaluated for the 42 resistance to Pythium aphanidermatum with the seed plate assay.

Table 3. Percent of plant stands for the 84 F2:6 lines and parents evaluated for the 45 resistance to Pythium aphanidermatum with the infested vermiculite assay. Table 4. Summary of single nucleotide polymorphism (SNP) markers used to screen 49 the 84 F2:6 lines derived from ‘Archer’ x ‘Hutcheson’ cross. Table 5. Qualitative trait loci for partial resistance to Pythium aphanidermatum from 56 84 F2:6 lines derived from ‘Archer’ X ‘Hutcheson cross that were mapped using Single Marker Analysis (SMA) using seed plate assay. Table 6. Qualitative trait loci for partial resistance to Pythium aphanidermatum from 57 84 F2:6 lines derived from ‘Archer’ X ‘Hutcheson’ cross that were mapped using Single Marker Analysis (SMA) using the infested vermiculite assay. Table 7. Qualitative trait loci for partial resistance to Pythium aphanidermatum from 58 84 F2:6 lines derived from an ‘Archer’ X ‘Hutcheson cross that were mapped using Composite interval mapping (CIM) using three phenotyping methods. Chapter III Table 1. Frequency of isolation of recovered across 10 crop rotation 79 system. Table 2. Isolates of five Pythium spp. recovered with soybean, corn and rice as the last 81 crops across the 10 rotation system. Table 3. Analysis the variance of estimated dead seeds on corn, soybean, rice and 82 wheat Table 4. Dead seeds (logit transformed) of soybean tested with five Pythium species 83 on soybean. Table 5. Dead seeds (logit transformed) of soybean tested for forty nine Pythium 84 species isolates. Table 6. Dead seeds (logit transformed) of corn for five for Pythium species. 85

Table 7. Dead seeds (logit transformed) of corn tested with forty-nine Pythium spp. 86 Isolates.

Table 8. Dead seeds (logit transformed) of rice tested with five Pythium species. 87

Chapter IV Table 1. Location, GPS coordinates, of the six fields where soil was collected. 105 Table 2. Analysis the variance of number of isolates of six Pythium species recovered 107 across three locations, two temperature and two rotation systems. Table 3. Richness, abundance and Shannon indices for six Pythium species recovered 109 from two locations, two temperatures and two rotation systems. Table 4. Analysis of variance for richness, abundance and Shannon index. 110

List of Figures

Chapter II Figure 1. Frequency distribution of Pythium resistance in a population derived 48 from ‘Archer’ (P1) x ‘Hutcheson’ (P2) cross evaluations a) Seed plate assay and b) Infested vermiculite.

Figure 2a. Linkage map of the chromosomes 1, 2 and 3 for the 84 F2:6 lines using 50 the 1269 polymorphic SNPs in Archer x Hutcheson across the 20 chromosomes.

Figure 2b. Linkage map of the chromosomes 4, 5, 6 and 7 for the 84 F2:6 lines 51 using the 1269 polymorphic SNPs in Archer x Hutcheson across the 20 chromosomes.

Figure 2c. Linkage map of the chromosomes 8, 9 and 10 for the 84 F2:6 lines using 52 the 1269 polymorphic SNPs in Archer x Hutcheson across the 20 chromosomes.

Figure 2d. Linkage map of the chromosomes 11, 12, 13 and 14 for the 84 F2:6 lines 53 using the 1269 polymorphic SNPs in Archer x Hutcheson across the 20 chromosomes.

Figure 2e. Linkage map of the chromosomes 15, 16, 17 and 18 for the 84 F2:6 lines 54 using the 1269 polymorphic SNPs in Archer x Hutcheson across the 20 chromosomes.

Figure 2f. Linkage map of the chromosomes 19 and 20 for the 84 F2:6 lines using 55 the 1269 polymorphic SNPs in Archer x Hutcheson across the 20 chromosomes. Figure 3. Composite interval mapping using single nucleotide polymorphism 59 markers for quantitative trait loci identified for seed germination in chromosome 4 with a) seed plate assay and b) infested vermiculite assay. Figure 4. Composite interval mapping using single nucleotide polymorphism 60 markers for quantitative trait loci identified in chromosome 7 with a) seed plate assay and b) infested vermiculite assay.

Chapter III Figure 1. Number of isolates for Pythium species collected in 11-year rotation 80 systems.

Chapter IV

Figure 1. Total of oomycetes isolates recovered across three locations, two 106 temperature and two rotation systems. Figure 2. Number of isolates of six Pythium species by location. 108 Figure 3. Abundance index of six Pythium species across two temperatures and 111 two crop rotations. Abundance was calculated as the sum of all the isolates in the location x temperature x crop rotation combination. Figure 4. Shannon index of six Pythium species across two temperatures and two 112 crop rotations. Species diversity in each location, crop and temperature was calculated using the Shannon index, H’= -Ʃ pi ln pi, where H’ is the species diversity score and pi is the proportion of individuals in the ith species.

I Introduction

Seedling diseases in Soybean

Seedling diseases are a major constraint to soybean production in Arkansas. Seedling diseases caused estimated losses of 8.7, 8.9 and 10.7 % in yield to Arkansas producers in 2011,

2012, and 2013, respectively (Heatherly, 2014, Data provided by Dr. S.R. Koenning, North

Carolina State University). These diseases can reduce stands, plant vigor and sometimes may requiring replanting (Yang, 1999). Soybean production in Arkansas in 2013 was 3.84 million metric tons of soybean, planted in 1.4 million hectares (USDA-NASS, 2013). Most soybean production in Arkansas occurs in the eastern part of the state where soils are predominantly alluvial with poor drainage, favoring seedling diseases (Kirkpatrick et al., 2006a). A number of soilborne pathogens are associated with seedling disease, primarily the Oomycetes Pythium spp. and sojae, and the fungi Fusarium spp. and (Schmitthenner,

1999; Yang, 1999; Wilcox, 1987). These pathogens may act singly or as a complex.

Importance of Pythium spp.

Pythium is the predominant pathogen group causing seedling diseases of soybean in

Arkansas (Kirkpatrick et al., 2006a; Rosso, 2007; Avanzato, 2011 and Urrea et al., 2013). This is made up of 100 species, 13 of which have been associated with soybean (Lévesque, et al, 2010; Yang, 1999; Dorrance et al., 2004; Broders et al., 2007; Bates et al., 2008, Avanzato

2011). These species are active across a range of environmental conditions resulting in seedling diseases being a threat to soybean production whenever are planted.

Pythium is a cosmopolitan genus; most species are soil inhabitants, while others prefer fresh water or marine aquatic environments (Van der Plaats-Niterink, 1981). Most Pythium spp. are saprophytes or facultative plant pathogens that cause losses to a wide variety of hosts, within

1 this genus P. aphanidermatum and P. ultimum have been reported to have broad host ranges

(Hendrix and Campbell, 1973). Other species such as P. oligandrum and P. nunn have been reported as biological control agents of plant pathogens (Berry et al., 1993). Pythium spp. reproduce both sexually and asexually via oospores and zoospores from sporangia, respectively.

Under favorable environmental conditions, Pythium initiates the process of infection that starts when survival structures in the soil such oospores or sporangia germinate. These structures germinate via a germ tube in response to exogenous carbohydrates and amino acids produced by the plant or volatile seed exudates produced during seed germination (Kerr, 1964; Nelson, 1990).

The germ tube grows towards these sources of nutrients and colonizes the host tissue. Sporangia also may germinate indirectly producing zoospores, which encyst and then germinate. Seeds form susceptible cultivars may not even germinate in the presence of the pathogen, or seed more distant a more resistant cultivar may overcome the colonization by the pathogen to produce a seedling. Pythium spp. also can cause post emergence damping-off and are associated with root pruning that leads to reduction of plant vigor and yields, but is not lethal to mature .

Because of the wide range of hosts and distribution, the lack of visible symptoms and the wide number of species, it seems that the reductions in yield due to this pathogen has been underreported and not fully appreciated (Martin, 2009).

In Arkansas the primary Pythium spp. are Pythium sylvaticum, followed by P. ultimum,

P. aphanidermatum, P. irregulare (Kirkpatrick et al., 2006a; Rosso, 2007; Bates et al., 2008).

Avanzato (2011) identified isolates of Pythium to species by morphological and molecular techniques and evaluated initially virulence with an in vitro pathogenicity seed assay. Nine

Pythium species were the most frequently recovered: P. sylvaticum, P. irregulare, P. ultimum, P. spinosum, P. mamillatum, P. dissotocum, P. accanthicum, P. attrantheridium, and P.

2 oligandrum. These studies showed that Pythium is the predominant group involved in seedling diseases in soybean fields in Arkansas.

Besides soybeans Arkansas ranks first among the six major rice-producing states, accounting for approximately 48 percent of the U.S. rice production. Rice production is approximately 4.04 million metric tons of rice in 607,028 hectares. Rice production is also concentrated in the eastern half of the state, which makes rice also prone to seedling diseases. In

Arkansas soybean-rice is a common rotation (Hristovska et al., 2011). In 2009, 68 % of the rice produced in Arkansas was rotated with soybean (Wilson et al., 2009). Pythium spp. causes stand-establishment problems in rice in Arkansas (Eberle et al., 2008; Rothrock et al., 2005).

Eberle (2008), reported that P. arrhenomanes and P. irregulare were the most common and virulent pathogens isolated from soil from 20 producers fields in Arkansas. Less common species were P. torulosum. P. catenulatum, and P. diclinum.

Pythium spp. are also known as a wheat pathogens (Vanterpool and Truscott, 1932;

Vanterpool, 1938; Chamswarng and Cook, 1985; Higginbotham, 2004). The main species known to cause Pythium root rot on wheat are: P. aristospurum, P. arrhenomanes, P. graminicola, P. ultimum, P. heterothallicum, P. torulosum (Paulitz, 2010). Pythium spp. in the Pacific Northwest cause root rot, decreasing root mass and leading to poor nutrient uptake resulting in yield losses

(Chamswarng and Cook, 1985; Higginbotham, 2004). The most common Pythium species causing root rot are P. ultimum var. ultimum, P. ultimum var. sporangiifereum, P. aristosporum,

P. volutum, P. torulosum, P. irregulare, and P. sylvaticum among others (Chamswarng and

Cook, 1950). In Arkansas preliminary studies also documented the importance of Pythium spp. in wheat (Milus and Rothrock, 1989; Milus et al., 1992; Rhoads et al., 1993).

3 Pythium spp. are commonly isolated from corn and have been reported as the most important pathogens associated with poor stand establishment in corn, causing pre and post emergence damping off (Deep and Lipps, 1996; Chen, 1999). In Ohio Pythium causes corn seed and seedling blight. There are at least 14 Pythium species that were known to cause seedling blight and root rot: P. acanthicum, P. adhaerens, P. aphanidermatum, P. arrhenomanes, P. graminicola, P. irregulare, P. paroecandrum, P. pulchrum, P. rostratum, P. splendens, P. tardicrescens, P. ultimum and P. vexans (Chen, 1999). Deep and Lipps (1996), reported that P. arrhenomanes was the primary cause of Pythium root rot of corn. Dorrance et al., (2004) reported that the four most common species recovered from three corn fields in Ohio were: P. cantenulatum, P. irregulare, P. paroecandrum, and P. torulosum, with isolation and pathogenicity depending on the location and species. Changes in earlier planting dates (Saab,

2005) and crop management such as reduced tillage in corn may have caused the shifts in

Pythium population in Ohio (Van Doren and Triplett, 1973; Broders et al., 2007). Broders et al.,

(2007) investigated Pythium spp. associated with seed and seedlings in corn and soybeans in

Ohio by conducting a survey 42 production fields. Eleven Pythium spp. were identified: P. attrantheridium, P. dissotocum, P. echinulatum, P. graminicola, P. inflatum, P. irregulare, P. helicoides, P. torulosum, P. ultimum var. ultimum and P. ultimum var. sporangiiferum were the most commonly recovered and pathogenic to corn and soybeans. In Arkansas there is little information available on the effect of Pythium on corn.

Effect of crop rotation on Pythium spp.

Diverse crop rotations have been shown to reduce the populations of soilborne pathogens and the use of a single crop for several years results in the increase of population density of a specific pathogen adapted to that crop (Christen and Sterling, 1995). Long-term rotation has been

4 used to reduce the pressure of plant diseases. However, pathogens such Pythium, Rhizoctonia and Sclerotinia that have broad host ranges and long-term survival structures are more challenging to manage by crop rotation (Krupinsky et al., 2002).

Crop rotation has been reported to influence Pythium population density. Davis and

Nunez (1999) studied the influence of crop rotation on Pythium and Rhizoctonia-induced carrot root dieback. They planted continuous carrot or rotated carrot with cotton, alfalfa, barley, onions, or fallow. After two years of these rotations, it was found that Pythium populations were greater when carrots were followed by alfalfa and barley than with any of the other crops in 1 year of the study. Populations of Rhizoctonia were greater when carrots were followed by cotton or alfalfa than other crops in the two years of the study (Davis and Nunez, 1999). Guo et al. (2005) studied the effect of a 4-year crop rotation of canola, wheat and flax and tillage systems on blackleg disease of canola, caused by Leptosphaeria maculans, and found that disease incidence and severity on stems were lower when canola was rotated with wheat and wheat and flax in tillage and non-tillage systems. Likewise, Hwang et al., (2009) evaluated the effect of long-term crop rotation on the effect of Fusarium, Pythium and Rhizoctonia populations on canola seedling establishment under controlled conditions in two locations in Saskatchewan, Canada. In the first year of the study the authors found that from soil collected in 2006 seedling emergence was higher in the three crop rotation (pea- canola- wheat) compared to two crop rotations (canola- wheat or pea wheat). Pankhurst et al., (1995) studied the effect of long-term crop rotation and tillage practices on Pythium population densities in wheat in South Australia. In this study there were three crop rotations: continuous-wheat, pasture-wheat and lupins-wheat. Three tillage practices were also compared: conventional cultivation, reduced cultivation and direct drilling.

They found that tillage and crop rotation had a significant effect in the number and distribution

5 of Pythium populations. They found the highest Pythium propagule density in the first 20 cm of soil, and the Pythium population density was higher in the continuous wheat rotation in soils subjected to direct-drilling compared to soil subjected to conventional cultivation. Similar results were found with pasture-wheat and lupine-wheat rotations. Likewise, Zhang and Yang (2000) studied the effect of corn- soybean long –term rotation effect on soilborne pathogens. They reported that 71 % of the total isolates were pathogenic in different levels to both corn and soybean and that the disease index on soybean is highly correlated with the disease index on corn. Long-term rotation and short-term rotations may have an effect on changing Pythium populations.

Environmental factors affecting Pythium spp.

It has been reported that infection by Pythium spp. is dependent on environmental factors.

Temperature and soil moisture (Allen et al., 2004; Hendrix and Campbell, 1973; Martin, 1996;

Kirkpatrick, et al., 2006b; Rothrock et al., 2012) have significant effects on the development of the Pythium disease severity. Pythium spp. are more prevalent in wet soils than in soils with lower soil moisture (Martin, 1996). Soil moisture affects the motility of the zoospores, which require free water (Bainbridge, 1970). It has been reported that high soil moisture favored saprophytic activity of P. irregulare, P. vexans and P. ultimum, which grew well when the pore spaces are filled with water, because the pathogens are highly tolerant of low oxygen levels

(Hendrix and Campbell, 1973). Kirkpatrick et al. (2006b) reported that the only pathogen group that increased in frequency from the roots of flooded soybeans were Pythium spp. Besides soil moisture, temperature is also important (Roncadori and McCarter, 1971; Hendrix and Campbell,

1973; Nannayakkara, 2001; Martin, 1996; Rothrock et al., 2012). In general, P. irregulare, P. spinosum and P. ultimum are damaging at temperatures of 15 to 20 ˚C, while, P. myriotylum, P.

6 aphanidermatum, P. arrhenomanes, P. polytylum, P. carolinianum, and P. volutum are damaging at higher temperatures. The optimal temperature for P. myriotylum and P. aphanidermatum has been reported to be 32˚C (Hendrix and Campbell, 1973; Martin, 1996). These differences in temperature range may determine the dominant Pythium species in any geographic area or within a field during the growing season (Martin, 1996).

Pythium spp. composition has been reported to vary among locations (soils). Dorrance et al. (2004) recovered Pythium spp. from three different locations in Ohio using both soybean and corn as a bait. The main Pythium species isolated for these fields were: P. cantenulatum, P. irregulare, P. paroecandrum, P. splendens and P. torulosum. A total of 129, 85 and 38 isolates were recovered from Defiance, Sandusky, and Wood County respectively. In Defiance, P. torulosum, P. spendens, and P. irregulare accounted for 40 and 38 and 22% of the isolates respectively. On the other hand, in Sanduski, P. splendans accounted for 72 % of the isolates and in Wood county P. cantenulatum and P. splendens accounted for 20 and 40 % of the isolates respectively. Moreover, Broders et al. (2009) identified 21 Pythium species from 88 locations in

Ohio representing fields planted with corn and soybean. From the 21 species, 6 species represented 40% of the total species, P. irregulare was the most isolated species, followed by P. inflatum, and P. torulosum. In the same study the authors using a canonical discriminant analysis

(CDA) found that the total number of isolates were grouped in five major communities, which differed among locations based on soil properties such as pH, calcium, magnesium, organic matter (OM) and cation exchange capacity (CEC).

Management of Pythium spp.

Fungicide seed treatments. One of the widely used management strategies for the control of Pythium species is the use of fungicide seed treatments, metalaxyl or its active isomer

7 mefenoxam have been a widely used active ingredient for the control of Oomycetes (Cohen and

Coffey, 1986). Field test performed in Arkansas in different locations over 3 years showed that metalaxyl was the most effective fungicide (Poag, 2005), however in more recent years field and growth chamber tests have shown that broad spectrum fungicide seed treatments were the best for the control of soybean seedling diseases and trifloxystrobin + metalaxyl was the most consistently effective fungicide in a test conducted during two years (Popp et al., 2010, Urrea et al., 2013). The limitations of the use of seed treatments is the inconsistent results of efficacy in field and controlled environment studies and the fact that most of the seed treatments only protect the seed for short period of time (10 to 14 days after planting) but not the emerging root

(Paulsrud and Montgomery, 2005).

Soybean resistance to Pythium spp. An alternative control to fungicides is the use of plant resistance. In soybean, most of the research on resistance against pathogens, has been done on Phytophthora root and stem rot caused by Phytophthora sojae (Sandhu et al, 2005).

Fifteen single dominant resistant genes (Rps) to Phytophthora root rot have been identified in soybean (Dorrance, et al., 1999; Dorrance et al., 2004; Dorrance and Grunwald, 2009; Dorrance, et al., 2009) using a hypocotyl inoculation technique (Dorrance, et al, 2004; Gordon et al, 2007).

These Rps genes are race specific and over 55 races of P. sojae have been described (Dorrance, et al, 1999; Dorrance et al., 2004; Dorrance and Grunwald, 2009; Dorrance, et al., 2009). Single gene resistance has been very effective in the field, but has led to the development of races of P. sojae requiring the introduction of new genes for resistance. In addition to single gene resistance, there is also multigenic partial resistance that affects all races of P. sojae (McDonald and Linde,

2002).

8 Compared to resistance to P. sojae, less research has been done on resistance to Pythium spp. Keeling (1974) reported that the soybean cultivar ‘Semmes’ was more resistant than ‘Hood’.

This difference in reaction to P. ultimum was related to higher concentrations of soluble carbohydrate in exudates of germinating seed in Hood than in Semmes. Griffen (1990) reported that the cultivar ‘Dare’, had significantly higher emergence in P. ultimum infested soil than the cultivar ‘Essex’, suggesting a greater level of Pythium resistance in Dare than Essex.

In Arkansas, Pythium spp. were the only pathogen group that increased with flooding treatments and the cultivar Archer had greater stands than any other cultivar when flooded

(Kirkpatrick et al., 2006a). Archer has been reported to be flood tolerant (Cianzio, 1991). In greenhouse experiments, Archer had significantly greater stands than the cultivar Hutcheson when in direct contact with inoculum of P. ultimum demonstrating that Archer also had resistance to P. ultimum (Kirkpatrick et al., 2006b). Using the inoculum layer technique

Nanayakkara (2001), reported that Archer also had resistance to P. vexans, P. aphanidermatum

P. ultimum, and P. irregulare. Archer has two genes for resistance to Phytophthora sojae, Rps 6 and Rps1k, which come from its parents, ‘PRX54-49’ and ‘Williams 82’, respectively (Cianzio,

1991). To determine if Pythium resistance was associated with these Phytophthora resistance genes, Bates et al. (2004) tested a set of differential cultivars containing specific resistant genes for P. sojae along with Archer and Hutcheson in vermiculite infested with Pythium aphanidermatum. Stands were significantly higher with Archer and Williams 82 (Rps 1k) than all the other cultivars which had stands that were not significantly different than Hutcheson, including the line with Rps 6. This suggested that Archer resistance to Pythium spp. could be associated with Rps1k gene. Using the hypocotyl inoculation method developed to identify single gene resistance to Phytophthora sojae, Rosso et al. (2008) reported that this method resulted in

9 clear differences between Archer and Hutcheson with Pythium aphanidermatum. Applying this approach to 86 F2:4 lines from a cross between Archer with Hutcheson the same criteria for resistance, susceptible, and segregating that had been developed for Phytophthora sojae, they found that the resulting reactions to Pythium aphanidermatum fit the model for a single dominant gene in Archer. This gene was designated as Rpa 1. Screening the lines with 88 SSR, representing all the soybean molecular linkage groups (MLG’s), two markers, Satt114 and

Satt510 located on the MLG F were associated with Rpa1 and were estimated to be 15.5 cM and

10.6 cM from the gene. This soybean population was also screened with race 7 of Phytophthora sojae to identify the Rps1k gene. The segregation of the Rps 1k gene was completely different than the Rpa 1 gene indicating that Rps 1k was not the source of Pythium resistance. Single gene plant resistance against Pythium has also been reported to P. aphanidermatum, P. ultimum and P. inflatum in periwinkle, beans and , respectively (Kulkarni and Baskaran, 2002; Otsyula,

2003; Yang et al., 2005).

Multigenic resistance to Pythium spp. by the identification of quantitative trait loci

(QTLs) have been reported. Resistance to P. ultimum in snap bean was first reported in 1977

(York et al., 1977). Navarro et al. (2008), reported five QTLs in linkage groups B6, B3 and B7 associated with root rot resistance. In soybean, 192 F2:3 lines from two populations: ‘OHS 303’

(OHS 303 x (Williams x PI424354)) and 127 F2:3 lines from ‘Denisson’ x (‘Denisson’ (Williams x PI424354)), were screened against P. irregulare using infested vermiculite (Ellis et al., 2013).

Pythium disease was accessed on root rot weight and root rot scores. The lines were screened with 74 SSR and 384 SNP markers resulting in two putative QTL associated with resistance to P. irregulare in the OHS 303 population on chromosomes 1 and 6 with composite interval mapping

(CIM) analysis and on chromosomes 5, 13, 14 and 20 using Internal mapping (IM) analysis for

10 root weight and root rot score. In the Denison population a putative QTL was identified in chromosomes 8 and 11 for root weight and in chromosome 13 for root rot score using CIM.

Overall, QTLs were mapped on chromosome 1, 6, 8, 11 and 13 for root weight and root discoloration.

The identification of these QTL for resistance to P. irregulare were possible because of the availability of thousands of single nucleotide polymorphism (SNP) makers in soybean. Also, since the seed plate (Avanzato, 2011) and the infested vermiculite assays (Rosso, 2007) have been shown to consistently distinguish differences in cultivar resistance and evaluate the seed rot phase of Pythium diseases (Rosso et al., 2008) it was decided that the Archer by Hutcheson population screened by Rosso et al. (2008) be reanalyzed to better understand the genetics of

Pythium resistance in Archer in the seed rot phase.

Research justification and objectives

Pythium is an important group of pathogens associated with stand losses in soybean in

Arkansas (Kirkpatrick et al., 2006a; Rosso, 2007; Avanzato, 2011). Several management strategies are used for the control of Pythium, the most common are cultural practices and fungicide seed treatments because resistance cultivars are not available. In Arkansas, evidence of resistance has been found in the soybean cultivar Archer against Pythium aphanidermatum with the hypocotyl inoculation method (Rosso et al., 2008), however the resistance in the seed rot phase of Pythium aphanidermatum has not been characterized in Arkansas. The aim for the objective one of this research was, to characterize the resistance of soybean to Pythium aphanidermatum with seed plate assay and infested vermiculite assay.

Pythium are an ecologically diverse group of microorganisms that are found in virtually all soils. The more than 100 species in this genus have a wide range of environmental and host

11 preferences, but little is known about the effect of long-term rotation on Pythium communities.

The hypothesis for the second objective of this study is that long-terms rotations have an effect on changing Pythium populations. The aims of this objective were i) to characterize Pythium populations from soil of a 11-year rotation study and ii) test isolates of the main Pythium species for pathogenicity to the four crops in the study.

Two major rotation in Arkansas for soybean is rice and the soybean-rice rotation is a common rotation. The production of these crops is concentrated in alluvial soils with poor drainage that are often saturated. Most fields are planted with soybean from April through June and with rice mainly in April or May (Faw and Johnston, 1975; Faw and Porter, 1981). Seedling diseases occur whenever these crops are planted. The hypothesis for the third objective, was that

Pythium populations are influenced by the soybean-rice rotation commonly used in Arkansas compared to continuous soybean. Also, since different species of Pythium are active over different temperatures range, temperature may affect the importance of Pythium species. The aims of this objective were: i) to determine the effect of continuous soybean and soybean-rice rotation on Pythium spp. diversity in several locations in Arkansas, and ii) determine the effect of temperature on the recovery of Pythium spp.

12 References

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18 II Characterization seed rot the resistance in soybean to Pythium aphanidermatum

Abstract

Pythium is important group of pathogens associated with stand losses in soybean in Arkansas

(Kirkpatrick et al., 2006a; Rosso, 2007; Avanzato, 2011). Several management strategies are used for the control of Pythium, the most common are cultural practices and fungicide seed treatments. Resistance cultivars are not available. In Arkansas, evidence of resistance has been found in the soybean cultivar Archer against Pythium aphanidermatum using the hypocotyl inoculation method (Rosso et al., 2008), however the resistance in the seed rot phase of P. aphanidermatum has not been characterized in Arkansas. The objective of this research was to characterize the resistance to soybean seed rot caused by Pythium aphanidermatum. Resistance to seedling disease caused by P. aphanidermatum was characterized in 84 F2:6 soybean lines derived from a cross of ‘Archer’ (resistant parent) and ‘Hutcheson’ (susceptible parent). The lines and parents were evaluated with two assays: seed plate and infested vermiculite greenhouse. Germination percentages with the seed plate and plant stands with the infested vermiculate assay were recorded after 7 and 14 days after planting respectively. The lines were then assayed with 5,403 single nucleotide polymorphism (SNP) molecular markers and the results compared to the assay data. Seed germination and plant stands for the Archer x

Hutcheson population fit the model for quantitative resistance indicating that these are a quantitative traits controlled by multiple genes or quantitative trait loci (QTL). Two QTLs were identified on chromosomes 4 and 7 with the two phenotyping methods. The QTL on chromosome 4 explained 8.29 and 13.76 % of the variation and the QTL on chromosome 7 explains 4.5 and 13.85 % of the variation with the seed plate and infested vermiculite assays,

19 respectively. These results will aid in breeding for resistance to Pythium spp. Resistance to seed rot in this population differs from the previous reported resistance for postemergence disease.

Introduction

Seedling diseases are a major constraint to soybean production in Arkansas. Seedling diseases caused an estimated 8.7, 8.9 and 10.7 % loss in yield to Arkansas producers in 2011,

2012, and 2013 respectively (Heatherly, 2014, Data provided by Dr. S.R. Koenning, North

Carolina State University). These diseases can reduce stands and plant vigor and sometimes require replanting (Yang, 1999). Most soybean production in Arkansas occurs in the eastern part of the state where soils are predominantly alluvial with poor drainage, favoring seedling diseases

(Kirkpatrick et al., 2006a). A number of soilborne pathogens are associated with seedling disease, primarily the Oomycetes Pythium spp. and Phytophthora sojae, and the fungi Fusarium spp. and Rhizoctonia solani (Schmitthenner, 1999; Yang, 1999; Wilcox, 1987). These pathogens may act singly or as complexes. Pythium spp. is the predominant pathogen group causing seedling diseases of soybean in Arkansas (Kirkpatrick et al., 2006a, Rosso, 2007; Avanzato,

2011; Urrea et al., 2013). This genus is made up of over 100 species, numerous species have been associated with soybean (Yang, 1999; Dorrance et al., 2004; Broders et al., 2007; Bates et al., 2008; Lévesque et al., 2010; Avanzato, 2011). These species are active across a range of environmental conditions resulting in seedling diseases being a threat to soybean production whenever soybeans are planted. One of the widely used management strategies for the control of

Pythium species is the use of fungicide seed treatments, metalaxyl or its active isomer mefenoxam have been a widely used active ingredient for the control of Oomycetes (Cohen and

Coffey, 1986). Field test performed in Arkansas in different locations over 3 years showed that metalaxyl was the most effective fungicide (Poag et al., 2005), however in more recent years

20 field and growth chamber test showed that broad spectrum fungicide seed treatments were the best for the control of soybean seedling diseases and trifloxystrobin + metalaxyl was the most consistently effective fungicide in a test conducted during two years (Popp et al., 2010; Urrea et al., 2013). The limitations of the use of seed treatments is the inconsistent results of efficacy in field and controlled environment studies and the fact that most of the seed treatments only protect the seed for short period of time (10 to 14 days after planting) but not the emerging root

(Paulsrud and Montgomery, 2005).

An alternative control to fungicide is the use of plant resistance. In soybean, most of the research on resistance against Oomycete pathogens, has been done on Phytophthora root and stem rot caused by Phytophthora sojae (Sandhu et al., 2005). Fifteen single dominant resistant genes (Rps) to Phytophthora root rot have been identified in soybean (Dorrance, et al, 1999;

Dorrance et al, 2004; Dorrance and Grunwald, 2009; Dorrance, et al., 2009) using a hypocotyl inoculation technique (Dorrance, et al., 2004; Gordon et al, 2007). These Rps genes are race specific and over 55 races of P. sojae have been described (Dorrance et al, 1999; Dorrance et al,

2004, Dorrance and Grunwald, 2009; Dorrance et al., 2009). Single gene resistance has been very effective in the field, but has led to the development of races of P. sojae requiring the introduction of new genes for resistance. In addition to single gene resistance, there is also multigenic partial resistance that affects all races of P. sojae (McDonald and Linde, 2002).

Compared to resistance to P. sojae, less research has been done on resistance to Pythium spp. Keeling (1974) reported that the soybean cultivar ‘Semmes’ was more resistant than ‘Hood’.

This difference in reaction to P. ultimum was related to higher concentrations of soluble carbohydrate in exudates of germinating seed in Hood than in Semmes. Griffen (1990) reported that the cultivar ‘Dare’, had significantly higher emergence in P. ultimum infested soil than the

21 cultivar ‘Essex’, suggesting a greater level of Pythium resistance in Dare than Essex. In

Arkansas, Pythium was the only pathogen group that increased with flooding treatments and the cultivar Archer had greater stands than any other cultivar when flooded (Kirkpatrick et al,

2006a). Archer was reported to be flood tolerant (Cianzio et al., 1991). In greenhouse experiments using an inoculum layer technique, Archer had significantly greater stands than the cultivar Hutcheson when in direct contact with inoculum of P. ultimum demonstrating that

Archer also had resistance to P. ultimum (Kirkpatrick et al., 2006b). Using the inoculum layer technique Nanayakkara, (2001) reported that Archer also had resistance to P. vexans, P. aphanidermatum P. ultimum, and P. irregulare. Archer has two genes for resistance to

Phytophthora sojae, Rps 6 and Rps 1k, which come from its parents, ‘PRX54-49’ and ‘Williams

82’, respectively (Cianzio et at., 1991). To determine if Pythium resistance was associated with these Phytophthora resistance genes, Bates et al. (2004) tested a set of differential cultivars containing specific resistant genes for P. sojae in the Williams 82 background along with Archer and Hutcheson in vermiculite infested with Pythium aphanidermatum. Stands were significantly higher with Archer and Williams 82 (Rps 1k) than all the other cultivars which had stands that were not significantly different than Hutcheson including the line with Rps 6. This suggested that

Archer resistance to Pythium spp. could be associated with Rps 1k gene. Using the hypocotyl inoculation method developed to identify single gene resistance to Phytophthora sojae, Rosso et al., (2008) reported that this method resulted in clear differences between Archer and Hutcheson with Pythium aphanidermatum. Applying this approach to 86 F2:4 lines from a cross between

Archer with Hutcheson the same criteria for resistance, susceptible, and segregating that had been developed for Phytophthora sojae, they found that the resulting reactions to Pythium aphanidermatum fit the model for a single dominant gene in Archer. Screening the lines with 88

22 SSR, representing all the soybean molecular linkage groups (MLG’s) found two markers,

Satt114 and Satt510, located on the MLG F were associated with Rpa1 and were estimated to be

15.5 cM and 10.6 cM from the gene. This gene was designated as Rpa1. This soybean population was also screened with race 7 of Phytophthora sojae to identify the Rps1k gene. The segregation of the Rps 1k gene was completely different than the Rpa 1 gene indicating that Rps 1k was not the source of Pythium resistance. Single gene plant resistance against Pythium has also been reported to P. aphanidermatum, P. ultimum and P. inflatum in periwinkle, beans and maize, respectively (Kulkarni and Baskaran, 2003; Otsyula, et al., 2003; Yang et al., 2005).

Multigenic resistance to Pythium spp. by the identification of quantitative trait loci

(QTLs) have been reported. Resistance to P. ultimum in snap bean was first reported in 1977

(York et al., 1977). In 2008, Navarro et al, reported five QTLs in linkage groups B6, B3 and B7 associated with this root rot resistance. In soybean, 192 F2:3 lines from two populations: ‘OHS

303’ (OHS 303 x (Williams x PI424354)) and 127 F2:3 lines from ‘Denisson’ x (‘Denisson’

(Williams x PI424354)), were screened against P. irregulare using infested vermiculite (Ellis et al., 2013). Pythium disease was accessed based on root rot weight and root rot scores. The lines were screened with 74 SSR and 384 SNP markers resulting in two putative QTL associated with resistance to P. irregulare in the OHS 303 population on chromosomes 1 and 6 with composite interval mapping (CIM) analysis and on chromosomes 5, 13, 14 and 20 using Internal mapping

(IM) analysis for root weight and root rot score. In the Denison population a putative QTL was identified in chromosomes 8 and 11 for root weight and in chromosome 13 for root rot score using CIM. Overall, QTLs were mapped on chromosome 1, 6, 8, 11 and 13 for root weight and root discoloration. The identification of these QTL for resistance to P. irregulare were possible

23 because of the availability of thousands of single nucleotide polymorphism (SNP) makers in soybean.

The seed plate (Avanzato, 2011) and the infested vermiculite assays (Rosso, 2007) have been shown to consistently distinguish differences in cultivar resistance to the seed rot phase of

Pythium diseases. The research at this point have examined resistance to root rot and post emergence damping-off of soybean. However, most of the Pythium problems in soybean are associated with seed rot. It was decided that the Archer by Hutcheson population screened by

Rosso et al. (2008) be reanalyzed to better understand the genetics of Pythium resistance in

Archer to the seed rot phase. The objectives of this research was to characterize of the resistance in Archer to P. aphanidermatum using these two screening methods and single nucleotide markers.

Materials and methods

Isolate

Pythium aphanidermatum isolate 64 used in this study was collected from a soybean field at the Pine Tree Research Station Colt, Arkansas (Kirkpatrick, 2006a) and was used in previous studies in pathogenicity tests and in the characterization of the resistance in Archer

(Nannayakara, 2001; Bates et al., 2008; Rosso et al., 2008 and Avanzato, 2011). The isolate was stored on cornmeal agar (CMA, BBL ™ Becton, Dickson and company Sparks, MD, USA) plugs of the isolate have been stored in vials with distilled autoclaved water, stored at room temperature (23 - 25°C).

Plant material for mapping

24 The Archer x Hutcheson population previously characterized by Rosso et al, (2008) was used in this study. There were 84 F2:6 lines in this population. Archer seed was from the original seed and Hutcheson seed was from the Foundation seed.

Phenotypic data

Resistance to P. aphanidermatum was evaluated using the seed plate assay and the infested vermiculate greenhouse assay. The seed plate method was originally described by

Broders et al., (2009) and modified by Avanzato (2011). Isolate 64 was grown on CMA for 4 to

5 days, a 0.5 cm2 plug was transferred onto the center of 9-cm petri dish containing 2% water agar (Agar gelidium, Moor Agar, Incorporated) and incubated for 4 days. A 0.5-cm layer of autoclaved vermiculite (Medium vermiculite, Sun Gro®, Belleve Washington, USA) was placed on top of the agar and 10-ml of autoclaved distilled water was added. Ten seeds of each line or cultivar were surface sterilized with 70% ethanol for 3 minutes, blotted dry on autoclaved paper towels and then placed on the plates. Plates were covered with aluminum foil to exclude light and incubated at room temperature (23 - 25°C). After seven days seed germination was assessed from each plate. There were three replications per line, the control was treated the same, but without P. aphanidermatum. The experimental design was a randomized complete design with three repetitions and the experiment was conducted twice.

Based on preliminary studies, a modified V8 juice-vermiculite medium infestation technique was used to screen the lines in the greenhouse (Rosso, 2007). Inoculum was produced with 250-ml of autoclaved vermiculite amended with 125-ml of V-8 juice broth (50 ml V8- juice®, 0.75 g of CaCO3, and 200 ml of water) and mixed in 12 x 24 cm transparent autoclavable polypropylene bags (Clavies®, Pequannock, New Jersey, 07440 USA). The bags were autoclaved for 40 min and then re-autoclaved 24 h later for 40 min. The isolate was grown on

25 CMA for 5 days at room temperature. Agar from half a plate was cut into small pieces (< 0.5 cm2) and transferred to a bag containing the 250-ml V8 juice-vermiculite medium, mixed and incubated for 7 days at room temperature. Each bag was covered with aluminum foil to exclude light. Bags were mixed every other day to ensure even colonization by the pathogen. After seven days, inoculum was mixed with sterile vermiculite at a 1:16 (v/v) ratio and 20-liters of the mixture were transferred to 37.85-liters polyethylene bags and the bags placed in 114-liters black plastic bags to exclude light an incubated at room temperature. The inoculated vermiculite was mixed every other day for 10 days to allow the pathogen population to stabilize. The control was

V8-vermiculite medium mixed with CMA without P. aphanidermatum.

Inoculum concentration was quantified by mixing 25 g of the infested vermiculite medium, oven-dry weight equivalent (odw) with 235 ml of sterile dilute water agar (0.2%

BactoTM Agar) in a 500-ml flask for a total volume of 250 ml. The flask was agitated on a wrist- action shaker for 20 min and then 10 ml of this suspension was added to 90 ml of dilute water agar and thoroughly mixed. One ml of the 1/10 and 1/100 suspensions were spread over the surface of each of six 9-cm diameter Petri plates per dilution containing the selective medium,

P5ARP (Jeffers and Martin, 1986). Plates were incubated in the dark at room temperature for 24 to 48 h, and colonies of Pythium were counted and the number of CFU’s per g of vermiculite

(odw) calculated. After the incubation period, 473.17-cm3 Styrofoam cups were filled with the infested vermiculite. Each cup had 4, 5-mm holes in the bottom to allow drainage. Cups were watered from the bottom until saturation and left to stabilize overnight. Ten seeds of each cultivar were then planted in each cup. Seeds were planted at a depth of 2-cm then covered with infested vermiculite. There were four cups per line and one control. The control was treated the same as the infested cups and was randomized in the test. The experiment was conducted twice.

26 After planting, the cups were placed in a greenhouse at average temperature of 23.7 °C. Soil water was monitored with a Watermark Soil Moisture Sensor (Spectrum Technologies, Inc.

Plainfield, Illinois) and temperature sensors and recorded with a data logger (Watchdog 400®,

Spectrum Technologies, Inc. Plainfield, Illinois). When the soil water potential reached -20 J/kg, cups were watered from the bottom. Plant stands were recorded 14 days after planting. The first planting was on November 6 and second planting was on November 21, 2013. The experimental design was a completely randomized design with four replications and the experiment was conducted twice.

Genotyping data

DNA extraction. The 84 (F2:6) lines and the parents were grown in the greenhouse for two weeks in 473.17-cm3 Styrofoam. There were 3 cups per line and 10 seed were planted in each cup. Tissue samples were collected from 12 to 15 leaflet at the V2 growth stage (Fehr et al,

1971) about two weeks after planting and placed in paper bags temporally stored in a cooler, after 1 to 2 hours the samples were placed at -80 °C. The tissue was ground with a mortar and pestle in approximately 200-ml of liquid nitrogen, transferred to 2 ml Eppendorf® tubes and stored at -80 ° C until DNA extraction. Total genomic DNA was extracted using the CTAB method (Kisha et al., 1997). The sample was thawed and 0.5 ml of the ground tissue transfer to 2 ml polypropylene centrifuge tubes and 750 µl of DNA extraction buffer was added. The tubes were mixed with a vortex mixer (PV-1 Grant-bio, Grant instruments Ltd. Shepreth.

Cambridgeshire, SG8 6GB, England) for 5 seconds and incubated at 65 °C for 60 minutes and inverted every 10 min. The tubes were cooled for 10 minutes on ice and then filled with 750 µl of chloroform: isoamyl alcohol (24:1) and inverted approximately 40 times to mix. Tubes were spun at 12,000 rpm for 15 min at room temperature. The aqueous layer was transferred into a 1.5

27 ml polypropylene Eppendorf® tube and 1ml of 95% ethanol was added to each tube and spun at

12,000 rpm for 5 minutes at room temperature. The ethanol was poured off and 1 ml of 75% ethanol was added to wash DNA pellet and then poured off. The DNA pellet was air dried overnight at room temperature. The next day, 200µl of 0.1 TE buffer was added to each tube and keep at room temperature over night to dissolve the DNA. The DNA was stored at -20 °C until needed.

Single Nucleotide polymorphism (SNP) genotyping. Once the DNA was extracted from all the lines, the samples were sent to Dr. Dechun Wang laboratory at Michigan State

University, East Lansing, MI, for the analysis with 5403 SNP markers (National Library of

Medicine 2012) using the BARC MSU Soy6k Illumina Infunium Genotyping HD Beadchip

(652K) on Illumina iScan (Illumina, San Diego, CA). SNP analysis was performed on 4-µL samples containing a concentration between 50 and 100 ng/µL of DNA. Intensities of the fluorescence were distinguished using the Illumina iScan TM Reader, and alleles for each SNP locus were named using Illumina’s Bead Studio TM software (Illumina, San Diego, CA, v3.2.23). Genotypes were identified using the program Genome Studio (Illumina, San Diego,

Calif. USA). For each SNP marker, the genotype data represent three possible genotypes AA

(homozygote), AB (heterozygote), and BB (homozygote) (Akond et al., 2013). Each SNP in every line was used to construct the genetic maps.

Data analysis

Phenotyping data. Plant stands and germination percentages were analyzed using the

GLM procedure (SAS version 9.4, SAS Institute Inc. Cary, NC. USA). Both runs of each test were combined for this analysis. Weighted mean for plant stand and germiantion was calculated as: (seed germination or plant stands in each replication/ non-inoculated control)*100.

28 To determine heritability of disease response, broad sense heritability (H2) was calculated with the TYPE3 sum of squares, with the following formula (Sleper and Poehlman, 2006):

2 2 2 2 2 H = ơ G/ (ơ G + ơ GEI/ e + ơ E/ re)

2 2 2 Where ơ G, ơ GEI , ơ E are genotypic variance, genotype-by-environment variance and error variance, respectively, and e and r are the number or environments or runs (n=2) and replications

(n=3 seed plate assay and n= 4 for infested vermiculate).

Genetic maps and QTL mapping. Genetic maps were constructed with the SNPs markers data from the Illumina’s Bead Studio TM software, genetic maps were constructed using

JoinMap 4.0 (Van Ooijen, 2006). Regression mapping for each chromosome was performed with a Haldane function with the default parameters. For QTL detection, single marker analysis

(SMA) and composite interval mapping (CIM) were carried out by WinQTL Cartographer 2.5

(Basten et al., 1999). CIM with forward and backward regression using1000 permutation with a walk speed of 1cM was used to determine QTL positions at experiment-wise α= 0.05 (Churchill and Doerge, 1994) and a maximum of 18 control markers. MapChart (Voorrips, 2002) was used to construct the chromosome maps and LOD (Logarithm of odds) plots based on JoinMap 4.0 and WinQTL Cartographer 2.5. The identified QTL were described as ‘major’ and ‘minor’ based on the proportion of phenotypic variation the QTL explains (R2 ), it is a major QTL if it accounts for large variation (>10%) and minor if it accounts for less than 10 % of the phenotypic variation

(Boopathi, 2013).

Results

Phenotypic data

There were significant differences (P ≤ 0.0001) in disease reactions among the 84 F2:6 lines derived from ‘Archer’ X ‘Hutcheson cross (Table 1) using the two phenotyping assays. In

29 the seed plate assay, germination ranged from 3.7 to 85% (Table 2). Archer averaged 36.85 % germination while Hutcheson averaged 3.7 % germination. There were 13 lines that were not significantly different that the most resistant line with the seed plate assay. Plant stands ranged from 93.75 to 20.79% (Table 3). Archer averaged 72.5 % emergence and Hutcheson averaged

20%. There were 26 lines that were not significantly different than the most resistant line with the infested vermiculite assay. In the greenhouse infested vermiculate study the population densities of Pythium aphanidermatum averaged 70.39 cfu/cc of infested vermiculite. The heritability estimates for the seed plate assay and the infested vermiculite assay was 0.8534 and

0.6955, respectively.

Quantitative trait loci mapping

Seed germination and plant stands for the Archer x Hutcheson population fit the model for quantitative resistance indicating that these are quantitative traits controlled by multiple genes or QTLs (Figure 1a and 1b). Of the 5403 SNP makers distributed among the 20 soybean chromosomes, 23.5 % (1269 SNPs) loci were polymorphic between Archer and Hutcheson, the parental genotypes. The linkages maps were constructed with 889 markers, which were mapped on 20 chromosomes, representing 695 unique loci (Table 4). The linkage map covered 3956.33 cM and the average distance of each loci was about 6.036 cM loci (Figure 2 a-f).

In the Single Marker Analysis, there were 54 significant markers (P ≤ 0.05) that were associated with resistance to Pythium aphanidermatum on chromosomes 1, 4, 8, 9, 12, 15, 16, 18 and 20 with the seed plate assay (Table 5) and 26 significant makers on chromosomes 1, 4, 9, 12,

14, 16 and 18 with the infested vermiculite assay (Table 6).

Composite Interval Mapping detected two major and two minor QTLs on chromosomes 4 and 7 across the two phenotyping methods (Table 7). Two P. aphanidermatum resistance QTL

30 on chromosome 4 and 7 were identified with the seed plate assay and with the infested vermiculite assay.

Based on seed germination using the seed plate assay, a minor QTL was mapped on chromosome 4, flanked by ss715589319 and ss715588524 linked to ss715589319. This QTL was mapped at 51 cM, had a LOD of 4.14 and explained 8.13 % of phenotypic variation for seed germination (Figure 3a). Based on plant stands using the greenhouse assay, a major QTL was identified in chromosome 4 flanked by flanked by ss715589319 and ss715588524 linked to ss715589319. This QTL was mapped at 49 cM, had a LOD of 6.12 and explained 13.76 % of phenotypic variation for plant stands (Figure 3b).

Based on seed germination a minor QTL was identified in chromosome 7, linked to ss715598762 at 121.21 cM, with a LOD of 3.12 and explaining 4.5% of phenotypic variation on seed germination (Figure 4a). In the same chromosome 7 a major QTL was identified in chromosome 7, linked to ss715598762 at 118.20 cM, with a LOD of 5.55 and explaining 13.

85% of phenotypic variation on plant stands (Figure 4b).

Discussion

Partial resistance to Pythium aphanidermatum was found in Archer x Hutcheson F 2:6 population using the seed plate assay and the infested vermiculite assay. Both methods separated the resistant parent Archer and the susceptible parent Hutcheson. The progeny of Archer x

Hutcheson ranged, continuously from low to high disease reactions and the histogram of percentage of both traits approximate a normal curve. These are characteristic of quantitative resistance, implying many genes or QTLs. On the other hand, qualitative traits fall into fewer characteristics, resistant or susceptible phenotypes and can be grouped into a few classes and can

31 be easily distinguished. These traits are simply inherited and governed by one or few genes

(Sleper and Poehlman, 2006; Poland et al., 2009; St. Clair, 2010).

Heritability of quantitative resistance is defined as the proportion of the observed variation in a progeny that is inherited (Poehlman and Sleper, 1995). Breeders uses heritability to determine the effect of environment and genetic factors on the trait and make decisions on the selection process to follow to improve a trait. High hereditably value (>50%) means that the trait is less influenced by the environment and more influenced by the genetic effects, on the other hand, low values of heritability (≤ 30 %) means that the environment had a bigger effect the trait

(Boopathi, 2013). Broad sense heritability for the percentage of seed germination and plants stands were high: 85 % and 69% respectively, meaning that the proportion of observed reactions in seed germination and plant stands to Pythium aphanidermatum resistance in in the Archer x

Hutcheson F2:6 population are highly influenced by genetic factors rather than environmental factors,

Another important characteristic of a quantitative resistance is that the progeny fall outside the range of the parents (Sleper and Poehlman, 2006). In this study, some of the lines appeared more resistant than the resistant parent (Archer) and a few more susceptible that the susceptible parent (Hutcheson) indicating transgressive segregation in the population.

Transgressive segregation is a condition that is common in the inheritance of quantitative characters when the progeny contain new combination of multiple genes with more positive or negative effects than the parent. In this study, the quantitative trait loci mapping using Composite

Interval Mapping (CMI) analysis detected two stable QTL for resistance on chromosomes 4 and

7, with the two phenotyping methods. Two resistance major QTL were found on chromosome 4

32 and 7 using with the infestation assay and two resistant minor QTL were found with the seed assay.

In this study the two major QTLs were found with the infested vermiculate assay on chromosome 4 linked to ss715589319 contributing 13.76% to the resistance to Pythium aphanidermatum and on chromosome 7 liked to ss715598762 contributing 13.85 % to P. aphanidermatum resistance. The two minor QTLs were identified with seed plate assay on the same chromosomes 4 and 7 and linked to the same marker as the major QTLs, these minor QTLs contributed 8.29% and 4.5 % on chromosomes 4 and 7 respectively to the resistance to Pythium aphanidermatum. This research confirmed two stable QTL in chromosome 4 and 7 identified with the two methods.

Although, QTLs for resistance to Pythium aphanidermatum have not been reported before on soybean chromosome 4 and 7, there are reported QTLs for resistance to other diseases in areas near these QTLs. On Chromosome 4, there are two QTLs for the partial resistance to

Phytophthora sojae (Lee et al., 2013) and five QTLs for resistance to soybean cyst nematode

(Yue et al., 2001). Likewise, on chromosome 7, there is one QTL reported for resistance to resistance to soybean cyst nematode (Ferreira, 2011).

Single gene resistance and multigenic resistance have been reported for different Pythium species on different crops: P. aphanidermatum, P. ultimum and P. inflatum in soybean, periwinkle, beans and maize, respectively (Kulkarni and Baskaran, 2002; Otsyula, 2003; Yang et al., 2005).

Multigenic resistance to Pythium spp. by have been reported. York reported Quantitative resistance in snap bean for seed decay and pre-emergence damping- off caused by Pythium ultimum (York, 1977). Navarro et al., (2008) reported a major Quantitative trait loci in snap bean

33 linkage group B6 to snap been root rot caused by Pythium ultimum and f. sp. phaseoli.

In soybean, single gene resistance against Pythium aphanidermatum has been reported on soybean by Rosso et al., (2008) using the hypocotyl inoculation method developed to identify single gene resistance to Phytophthora sojae. Rosso et al., (2008) reported that this method resulted in clear differences between Archer and Hutcheson with Pythium aphanidermatum.

Applying this approach to 86 F2:4 lines from a cross between Archer with Hutcheson the same criteria for resistance, susceptible, and segregating that had been developed for Phythopthroa sojae, they found that the resulting reactions to Pythium aphanidermatum fit the model for a single dominant gene in Archer. This gene was designated as Rpa1. Screening the lines with 88

SSR, representing all the soybean molecular linkage groups (MLG’s), two markers, Satt114 and

Satt510 located on the MLG F were associated with Rpa1 and were estimated to be 15.5 cM and

10.6 cM from the gene. Since in this study QTL in the MGL F were not found, there are different resistances mechanism of resistance to Pythium diseases on soybean where the seeds the seedlings are expose to the infection of Pythium.

Ellis et al., (2013) reported QTL for partial resistance to P. irregulare in two populations,

QTL were found on chromosomes 1, 6, 8, 11 and 13 for root weight and root discoloration.

However, the QTL identified in this study were located on different chromosomes than Ellis et al., (2013), this could be due to the screening techniques. They evaluated root discoloration and root weight which impact more the seedling in the root rot phase but did not assessed stands. The seed assay and infested vermiculite assessed the seed rot phase of Pythium diseases on soybean.

Seed plate assay and infested vermiculite assay were used in this study to screen for resistance in the Archer x Hutcheson population to Pythium aphanidermatum. Both methods

34 separated the resistant parent Archer and the susceptible parent Hutcheson. To be valid a technique must be reproducible but also represent field conditions. The seed plate assay technique is cheap and fast screening technique that does not required a lot of laboratory space.

On the other hand, infested vermiculite technique is more time consuming, labor intense and needs large greenhouse space; however, this technique allow for a more realistic scenario in how

Pythium spp. infect soybean in the field conditions. In this study the correlation between the two screening techniques was low (R2= 34%). When 20 of the most resistant lines were compared with the two techniques the seed assay only predicted 45% of the lines that were the more resistant with the invested vermiculite method. Although, the seed assay did not accurately predict resistant lines based on the vermiculite assay, to be validated a preliminary screening method for Pythium species, it needs to be tested in the future with more advance populations and compared with field performance.

Overall, we found that seed germination and plant stands for the Archer x Hutcheson population fit the model for quantitative resistance indicating that these are a quantitative traits controlled by multiple genes or QTLs. The two phenotyping methods evaluated soybean seed rot and separated the parents. Two stable QTL were found on chromosome 4 and 7 using the infested vermiculite assay and seed plate assay respectively, showing that these two assays separated the parents and lines. The QTL on chromosome 4 and 7 were identified at similar locations using the seed plate and the infestation assays making them strong candidates for further research.

Factors that may contribute to expand QTL detection to resistance to Pythium aphanidermatum could be the use of a bigger population (>250 lines), more advance population

35 (RIL) and more SNP makers in order to be able to find more QTLs for resistance to Pythium aphanidermatum in soybeans.

Conclusion

Overall, the research demonstrated that seed germination and plant stands for the Archer x Hutcheson population fit the model for quantitative resistance indicating that these are quantitative traits controlled by multiple genes or QTLs. The two phenotyping methods evaluated soybean seed rot and separated the parents. Two stable QTL were found on chromosome 4 and 7 using the infestation assay seed plate assay respectively, showing that these two assays separated the parents and lines. The QTL on chromosome 4 and 7 were identified at similar locations using the seed plate and the infestation assays making them strong candidates for further research.

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Yue, P., Arelli, P., Sleper, D.A. 2001. Molecular characterization of resistance to Heterodera glycines in soybean PI 438489B. Theor. Appl. Genet. 102(6-7):921-928.

40 Table 1. Analysis of variance of seed plate and infested vermiculite assay with Pythium aphanidermatum of 84 F2:6 lines and the parents, Archer and Hutcheson.

Degrees of freedom P-value Source Seed plate Infested Seed plate Infested vermiculite vermiculite Model 343 429 <.0001 <.0001 Run 1 1 <.0001 <.0001 Line 85 85 <.0001 <.0001 Rep 2 3 0.3944 0.006 Run x line 85 85 <.0001 0.0006 Line x rep 170 255 0.1918 0.7747 Error 149 258

41 Table 2. Seed germination (%) for the 84 F2:6 lines an2d parents evaluated for the resistance to Pythium aphanidermatum with the seed plate assay.

Line Seed germination mean (%)w LR-23 85.00 AX LR-128 85.00 A LR-291 83.5 AB LR-343 78.33 ABC LR-300 78.22 ABC LR-4 71.67 ABCD LR-175 71.67 ABCD LR-1 69.21 ABCDE LR-73 68.00 ABCDEF LR-66 65.78 ABCDEFG LR-64 64.50 ABCDEFGH LR-7 63.89 ABCDEFGH LR-93 62.14 ABCDEFGHI LR-283 61.67 BCDEFGHI LR-275 61.67 CDEFGHI LR-22 60.44 CDEFGHIJ LR-106A 60.00 CDEFGHIJ LR-87 57.50 CDEFGHIJK LR-110 55.56 CDEFGHIJKL LR-220 54.00 DEFGHIJKLM LR-264 53.33 DEFGHIJKLM LR-302 52.78 DEFGHIJKLM LR-301 51.67 DEFGHIJKLMN LR-101 51.67 DEFGHIJKLMN LR-187 51.16 DEFGHIJKLMNO LR-77 50.00 DEFGHIJKLMNOP LR-142 50.00 DEFGHIJKLMNOP LR-144 49.69 DEFGHIJKLMNOP w. Weighted mean for seed assay for each experiment with three replications, conducted twice. Weighted mean seed germination represents (seed germination in each replication/ non-inoculated control)*100). x. Values followed by the same letter and case were not significantly different according with the Fisher’s protected least significant different (LSD) at P =0.05 y. Resistant parent cultivar z. Susceptible parent cultivar

42 Table 2. Seed germination (%) for the 84 F2:6 lines and parents evaluated for the resistance to Pythium aphanidermatum with the seed plate assay (Cont.)

Line Seed germination mean (%)w LR-261 48.38 EFGHIJKLMNOPX LR-309 47.22 EFGHIJKLMNOPQ LR-28 47.08 EFGHIJKLMNOPQ LR-2 46.03 FGHIJKLMNOPQR LR-178 45.00 GHIJKLMNOPQRS LR-63 45.00 GHIJKLMNOPQRS LR-65 44.89 GHIJKLMNOPQRS LR-216 44.67 GHIJKLMNOPQRS LR-344 43.75 GHIJKLMNOPQRS LR-316 43.33 GHIJKLMNOPQRS LR-56 43.33 GHIJKLMNOPQRS LR-349 41.67 HIJKLMNOPQRS LR-277A 40.00 IJKLMNOPQRST LR-242 40.00 IJKLMNOPQRST LR-277B 40.00 IJKLMNOPQRST LR-199 39.50 IJKLMNOPQRST LR-209 38.61 IJKLMNOPQRSTU LR-278 37.73 JKLMNOPQRSTUV LR-75 37.59 JKLMNOPQRSTUV LR-202 37.08 KLMNOPQRSTUVW Archer y 36.85 KLMNOPQRSTUVW LR-13 36.85 KLMNOPQRSTUVW LR-284 35.19 KLMNOPQRSTUVW LR-201 35.00 KLMNOPQRSTUVW LR-29 33.70 LMNOPQRSTUVWX LR-207 32.41 MNOPQRSTUVWX LR-176 31.67 MNOPQRSTUVWXY LR-272 29.63 NOPQRSTUVWXYZ LR-203 28.89 OPQRSTUVWXYZa LR-36 28.33 OPQRSTUVWXYZaX LR-45 28.33 OPQRSTUVWXYZa LR-234 28.33 OPQRSTUVWXYZa LR-38 26.67 PQRSTUVWXYZa LR-145 26.67 PQRSTUVWXYZa LR-158 25.74 PQRSTUVWXYZab LR-89 24.76 QRSTUVWXYZab

43 Table 2. Seed germination (%) for the 84 F2:6 lines and parents evaluated for the resistance to Pythium aphanidermatum with the seed plate assay (Cont.).

Line Seed germination mean (%)w LR-97 24.34 QRSTUVWXYZab LR-237 24.17 RSTUVWXYZab LR-188 24.07 RSTUVWXYZab LR-52 23.33 RSTUVWXYZab LR-184 23.13 RSTUVWXYZab LR-229 22.92 STUVWXYZab LR-198 18.75 TUVWXYZab LR-37 18.70 TUVWXYZab LR-43 18.33 TUVWXYZab LR-8 17.86 TUVWXYZab LR-71 16.43 UVWXYZab LR-189 15.00 VWXYZab LR-12 14.44 WXYZab LR-215 11.67 XYZab LR-62 11.67 XYZab LR-235 11.11 XYZab LR-98 9.26 YZab LR-19 8.33 Zab LR-181 8.33 Zab LR-25 6.67 ab LR-208 3.70 b Hutcheson z 3.70 b

44 Table 3. Plant stands (%) for the 84 F2:6 lines and parents evaluated for the resistance to Pythium aphanidermatum with the infested vermiculite assay.

Line Plant stands (%)w LR-22 93.75 Ax LR-65 92.50 AB LR-93 91.43 ABC LR-1 90.00 ABCD LR-128 88.75 ABCDE LR-66 86.94 ABCDEF LR-283 86.25 ABCDEFG LR-2 85.69 ABCDEFGH LR-56 85.00 ABCDEFGHI LR-7 84.44 ABCDEFGHIJ LR-110 83.75 ABCDEFGHIJ LR-28 83.75 ABCDEFGHIJ LR-344 82.70 ABCDEFGHIJK LR-75 82.54 ABCDEFGHIJK LR-64 82.50 ABCDEFGHIJK LR-13 82.22 ABCDEFGHIJKL LR-101 80.62 ABCDEFGHIJKLM LR-23 80.56 ABCDEFGHIJKLM LR-277A 80.00 ABCDEFGHIJKLMN LR-216 80.00 ABCDEFGHIJKLMN LR-278 79.86 ABCDEFGHIJKLMN LR-142 78.89 ABCDEFGHIJKLMNO LR-77 78.75 ABCDEFGHIJKLMNO LR-4 78.75 ABCDEFGHIJKLMNO LR-8 78.12 ABCDEFGHIJKLMNOP LR-178 77.50 BCDEFGHIJKLMNOPQ LR-202 76.25 BCDEFGHIJKLMNOPQ w. Weighted mean for plant stands for each experiment with four replications, conducted twice. Weighted mean plant stands represents ((plant stands in each replications/ non-inoculated control)*100). x. Values followed by the same letter and case were not significantly different according with the Fisher protected least significant different (LSD) at P =0.05 y. Resistant parent cultivar z. Susceptible parent cultivar

45 Table 3. Plant stands (%) for the 84 F2:6 lines and parents evaluated for the resistance to Pythium aphanidermatum with the infested vermiculite assay (Cont.).

Line Plant Stands mean (%)w LR-176 76.53 BCDEFGHIJKLMNOPQX LR-63 76.25 CDEFGHIJKLMNOPQ LR-106 76.25 CDEFGHIJKLMNOPQ LR-277B 76.11 CDEFGHIJKLMNOPQ LR-73 75.00 DEFGHIJKLMNOPQR LR-144 75.00 DEFGHIJKLMNOPQR LR-158 75.00 DEFGHIJKLMNOPQR LR-87 74.29 DEFGHIJKLMNOPQR LR-97 74.03 DEFGHIJKLMNOPQR LR-242 73.75 EFGHIJKLMNOPQR LR-209 72.81 EFGHIJKLMNOPQR LR-36 72.78 EFGHIJKLMNOPQR Archery 72.50 FGHIJKLMNOPQR LR-275 72.22 FGHIJKLMNOPQRS LR-234 72.22 FGHIJKLMNOPQRS LR-261 72.22 FGHIJKLMNOPQRS LR-62 71.81 FGHIJKLMNOPQRS LR-62 71.25 FGHIJKLMNOPQRS LR-349 71.25 FGHIJKLMNOPQRS LR-300 71.25 FGHIJKLMNOPQRS LR-184 70.70 GHIJKLMNOPQRST LR-284 70.00 HIJKLMNOPQRST LR-301 70.00 HIJKLMNOPQRST LR-220 68.75 IJKLMNOPQRSTU LR-45 68.75 JKLMNOPQRSTU LR-188 68.61 JKLMNOPQRSTU LR-175 68.61 JKLMNOPQRSTU LR-98 67.35 KLMNOPQRSTU LR-89 67.14 KLMNOPQRSTU LR-52 66.25 LMNOPQRSTUVX LR-203 65.83 MNOPQRSTUVW LR-199 64.58 MNOPQRSTUVW LR-264 64.31 NOPQRSTUVW LR-215 62.78 OPQRSTUVWX LR-145 61.94 PQRSTUVWXY

46 Table 3. Plant stands (%) for the 84 F2:6 lines and parents evaluated for the resistance to Pythium aphanidermatum with the infested vermiculite assay (Cont.).

Line Plant stands (%)w LR-302 61.43 QRSTUVWXY LR-237 59.72 RSTUVWXYZ LR-207 56.25 STUVWXYZa LR-201 56.25 STUVWXYZa LR-291 55.00 TUVWXYZa LR-71 54.92 TUVWXYZa LR-316 53.75 UVWXYZa LR-25 53.75 UVWXYZa LR-43 50.64 VWXYZab LR-37 50.00 WXYZab LR-187 49.99 WXYZab LR-181 49.99 WXYZab LR-208 47.50 XYZab LR-198 47.36 XYZab LR-38 47.14 XYZab LR-229 46.53 XYZab LR-189 45.83 YZab LR-235 43.65 Zab LR-272 41.39 ab LR-29 40.36 abc LR-12 36.51 bcd LR-309 36.11 bcd LR-19 25.00 cd Hutchesonz 20.79 d

47

(a) 20 (b) P1 30 18 P1 16 25 14 12 20 P2 10 15

8 No. ofNo. lines 6 ofNo. lines 10 P2 4 5 2 0 0 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100 Seed germiantion (%) Plant stands (%)

Figure 1. Frequency distribution of Pythium resistance in a population derived from ‘Archer’ (P1) x ‘Hutcheson’ (P2) cross evaluations: a) Seed plate assay and b) Infested vermiculite.

48 Table 4. Summary of single nucleotide polymorphism (SNP) markers used to screen the 84 F2:6 lines derived from ‘Archer’ x ‘Hutcheson’ cross.

Average No. SNP z No SNP locus Length distance Chromosome MLG y marker located (cM)) between SNP mapped separately loci (cM) 1 D1a 246.98 38 28 6.46 2 D1b 375.21 117 75 5.26 3 N 241.40 46 37 6.35 4 C1 154.53 25 21 7.99 5 A1 135.82 29 25 5.66 6 C2 136.85 33 26 5.47 7 M 236.93 55 42 5.76 8 A2 199.51 39 31 6.86 9 K 272.35 51 63 5.34 10 0 68.59 21 18 4.03 11 B1 136.30 17 15 9.73 12 H 236.40 53 43 6.17 13 F 57.71 32 24 2.83 14 B2 250.73 43 39 5.70 15 E 273.20 68 50 5.55 16 J 207.86 59 42 4.95 17 D2 226.98 66 44 5.05 18 G 139.52 32 24 6.07 19 L 144.23 35 23 6.55 20 I 215.21 30 25 8.96 Average 197.82 44.45 34.75 6.04 Total 3956.33 889.00 695.00 120.73 y MLG, molecular linkage groups z SNP, single nucleotide polymorphism

49 Chromosome-1 Chromosome-2 [1] Chromosome-2 [2] Chromosome-3

cM Chr.1 cM Chr.2 cM Chr.3

203.3 ss715581222 0.0 ss715578810 0.0 ss715583404 0.0 ss715586464 213.2 ss715581250 ss715586487 4.5 ss715583506 0.3 13.3 ss715579040 ss715579081 217.0 ss715581250 5.8 ss715586508 6.9 ss715583906 13.4 ss715579057 248.3 ss715581428 9.8 ss715586460 7.1 ss715582795 18.1 ss715579291 248.8 ss715581587 11.6 ss715586432 ss715583999 9.2 ss715581484 ss715581566 16.5 ss715586323 ss715583314 10.5 249.1 ss715581519 25.1 ss715586284 12.9 ss715584056 255.9 ss715581808 26.3 ss715586273 15.0 ss715584262 49.6 ss715579229 ss715579274 259.0 ss715581417 32.1 ss715586215 ss715581732 ss715581751 36.5 263.2 ss715581904 32.2 ss715586203 65.0 ss715579755 ss715579793 39.3 ss715581640 ss715581576 ss715581847 ss715581880 37.9 ss715586146 39.8 ss715581593 263.6 ss715581794 49.7 ss715586085 ss715581676 78.0 ss715579920 42.2 263.9 ss715581851 51.8 ss715586013 ss715581813 ss715585963 44.8 264.4 ss715581788 53.3 94.9 ss715579920 54.1 ss715582163 64.9 ss715585882 ss715579973 266.2 ss715581932 112.9 58.9 ss715582547 ss715582347 75.7 ss715585945 ss715585949 ss715579950 ss715582018 ss715581956 117.8 60.0 ss715582735 267.1 77.7 ss715586032 ss715579975 ss715582052 ss715581941 121.7 61.9 ss715582816 104.7 ss715585721 ss715579958 267.3 ss715582005 130.1 63.1 ss715583267 114.9 ss715585573 143.7 ss715580047 269.0 ss715582138 63.2 ss715583146 115.0 ss715585568 146.1 ss715580035 269.3 ss715582112 64.4 ss715582963 117.5 ss715585577 149.7 ss715580023 269.5 ss715582090 67.4 ss715583580 119.9 ss715585530 154.6 ss715580105 271.5 ss715582156 ss715582153 ss715585558 ss715585512 68.8 ss715583592 ss715583597 120.7 160.1 ss715580090 275.1 ss715582184 ss715585444 ss715585454 166.0 ss715580114 277.2 ss715582216 125.2 ss715585442 166.1 ss715580117 277.3 ss715582265 ss715582259 172.1 ss715580131 277.4 ss715582314 175.4 ss715586928 172.8 ss715580144 279.0 ss715582323 179.6 ss715586851 173.1 ss715580183 ss715580166 109.3 ss715583874 281.3 ss715582337 183.6 ss715586806 197.3 ss715580343 ss715583976 ss715584007 284.6 ss715582348 184.5 ss715586784 197.4 ss715580325 ss715580331 115.3 ss715583972 ss715583966 286.1 ss715582363 185.7 ss715586612 201.7 ss715580379 ss715583992 289.5 ss715582382 188.5 ss715586444 207.2 ss715580434 116.1 ss715584026 290.3 ss715582395 195.1 ss715585102 208.0 ss715580441 122.0 ss715584019 296.2 ss715582495 198.1 ss715584839 ss715584780 128.7 ss715584307 297.7 ss715582653 200.1 204.1 ss715584699 238.6 ss715580655 ss715580642 128.8 ss715580896 ss715584333 298.3 ss715582685 207.5 ss715584661 240.1 ss715580676 128.9 ss715584279 ss715582664 ss715582636 299.0 210.0 ss715584596 243.5 ss715580611 131.4 ss715584048 ss715582537 219.5 ss715584477 246.0 ss715580631 138.0 ss715584114 ss715584109 303.0 ss715582747 ss715584254 ss715584174 223.1 ss715587012 247.0 ss715580627 304.3 ss715582768 ss715586870 138.8 ss715584240 ss715584196 227.8 305.0 ss715582782 240.7 ss715585247 306.9 ss715582813 241.4 ss715584699 ss715584856 308.2 ss715582859 ss715582849 164.1 ss715581190 328.1 ss715583020 165.1 ss715581177 329.0 ss715583026 ss715583084 175.1 ss715581101 330.7 ss715583075 176.9 ss715581080 ss715581097 333.4 ss715583067 178.8 ss715581063 333.8 ss715583057 180.9 ss715581032 333.9 ss715583058 186.1 ss715581023 ss715581014 334.1 337.3 ss715583112 348.1 ss715583207 349.7 ss715583239 370.6 ss715583239 371.5 ss715583466 375.2 ss715583499

Figure 2a. Linkage map of the chromosomes 1, 2 and 3 for the 84 F2:6 lines using the 1269 polymorphic SNPs in Archer x Hutcheson across the 20 chromosomes.

50 Chromosome-6 Chromosome-4

Chromosome-5 Chromosome-7 cM Chr.4 cM Chr.5 cM Chr.6 cM Chr.7

0.0 ss715595144 0.0 ss715590209 0.0 ss715596455 ss715596467 0.0 ss715589058 2.8 ss715590291 2.1 ss715595131 1.1 ss715596475 4.4 ss715590408 5.5 ss715595155 3.8 ss715596463 ss715596463 4.9 ss715590418 10.6 ss715595211 4.9 ss715596444 5.2 ss715590416 ss715596359 ss715596350 15.6 ss715589167 7.2 ss715590557 8.0 ss715596372 19.5 ss715589227 9.7 ss715590624 9.5 ss715590607 ss715596365 19.6 ss715589231 9.8 11.3 17.1 ss715590624 12.5 ss715596305 25.0 ss715589319 17.5 ss715590681 35.1 ss715594933 12.7 ss715596294 25.2 ss715590773 47.7 ss715594704 27.7 ss715598896 39.0 ss715590841 49.0 ss715594881 29.3 ss715598967 50.2 ss715595003 37.9 ss715598725 47.3 ss715590919 52.9 ss715594872 38.9 ss715598722 54.9 ss715594771 41.7 ss715598608 57.9 ss715591016 59.7 ss715591065 55.0 ss715594730 42.6 ss715598582 63.7 ss715591092 55.2 ss715594746 42.7 ss715598558 64.5 ss715591101 ss715591118 58.0 ss715594836 43.9 ss715598498 ss715598483 64.7 ss715591134 ss715594820 45.3 ss715598450 69.4 ss715591134 58.2 60.4 ss715594630 46.8 ss715598506 61.9 ss715594654 47.8 ss715598514 85.2 ss715591234 62.1 ss715594677 49.3 ss715598478 51.1 ss715598528 90.6 ss715591194 ss715594594 ss715594609 91.7 ss715588524 63.7 ss715594622 53.1 ss715598534 105.8 ss715588639 100.8 ss715591417 55.3 ss715598573 ss715598589 ss715591342 64.6 ss715594576 ss715588649 101.6 ss715598743 108.1 101.8 ss715591330 70.4 ss715594510 63.5 ss715599044 109.7 ss715588669 102.4 ss715591335 87.3 ss715594793 69.6 71.7 ss715598980 118.2 ss715588706 88.6 ss715594860 72.4 ss715598941 118.7 ss715588716 116.6 ss715591991 92.1 ss715594989 119.0 ss715588738 75.2 ss715598871 120.9 ss715588722 82.3 ss715598826 128.2 ss715591790 121.7 ss715588741 83.2 ss715598762 ss715597129 123.9 ss715588806 135.8 ss715591659 142.9 126.8 ss715588830 191.3 ss715597765 129.1 ss715588903 ss715593895 ss715594002 192.7 ss715597930 ss715597887 124.0 ss715597201 ss715597185 132.3 ss715588919 ss715594135 200.2 ss715597135 134.4 ss715588949 129.6 ss715593820 200.3 203.5 ss715597092 ss715597050 134.6 ss715588940 ss715588940 129.7 ss715593829 204.7 ss715596938 ss715596978 139.3 ss715589027 132.1 ss715593858 204.9 ss715597003 ss715589007 136.8 ss715594029 ss715593914 143.6 205.3 ss715597015 ss715588972 147.3 208.5 ss715596861 154.5 ss715589030 220.0 ss715596094 220.8 ss715596176 223.2 ss715595905 236.0 ss715596359 236.9 ss715596704

Figure 2b. Linkage map of the chromosomes 4, 5, 6 and 7 for the 84 F2:6 lines using the 1269 polymorphic SNPs in Archer x Hutcheson across the 20 chromosomes.

51 Chromosome-8 Chromosome-10 Chromosome-9 [1] Chromosome-9 [2]

cM Chr.8 cM Chr.9 cM Chr.10 0.0 ss715602449 151.1 ss715604428 0.8 ss715602368 0.0 ss715603826 ss715603786 152.5 ss715604156 0.0 ss715607944 156.5 ss715603985 158.4 ss715603850 ss715603788 33.5 ss715602108 160.3 ss715603731 37.3 ss715602097 12.8 ss715603736 161.8 ss715603674 6.8 ss715608087 39.4 ss715602072 168.5 ss715603523 8.8 ss715608151 169.3 ss715603594 40.4 ss715602088 21.1 ss715603646 10.9 ss715608145 ss715602131 170.0 ss715603561 41.7 ss715603506 28.4 ss715603663 171.0 13.8 ss715608078 ss715608102 42.3 ss715602115 ss715603464 32.2 ss715603573 ss715603610 174.1 ss715601519 ss715603398 17.0 ss715608043 76.7 34.9 ss715603524 177.1 ss715603357 ss715607993 ss715608012 76.8 ss715601530 36.0 ss715603435 179.5 18.0 79.7 ss715601723 184.6 ss715603094 ss715608035 ss715607955 83.2 ss715601537 47.6 ss715603084 85.7 ss715601199 51.7 ss715603210 85.8 ss715601386 55.5 ss715605391 26.1 ss715607917 57.7 ss715605352 203.2 ss715603014 89.6 ss715601489 ss715601502 28.6 ss715607890 ss715605445 ss715603023 29.3 ss715607879 91.3 ss715601127 61.1 ss715605439 ss715607862 91.5 ss715601281 61.2 ss715602993 32.1 106.8 ss715600456 ss715600381 68.9 ss715605405 ss715600223 ss715600304 72.5 ss715605384 221.4 ss715605374 107.4 223.0 ss715605251 107.5 ss715600168 74.0 ss715605404 77.6 ss715603002 228.1 ss715603033 110.0 ss715600190 230.3 ss715603092 110.4 ss715600388 86.1 ss715603006 ss715600153 86.3 ss715605418 111.6 90.2 ss715605316 112.6 ss715600140 90.3 ss715605318 48.0 ss715607686 133.5 ss715599914 94.7 ss715605333 142.8 ss715599872 95.0 ss715605274 145.2 ss715599877 96.3 ss715605303 53.4 ss715607633 96.4 ss715605259 54.7 ss715607595 146.8 ss715599862 104.4 ss715605225 56.7 ss715607624 155.3 ss715599787 111.6 ss715605238 156.0 ss715599768 112.8 ss715605256 57.9 ss715607638 156.7 ss715599743 ss715603072 156.9 ss715599753 128.3 ss715605106 272.3 157.6 ss715599734 129.9 ss715605069 132.1 ss715605087 164.7 ss715599658 133.2 ss715605125 68.6 ss715607526 136.3 ss715605167 ss715599406 139.8 ss715605111 198.8 140.0 ss715605104 199.5 ss715599349 146.2 ss715604708 147.2 ss715604608 148.1 ss715604784

Figure 2c. Linkage map of the chromosomes 8, 9 and 10 for the 84 F2:6 lines using the 1269 polymorphic SNPs in Archer x Hutcheson across the 20 chromosomes.

52 Chromosome-11 C13 Chromosome-14 Chromosome-12 cM Chr 11 cM Chr 12 cM Chr 13 cM Chr 14

0.0 ss715610617 0.0 ss715613081 0.0 ss715614558 0.0 ss715619591 4.7 ss715613114 1.9 ss715619194 9.4 ss715613101 3.0 ss715614710 5.0 ss715619466 14.8 ss715610298 17.2 ss715613179 6.9 ss715619453 6.3 ss715614830 15.5 ss715610355 29.9 ss715613250 8.6 ss715619435 32.9 ss715613271 11.1 ss715614764 10.2 ss715619422 33.0 ss715613294 11.3 ss715614907 25.6 ss715610315 12.5 ss715614914 ss715614755 19.0 ss715619356 ss715619377 26.9 ss715610337 35.0 ss715613298 14.6 ss715614983 20.0 ss715619397 37.9 ss715613333 15.8 ss715615024 20.7 ss715619343 ss715619377 33.8 ss715610369 45.2 ss715613460 16.2 ss715615049 22.6 ss715619203 38.7 ss715610270 58.5 ss715613669 17.7 ss715615064 24.6 ss715619194 58.8 ss715613666 22.2 ss715615118 23.3 ss715615093 29.2 ss715619151 60.8 ss715613671 24.5 ss715615158 74.5 ss715618180 61.8 ss715613628 24.6 ss715615164 ss715615170 ss715618096 63.5 ss715613672 25.9 ss715615257 76.9 ss715618189 66.9 ss715613704 27.2 ss715615217 79.5 29.2 ss715615195 ss715617935 68.6 ss715613710 83.2 29.4 ss715615185 ss715617948 69.1 ss715611377 83.8 72.6 ss715609472 30.3 ss715615228 84.5 ss715617932 70.5 ss715611375 ss715615266 74.4 ss715609475 31.5 70.7 ss715611389 ss715611397 33.1 ss715615320 77.3 ss715609538 71.3 ss715611380 33.3 ss715615361 77.4 ss715609463 ss715615363 117.2 ss715619849 73.8 ss715611379 35.2 77.5 ss715609489 37.0 ss715615361 119.3 ss715619836 76.6 ss715613702 85.2 ss715609881 37.2 ss715615328 139.4 ss715619801 ss715611336 ss715615381 87.0 ss715609695 78.4 38.8 160.3 ss715619811 ss715611391 43.2 ss715615438 91.8 ss715610029 84.5 163.4 ss715619783 ss715611383 43.3 ss715615420 ss715615423 92.6 ss715609975 84.6 169.2 ss715619721 ss715611451 ss715611453 170.1 ss715619714 91.0 ss715611482 ss715611431 192.2 ss715619495 ss715611467 91.1 196.7 ss715618699 ss715611733 103.7 197.3 ss715618631 109.3 ss715612070 198.7 ss715618446 ss715618556 57.7 ss715615590 112.9 ss715611920 199.0 ss715618524 117.4 ss715612244 201.9 ss715618305 123.8 ss715612301 207.2 ss715618374 125.7 ss715612250 211.9 ss715619054 142.2 ss715612380 ss715612408 136.3 ss715609929 214.9 ss715618785 143.7 ss715612456 216.8 ss715618256 157.0 ss715612589 222.3 ss715618082 158.7 ss715612596 222.4 ss715618096 161.2 ss715612608 233.6 ss715618057 170.6 ss715612652 243.1 ss715617826 178.8 ss715612715 250.7 ss715617539 189.8 ss715612859 ss715612847 190.7 ss715612853 196.7 ss715612794 207.2 ss715612810 211.3 ss715612742 236.4 ss715612621

Figure 2d. Linkage map of the chromosomes 11, 12, 13 and 14 for the 84 F2:6 lines using the 1269 polymorphic SNPs in Archer x Hutcheson across the 20 chromosomes.

53 C18

cM Chr 15 Chromosome-16cM Chr 16 cMC17 Chr 17 cM Chr 18 Chromosome-15 0.0 ss715622605 0.0 ss715625603 0.0 ss715627848 0.0 ss715631530 5.3 ss715622494 1.4 ss715625674 1.6 ss715627837 2.1 ss715631455 ss715631507 1.5 ss715625698 ss715631502 ss715631484 7.0 ss715622476 3.1 ss715627826 2.9 ss715622629 30.9 ss715624634 3.5 ss715631473 21.1 7.2 ss715627808 ss715622631 33.6 ss715624189 21.2 ss715627791 22.6 ss715622616 45.2 ss715625101 8.0 ss715625139 ss715628776 22.9 ss715622584 46.0 10.8 ss715627791 25.3 48.9 ss715625103 ss715632703 27.0 ss715622555 12.9 ss715627762 27.5 50.2 ss715625087 29.8 ss715632686 28.3 ss715622564 15.0 ss715627750 59.8 ss715625161 31.0 ss715632667 29.3 ss715622545 21.5 ss715627700 64.9 ss715625212 32.9 ss715632657 ss715622509 22.4 ss715627681 31.2 66.9 ss715625224 37.1 ss715632637 ss715622415 ss715622404 ss715627677 37.7 71.3 ss715625261 22.7 38.0 ss715632624 ss715621827 ss715621805 77.4 78.4 ss715625369 23.7 ss715627688 39.7 ss715632589 78.9 ss715621662 79.5 ss715625333 24.5 ss715627663 41.9 ss715632589 80.2 ss715621618 ss715621598 81.4 ss715625377 ss715627640 ss715627656 44.9 ss715632537 ss715632542 80.3 ss715621514 ss715621562 83.9 ss715625343 24.8 ss715627637 88.2 ss715625453 96.5 ss715621054 46.5 ss715627489 89.6 ss715625498 62.5 ss715631531 100.6 ss715620923 46.8 ss715627477 100.7 ss715620977 112.6 ss715623946 ss715623912 66.8 ss715631061 ss715631000 ss715623979 49.5 ss715627445 ss715627461 101.4 ss715621000 113.5 116.6 ss715623936 ss715627432 102.3 ss715621025 52.2 121.7 ss715623903 ss715620841 68.8 ss715627016 106.6 141.4 ss715624314 106.7 ss715620749 69.7 ss715627055 ss715624241 ss715624262 ss715627021 108.5 ss715620797 142.4 ss715624288 ss715624228 70.8 112.9 ss715620795 157.9 ss715624415 ss715624423 70.9 ss715627000 113.5 ss715620752 158.8 ss715624394 72.5 ss715626938 97.0 ss715629866 115.2 ss715620709 160.1 ss715624452 74.2 ss715626795 97.1 ss715629539 115.4 ss715620724 163.5 ss715624478 74.3 ss715626903 99.9 ss715629217 115.8 ss715620651 173.3 ss715624508 74.4 ss715626795 101.7 ss715629677 179.5 ss715624527 116.5 ss715620564 ss715626733 ss715626623 ss715629979 181.4 ss715624520 76.7 112.9 118.7 ss715620674 ss715626744 ss715626711 113.8 ss715629957 120.4 ss715620674 ss715620540 181.6 ss715624576 ss715624596 76.8 ss715626724 119.7 ss715630057 ss715630025 127.0 ss715620460 182.8 183.7 ss715624632 ss715624640 78.2 ss715626572 ss715626528 121.4 ss715630006 127.9 ss715620463 184.1 ss715624657 109.7 ss715626059 147.1 ss715620263 187.0 ss715624670 ss715625974 148.3 ss715620304 112.0 187.2 ss715624738 115.1 ss715625893 151.3 ss715620351 188.5 ss715624781 ss715629677 ss715630339 115.2 ss715625886 ss715625880 139.5 156.1 ss715620330 195.6 ss715624834 ss715624823 ss715630175 157.8 ss715620326 195.7 ss715624847 143.3 ss715625718 159.1 ss715620316 196.8 ss715624856 ss715624862 150.8 ss715628380 ss715628372 ss715620288 ss715624888 ss715624875 154.6 ss715628367 160.2 200.5 160.4 ss715620292 ss715624906 ss715624899 160.4 ss715628336 163.2 ss715620237 201.3 ss715624913 161.5 ss715628332 ss715624939 ss715624938 164.9 ss715620199 204.4 161.8 ss715628328 165.9 ss715620185 206.4 ss715624964 207.9 ss715624993 163.2 ss715628324 169.6 ss715623324 166.0 ss715628309 171.1 ss715623306 166.1 ss715628311 ss715623260 174.3 166.2 ss715628316 182.9 ss715623130 ss715623122 167.3 ss715628286 ss715628259 188.6 ss715623114 ss715628234 ss715628242 194.6 ss715623081 168.3 199.4 ss715623012 170.4 ss715628226 214.6 ss715622869 193.3 ss715627931 232.0 ss715621969 ss715621953 199.8 ss715627969 232.3 ss715621926 201.4 ss715628043 232.5 ss715621908 202.9 ss715627958 232.8 ss715621859 205.1 ss715627890 245.6 ss715621633 208.6 ss715626718 271.0 ss715620179 221.1 ss715626457 273.2 ss715623130 222.0 ss715626413 227.0 ss715626431

Figure 2e. Linkage map of the chromosomes 15, 16, 17 and 18 for the 84 F2:6 lines using the 1269 polymorphic SNPs in Archer x Hutcheson across the 20 chromosomes.

54 C19 C20

cM Chr 19 cM Chr 20

0.0 ss715635977 0.0 ss715638966 3.7 ss715636924 5.7 ss715637218 11.9 ss715636065 21.8 ss715639040 ss715639060 39.8 ss715636773 40.2 ss715636665 26.6 ss715635925 42.4 ss715636828 42.9 ss715636751 ss715636817 37.8 ss715635844 44.5 47.6 ss715636691 65.0 ss715637264

66.1 ss715635477 ss715635506 67.9 ss715635494 ss715635477 68.0 ss715635433 ss715635454 72.5 ss715635335 ss715635292 ss715635276 72.6 ss715635292 ss715635313 72.9 ss715635349 74.5 ss715635250 135.6 ss715637832 75.1 ss715635361 136.9 ss715637792 ss715637808 76.3 ss715635361 ss715635304 137.1 79.0 ss715635198 157.2 ss715638045 80.2 ss715635181 165.3 ss715638030 86.1 ss715635129 167.5 ss715638028 ss715638047 92.2 ss715635117 ss715635104 179.3 ss715638104 102.7 ss715635043 ss715635070 187.3 ss715638163 107.6 ss715634990 192.9 ss715638333 107.9 ss715634978 197.5 ss715638352 110.2 ss715634961 201.2 ss715638414 119.1 ss715634802 206.5 ss715638506 125.5 ss715634777 209.0 ss715638607 135.3 ss715634720 213.1 ss715638616 ss715638618 142.8 ss715634713 213.8 ss715638624 144.2 ss715634723 215.2 ss715638650

Figure 2f. Linkage map of the chromosomes 19 and 20 for the 84 F2:6 lines using the 1269 polymorphic SNPs in Archer x Hutcheson across the 20 chromosomes.

55 Table 5. Qualitative trait loci for partial resistance to Pythium aphanidermatum from 84 F2:6 lines derived from ‘Archer’ X ‘Hutcheson cross that were mapped using Single Marker Analysis (SMA) using seed plate assay. Chromosome SNP Maker Position P-value 1 ss715579229 49.61 0.0248 1 ss715579274 49.61 0.0248 4 ss715589227 19.54 0.0375 4 ss715589231 19.56 0.0374 8 ss715599862 146.80 0.0447 8 ss715599406 198.81 0.0247 8 ss715599349 199.51 0.0311 9 ss715605405 68.91 0.0219 9 ss715605384 72.52 0.0097 9 ss715605404 73.99 0.0134 9 ss715603002 77.58 0.0257 9 ss715605333 94.73 0.0478 9 ss715605274 95.00 0.0484 9 ss715605225 111.55 0.0365 9 ss715605238 112.81 0.0312 9 ss715605256 128.26 0.0433 12 ss715613628 61.76 0.0485 12 ss715613704 66.87 0.0309 15 ss715621805 77.44 0.0334 15 ss715621662 78.85 0.0155 15 ss715621618 80.19 0.0106 15 ss715621598 80.22 0.0106 15 ss715621514 80.27 0.0105 15 ss715621562 80.27 0.0105 16 ss715624394 158.82 0.0483 16 ss715624452 160.10 0.0431 18 ss715631530 0.0 0.0036 18 ss715631455 2.12 0.0084 18 ss715631455 2.12 0.0084 18 ss715631502 2.88 0.0058 18 ss715631484 2.89 0.0058 18 ss715631473 3.54 0.0063 20 ss715638616 213.10 0.0448 20 ss715638618 213.19 0.0447 20 ss715638624 213.78 0.0259

56 Table 6. Qualitative trait loci for partial resistance to Pythium aphanidermatum from 84 F2:6 lines derived from ‘Archer’ X ‘Hutcheson’ cross that were mapped using Single Marker Analysis (SMA) using the infested vermiculite assay. Chromosome SNP Maker Position P-value 1 ss715579229 49.61 0.0332 1 ss715579274 49.62 0.0303 1 ss715579920 78.00 0.0211 1 ss715579950 117.80 0.0381 4 ss715589319 24.97 0.0441 9 ss715605404 73.99 0.0382 12 ss715612652 170.59 0.0032 14 ss715618699 196.67 0.0248 14 ss715618631 197.34 0.0289 14 ss715618446 198.65 0.0355 14 ss715618556 198.65 0.0355 14 ss715618524 199.00 0.0343 14 ss715618305 201.91 0.0297 14 ss715618785 207.16 0.0283 14 ss715618256 214.90 0.0337 14 ss715618082 216.85 0.0435 14 ss715618096 222.34 0.0271 14 ss715618057 222.38 0.0107 16 ss715624634 30.92 0.0293 16 ss715624189 33.58 0.0205 18 ss715631530 0.0 0.0004 18 ss715631455 2.12 0.0016 18 ss715631455 2.12 0.0016 18 ss715631502 2.88 0.0018 18 ss715631484 2.89 0.0018 18 ss715631473 3.54 0.0024

57 Table 7. Qualitative trait loci for partial resistance to Pythium aphanidermatum from 84 F2:6 lines derived from an ‘Archer’ X ‘Hutcheson cross that were mapped using Composite interval mapping (CIM) using two phenotyping methods. Chromosome Estimated Position Nearest CIM Explained Trait Intervals (cM) (cM) marker LOD variation (%) 4 24.98 - 90.98 51.00 ss715589319 4.13 8.29 SA y 4 24.98 - 90.98 49.00 ss715589319 6.13 13.76 IV z 7 116.18 - 127.18 121.20 ss715598762 3.12 4.50 SA 7 83.19 - 142.18 118.20 ss715598762 5.50 13.85 IV y Seed assay (SA) z Infested vermiculite (IV)

58 Ch4-IV Ch4-SA a) b) cM Markers cM Markers

0 2 4 6 8

0 2 4 6 0.0 ss715589058 0.0 ss715589058 15.6 ss715589167 15.6 ss715589167 19.5 ss715589227 19.5 ss715589227 19.6 ss715589231 19.6 ss715589231 25.0 ss715589319 25.0 ss715589319

QTL1 QTL2

QTL1 QTL2

91.7 ss715588524 91.7 ss715588524 105.8 ss715588639 105.8 ss715588639 108.1 ss715588649 108.1 ss715588649 109.7 ss715588669 109.7 ss715588669 118.2 ss715588706 118.2 ss715588706 118.7 ss715588716 118.7 ss715588716 119.0 ss715588738 119.0 ss715588738 120.9 ss715588722 120.9 ss715588722 121.7 ss715588741 121.7 ss715588741 123.9 ss715588806 123.9 ss715588806 126.8 ss715588830 126.8 ss715588830 129.1 ss715588903 129.1 ss715588903 132.3 ss715588919 132.3 ss715588919 134.4 ss715588949 134.4 ss715588949 134.6 ss715588940 134.6 ss715588940 139.3 ss715589027 139.3 ss715589027 143.6 ss715589007 143.6 ss715589007 147.3 ss715588972 147.3 ss715588972 154.5 ss715589030 154.5 ss715589030

Figure 3. Composite interval mapping using single nucleotide polymorphism markers for quantitative trait loci identified on chromosome 4 with a) seed plate assay and b) infested vermiculite.

59 a) b)

cMCh7-SA Markers cMCh7-IV Markers

0.0 ss715596455 0.0 ss715596455 1.1 ss715596475 1.1 ss715596475 3.8 ss715596463 3.8 ss715596463

0

2

4

6

8 4.9 ss715596444 4.9 ss715596444 0 2 4 6 8.0 ss715596359 8.0 ss715596359 27.7 ss715598896 27.7 ss715598896 29.3 ss715598967 29.3 ss715598967 37.9 ss715598725 37.9 ss715598725 38.9 ss715598722 38.9 ss715598722 41.7 ss715598608 41.7 ss715598608 42.7 ss715598558 42.7 ss715598558 49.3 ss715598478 49.3 ss715598478 51.1 ss715598528 51.1 ss715598528 53.1 ss715598534 53.1 ss715598534 55.3 ss715598573 55.3 ss715598573 63.5 ss715598743 63.5 ss715598743 69.6 ss715599044 69.6 ss715599044 71.7 ss715598980 71.7 ss715598980 72.4 ss715598941 72.4 ss715598941 75.2 ss715598871 75.2 ss715598871 82.3 ss715598826 QTL3 82.3 ss715598826

QTL4 83.2 ss715598762 83.2QTL3ss715598762

142.9 ss715597129 142.9 ss715597129

191.3 ss715597765 191.3 ss715597765 192.7 ss715597930 192.7 ss715597930 200.2 ss715597201 200.2 ss715597201 203.5 ss715597092 203.5 ss715597092 204.7 ss715596938 204.7 ss715596938 205.3 ss715597015 205.3 ss715597015 208.5 ss715596861 208.5 ss715596861 220.0 ss715596094 220.0 ss715596094 220.8 ss715596176 220.8 ss715596176 223.2 ss715595905 223.2 ss715595905 236.9 ss715596704 236.9 ss715596704

Figure 4. Composite interval mapping using single nucleotide polymorphism markers for quantitative trait loci identified on chromosome 7 with a) seed plate assay and b) infested vermiculite.

60 III Effect of crop rotation on Pythium spp. population composition in Arkansas soybean

fields

Abstract

Pythium is an ecologically diverse group of microorganisms that are found in virtually all soils. The more than 100 species in this genus have a wide range of environmental and host preferences, but little is known about the effect of crop rotation on Pythium communities. To understand the effect of crop rotation on species diversity, soil was collected from plots following long-term rotation treatments including the crops rice, corn, soybean and wheat. Soil from each plot was placed in cups, wetted to saturation, planted with ten seeds of the soybean cultivar Hutcheson, and incubated at 25oC. After three days, seeds were collected and washed in running water and placed on 2 % water agar and hyphal tips transferred to a Pythium selective medium. Molecular identification was performed by sequencing the ITS region and Blast analysis to a curated reference database. Pathogenicity tests were conducted with forty nine

Pythium isolates in four species on soybean, corn, wheat and rice using a seed plate assay. A total of 320 isolates were identified representing 12 species, 16 isolates could not be identified to known species and were reported as Pythium spp. Overall, the most frequently recovered species were P. spinosum, P. irregulare, P. pareocandrum, P. sylvaticum and Pythium. spp. There were significant differences in number of Pythium isolates from the ten rotation systems with the rice

(wheat)-soybean (wheat) rotation having the highest recovery of these six Pythium spp. with the rice (wheat)-rice (wheat) rotation having the lowest recovery. Pythium sylvaticum was suppressed when rice was the last crop compared with soybean and corn. All Pythium isolates were highly virulent on soybean and there were significant differences in isolates within species.

On rice, Pythium spp. and P. spinosum were the most virulent causing between 70 to 100 %

61 reduction in seed germination. On corn, the different Pythium species caused from 0 – 13 % reduction in seed germination and the pathogenicity vary in isolates within species. Knowing the

Pythium species diversity in the different rotation systems of the most important crops in

Arkansas would help the growers to select the appropriate management options.

Introduction

Pythium is an ecologically diverse group of microorganisms that are found in virtually all soils. The more than 100 species in this genus have a wide range of environmental and host preferences (Lévesque and de Cock, 1994). Pythium spp. have been reported as important root and seed pathogens causing diseases on important economic crops, including soybean, corn, rice and wheat (Zhang and Yang, 2000). Arkansas produces 3.84 million metric tons of soybean, 4.04 million metric tons of rice, 4.14 million metric tons of corn (USDA - NASS, 2013) and 505 thousand metric tons of wheat in 2013 (Arkansas Wheat Promotion Board). All of these crops are prone to diseases cause by Pythium spp.

Seedling diseases in soybean can reduce stands, plant vigor and yield and sometimes may require replanting (Yang, 1999). Seedling diseases caused an estimated of 8.7, 8.9 and 10.7 % loss in yield to Arkansas producers in 2011, 2012, and 2013, respectively (Heatherly, 2014, Data provided by Dr. S.R. Koenning, North Carolina State University). Seedling diseases are caused by a number of soilborne pathogens that may work singly or as a complex. The primary groups of pathogens involved in soybean seedling diseases are Pythium spp, Fusarium spp., Rhizoctonia solani and Phytophthora sojae (Schmitthenner, 1999; Yang, 1999; Wilcox, 1987). Pythium is the primary group of pathogens involved in soybean seedling diseases (Nelson, 1990;

Schmitthenner, 1999; Yang, 1999; Wilcox, 1987). Pythium causes seed rot and post emergence

62 damping-off. Pythium is also associated with root pruning that leads to a reduction of plant vigor and yields, but generally it is not lethal to mature plants (Martin, 2009).

In Arkansas the primary Pythium spp. are Pythium sylvaticum, followed by P. ultimum,

P. aphanidermatum and P. irregulare (Kirkpatrick et al., 2006; Rosso, 2007; Bates et al., 2008).

More extensive research by Avanzato (2011) identifying isolates of Pythium to species by morphological and molecular techniques and evaluating virulence initially with an in vitro pathogenicity seed assay, found nine Pythium species were the most recovered: P. sylvaticum, P. irregulare, P. ultimum, P. spinosum, P. mamillatum, P. dissotocum, P. accanthicum, P. attrantheridium, and P. oligandrum. These studies showed that Pythium is the predominant group involved in seedling diseases in soybean fields in Arkansas.

Besides soybean, Arkansas is the largest rice producer in the United States. Pythium spp. cause stand-establishment problems on rice in Arkansas (Eberle et al., 2008; Rothrock et al.,

2005). Eberle et al, (2008) reported that P. arrhenomanes and P. irregulare were the most common and virulent pathogens isolated from soil from 20 producers fields in Arkansas. Less common species were P. torulosum. P. catenulatum, and P. diclinum.

Pythium spp. also are known as wheat pathogens (Vaterpool and Truscott, 1932;

Vaterpool, 1938; Chamswarng and Cook, 1985; Higginbotham, 2004). The main species known to cause Pythium root rot on wheat are: P. aristospurum, P. arrhenomanes, P. graminicola, P. ultimum, P. heterothallicum, P. torulosum (Paulitz, 2010). Pythium spp. in the Pacific Northwest cause root rot, decreasing root mass and leading to poor nutrient uptake resulting in yield losses

(Chamswarng and Cook, 1985; Higginbotham, 2004). The most common Pythium species causing root rot are P. ultimum var. ultimum, P. ultimum var. sporangiifereum, P. aristosporum,

P. volutum, P. torulosum, P. irregulare, and P. sylvaticum complex among others (Chamswarng

63 and Cook, 1985). In Arkansas preliminary studies also have established the importance of

Pythium on wheat (Milus and Rothrock, 1989; Milus et al., 1992; Rhoads et al., 1993).

Pythium spp. are commonly isolated from corn and have been reported as the most important pathogens associated with poor stand establishment in corn, causing pre and post emergence damping-off (Deep and Lipps, 1996; Dodd and White, 1999). In Ohio Pythium causes corn seed and seedling blight. There are at least 14 Pythium species that were known to cause seedling blight and root rot: P. acanthicum, P. adhaerens, P. aphanidermatum, P. arrhenomanes, P. graminicola, P. irregulare, P. paroecandrum, P. pulchrum, P. rostratum, P. splendens, P. tardicrescens, P. ultimum and P. vexans (Chen, 1999). Deep and Lipps (1996), reported that P. arrhenomanes was the primary cause of Pythium root rot of corn. Dorrance et al.

(2004) reported that the four most common species recovered from three corn fields in Ohio were: P. cantenulatum, P. irregulare, P. paroecandrum, and P. torulosum, with isolation and pathogenicity depending on the location and species. Changes to earlier planting dates (Saab,

2005) and in crop management, such as reduced tillage, in corn may have caused shifts in

Pythium populations in Ohio (Van Doren and Triplett, 1973; Broders et al., 2007). Broders et al.

(2007) investigated Pythium spp. associated with seed and seedlings in corn and soybeans in

Ohio by conducting a survey of 42 production fields. Eleven Pythium spp. were identified: P. attrantheridium, P. dissotocum, P. echinulatum, P. graminicola, P. inflatum, P. irregulare, P. helicoides, P. sylvaticum, P. torulosum, P. ultimum var. ultimum and P. ultimum var. sporangiiferum and P. dissotocum were the most commonly recovered and pathogenic to corn and soybeans. In Arkansas there is little information available on the effect of Pythium on corn.

Diverse crop rotations have been shown to reduce the populations of soilborne pathogens and the use of a single crop for several years generally results in the increase of population

64 density of specific pathogens adapted to that crop (Christen and Sterling, 1995). Long-term rotation has been used to reduce the pressure of plant diseases. However, pathogens such

Pythium, Rhizoctonia and Sclerotinia that have a broad host range and long-term survival structures are more challenging to manage by crop rotation (Krupinsky et al., 2002).

Crop rotation has been reported to influence Pythium population density. Davis and

Nunez (1999) studied the influence of crop rotation on Pythium- and Rhizoctonia-induced carrot root dieback. They rotated carrot with cotton, alfalfa, barley, onions, continuous carrots or fallow

After two years of these rotations, it was found that Pythium populations were greater when carrots were followed by alfalfa and barley than with any of the other crops in 1 year of the study; and populations of Rhizoctonia were greater when carrots were followed by cotton or alfalfa than other crops in the two years of the study (Davis and Nunez, 1999). Guo (2005) studied the effect of a 4-year crop rotation of canola, wheat and flax and tillage systems on blackleg disease of canola, caused by Leptosphaeria maculans, and found that disease incidence and severity on stems were lower when canola was rotated with wheat and wheat and flax in tillage and non-tillage systems. Likewise, Hwang et al. (2009) evaluated the effect of long-term crop rotation on the effect of Fusarium, Pythium and Rhizoctonia populations on canola seedling establishment under controlled conditions in two locations in Saskatchewan, Canada. In the first year of the study the authors found that for soil collected in 2006 seedling emergence was higher in the three crop rotation (pea- canola- wheat) compared to two crop rotations ( canola-wheat or pea wheat). Pankhurst et al. (1995) studied the effect of long-term crop rotation and tillage practices on Pythium population densities in wheat in South Australia. In this study there were three crop rotations: continuous-wheat, pasture-wheat and lupins-wheat, three tillage practices were also compared: conventional cultivation, reduced cultivation and direct drilling. They found

65 that tillage and crop rotation had a significant effect in the number and distribution of Pythium populations. They found the highest Pythium propagule density in the first 20 cm of soil, and the

Pythium population density was higher in the continuous wheat rotation in soils subjected to direct-drilling compared to soil subjected to conventional cultivation. Similar results were found with pasture-wheat and lupine-wheat rotations. Likewise, Zhang and Yang (2000) studied the effect of corn- soybean long –term rotation effect on soilborne pathogens. They reported that 71

% of the total isolates were pathogenic in different levels to both corn or soybean and that disease index on soybean was highly correlated with the disease index on corn.

The hypothesis for this study is that long-term rotation has an effect on Pythium populations. The objectives of this study were i) to characterize Pythium populations from soil of a 11-year rotation study and ii) test isolates of the main Pythium species for pathogenicity to the four crops in the study.

Materials and Methods

Sample collection

Soil was collected in August 2011 from an 11-year rotation study that was conducted at the University of Arkansas Rice Research and Extension Center (RREC) in Stuttgart, AR from

1999 to the spring 2011. Ten rotation systems were established in 1999 on a Dewitt silt loam soil

(fine, smectitic, thermic, Typic Albaqualf, NRCS, 2008): continuous rice (R), rice-soybean (RS), soybean-rice (SR), rice–corn (RC), corn-rice (CR), rice- (winter wheat) [R(W)] , rice- (winter wheat)-soybean-(winter wheat) [R(W)S(W)], soybean (winter wheat)-rice -(winter wheat)

[S(W)R(W)], rice-soybean-corn (RSC) and rice-corn-soybean (RCS). The study was a randomized complete block design with four replications. A 3-cm probe was used to collect soil samples, a total of 1000 g soil from the top 15-cm in the center two rows of each plot and

66 brought to the University of Arkansas campus in Fayetteville, AR and were maintained at 4°C until processed.

Baiting and isolation

From each soil sample, 250-ml of soil were placed in 10.6 cm plastic containers, soil was wetted to saturation (0 Jules/Kg) and, planted with ten seeds of the Pythium susceptible soybean cultivar ‘Hutcheson’ (Kirkpatrick et al., 2006; Bates et al, 2008; Rosso et al., 2008; Avanzato,

2011), and incubated in a growth chamber at 25°C. After three days, seeds were collected and washed in running water for 20 minutes, dried on paper towels and placed on 2 % water agar

(Agar gelidium, Moor Agar, Incorporated). After two days, hyphal tips of colonies growing from the seed were transferred and maintained on the Pythium semi-selective medium P5ARP (Jeffers and Martin, 1986). The experimental design for the baiting experiments was a completely randomized design with ten seeds per container and four repetitions per rotation, one for each field plot.

Molecular identification

Pure cultures of Pythium spp. were sent to Dr. Martin Chilvers at Michigan State University for high-throughput identification. Isolates were grown on 50 mL of V8-juice broth for 10 d, then mycelia was harvested, lyophilize overnight, and ground. A total of 100 mg of ground mycelia were resuspended on 800 µL of CTAB buffer (AutoGen AG00121, AutoGen Inc.) and incubated for 1 h at 65ºC. A phenol-chloroform automated DNA extraction was performed using the AutoGen

850 system (AutoGen Inc., Holliston, MA). The Internal Transcribed Spacer (ITS) 1 and 2 of rDNA were amplified with primers ITS6 and ITS4 (Cooke et al., 2000) for all the isolates and ITS sequences were compared to a curated database of Oomycetes for identification (Robideau et al.,

2011). There were sixteen Pythium isolates that had different sequences that did not match any of

67 the other Pythium spp. reported in the curated database; thus, they were designated in a Pythium spp. group.

Pathogenicity tests

Isolates. Fifty-two Pythium isolates from the collection were evaluated for their virulence using a modified Petri dish seed assay (Broders et al., 2007; Avanzato, 2011). Of the 53 isolates evaluated, ten isolates each were of Pythium irregulare, , , and Pythium sylvaticum, nine isolates of Pythium spp., and two isolates of Pythium aff. dissotocum, one isolate of each of Pythium coloratum and Pythium kunmingense.

Cultivars. Untreated seed of the soybean cultivar ‘Hutcheson’, the rice cultivar ‘Wells’, the wheat cultivar ‘Ricochet’ and the corn cultivar ‘DKC64-69’ were used.

Petri dish seed assay. A seed plate method was used to test the pathogenicity of each

Pythium isolate on each of the four crops: soybean, rice, wheat and corn following a modified protocol from Avanzato (2011). Each Pythium isolate was grown in Corn Meal Agar (CMA)

(BBL ™ Becton, Dickson and company Sparks, MD, USA) for 4 days, a 0.5 cm2 plug was transfer onto the center of 9-mm petri dish containing 2% Water agar (Agar gelidium, Moor

Agar, Incorporated). After four days a layer of 0.5-cm autoclaved vermiculite (medium vermiculite, Sun Gro®, Belleve Washington, USA) was placed on the agar and wetted with 10- ml of sterile distilled water. Ten seeds of each crop were surface disinfested with 70% ethanol for 3 minutes, allowed to dried and then placed onto the plates. There were three replications per crop. The control was plain water agar without Pythium. Plates were covered with aluminum foil and incubated at room temperature (23 to 25°C). After seven days, seed germination was assessed on each plate. The experimental design was a completely randomized design with three

68 replications per Pythium isolate and crop and the experiment was repeated twice. There was one control per isolate and it was treated the same, but without Pythium.

Statistical analysis

The total number of oomycete isolates was analyzed by the generalized linear mixed models (GLIMIX) procedure by SAS Inc. (SAS version 9.4, SAS Institute Inc. Cary, NC. USA) using the Poisson distribution. The differences among means were determined by the P-value.

Isolates were treated as counts with the Poison distribution. The total number of molecularly identified Pythium isolates was analyzed by the generalized linear mixed models (GLIMIX) procedure by SAS Inc. (SAS institute Inc. Cary, USA). The differences among means were determined by the P-value. Isolates were treated as a counts with the Poison distribution.

The in-vitro pathogenicity data were analyzed with the logit analysis where that were adjusted with the control and proportion of dead seeds were transformed with the logit model. Only forty- nine isolates of Pythium were included in the statistical analysis.

Results

Isolation and identification

A total of 765 oomycete isolates were recovered across the ten rotation systems. There were significant differences among rotations (P = 0.0031). The corn-rice rotation had the highest number of isolates, followed by the rice (wheat)-soybean (wheat) and rice-soybean, rice-corn- soybean and continuous rice rotations. The rotation system that had the lowest number of isolates was rice (wheat)-rice (wheat) (Table 1).

A total of 320 isolates were molecularly identified. The 320 species were composed of 12

Pythium species plus 16 isolates that could not be identified to species, these were placed in the

Pythium. spp., group (Figure 1). The most frequently recovered species were P. spinosum, P.

69 irregulare, P. pareocandrum, P. sylvaticum and Pythium. spp. Moreover, P. irregulare, P. spinosum, P. sylvaticum, P. paroecandrum were the most frequently recovered and were recovered from 100 % of the rotations, while Pythium spp. was recovered for 60 % of the rotations. Frequency of occurrence was only calculated for the main six species because these species accounted for almost 93 % of all the isolates recovered.

Crop rotation

When the rotations were grouped as the last crop grown during the summer of 2010: corn, rice or soybean, there were significant differences of Pythium species across and within a crop (Table 2). When rice was the last summer crop, there were significantly less isolates recovered of P. sylvaticum and Pythium. spp., compared when the last crop was soybean or corn.

For corn, Pythium spp., was significantly less recovered than the other four Pythium species. For rice, P. irregulare and P. spinosum were the most frequently recovered species followed by P. paroecandrum, P. sylvaticum and Pythium spp., respectively. For soybean, P. sylvaticum, P. paroecandrum and P. irregulare were the most frequently recovered species followed by P. spinosum and Pythium. spp. (Table 2)

Pathogenicity tests

There were significant differences in dead seed among Pythium species and isolates within a species with corn, soybean or rice (Table 3). In soybean, all Pythium species were highly virulent and there were significant differences in level of dead seed among each of the species (Table 4). Pythium spp., caused the highest level of dead seeds, followed by P. paroecandrum, P. sylvaticum. There were significant differences in pathogenicity among isolates within each species (Table 5). Based on proportion of dead seeds, isolates were divided into two

70 groups with Pythium spp., three groups with P. paroecandrum and P. sylvaticum and four groups with P. spinosum and P. irregulare.

With corn, all Pythium species had low virulence compared to soybean. There were significant differences among the five Pythium species (Table 9). Pythium spp., was the most virulent, followed by P. paroecandrum and P. sylvaticum. The less pathogenic species in corn were P. spinosum and P. irregulare. There were significant differences in virulence within species (Table 7). Based on virulence, isolates fell into two groups with Pythium spp., P. paroecandrum and P. spinosum and into three groups with P. irregulare. There were not significant differences among isolates with P. sylvaticum.

With rice, there was a significant differences among species with Pythium spp., and P. spinosum being significantly more virulent than P. irregulare, P. paroecandrum and P. sylvaticum (Table 8). There were no significant differences among isolates within the species.

With wheat, there were no significant differences in pathogenicity among species or isolates

(Table 3). The average logit of number of dead seeds in wheat across all cultivars was 0.0070.

Discussion

Crop had an impact on Pythium populations in this study. Corn- rice rotation had the highest incidence of infection, followed by the rice (wheat)-soybean (wheat) and rice-soybean, rice-corn-soybean and continuous rice rotations. P. irregulare, P. spinosum, and P. paroecandrum were the most recovered species across the 10 rotations. Long-term rotation have been reported to have an effect on Pythium species in crops such as corn, soybean and canola

(Zhang et al., 1998; Zhang and Yang, 2000; Hwang et al., 2009). Zhang et al., (1998) studied the effect of three-long term rotations systems: continuous corn, continuous soybean, and corn soybean rotation on Pythium species abundance in two fields 200 km apart. Baiting with

71 cucumber seed, they found that indices of Pythium isolates recovered from corn- soybean and soybean- soybean were higher than those from corn-corn rotations fields. Also they found that P. torulosum and P. ultimum were isolated in greater amounts than P. paroecandrum and P. spinosum. Hwang et al., (2009) reported that Hwang et al., (2009) evaluated the effect of long- term crop rotation on the effect of Fusarium, Pythium and Rhizoctonia populations on canola seedling establishment under controlled conditions in two locations in Saskatchewan, Canada. In the first year of the study the authors found that from soil collected in 2006 seedling emergence was higher in the three crop rotation (pea- canola- wheat) compared to two crop rotations

(canola-wheat or pea wheat). Similar to our study, these studies above show the effect to crop rotation on soilborne pathogens.

P. sylvaticum and Pythium spp. were more suppressed than other species when rice was the last crop than when corn or soybean were the last summer crops. Pythium sylvaticum is a heterothallic species, which does not produce long-lived sexual spores, Oospores, unless it has the other mating type and does not produce zoospores (Pratt and Green, 1973). In flooded conditions for rice production, Pythium sylvaticum may have difficulty to infect or produce long term survival structures to survive until the next summer crop. This may explain why it was not frequently recovered after rice. On the other hand, Pythium species also vary in this study, among last summer crops. P. irregulare, P. sylvaticum, P. paroecandrum were highly significantly recovered when the rice was the last crop. These results suggested that there may be a species crop adaptation or environmental factors that are favoring certain species over others

(Zhang and Yang, 2000; Eberle et al., 2008; Wei et al., 2011).

The pathogenicity assay using the seed plate assay with forty-nine isolates over four crops, showed that Pythium species were pathogenic on soybean, corn and rice and virulence

72 differed within crops. Pythium spp., and P. spinosum were the most pathogenic species in soybean and corn while in rice Pythium spp. and P. spinosum were the most virulent species. P. irregulare and P. spinosum isolates significantly differed in levels of virulence in soybean and corn. These results agreed with Martin and Loper (1990) that reported that virulence of Pythium species can be isolate specific. Zhang and Yang (2000), in a long-term rotation also found that isolates within Pythium species had differences in virulence on corn and soybean. Hoppe and

Middleton (1950) reported that isolates of Pythium ultimum differed in their ability to cause death corn. Likewise, Wei et al., (2010) reported significant differences in two soybean cultivars in percentage of seed rot among isolates within P. aphanidermatum, P. arrhenomanes, P. irregulare, and P. macrosporum. All Pythium isolates in this study were baited with the susceptible soybean cultivar Hutcheson, which may have influenced results by possibly isolates that were selective more virulent on soybean than other crops.

The group of sixteen Pythium isolates that did not match any of the known Pythium spp., sequences available in the curated database of Oomycetes (Robideau et al., 2011) were designated as Pythium spp. These isolates may belong to two new Pythium species that have not been reported before based on the ITS sequences of the rDNA, and are in the description process

(Rojas Alejandro, personal communication). Although, these isolates represent only 5 % of the total isolates, they were recovered in 60 % of the rotations. These group of isolates may have a role in Pythium diseases because most of them were highly virulent on soybean and rice.

Research will continue to understand more in depth the role of these two new species in Pythium diseases in Arkansas.

73 Conclusion

Previous crop had an impact on Pythium population in this study. Corn- rice rotation had the highest incidence of infection, followed by the rice (wheat)-soybean (wheat) and rice-soybean, rice-corn-soybean and continuous rice rotations. P. irregulare, P. spinosum, and P. paroecandrum were the most recovered species across the 10 rotations. Pythium spp., and P. spinosum were the most virulent species on soybean and corn, while in rice Pythium spp., and P. spinosum were the most virulent species. P. irregulare and P. spinosum isolates significantly differed in levels of virulence in soybean and corn.

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78 Table 1. Isolation frequency of Oomycetes recovered across 10 crop rotation system x

Rotation Isolates y Corn- rice 25.99 a z Rice(wheat)_soybean(wheat) 22.99 ab Rice_soybean 22.50 ab Rice_corn_soybean 19.75 abc Continuous rice 19.25 abc Soybean(wheat)_rice(wheat) 18.75 bc Rice_soybean_corn 18.75 bc Soybean_rice 17.25 bc Rice_corn 15.99 c Rice(wheat)_rice(wheat) 9.99 d x The total number of oomycetes isolates was analyzed by generalized linear mixed models (GLIMIX) procedure by SAS Inc. using the Poisson distribution. The differences among means were determined by the P-value. Isolates were treated as a counts with the Poison distribution. y Values are the mean of number of isolates across the 10 rotation systems. z Means in a column followed by the same letter are not significantly different, according with P-value calculated with the Poisson distribution

79

120

100

80

60

No of isolates ofisolates No 40

20

0

Pythium spp.

Figure 1. Number of isolates for Pythium species collected in 11-year rotation systems.

80 Table 2. Isolates of five Pythium spp. recovered with soybean, corn and rice as the last crops w across the 10 rotation systems .

Last Pythium spp. crop P. irregulare P. spinosum P. paroecandrum P. sylvaticum P. spp. Corn 5.50 xAy az 6.0 A a 4.5Aa 10.0 Aa 1.5 AB b Rice 7.40Aa 9.6A a 6.0 A b 2.8 Bc 1.0 B d Soybean 10.33Aa 6.33A bc 9.33 Aa 9.33 Aa 3.0 Ac w Isolates recovered from the last summer crops: soybean rice and corn were analyzed by generalized linear mixed models (GLIMIX) procedure by SAS Inc. using the Poisson distribution. The differences among means were determined by the P-value. Isolates were treated as a counts with the Poison distribution. x Values are the mean of number of isolates across six Pythium species and three crops. y Means for same Pythium species and different crops followed by the same capital letter are not significant different (P < 0.05). z Means for the same crop and different Pythium species followed by the same lower case are not significantly different (P < 0.05).

81

Table 3. Analysis the variance of estimated dead seeds on corn, soybean, rice and wheat.

Effect P- valuez Soybean Corn Rice Wheat Pythium species <0.001 <0.001 0.0002 0.7089 Isolates (species) <0.001 0.0177 1.00 0.9907 z Dead seeds for corn, rice, soybean and where was analyzed by generalized linear mixed models (GLIMIX) procedure by SAS Inc. Proportion of dead seeds was transformed with the logit function.

82 Table 4. Dead seeds (logit transformed) of soybean tested with five Pythium species on soybean.

Pythium spp. Dead seedsX

Pythium spp. 319.25Y a Z P. paroecandrum 119.22 b P. sylvaticum 102.60 c P. spinosum 27.93 d P. irregulare 21.74 e x Proportion of dead seeds from 30 seeds of soybean across forty nine Pythium isolates y Values are the estimated logit transformed of number of isolates across six Pythium species and three crops. z Means for the different Pythium species followed by the same lower case are not significantly different (P< 0.05)

83 Table 5. Dead seeds (logit transformed) of soybean tested for forty nine Pythium species isolates

Pythium spp. Pythium. spp. P. paroecandrum P. sylvaticum P. spinosum P. irregulare Isolate Estimatey Isolate Estimate Isolate Estimate Isolate Estimate Isolate Estimate 9 321.7 az 9 152.80 a 8 131.88 a 7 40.11 a 8 50.12 a 8 321.7 a 6 152.80 a 6 131.88 a 6 40.11 a 9 50.12 a 7 321.7 a 5 152.80 a 5 131.88 a 5 40.11 a 5 50.12 a 6 321.7 a 4 152.80 a 4 131.88 a 4 40.11 a 1 38.14 b 5 321.7 a 3 152.80 a 3 131.88 a 1 40.00 a 2 21.83 c 4 321.7 a 2 152.80 a 2 131.88 a 8 37.08 b 7 2.91 d 3 321.7 a 1 152.80 a 1 131.88 a 2 18.32 c 3 1.85 d 2 321.7 a 10 121.68 a 10 100.78 b 9 18.33 c 4 1.85 d 1 299.67 b 7 1.34 b 9 1.08 c 10 3.08 d 10 1.17 d 8 -0.43 c 7 0.99 c 3 1.98 d 6 -0.65 d y Proportions of dead seeds from 30 seeds of soybean z Means for the same Pythium species followed by the same lower case are not significantly different (P< 0.05)

84 Table 6. Dead seeds (logit transformed) of corn for five for Pythium species Pythium spp. Dead seeds y Pyhtium spp. -5.30 a z P. paroecandrum -7.11 b P. sylvaticum -7.64 b P. spinosum -9.84 c P. irregulare -19.11 d y Proportions of dead seeds from 30 seeds of corn across forty nine Pythium isolates z Means for the different Pythium species followed by the same lower case are not significantly different (P< 0.05)

85 Table 7. Dead seeds (logit transformed) of corn tested with forty-nine Pythium spp. isolates.

Pythium spp. Pythium. spp. P. paroecandrum P. sylvaticum P. spinosum P. irregulare Isolate Estimatey Isolate Estimate Isolate Estimate Isolate Estimate Isolate Estimate 9 -1.87 az 4 -2.19 a 6 -2.19 a 4 -2.19 a 4 -2.63 a 8 -2.02 a 3 -2.63 a 4 -2.63 a 2 -2.94 a 3 -3.36 a 5 -2.17 a 1 -3.36 a 8 -2.19 a 3 -3.36 a 9 -3.36 a 3 -2.39 a 6 -3.36 a 2 -2.94 a 1 -3.36 a 2 -4.07 a 4 -2.94 a 7 -3.36 a 5 -3.36 a 5 -4.07 a 5 -4.07 a 6 -2.94 a 9 -3.36 a 1 -4.07 a 6 -4.07 a 10 -21.17 b 2 -3.36 a 10 -3.36 a 3 -4.07 a 10 -2.63 a 1 -38.11 c 1 -4.07 a 2 -4.07 a 9 -4.07 a 7 -25.51 b 6 -38.11 c 7 -25.98 b 5 -25.33 b 10 -4.07 a 8 -25.51 b 7 -38.11 c 8 -25.33 b 7 -17.24 a 9 -25.51 b 8 -38.11 c y Proportions of dead seeds from 30 seeds of corn z Means for the same Pythium species followed by the same lower case are not significantly different (P< 0.05)

86 Table 8. Dead seeds (logit transformed) of rice tested with five Pythium species. Pythium spp. Dead seeds Pythium spp. 16.11 az P. spinosum 0.83 a P. irregulare -0.62 b P. paroecandrum -1.11 b P. sylvaticum -1.25 b y Proportions of dead seeds from 30 seeds of rice z Means for the different Pythium species followed by the same lower case are not significantly different (P< 0.05)

87 IV Effect of crop rotation, location and isolation temperature on Pythium spp. population

composition in Arkansas

Abstract

Pythium spp. are an important group of pathogens associated with stand losses in soybean and rice. Soybean rice rotations are a common rotation cropping systems in Arkansas. The objectives of this study were: i) to determine the effect of continuous soybean and soybean-rice rotation on Pythium spp. diversity in several locations in Arkansas, and ii) determine the effect of temperature on the recovery of Pythium. Soils from a soybean-rice and a soybean- soybean rotation were collected from three locations in 2012, placed in cups, wetted to saturation, planted with ten seeds of the soybean cultivar Hutcheson, and incubated at 20oC or 30oC. After three days, seeds were collected and washed in running water and placed on CMA-PARP+B medium..

DNA was extracted and the ITS region sequenced followed by Blast analysis to a curated reference database was done. A total of 275 isolates were identified representing 25 species. The most frequently recovered species were Pythium irregulare, P. pareocandrum, P. sylvaticum, P. corolatum and P. spinosum. Location had a large effect on Pythium population composition and diversity. Pythium irregulare and P. paroecandrum were the most recovered species from all the locations and were recovered the most, from Stuttgart soils. Pythium coloratum and P. ultimum var. ultimum were mainly recovered from Keiser soils and P. spinosum was mostly isolated from

Pine Tree soils. The recovery of P. sylvaticum was similar among the three locations. Overall, populations of Pythium spp. varied the most among locations, but were not influenced by the previous crop or the isolation temperature. Location had a significant effect with abundance and shannon indices but not with the richness index. Distinctive species prevailed in each of the three locations across two temperature and two rotation systems in Arkansas when baited from soil

88 using soybean seed. Overall, Location has an effect of Pythium species population and that this effect may be related with physical and chemical soil properties, furthermore more studies are needed in Arkansas to understand the soil ecology affecting Pythium species population in

Arkansas.

Introduction

Pythium is the predominant group involved in seedling diseases in soybean fields in

Arkansas. Pythium diseases in soybean can rot seed, reduce stand, plant vigor and yield and may require replanting (Yang, 1999). The primary Pythium spp. are Pythium sylvaticum, followed by

P. ultimum, P. aphanidermatum, P. irregulare (Kirkpatrick et al., 2006a; Rosso, 2007; Bates et al., 2008). Avanzato (2011) identified nine Pythium species, P. sylvaticum, P. irregulare, P. ultimum, P. spinosum, P.mamillatum, P.dissotocum, P. accanthicum, P. attrantheridium, and P. oligandrum from soybean seeds and seedlings.

In rice, seedling diseases are commonly associated with Fusarium spp. Achya spp., and

Pythium spp. (Rush, 1992). Moreover, Pythium spp. have been reported the most important pathogen involved in rice stand-establishment problems (Cother and Guilbert, 1993) and causes important rice stand-establishment problems in Arkansas (Eberle et al., 2008; Rothrock et al.,

2005). Eberle et al., (2008) reported Pythium arrhenomanes and P. irregulare were the most common and virulent pathogens isolated from soil from 20 producers fields. Less common species were P. torulosum. P. catenulatum, and P. diclinum. In the same study they conducted a controlled pathogenicity test in sterilized vermiculite using two environments showing that

Pythium arrhenomanes and P. irregulare caused greater stand losses than the other Pythium species.

89 Pythium is a cosmopolitan pathogen; most species are soil inhabitants, while others prefer fresh water or marine aquatic environments (Van der Plaats-Niterink, 1981). Most

Pythium spp. are saprophytes or facultative plant pathogens that cause losses to a wide variety of hosts, within this genus P. aphanidermatum and P. ultimum have been reported to have broad host ranges (Hendrix and Campbell, 1973). Other species such P. oligandrum and P. nunn have been reported as biological control agents (Berry et al., 1993). Pythium spp. reproduce both sexually and asexually via oospores and zoospores from sporangia. Under favorable environmental conditions, Pythium initiates the process of infection that starts when survival structures in the soil such oospores or sporangia germinate. These structures germinate via a germ tube in response to exogenous carbohydrates and amino acids produced by the plant or volatile seed exudates produced during seed germination (Kerr, 1964; Nelson, 1990). The germ tube grows towards these sources of nutrients and colonizes the host tissue. Sporangia also may germinate indirectly producing motile zoospores, which encyst and germinate. Seeds form susceptible cultivars may not even germinate in the presence of the pathogen, on seed more distant to inoculum or for a more resistant cultivar, the plant may overcome the colonization by the pathogen to produce a seedling. Pythium spp. also can cause post emergence damping-off and are associated with root pruning that leads to reduction of plant vigor and yields, but is not lethal to mature plants. Because of the wide range of hosts and distribution, the lack of visible symptoms and the wide number of species, it seems that the reductions in yield due to this pathogen has been underreported and not fully appreciated (Martin, 2009).

It has been reported that infection by Pythium spp. is dependent on environmental factors.

Temperature and soil moisture (Hendrix and Campbell, 1973; Martin, 1996; Allen et al., 2004;

Kirkpatrick, et al., 2006b; Rothrock et al., 2012) have significant effects on the development of

90 the pathogens and in the expression of virulence (Martin, 1996). Pythium spp. are more prevalent in wet soils than in soils with lower soil moisture (Martin, 1996). Soil moisture affects the motility of the zoospores, which require free water (Bainbridge, 1970). It has been reported that high soil moisture also favored saprophytic activity of P. irregulare, P. vexans and P. ultimum, which grew well when the pore spaces are filled with water, because these pathogens are highly tolerant of low oxygen levels (Hendrix and Campbell, 1973). Kirkpatrick et al. (2006b) reported that the only pathogen group that increased in frequency from the roots of flooded soybeans were

Pythium spp. Besides soil moisture, temperature is also important (Roncadori and McCarter,

1971; Hendrix and Campbell, 1973; Martin, 1996; Nannayakkara, 2001; Rothrock et al., 2012).

In general, P. irregulare, P. spinosum and P. ultimum are damaging at temperatures of 15 to

20˚C. While, P. myriotylum, P. aphanidermatum, P. arrhenomanes, P. polytylum, P. carolinianum, P. volutum, are damaging at higher temperatures. The optimal temperature for P. myriotylum and P. aphanidermatum has been reported to be 32˚C (Hendrix and Campbell, 1973;

Martin, 1996). These differences in temperature range may determine the dominant Pythium species in any geographic area or within a field during the growing season (Martin, 1996).

Pythium spp. composition has been reported to vary among locations (soils). Dorrance et al., (2004) recovered Pythium spp. from three different locations in Ohio using both soybean and corn as a bait. The main Pythium species isolated for these fields were: P. cantenulatum, P. irregulare, P. paroecandrum, P. splendens and P. torulosum. A total of 129, 85 and 38 isolates were recovered from Defiance, Sandusky, and Wood County respectively. In Defiance, P. torulosum P. spendens and P. irregulare accounted for 40 and 38 and 22% of the isolates respectively. On the other hand, in Sanduski, P. splendans accounted for 72 % of the isolates and in Wood county P. cantenulatum and P. splendens accounted for 20 and 40 % of the isolates

91 respectively. Moreover, Broders et al., (2009) identified 21 Pythium species from 88 locations in

Ohio representing fields planted with corn and soybean. From the 21 species, 6 species represented 40% of the total species, P. irregulare was the most isolated species, followed by P. inflatum, and P. torulosum. In the same study, the authors using a canonical discriminant analysis (CDA) found that the total number of isolates were grouped in five major communities, which differed among locations based on soil properties such as pH, calcium, magnesium, organic matter (OM) and cation exchange capacity (CEC).

In Arkansas, soybean and rice production are concentrated in alluvial soils with poor drainage that are often saturated. Most fields are planted with soybean from April through June and with rice mainly in April or May (Faw and Johnston, 1975; Faw and Porter, 1981). Seedling diseases occur whenever these crops are planted.

Rotation of crops is a very common practice for the control of soilborne pathogens and it has been reported that rotation also may have an influence in the population density and diversity of Pythium spp. (Hoppe and Middleton, 1950; Schmitthenner, 1962; Pankhurst et al., 1995).

Zhang and Yang (2000) studied the effect of long –term corn- soybean rotation on soilborne pathogens. They reported that 71 % of the total isolates were pathogenic in different levels to both corn or soybean and that disease index on soybean was highly correlated with the disease index on corn.

Two major crops in Arkansas are soybean and rice. Soybean production in Arkansas in

2013 was 3.84 million metric tons of soybean, planted in 1.4 million hectares (USDA- NASS,

2014). The main production area is concentrated in the Delta area on alluvial soils that are prone to seedling disease. Besides soybeans Arkansas ranks first among the six major rice-producing states, accounting for approximately 48 percent of the U.S. rice production. Rice production is

92 approximately 4.04 million metric tons of rice on 607,028 hectares. Rice production is also concentrated in the eastern half of the state, which makes rice also prone to seedling diseases. In

Arkansas soybean-rice is a common rotation (Hristovska et al., 2011) However rice is a more profitable crop than soybean. It is commonly rotate with soybeans to control red rice, which is a weed relative to rice. In 2009, 68 % of the rice produced in Arkansas was rotated with soybean

(Wilson et al., 2009).

The hypothesis in this study, was that Pythium populations are influenced by the soybean-rice rotation commonly used in Arkansas compared to continuous soybean. Also, since different species of Pythium are active over different temperatures ranges, baiting temperature may affect which Pythium species are isolated. The objectives of this study were: i) to determine the effect of continuous soybean and soybean-rice rotation on Pythium spp. diversity in several locations in Arkansas, and ii) determine the effect of temperature on the recovery of Pythium spp.

Materials and Methods

Soil collection

Soil samples were collected from three different locations in Arkansas: Northeast

Research and Extension Center at Keiser, Arkansas; Pine Tree, Experimental Station, at Colt,

Arkansas, and at the Rice Research and Extension Center (RREC) in Stuttgart, Arkansas. The field history and characteristics of these three locations differed: Keiser is a Sharkey silty clay soil, taxonomically classified as very-fine, smectitic, thermic chromic epiaquerts that had been cropped with agricultural crops; Pine Tree is a silt loam taxonomically classified as fine-silty mixed, active, thermic Aquic Fraglossuadalfs soil that was a swamp and forest converted into agricultural use, and Stuttgart is a Dewitt silt loam, taxonomically classified as fine, smectitic,

93 thermic Typic Albaqualfs soil which was a prairie converted into agricultural use (USDA-

NRCS, 2012).

Soil sampling

Soil samples in each location were collected from two different fields in July 2012, the fields had been under two different rotations: soybean –soybean or rice-soybean for the last two years. Soil samples were collected following a “Z” pattern from the first 15 cm of the soil profile, there were 30 collection points in each field (Table 1). Soil was kept in a cooler and transported to the main campus in Fayetteville, AR, where the samples were kept at 4°C until used.

Isolation

Soil from each location was placed in 473-milliliter Styrofoam containers (Dart ®, Dart

Container Corporation, Mason MI, 48854) and filled with 150 g oven dry weight of soil.

Containers were placed in trays and wetted close to saturation (0 Jules/kg) with deionized water from the bottom and allowed to equilibrate overnight and, planted with ten seeds of the susceptible soybean cultivar ‘Hutcheson’. There were five cups per location, temperature and rotation. Trays were placed at 20 or 30 °C. The two temperatures represent the early and late planting dates in Arkansas (Rosso, 2007). After three days, seeds were collected and washed in running water for 30 minutes and plated onto Pythium semi-selective medium CMA+ PARPB

(Rojas et al, 2012). There was one seed per petri plate and 50 seeds per location-rotation- temperature combination. Petri plates were cover with aluminum foil and maintained in the dark.

After two days hyphal tips were transferred to CMA+ PARPB. Twenty five isolates per location, temperature and rotation combination, in total 300 isolates were sent for molecular identification to Dr. Martin Chilvers in Michigan State University.

94 Molecular identification

Isolates were grown on 50 mL of V8-juice broth for 10 d, then mycelia was harvested, lyophilize overnight, and ground. A total of 100 mg of ground mycelia were resuspended on 800

µL of CTAB buffer (AutoGen AG00121, AutoGen Inc.) and incubated for 1 h at 65ºC. A phenol- chloroform automated DNA extraction was performed using the AutoGen 850 system (AutoGen

Inc., Holliston, MA). The Internal Transcribed Spacer (ITS) 1 and 2 of rDNA were amplified with primers ITS6 and ITS4 (Cooke, et al, 2000) for all the isolates. ITS sequences were compared to a curated database of Oomycetes for identification (Robideau et al., 2011).

Experimental design and statistical analysis

The experimental design was a completely randomized design. The effect of location, temperature and rotation on Pythium species population was analyzed with Linear Mixed Model

GLIMMIX procedure in SAS package (version 9.4, SAS Institute Inc. Cary, NC. USA.

Abundance, richness and shannon indices were calculated and analyzed with Linear Mixed

Model GLIMMIX procedure in SAS package (version 9.4, SAS Institute Inc. Cary, NC. USA.

Each Pythium isolate was considered as a count and isolates were analyzed with the Poisson distribution for the richness and abundance. Shannon index data was analyzed with Gaussian distribution. Abundance in each location, crop and temperature was calculated as the sum of all the isolates in the location x temperature x crop rotation combination (Pielou, 1975). Richness was calculated as the presence of any of the species in any location x temperature x rotation combination (Pielou, 1975). Species diversity in each location, crop and temperature was calculated using the Shannon index, H’= Ʃ pi ln pi, where H’ is the species diversity score and pi is the proportion of individuals in the ith species (Krebs, 1999)

Results

95 Pythium spp. isolation and identification.

A total of 275 isolates were identified from soil from three locations, two rotations and two baiting temperatures in 2012. In total 25 species were identified (Figure 1). These included

264 isolates of Pythium species, ten isolates of Phytophthora species and one isolate of

Phytopythium sindhum which were recovery across the two locations, two temperatures and two rotation systems. The most recovered species were P. irregulare, P. pareocandrum, P. sylvaticum, P. coloratum, P. spinosum and P. ultimum var. ultimum.

Pythium species by location

There were significant differences in Pythium species among locations when the number of isolates of the main six Pythium groups were analyzed across the three locations, two temperatures and two rotations systems (Table 2). There was not a significant effect of baiting temperature or rotation system on Pythium population. Since location by species had a significant effect, data was analyzed across temperature and rotation which were treated as a replication to find the effect of location on Pythium species. In soil from Stuttgart, P. paroecandrum and P. irregulare were the most recovered Pythium species, followed by P. sylvaticum and P. spinosum, but P. ultimum var. ultimum and P. corolatum were not recovered from this location (Figure 2). In soil from Pine Tree, P. irregulare and P. spinosum were the most recovered Pythium species, followed by P. sylvaticum, P. paroecandrum and P. coloratum.

P. ultimum var. ultimum was not recovered from this location. In soil from Keiser, P. corulatum and P. ultimum var. ultimum were the most recovered species, followed by P. sylvaticum and P. irregulare; but, P. spinosum and P. paroecandrum were not recovered from this location.

Across rotations, P. irregulare and P. paroecandrum were recovered more from Stuttgart than Pine Tree and Keiser. P. irregulare more common in Pine Tree than Keiser. P. spinosum

96 was recovered more from Pine Tree, while, P. ultimum and P. coloratum were recovered more from Keiser soil.

Pythium species diversity

To study the diversity of Pythium spp., richness, abundance and shannon index were calculated for the more frequently recovered species: P. irregulare, P. pareocandrum, P. sylvaticum, P. coloratum, P. spinosum and P. ultimum var. ultimum across locations temperatures, and crop rotation (Table 3).

Location had a significant effect with abundance and shannon indices but not with the richness index (Table 4). With abundance, Stuttgart soil had significantly more Pythium species across temperatures and rotations than Keiser soil. Pine Tree did not have any significant differences with either Stuttgart or Keiser soils in abundance of Pythium species (Figure 3).

With Shannon index, Pine Tree soil had a higher diversity of Pythium spp. than the other two locations, however it was not significantly different than Keiser. Keiser soil did not have any significant differences with either Stuttgart or Pine Tree soils in diversity of Pythium species

(Figure 4).

Discussion

In this study location had a large effect on Pythium population composition and diversity.

Distinctive species prevailed in each of the three locations across two temperature and two rotation systems in Arkansas when baited from soil using soybean seed. Pythium irregulare was the most recovered species from Pine Three and Stuttgart but were recovered the most, from

Stuttgart soils. Pythium irregulare is a pathogen of both rice and soybean and has been recovered from seedlings of soybean and rice (Dorrance et al., 2004; Rosso, 2007; Broders et al., 2007;

Eberle et al., 2008 and Avanzato, 2011). It is the first time that P. pareocandrum have been

97 reported as one of the main Pythium species in Arkansas soils, but it has been reported to be a pathogen of soybean in Ohio (Dorrance et al., 2004). Pythium coloratum and P. ultimum var. ultimum were mainly recovered from Keiser soils and P. spinosum was mostly frequently isolated species from Pine Tree soils. There were not differences of recovery of P. sylvaticum among the three locations.

The soils of the three location had different physical and chemical properties: Keiser is a clay soil with a higher CEC and OM content, compared to soils from Pine tree and Stuttgart.

Pine Tree and Stuttgart soils were silt loam soils with average pH of 7.9 and 5.35, respectively, with similar contents of OM and CEC. Broders et al. (2009), reported strong association between abiotic soil components such as OM, CEC, Ca, Mg, percent of sand and percent of silt, disease indices (number of infected plants) and Pythium diversity. They reported that P. irregulare was the most frequently recovered species and the most abundant in locations with low levels of calcium, magnesium and OM, poorly drained and with limited species diversity. Based on the discriminated canonical analysis they suggested that species in community IF, which was dominated by P irregulare and P. inflatum, may outcompete other species as pH, and magnesium increase and OM, percent clay and field capacity decrease. Gomez-Aparicio et al.,

(2012) reported that soilborne pathogens abundance in the forest was influenced mainly by abiotic (soil texture) and biotic (tree and shrub species). They found a negative correlation of soil sand content on pathogen abundance and the effect varied among groups, the magnitude of texture effect was larger for Pythium spp. than for Phytophthora cinnamomi or Pythium speculum. Dick and Ali- Shtayeh (1986) reported that soil pH was the most important characteristic influencing Pythium community structure in parkland and farmland soils. They reported that the largest number of Pythium spp. propagules were found at soils with pH between

98 6.0 and 7.4 and that very acidic soils contained lower numbers of Pythium propagules. These results agreed with the results in this study, where soils from Stuttgart had an average lower pH

(5.35) than the other 3 locations and had the lowest diversity of Pythium species, while, Keiser that has an average neutral pH (7.9) had the highest diversity of Pythium species.

Temperature did not have an effect on the recovery of Pythium species in this study during the baiting period. Temperature has been reported as an important for Pythium infection

(Roncadori and McCarter, 1971; Hendrix and Campbell, 1973; Martin, 1996; Nanayakkara,

2001) and certain species such as P. irregulare, P. spinosum and P. ultimum are damaging at temperatures of 15 to 20˚C, while, P. myriotylum, P. aphanidermatum, P. arrhenomanes, P. polytylum, P. carolinianum, P. volutum, were damaging at high temperatures. It was thought that these differences in temperature range may influence Pythium species infecting soybean or rice, however it could not been demonstrated in this study. It may suggest that the Pythium species recovered in this study infected soybeans over a wide range of temperature. Also these results may suggest that soil moisture as reported previously, is a more important factor for Pythium diseases in soybean than temperature (Hendrix and Campbell, 1973; Martin, 1996; Allen et al.,

2004; Kirkpatrick, et al., 2006b).

A two year rotation of soybean-soybean and rice-soybean rotation system did not have an effect on Pythium recovery in this study. Long-term rotations have shown and effect on Pythium species population in crops such as canola (Hwang et al., 2009), wheat (Pankhust et al., 1995) and soybean (Zhang and Yang, 2000). But the effect of a short-term rotation in Pythium population have not been reported on soybean and rice. It is only been reported on carrots to

Pythium carrot dieback caused Pythium and Rhizoctonia. It may suggest that the two year

99 rotation is not enough time to have a measurable effect on Pythium populations or that the

Pythium population collected from the fields in this study were already adapted to both crops.

The Pythium species composition in this study differed from those recovered by Rosso

(2007) and Avanzato (2011), which reported that P. sylvaticum was the most recovered Pythium species from five and two locations in Arkansas, respectively. The differences in Pythium species in Arkansas between studies may be due to locations as we have shown here in this study but factors such as cropping systems, soil sample collection time and fields (molecular identification primers and databases may influence).

Conclusion

Overall, this study indicated that location has an effect of Pythium species population and that this effect may be related with physical and chemical soil properties, furthermore more studies are needed in Arkansas to understand the soil ecology affecting Pythium species population in Arkansas. Knowing the Pyhtium species diversity in each location in Arkansas soybean fields would help the growers to select the appropriate fungicide seed treatments.

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104 Table 1. Location, GPS coordinates, of the six fields where soil was collected.

Location GPS coordinates Rotation Pine Tree experimental station, at Colt , AR N35°07.533' W090° 57.137' Soybean - Rice Pine Tree experimental station, at Colt, AR N35°07.533' W090° 57.836' Soybean - Soybean Northeast research and extension center at Keiser, AR N35°39.934' W090° 04.980' Soybean - Rice Northeast research and extension center at Keiser, AR N35°39.714' W090° 04.762' Soybean - Soybean Rice Research and Extension Center, Stuttgart, AR N34°27.815' W091° 25.614' Soybean - Rice Rice Research and Extension Center, Stuttgart, AR N34°28.366' W091° 25.350 Soybean - Soybean

105 30

25

20

15

10 Number Number isolatesof

5

0

Pythium species

Figure 1. Total of oomycetes isolates recovered across three locations, two temperature and two rotation systems.

106 Table 2. Analysis the variance of number of isolates of six Pythium species recovered across three locations, two temperature and two rotation systems.

Effect df F-value P >F Location 2 2.21 <.0001 Temperature 1 0.19 0.6623 Rotation 1 0.07 0.7931 Species 5 6.66 <.0001 Location x species 10 7.65 <.0001 Temperature x species 5 0.96 0.456 Rotation x species 5 0.61 0.693

107 Keiser 12 a a Pine Tree 10 Stuttgart

8 b bc 6 bdc bcde bcde 4 cde cde cde Number Number isolatesof de de 2 e e e e e e 0

Pythium spp.

Figure 2. Number of isolates of six Pythium species by location. Means for the different or same Pythium species followed by the same lower case are not significantly different (P< 0.05).

108 Table 3. Richness, abundance and Shannon indices for six Pythium species recovered from two locations, two temperatures and two rotation systems.

Shannon Temperature Location Crop rotation Richnessx Abundance y Index (°C) (H')z Kaiser 20 Rice-Soybean 3 8 0.900 Kaiser 30 Rice-Soybean 4 16 1.070 Kaiser 20 Soybean-Soybean 4 16 1.355 Kaiser 30 Soybean-Soybean 4 18 0.761 Pine Tree 20 Rice-Soybean 5 18 1.426 Pine Tree 30 Rice-Soybean 5 22 1.375 Pine Tree 20 Soybean-Soybean 4 26 1.216 Pine Tree 30 Soybean-Soybean 5 15 1.395 Stuttgart 20 Rice-Soybean 4 24 0.761 Stuttgart 30 Rice-Soybean 3 25 0.909 Stuttgart 20 Soybean-Soybean 2 23 0.615 Stuttgart 30 Soybean-Soybean 3 25 0.974 x Richness was calculated as the presence of any of the species in any location x temperature x rotation combination. y Abundance was calculated as the sum of all the isolates in the location x temperature x crop rotation combination. z Species diversity in each location, crop and temperature was calculated using the Sha nnon index, H’= Ʃ pi ln pi, where H’ is the species diversity score and pi is the proportion of individuals in the ith species (Krebs, 1999)

109 Table 4. Analysis of variance w for richness, abundance and shannon index.

Richnessx Abundancey Shannon indexz Effect P- value Location 0.4890 0.0486 0.0213 Temperature 0.7767 0.7078 0.7749 Rotation 0.7767 0.5360 0.8652 w Data was analyzed with Linear Mixed Model GLIMMIX procedure in SAS package with a P < 0.05 x Richness was calculated as the presence of any of the species in any location x temperature x rotation combination. y Abundance was calculated as the sum of all the isolates in the location x temperature x crop rotation combination. z Species diversity in each location, crop and temperature was calculated using the Shannon index, H’= Ʃ pi ln pi, where H’ is the species diversity score and pi is the proportion of individuals in the ith species (Krebs, 1999)

110 30 Stutgart a 25 Pine Tree ab Keiser 20 b 15

10 Abundance Abundance Index 5

0 Stutgart Pine Tree Keiser

Location

Figure 3. Abundance index of six Pythium species across two temperatures and two crop rotations. Abundance was calculated as the sum of all the isolates in the location x temperature x crop rotation combination.

111

1.6 a 1.4

1.2 ab

1 b 0.8

0.6

Shannon Shannon index 0.4

0.2

0 Pinetree Keiser Stuttgart Location

Figure 4. Shannon index of six Pythium species across two temperatures and two crop rotations. Species diversity in each location, crop and temperature was calculated using the Shannon index, H’= -Ʃ pi ln pi, where H’ is the species diversity score and pi is the proportion of individuals in the ith species.

112 Overall Conclusion

Overall, we found that seed germination and plant stands for the Archer x Hutcheson population fit the model for quantitative resistance indicating that these are a quantitative traits controlled by multiple genes or QTLs. The two phenotyping methods evaluated soybean seed rot and separated the parents. Two stable QTL were found on chromosome 4 and 7 using the infestation assay and seed plate assay, respectively, showing that these two assays separated the parents and lines. The QTLs on chromosome 4 and 7 were identified at similar locations using the seed plate and the infestation assays, making them strong candidates for further research.

Crop history had an impact on Pythium populations for long-term rotation of the Rice

Research and Extension Center, Stuttgart, AR. Corn- rice rotation had the highest incidence of infection, followed by the rice (wheat)-soybean (wheat) and rice-soybean, rice-corn-soybean and continuous rice rotations. P. irregulare, P. spinosum, and P. paroecandrum were the most recovered species across the 10 rotations.

Location has an effect of Pythium species population. Distinctive species prevailed in each of the three locations across two temperature and two rotation systems in Arkansas when baited from soil using soybean seed. Pythium irregulare was the most recovered species from

Pine Three and Stuttgart but were recovered the most, from Stuttgart soils. The effect of location on Pythium species population in this study may be related with physical and chemical soil properties, furthermore more studies are needed in Arkansas to understand the soil ecology affecting Pythium species population in Arkansas. Knowing the Pyhtium species diversity in each location in Arkansas soybean fields would help the growers to select the appropriate fungicide seed treatments.

113

Appendix: Soil chemical and physical analysis from the three locations in Arkansas.

Location Rotation pH P K Ca Mg S Na Fe Mn Zn Cu B Keiser 1 Soy - soy 6.7 37 353 4262 948 4.8 45 315 28 3.4 4.5 0.8 Keiser 2 Soy- Rice 7.9 66 418 5244 1032 6.6 52 356 54 3.2 4.6 1.2 Pine Tree 1 Soy -soy 8.3 27 129 2671 395 8.9 61 296 184 2.8 1.3 0.4 Pine Tree 2 Soy - Rice 7.5 6 82 1646 342 22.4 54 634 78 1.5 0.3 0.4 Stuttgart 1 Soy -soy 5.6 9 107 933 173 8.3 64 535 160 0.7 0.7 0.3 Stuttgart 2 Soy -rice 5.1 28 180 1171 127 9.5 36 465 85 3.8 0.9 0.3

114

Appendix: Soil chemical and physical from the three locations in Arkansas Cont.

Location Rotation %sand %silt %clay %Total N %Total C OM CEC meq/100g soil Keiser 1 Soy - soy 17 30 54 0.13 1.29 2.22 20.14 Keiser 2 Soy - rice 9 34 56 0.15 1.41 2.43 22.32 Pine Tree 1 Soy - soy 4 77 20 0.08 0.68 1.13 13.29 Pine Tree 2 Soy - rice 1 83 16 0.11 1.25 2.15 10.59 Stuttgart 1 Soy - soy 3 79 19 0.11 0.99 1.71 10.87 Stuttgart 2 Soy - rice 3 80 17 0.11 0.97 1.67 9.55

115