Assessment of Resistance in Soybean to Pythium Ultimum and Sensitivity of Ohio’S

Home , Pythium, Pythium dissotocum, Pythium irregulare, Pythium ultimum

Assessment of Resistance in Soybean to Pythium ultimum and Sensitivity of Ohio’s

Diverse Pythium species towards Metalaxyl

THESIS

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in

the Graduate School of The Ohio State University

Christine Susan Balk

Graduate Program in Plant Pathology

The Ohio State University 2014

Master's Examination Committee:

Dr. Anne E. Dorrance, Advisor Dr. Pierce Paul Dr. Francesca Peduto Hand

Copyrighted by Christine Susan Balk 2014

Abstract

In Ohio, seedling blight caused by oomycetes is an annual problem in crop production.

More than 25 different species of Pythium have been identified that contribute to seed and seedling loss in Ohio; which is economically detrimental to soybean producers.

Several factors have been proposed that may contribute to the high incidence of seedling blights caused by Pythium. These include long term no-till production, changes in seed treatments, and environmental conditions that favor infection, which occurs shortly after planting. Host resistance and seed treatments are two management strategies that could be deployed for these pathogens. Pythium ultimum var. ultimum and Pythium ultimum var. sporangiiferum are aggressive Pythium species that are abundant in Ohio soils. There has not been an in-depth evaluation of resistance in soybeans towards Pythium ultimum.

The first objective of this study was to 1a) identify sources of resistance to these two varieties of Pythium ultimum. Much of the germplasm that was evaluated were previously identified as sources of resistance Phytophthora sojae and P. irregulare.

Therefore objective 1b) was to determine if genotypes resistant to the varieties of

Pythium ultimum are the same or different to each other as well as other oomycete pathogens Phytophthora sojae and P. irregulare. Multiple isolates of Pythium ultimum var. ultimum and Pythium ultimum var. sporangiiferum were used to evaluate germplasm to identify resistant soybean genotypes. ii

Metalaxyl and pyraclostrobin are fungicides currently used as seed treatments to manage

Pythium species. However, some species are insensitive to one or both of the active ingredients. A second objective was to determine which proportion of isolates within a species, were sensitive to metalaxyl. A collection of isolates of Pythium species was

evaluated on amended agar (127 isolates) or amended broth assays (292). The results from these studies may identify new sources of resistance to P. ultimum v. ultimum, and

P. ultimum v. sporangiiferum and if metalaxyl is an effective seed treatment to improve

current management and limit further losses.

Resistance to both varieties of P. ultimum was expressed as a reduction in root rot, higher

stands, and larger root mass in these cup assays. A high level of resistance was found in

soybean genotypes Dennison, Hutchinson, PI 424354, OSU038, OSU015, OSU028, PI

408225A, OSU049, OSU027 and OhioFG1 to P. ultimum var. sporangiiferum. While

Williams, Kottman, Streeter, and Wyandot; had high levels of resistance to P. ultimum v.

ultimum. Among the 298 genotypes that were evaluated, there was one that had high

levels of resistance to both species, which was Dennison.

There was growth on metalaxyl amended broth (100 ppm) for 96 of the 252 isolates

evaluated in this study from years previous to 2014. The sensitivity to metalaxyl was variable both among and within isolates for Pythium irregulare, Pythium sylvaticum,

Pythium torulosum, Pythium aphanidermatum, and Phytophthora sojae. There was

complete sensitivity to pyraclostrobin for all 127 Pythium or Phytophthora isolates tested. iii

Dedication

I would like to dedicate this thesis to my family and friends. My parents, Daniel and

Barbara Balk have been with me every step of the way; not only for this degree, but in life. They are such supportive and encouraging role models. My brother Tommy has been an inspiration to me to be myself, no matter who’s watching. My sister Melissa has taught me that if you want something, go get it. The sky is the limit.

Thank you all!

Acknowledgments

I would like to acknowledge Dr. Anne E. Dorrance in her constant leadership, support, and drive throughout this intense process. She has been an inspirational advisor. I’d also like to acknowledge Dr. Aswini Pai for pushing me throughout my undergraduate years and having faith in me at St. Lawrence University. I never would have started this path had she not put me on it. I’d like to acknowledge St. Lawrence University for a phenomenal liberal arts degree that I will forever be grateful for. Lastly, I’d like to acknowledge The Ohio State University for making this degree a reality in my life.

Vita

July 25, 1988 ...... Born- Buffalo, New York

June 2006 ...... Amherst Central High School

2010...... B.S. Biology, St. Lawrence University

2014...... M.S. Plant Pathology, The Ohio State

University

Fields of Study

Major Field: Plant Pathology

Table of Contents

Abstract ...... ii

Acknowledgments...... v

Vita ...... vi

List of Tables ...... x

List of Figures ...... xiii

Chapter 1: Assessment of Resistance in Soybean to Pythium ultimum and Effective

Rates of Metalaxyl Towards Ohio’s Diverse Pythium species ...... 1

Literature Review ...... 1

Chapter 2: Quantitative Resistance in Elite Soybean Germplasm to Pythium

ultimum var. ultimum and Pythium ultimum var. sporangiiferum ...... 12

Introduction ...... 12

Materials & Methods:...... 15

Pythium Isolates...... 15

Inoculum for greenhouse assays...... 15

Greenhouse Studies...... 16

vii

Soybean Genotypes...... 16

Data collection...... 17

Comparison between Pythium species...... 18

Data analyses...... 18

Results...... 18

Checks ...... 19

There were 10 genotypes used as controls across experiments. Conrad was excluded

from the final analysis of checks due to problems with the seed. The Virginia Tech

experiments were also excluded due to less disease in those 2 experiments. Once

analyzed, there was not an isolate x genotype interaction (Root score P-value=

0.9152, Root weight P-value= 0.2190, Appendix A)...... 19

Nested Association Mapping ...... 19

University of Missouri ...... 20

Virginia Tech Genotypes ...... 20

Genetic Gain ...... 21

Ohio State University Genotypes ...... 22

Comparison of isolates and genotypes ...... 22

viii

Discussion ...... 24

Chapter 3: Efficacy of Metalaxyl and Pyraclostrobin to Pythium species affecting soybean and corn in Ohio ...... 73

Introduction ...... 73

Materials & Methods ...... 75

Phytophthora and Pythium isolates ...... 75

Sensitivity to metalaxyl ...... 76

Statistical Analysis ...... 77

Sensitivity to pyraclostrobin ...... 77

Results ...... 78

Metalaxyl ...... 79

Pyraclostrobin ...... 80

Discussion ...... 80

Appendix A: ANOVA for checks & Metalaxyl and Pyraclostrobin Table...... 99

Appendix B: Protocols for Chapter 2 & 3 ...... 114

List of Tables

Table 1. Isolates of Pythium ultimum that originate in Ohio and were used to evaluate soybean genotypes for resistance in greenhouse cup assays...... 28

Table 2. Analysis of variance (ANOVA) for Nested Association Mapping population

evaluated for resistance to Will 1-6-7 (P. ultimum var. sporangiiferum) and Miami 1-3-7

(P. ultimum var. ultimum) in greenhouse assays...... 30

Table 3. Analysis of variance (ANOVA) for The University of Missouri population

evaluated for resistance to Will 1-6-7 (P. ultimum var. sporangiiferum) and Miami 1-3-7

(P. ultimum var. ultimum) in greenhouse assays...... 33

Table 4. Analysis of variance (ANOVA) Virginia Tech population evaluated for

resistance to Will 1-6-7 (P. ultimum var. sporangiiferum) and Miami 1-3-7 (P. ultimum var. ultimum) in greenhouse assays...... 35

Table 5. Analysis of variance (ANOVA) for Genetic Gain population evaluated for

resistance to Will 1-6-7 (P. ultimum var. sporangiiferum) and Miami 1-3-7 (P. ultimum var. ultimum) in greenhouse assays...... 37

Table 6. Analysis of variance (ANOVA) for the OSU population evaluated for resistance

to Will 1-6-7 (P. ultimum var. sporangiiferum) and Miami 1-3-7 (P. ultimum var.

ultimum) in greenhouse assays...... 39

Table 7. Comparison of the mean root score (1-5) for soybeans following inoculation

with four isolates of both Pythium ultimum var. ultimum, and Pythium ultimum var. sporangiiferum, in a growth chamber assay...... 40

Table 8. P. ultimum v. ultimum NAM population Means of lines evaluated...... 42

Table 9. NAM population means of lines evaluated with P. ultimum v. sporangiiferum. 44

Table 10. University of Missouri population inoculated with P. ultimum v. ultimum

(There was poor seed quality with some lines in the non-inoculated controls. This table

only includes lines that had growth in the non-inoculated control)...... 46

Table 11. University of Missouri population inoculated with P. ultimum v.

sporangiiferum (There was poor seed quality with some lines in the non-inoculated

controls. This table only includes lines that had growth in the non-inoculated control.). 48

Table 12. Overall means of Virginia Tech population inoculated with P. ultimum v.

ultimum...... 50

Table 13. Overall means of Virginia Tech population evaluated with P. ultimum v.

sporangiiferum...... 52

Table 14. Overall means of Genetic Gain population inoculated with Pythium ultimum v.

ultimum...... 54

Table 15. Overall means of Genetic Gain population inoculated with P. ultimum v.

sporangiiferum...... 57

Table 16. Overall means of The Ohio State University population inoculated with P. ultimum v. ultimum...... 60

Table 17. Overall means of The Ohio State University population inoculated with P. ultimum v. sporangiiferum...... 64

Table 18. Mean root weight and root rot score for the NAM experiments of both P. ultimum v. ultimum and P. ultimum v. sporangiiferum for genotype checks...... 68

Table 19. Mean root weight and root rot score for the University of Missouri experiments of both P. ultimum v. ultimum and P. ultimum v. sporangiiferum for genotype checks. .. 69

Table 20. Mean root weight and root rot score for the Virginia Tech experiments of both

P. ultimum v. ultimum and P. ultimum v. sporangiiferum for genotype checks...... 70

Table 21. Mean root weight and root rot score for the Genetic Gain experiments of both

P. ultimum v. ultimum and P. ultimum v. sporangiiferum for genotype checks...... 71

Table 22. Mean root weight and root rot score for the OSU experiments of both P. ultimum v. ultimum and P. ultimum v. sporangiiferum for genotype checks...... 72

Table 23. ANOVA for root score of checks. Excluding Conrad and the experiments for

Virginia Tech...... 100

Table 24. ANOVA for root weight of checks. Excluding Conrad and the experiments for

Virginia Tech...... 100

xii

List of Figures

Figure 1. The number of soybean genotypes of the Nested Association Mapping parent

population with (A) mean heights (mm) of 3 seedlings, (B) mean percent plant stand of 8

soybeans, (C) mean plant weight (g) of all seedlings, (D) mean root weight (g) of all

seedlings, and (E) root rot score of overall roots: (R – 1.0-2.0), moderate resistance (MR

–2.0-3.0), moderate susceptibility (MS- 3.0-3.8), and susceptibility (S- 3.8-5.0) to

Pythium ultimum var. ultimum and Pythium ultimum var. sporangiiferum (N= 48)...... 29

Figure 2. The number of soybean genotypes of the Missouri population with (A) mean

heights (mm) of 3 seedlings, (B) mean percent plant stand of 8 soybeans, (C) mean plant

weight (g) of all seedlings, (D) mean root weight (g) of all seedlings, and (E) root rot

score of overall roots: (R – 1.0-2.0), moderate resistance (MR –2.0-3.0), moderate

susceptibility (MS- 3.0-3.8), and susceptibility (S- 3.8-5.0) to Pythium ultimum var.

ultimum and Pythium ultimum var. sporangiiferum (N= 48)...... 32

Figure 3. The number of soybean genotypes of the Virginia Tech population with (A)

mean heights (mm) of 3 seedlings, (B) mean percent plant stand of 8 soybeans, (C) mean

plant weight (g) of all seedlings, (D) mean root weight (g) of all seedlings, and (E) root

rot score of overall roots: (R – 1.0-2.0), moderate resistance (MR –2.0-3.0), moderate

susceptibility (MS- 3.0-3.8), and susceptibility (S- 3.8-5.0) to Pythium ultimum var.

ultimum and Pythium ultimum var. sporangiiferum (N= 54)...... 34 xiii

Figure 4. The number of soybean genotypes of the Genetic Gain population with (A)

mean heights (mm) of 3 seedlings, (B) mean percent plant stand of 8 soybeans, (C) mean

plant weight (g) of all seedlings, (D) mean root weight (g) of all seedlings, and (E) root

rot score of overall roots: (R – 1.0-2.0), moderate resistance (MR –2.0-3.0), moderate

susceptibility (MS- 3.0-3.8), and susceptibility (S- 3.8-5.0) to Pythium ultimum var.

ultimum and Pythium ultimum var. sporangiiferum (N= 71)...... 36

Figure 5. The number of soybean genotypes of the OSU population with (A) mean

heights (mm) of 3 seedlings, (B) mean percent plant stand of 8 soybeans, (C) mean plant

weight (g) of all seedlings, (D) mean root weight (g) of all seedlings, and (E) root rot

score of overall roots: (R – 1.0-2.0), moderate resistance (MR –2.0-3.0), moderate

susceptibility (MS- 3.0-3.8), and susceptibility (S- 3.8-5.0) to Pythium ultimum var.

ultimum and Pythium ultimum var. sporangiiferum (N= 101)...... 38

Figure 6. Soybeans inoculated with Pythium spp. Root rot scores from the left to right:

1, 2, 3 and 4 where (R – 1.0-2.0), moderate resistance (MR – 2.0-3.0), moderate

susceptibility (MS- 3.0-3.8), and susceptibility (S- 3.8-5.0) to Pythium ultimum var.

ultimum and Pythium ultimum var. sporangiiferum...... 41

Figure 7. The Mean score of 131 Pythium irregulare isolates tested in potato carrot broth

visible macroscopically; 4= mycelium uniform, however, not full growth compared to it’s

non-amended control- quantified by 85-95% of control; 5= mycelium visible, uniform,

and equal to the PCB control (100% of control), after 24 and 48 hours of growth...... 83

Figure 8. The Mean score of 18 Pythium sylvaticum isolates tested in potato carrot broth amended with 100 ppm metalaxyl. Rated on a scale from 0-5: 0= no growth; 1= hyphae

only grew microscopically, sporadic hyphae grew from plug- No uniformity in hyphae

growth; 2= uniform hyphae growth, seen only microscopically; 3= uniform mycelium

visible macroscopically; 4= mycelium uniform, however, not full growth compared to it’s

non-amended control- quantified by 85-95% of control; 5= mycelium visible, uniform,

and equal to the PCB control (100% of control), after 24 and 48 hours of growth...... 84

Figure 9. The mean score of 7 Pythium torulosum isolates tested in potato carrot broth

amended with 100 ppm metalaxyl. Rated on a scale from 0-5: 0= no growth; 1= hyphae only grew microscopically, sporadic hyphae grew from plug- No uniformity in hyphae growth; 2= uniform hyphae growth, seen only microscopically; 3= uniform mycelium visible macroscopically; 4= mycelium uniform, however, not full growth compared to it’s non-amended control- quantified by 85-95% of control; 5= mycelium visible, uniform, and equal to the PCB control (100% of control), after 24 and 48 hours of growth...... 85

Figure 10. The mean score of 2 Pythium dissotocum isolates tested in potato carrot broth

amended with 100 ppm metalaxyl. Rated on a scale from 0-5: 0= no growth; 1= hyphae

only grew microscopically, sporadic hyphae grew from plug- No uniformity in hyphae xv

growth; 2= uniform hyphae growth, seen only microscopically; 3= uniform mycelium

visible macroscopically; 4= mycelium uniform, however, not full growth compared to it’s

non-amended control- quantified by 85-95% of control; 5= mycelium visible, uniform,

and equal to the PCB control (100% of control), after 24 and 48 hours of growth...... 86

Figure 11. The mean score of 4 Pythium attrantheridium isolates tested in potato carrot

broth amended with 100 ppm metalaxyl. Rated on a scale from 0-5: 0= no growth; 1=

hyphae only grew microscopically, sporadic hyphae grew from plug- No uniformity in

hyphae growth; 2= uniform hyphae growth, seen only microscopically; 3= uniform

mycelium visible macroscopically; 4= mycelium uniform, however, not full growth

compared to its non-amended control- quantified by 85-95% of control; 5= mycelium visible, uniform, and equal to the PCB control (100% of control), after 24 and 48 hours of growth...... 87

Figure 12. The mean score of 16 Pythium ultimum var. ultimum isolates tested in potato

carrot broth amended with 100 ppm metalaxyl. Rated on a scale from 0-5: 0= no growth;

1= hyphae only grew microscopically, sporadic hyphae grew from plug- No uniformity in hyphae growth; 2= uniform hyphae growth, seen only microscopically; 3= uniform mycelium visible macroscopically; 4= mycelium uniform, however, not full growth compared to its non-amended control- quantified by 85-95% of control; 5= mycelium visible, uniform, and equal to the PCB control (100% of control), after 24 and 48 hours of growth...... 88 xvi

Figure 13. The mean score of 7 Pythium ultimum v. sporangiiferum isolates tested in

potato carrot broth amended with 100 ppm metalaxyl. Rated on a scale from 0-5: 0= no

growth; 1= hyphae only grew microscopically, sporadic hyphae grew from plug- No

uniformity in hyphae growth; 2= uniform hyphae growth, seen only microscopically; 3=

uniform mycelium visible macroscopically; 4= mycelium uniform, however, not full

growth compared to its non-amended control- quantified by 85-95% of control; 5=

mycelium visible, uniform, and equal to the PCB control (100% of control), after 24 and

48 hours of growth...... 89

Figure 14. The mean score of 6 Pythium aphanidermatum isolates tested in potato carrot broth amended with 100 ppm metalaxyl. Rated on a scale from 0-5: 0= no growth; 1= hyphae only grew microscopically, sporadic hyphae grew from plug- No uniformity in hyphae growth; 2= uniform hyphae growth, seen only microscopically; 3= uniform mycelium visible macroscopically; 4= mycelium uniform, however, not full growth compared to it’s non-amended control- quantified by 85-95% of control; 5= mycelium visible, uniform, and equal to the PCB control (100% of control), after 24 and 48 hours of growth...... 90

Figure 15. The mean score of 6 Pythium inflatum isolates tested in potato carrot broth amended with 100 ppm metalaxyl. Rated on a scale from 0-5: 0= no growth; 1= hyphae only grew microscopically, sporadic hyphae grew from plug- No uniformity in hyphae growth; 2= uniform hyphae growth, seen only microscopically; 3= uniform mycelium xvii visible macroscopically; 4= mycelium uniform, however, not full growth compared to it’s non-amended control- quantified by 85-95% of control; 5= mycelium visible, uniform, and equal to the PCB control (100% of control), after 24 and 48 hours of growth...... 91

Figure 16. The mean score of 33 Phytophthora sojae isolates tested in potato carrot broth amended with 100 ppm metalaxyl. Rated on a scale from 0-5: 0= no growth; 1= hyphae only grew microscopically, sporadic hyphae grew from plug- No uniformity in hyphae growth; 2= uniform hyphae growth, seen only microscopically; 3= uniform mycelium visible macroscopically; 4= mycelium uniform, however, not full growth compared to it’s non-amended control- quantified by 85-95% of control; 5= mycelium visible, uniform, and equal to the PCB control (100% of control), after 24 and 48 hours of growth...... 92

Figure 17. Map of counties in Ohio where Pythium irregulare isolates were evaluated for sensitivity to metalaxyl. The number above the diagonal line is the number of insensitive isolates out of the total P. irregulare isolates tested in a broth assay of 100 ppm metalaxyl, a common active ingredient in seed treatment fungicides...... 93

xviii

Chapter 1: Assessment of Resistance in Soybean to Pythium ultimum and Effective

Rates of Metalaxyl Towards Ohio’s Diverse Pythium species

Literature Review

With over 4 million acres produced in the state, soybean [Glycine max (L.) Merr] is

Ohio’s number one crop, closely followed by corn (Beuerlein & Dorrance, 2005). The millions of acres of soybean planted in Ohio produce over 43 billion dollars (National

Agricultural Statistics Service, USDA, July 25, 2013). In 2011, the U. S. was the

number one soybean producer worldwide, totaling over 3 billion bushels (SoyStats.com

September 18, 2013). These numbers stress the economic importance of soybean not

only in Ohio, but also for the U.S. The primary goal of soybean producers is to obtain

the highest yield and the highest quality product annually. Seedling blights are the third

most important disease of soybean that impact yield in the U.S. (Wrather et al., 2010).

In Ohio, as well as other Midwest states, oomycetes contribute substantially to crop loss

every year, primarily through root rot and damping-off of seedlings (Broders et al., 2009;

Campa et al., 2010; Murillo-Williams and Pedersen, 2008; Zhang and Yang, 2000). No

till production systems, poor seed quality, and favorable environmental conditions, are all

factors that contribute to a high rate of infection by oomycetes (Wrather et al., 2010).

Due to the high clay content of production fields in Ohio, soil conditions are often

saturated and suitable for Pythium species to thrive (Broders et al., 2009; McGee, 1986).

Once a field becomes infested with Pythium, it is difficult to eradicate the pathogen due

to its robust overwintering structure, the oospore (Rosso et al., 2008).

Pythium species are classified as oomycetes. Oomycetes were once classified with fungi;

however, due to some key differences, they are now in the Kingdom Stramenopiles,

whereas fungi are in the Kingdom Eumycota (Kamoun et al., 2003; Van West et al.,

2003). The primary similarity between fungi and oomycetes is their mycelial growth

habit. The cell walls of a true fungus contain chitin and their hyphae have cross walls.

Oomycetes, have cell walls made of cellulose, β-3 glucans, and coenocytic hyphae

growth (do not contain cross walls). Fungi do thrive in water, however oomycetes

require it for growth and to infect (Kamoun et al., 2003; Schroeder et al., 2013).

Most oomycetes are capable of reproducing both sexually and asexually. They are able to reproduce under numerous environmental and physiological conditions. Sexual reproduction introduces genetic recombination, resulting in new alleles. To reproduce asexually, they form a structure called sporangium in which zoospores form. In Pythium species, a vesicle is formed within the sporangia, and zoospores are produced within this vesicle. For Phytophthora, there is no vesicle, as the zoospores are produced and released directly from the sporangia. Zoospores are motile, asexual spores that have two flagella. Not all oomycete species form zoospores, but most Pythium species do (Francis

et al., 1994). The flagella aid the zoospores in locomotion through water or saturated

soils (Kamoun et al., 2003; Schroeder et al., 2013; van West et al., 2003). The zoospores

are maintained in the sporangium until they are signaled by root exudates, to release and

swim to the root tissue (Van West et al., 2003).

Donaldson & Deacon (1992) studied chemical signaling, where they found that P.

aphanidermatum, P. catenulatum, and P. dissotocum were all attracted to roots using chemotaxis when amino acids and sugars were released. This study provided evidence that Pythium and other oomycetes utilize chemotaxis in order to find their host. This is important information leading to control of oomycetes. From their studies, they determined that all species respond to the same compounds, albeit that each species seems to respond to these compounds at different stages of the pathogen life cycle

(Donaldson & Deacon, 1992). After chemotaxis occurs and the zoospore locates its host plant, it encysts on the root, and then germinates. Enzymes are released that allow

penetration of the root for colonization, both inter- and intra- cellularly (van West et al.,

2003). As the area of colonization expands within the plant tissue, it gradually absorbs nutrients from the surrounding cells. While the oomycete grows, it produces spores

(oospores for homothallic species, or sporangia) and continues its reproduction cycles continuously until the host can no longer survive (van West et al., 2003).

Like all living organisms, sexual reproduction for an oomycete requires both male and female structures. The female structure is the oogonium, which holds the unfertilized

spores. The male structure is the antheridium, which fertilizes the oogonium (Kamoun et al., 2003; Schroeder et al., 2013). It is with this process that the oomycete is capable of

genetic recombination. If genetic recombination occurs in the root then there is the

possibility of complications with pathogen management if more aggressive strains

develop. The outcome of this could lead to resistance to fungicides and changes in

virulence to resistance genes (Francis & St. Clair, 1993, Olive, 1963).

Chamnanpunt et al. (2001), described mitotic gene conversion in Phytophthora sojae as

another means of genetic variability. Considering the close linkage between

Phytophthora and Pythium species this is cause for concern for all oomycetes causing

disease in soybeans. This is not genetic recombination, however, it is similar and it could

produce new genetic types, or lead to genetic recombination (Kamoun, 2003). For

example, host genotypes that are resistant may no longer have the same effect to new

genetic variants. For instance, with Phytophthora species and tobacco, tobacco is a not a

host. This is due to elicitins (also effectors) secreted by the oomycete. When these

elicitins are recognized by tobacco, the plant signals a hypersensitive response (HR)

causing programmed cell death of the cells that are infected to save the rest of the plant

(Kamoun, 2003). If something were to change with the HR response, there would be a possibility for the tobacco plant to become susceptible.

Soybean damping-off and seed rot caused by Pythium species are a problem throughout

the state of Ohio. The primary effects are decreased stands, which can lead to replanting,

and in many cases decreased yields (Broders et al., 2009; Campa et al., 2010; Murillo-

Williams & Pedersen, 2008; Zhang & Yang, 2000). Most species of Pythium are thought to thrive in cool, wet conditions. However, recent evidence from pathogenicity assays indicates that there are temperature differences within and among different Pythium spp.

(Ellis et al., 2013; Martin & Loper, 1999). Therefore, temperature is a key element to incorporate into any pathogenicity experiment (Rosso et al., 2008). The most aggressive

Pythium species affecting soybean in Ohio were Pythium irregulare, Pythium ultimum

var. ultimum, and Pythium ultimum var. sporangiiferum (Broders et al., 2007).

One of the primary reasons for the prevalence of seed and seedling blights caused by

Pythium in Ohio is due to the large number of fields with high clay content. Clay retains

water, and therefore, provides favorable environmental conditions, i.e. saturated soil

conditions, for zoospore development, and free water to locate soybean roots (Broders et

al., 2007). Numerous management strategies have been evaluated for management of

Pythium spp., albeit with some limited success: crop rotation, seed treatments, and host

resistance.

Crop rotation is one of several management strategies for numerous field crop diseases.

However, many pathogens, such as Pythium, have multiple hosts, meaning that crop

rotation is not as effective; at least not without another form of management along with it.

Zhang and Yang (1998, 2000) evaluated inoculum levels of Pythium in corn and soybean

crop rotation for three field seasons, and reported that the inoculum did not decrease.

They indicated that the reason for no change in inoculum levels was because both corn

and soybean are susceptible to the Pythium spp. in these fields.

Broders et al. (2007) identified numerous Pythium spp. that were able to cause seed rot on both corn and soybean. The majority of Pythium spp. have a broad host range while a few such as P. graminicola are limited to one or few hosts.

The overwintering structure of Pythium, the oospore, can also survive extreme conditions, and stay dormant in fields for years (Campa et al., 2010; Rosso et al., 2008).

The oospore makes it unlikely to rid any already infested field of the pathogen. While helpful in decreasing inoculum, other forms of management are necessary.

Fungicide seed treatments are a proven effective management strategy (Bradley, 2008;

Conley and Esker, 2012; Dorrance et al., 2004; Dorrance et al., 2009). Metalaxyl and

strobilurins are currently the primary fungicides for Pythium control. Metalaxyl, an

acylalanine fungicide targeted towards oomycetes, has been used for a long time as a

seed treatment application against Pythium species and Phytophthora sojae. The specific

mode of action for metalaxyl is that it inhibits ribosomal RNA synthesis (Cohen &

Coffey, 1986). As a result of the specific mode of action of metalaxyl, some Pythium

species have been identified that are insensitive to this chemistry (Broders et al., 2007;

Dorrance et al., 2004; Moorman et al., 2004; Olson et al., 2013). For some of these

species, it is not clear if they were always insensitive to metalaxyl or if they developed

this insensitivity following repeated exposure. Due to the increasing number of reports of insensitivity to metalaxyl, other fungicides have been studied for their effectiveness

against Pythium species.

Strobilurin fungicides, or Quinone outside Inhibitors (Qol), are another type of fungicide

used to manage Pythium as well as other fungal pathogens (Broders et al., 2007; Ypema

& Gold, 1999). This group of fungicides blocks electron transfer from the cytochrome

bc1 complex and the QoI site. This ultimately inhibits ATP synthesis, which prevents

spore germination, helping to reduce the rate of infection of Pythium species in the fields

(FRAC, 2013).

Originally derived from Strobilurus tenacellus, a wood rotting fungus, strobilurins were

eventually modified by scientists to be more stable in the environment (Vincelli, 2002).

They have a broad spectrum of pathogens, including oomycetes, but expanding to true

fungi such as rust (Vincelli, 2002; Ypema & Gold, 1999).

Strobilurins are effective as a preventative fungicide. Some isolates (not all), of the

species P. dissotocum, P. selbyii, P. delawarii, P. schmitthenerii, and P. torulosum were

resistant to mefenoxam (Broders et al., 2007). In some cases, isolates of the same species

reacted differently to the fungicide revealing even more difficulties with Pythium

management. In addition, there were differences among a few isolates of several

Pythium spp. for sensitivity towards strobilurin fungicides (Broders et al., 2007). For

example, P. irregulare, P. sylvaticum, and P. ultimum all grew on the media with

different strobilurin active ingredients. It is crucial to evaluate both the efficacy of metalaxyl and strobilurin fungicides as well as the effective rate against a larger collection of isolates. The most effective strobilurin towards 58 isolates representing 12 species evaluated by Broders et al. (2007) was pyraclostrobin.

In a previous study by Dorrance and McClure (2001), higher rates of metalaxyl provided more protection against infection by Phytophthora sojae. This raises the question about what rate of metalaxyl should be used for control of the diversity of Pythium species.

Broders et al. (2007), hypothesized that most Pythium species should be sensitive to the combination of metalaxyl and a strobilurin fungicide, as they inhibit growth using two different modes of action. Identifying the most effective rate of each of these fungicides is very important for the use of seed treatments as a management tool.

Past studies have proven that seed treatments are an effective form of management of seed and seedling blights in soybean; however, their effectiveness is dependent on cultivar, environmental conditions, and overall quality of the seed (Bradley, 2008;

Dorrance et al., 2009; Esker and Conley, 2012; McGee, 1986). Thus, seed treatments alone may not be effective in managing some Pythium species. However, if seed treatment is utilized with additional management strategies, the combination may be effective in limiting the development of seed and seedling blights. For example, combining seed treatment with a soybean cultivar that has partial resistance to the

predominant Pythium spp, may be an effective disease management strategy. Albeit,

partial resistance in soybean towards Pythium has not been studied in depth.

Resistance in soybeans is a key management strategy for oomycetes; it’s very effective.

There have been several recent studies that have focused on identifying and

characterizing sources of resistance to Pythium spp. Kirkpatrick et al. (2006), identified

resistance to Pythium ultimum in soybean cultivar Archer (Bates et al., 2008). In a recent

study by Ellis et al. (2013), resistance to Pythium irregulare was evaluated in 65

genotypes and identified the highest level of resistance in PI 424354. Since most fields

have more than one Pythium species, it would be ideal to find a cultivar that has

resistance to many Pythium species.

Resistance to pathogens can be examined in a number of different ways. Breeders and

pathologists often search for resistance just in elite germplasm, advanced breeding lines,

etc. (Stoskopf et al., 1993). Once resistance is characterized, these populations are mapped for resistance genes. This is why genotypes of specifically developed populations are used for these resistance evaluations. Thus, the importance of identifying resistance in elite soybean lines such as Genetic Gain, NAM parents, as well as soybean genotypes that have resistance towards other root pathogens (Dorrance et al., unpublished data; Ellis et al., 2013). If quantitative resistance is found to Pythium ultimum var. ultimum and

Pythium ultimum var. sporangiiferum, then these lines will need to be evaluated against

other key Pythium species.

Therefore the first objectives of this study were to: i) to identify soybean genotypes with resistance towards Pythium ultimum var. ultimum and Pythium ultimum var. sporangiiferum among parental lines that have served as elite germplasm (genetic gain) as well as parents for population development; and ii) compare the response of specific genotypes to Pythium ultimum var. ultimum and Pythium ultimum var. sporangiiferum to identify if resistance is conferred to both species.

In this project, the efficacy of both metalaxyl and pyraclostrobin was also evaluated towards a collection of Pythium species recovered from fields where seed disease is common. Approximately 292 isolates were screened first for sensitivity to metalaxyl using an amended broth assay as a preliminary screen. The second assay evaluated sensitivity to pyraclostrobin fungicide at the current commercial rate of 234.38 ppm (0.6 fl oz/cwt). Approximately 127 Pythium isolates were screened for sensitivity to pyraclostrobin.

Objective 1. Identify sources of resistance towards Pythium ultimum var. ultimum and

Pythium ultimum var. sporangiiferum in soybean.

Hypotheses to be tested:

Resistance towards these two species will be expressed as a reduction in overall root rot, and soybean genotypes will express similar levels of resistance towards both species.

Objective 2. Identify fungicides with efficacy towards the key Pythium spp. causing seed

and seedling blight of soybean in Ohio.

A. Confirm which isolates of species of Pythium are sensitive and insensitive to

metalaxyl and pyraclostrobin.

Hypothesis to be tested: There will be differences in response to metalaxyl and pyraclostrobin between Pythium and Phytophthora isolates.

Chapter 2: Quantitative Resistance in Elite Soybean Germplasm to Pythium

ultimum var. ultimum and Pythium ultimum var. sporangiiferum

Introduction

With over 4 million acres produced in the state, soybean [Glycine max (L.) Merr] is

Ohio’s number one crop, which is closely followed by corn [Maize- L. Zea mays L.]

(Beuerlein & Dorrance, 2005; National Agricultural Statistics Service, 2013). The

primary goal of soybean producers is to obtain the highest yield and the highest quality

product annually. Soybean damping-off and seed rot caused by Pythium species is a

problem throughout the state of Ohio. The primary effects are decreased stands, which

can lead to replanting, and in many cases decreased yields (Broders et al., 2009; Campa

et al., 2010; Murillo-Williams & Pedersen, 2008; Zhang & Yang, 2000). Most species of

Pythium are thought to thrive in cool, wet conditions. However, evidence from

pathogenicity assays indicated that there are temperature differences within and among

different Pythium spp. (Ellis et al., 2013; Martin & Loper, 1999). There have been more

than 25 Pythium species identified that affect soybean in Ohio as seed and seedling

pathogens (Broders et al., 2007, 2009; Dorrance et al., 2004; Ellis et al., 2012 ). Among

the most aggressive and prevalent were Pythium irregulare, Pythium ultimum var.

ultimum, and Pythium ultimum var. sporangiiferum (Broders et al., 2009).

Resistance in soybeans is a key management strategy for some oomycetes (i.e.

Phytophthora sojae), but very few studies have focused on identifying and characterizing

resistance towards Pythium spp.. Kirkpatrick et al. (2006), identified resistance to

Pythium ultimum in the soybean cultivar Archer. In a recent study by Ellis et al. (2013),

resistance to Pythium irregulare was evaluated in 65 genotypes; close to one third of

these had moderate to high resistance. The highest level of resistance was identified in

PI424354. Since most fields have more than one Pythium species, it would be ideal to

find a source of resistance to many Pythium species.

Sources of resistance to pathogens can be identified in a number of different ways.

Breeders and pathologists often search for resistance first in elite germplasm and

advanced breeding lines (Stoskopf et al., 1993). The Ohio State University and other

public land grant universities have also developed a large number of soybean mapping

populations that are segregating for different traits. The soybean genotypes, which were

used as parents of specifically developed populations, are a priority for these resistance

evaluations. If the parents used to develop a population have a differential response

towards one or more of the Pythium spp., this will allow for rapid identification of loci

that are associated with resistance. This identification is called quantitative trait locus

(QTL), and is important for selecting parents with quantitative resistance to pathogens

(Varshey et al., 2014).

For example, the genetic gain population is a group of soybean cultivars that represent the highest yielding genotype for each decade since 1924. These were used in a study to assess grain yield, which has increased over 23 Kg ha -1 (Wilson et al., 2014). This increase in yield was partially due to genetic advances but also to better overall agronomic practices (Wilson et al., 2014).

The Nested Association Mapping (NAM) population is a group of soybean genotypes that were all crossed to one parent to develop populations that will have a high-resolution map to identify QTL alleles. The soybean NAM population originates from 40 parents that were selected for high yield and drought tolerance. These soybean genotypes are also very desirable to screen for resistance because they will be ideal for finding QTL linkages

(Diers et al., personal communication, June 2014).

Therefore the objectives of this study were to: i) to identify soybean genotypes with resistance towards Pythium ultimum var. ultimum and Pythium ultimum var. sporangiiferum among parental lines that have served as elite germplasm (genetic gain) as well as parents for population development; and ii) compare the response of specific genotypes to Pythium ultimum var. ultimum and Pythium ultimum var. sporangiiferum to identify if resistance is conferred to both species.

Materials & Methods:

Pythium Isolates.

Pythium ultimum var. ultimum and Pythium ultimum var. sporangiiferum were isolated

from soybeans in 2005 by Kirk Broders from different locations in Ohio and are listed in

Table 1. The isolates were stored at 15°C on Potato Carrot Agar (PCA) in Whatman

vials, until used. Two isolates (Will 1-6-7: P. ultimum var. sporangiiferum, and Miami

1-3-7: P. ultimum var. ultimum) were selected for evaluation of resistance in soybean.

Each isolate’s species identification was verified by both morphological features and

comparison of the internal transcribed spacer (ITS) sequence. For ITS, each isolate was

grown in potato-carrot broth for 3 days in the dark. The mycelium was crushed in an

extraction buffer using a pestle in a 1.5 ml eppendorf tube and DNA extracted using the

DNeasy Plant Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s

instructions. After extracting the DNA, it was cleaned up using Polymerase Chain

Reaction (PCR). PCR selects for a specific part of DNA for sequencing. In this case, we used PCR to get the ITS region for sequencing. DNA quantity and quality was measured with a NanoDrop 1000 (Fisher Scientific, Wilmington, DE), diluted to 5 ng/ul, and sequenced at the MCIC in Wooster, Ohio.

Inoculum for greenhouse assays.

Inoculum of each isolate was grown in Spawn bags (Myco Supply, Pittsburgh, PA)

containing 950 ml of ‘Clean Play Sand’ (Quikrete, Ravenna, Ohio), 50 ml cornmeal

(Quaker Oats Company, Chicago, IL), and 250 ml of deionized water. Each bag was mixed and sterilized for an hour on two consecutive days, prior to inoculation. For each isolate, eight 5-mm plugs from a 3-4 day culture grown on Potato carrot agar (PCA) at

20°C, were placed into a bag. Each bag was sealed with a sealer-electrical impulse

(Harbor Freight Tools; Calabasas, CA). To ensure even mycelial growth, the inoculum bags were mixed manually every other day for ten days.

Greenhouse Studies.

To test the soybean genotypes for resistance, a single spawn bag was mixed with 4-liters of fine vermiculite (Perlite Vermiculite Packaging Industries, North Bloomfield, Ohio) in a 1:4 ratio, making approximately 15 cups per bag. Three hundred ml of the inoculum mixture was placed on top of 100 ml of coarse vermiculite in a 500 ml cup. The cups were watered 3 times, over 24 hours, prior to planting. Eight seeds of each line was planted directly on inoculum mixture and covered with another 100 ml of coarse vermiculite. After planting, cups were watered 3 times a day to ensure saturation to favor Pythium growth. The greenhouse temperature was set for 22 °C, and the humidity was set for 20%. However, these conditions varied.

Soybean Genotypes.

There were 91 soybean genotypes from the Ohio State University (OSU) breeders (Table

16 & Table 17), 43 from the Nested Association Mapping (Table 8 & Table 9), 62 from the Genetic Gain study (Table 14 & Table 15), 41 from University of Missouri breeders

(Table 10 & Table 11), and 44 from Saghai Maroof at Virginia Tech (Table 12 & Table

13). Each group of soybean genotypes was evaluated separately. There were 10 soybean genotypes that were included in every experiment. It is crucial to include these checks

across all experiments to ensure they are responding the same to the inoculum throughout

time.

Each experiment was arranged in a randomized split-plot design to prevent cross

contamination among the inoculum (Pythium ultimum var. ultimum, Pythium ultimum

var. sporangiiferum, and non-inoculated), which was the main plot, and soybean

genotype, which was the sub-plot. The treatment (isolate x genotype) was replicated 3

times within each experiment. Each experiment was repeated once so that every genotype was evaluated at least 2 times.

Data collection.

Approximately 2 weeks after planting, or VC growth stage, the plants were removed from cups and roots were washed to remove inoculum. The soybeans were wrapped in a paper towel and placed in a 4 °C cold room. Data collected on these plants after washing included: height of 3 plants, total final stand, plant weight, root weight and root rot score

(within 48 hours of washing). The root rot scale was from 1-5, where 1= all roots healthy, with no symptoms on root system; 2= 1-20% of root system has visible lesions on lateral roots; 3= 21-75% of roots showing visible symptoms, with symptoms

beginning to show on tap root; 4= 76-100% of roots infected with symptoms on lateral

roots and tap root; and 5= complete root rot, no germination of seed (Figure 6).

Comparison between Pythium species.

Once all the genotypes were tested at least twice for resistance, two genotypes representing each resistance class (highly resistant, moderately resistant, moderately

susceptible, and susceptible) were selected for each species. The same assays and data

collection were repeated as before, but with 4 representative isolates of Pythium ultimum

var. ultimum (Wyan 1-1-9, Miami 1-3-7, Miami 1-3-14, and Pick 1-3-5) and Pythium ultimum var. sporangiiferum (Will 1-6-7, Fay 1-1-3, Erie 2-4-2, and Miami 1-1-5).

Data analyses.

Each set or group of soybean genotypes was analyzed separately. An Analysis of

Variance on the combined experiments for each set of soybean genotypes was done using

PROC GLM in SAS (Version 9.3; SAS Institute Inc.). Mean separation among genotypes was based on Fishers protected least significant difference (LSD) (P≤0.05).

Results.

Approximately 300 soybean genotypes were evaluated for resistance to Pythium ultimum var. ultimum and Pythium ultimum var. sporangiiferum. Each of the soybean genotypes developed seed or root rot to each of the isolates, albeit at different levels in all experiments. There were highly significant differences among the genotypes for the root

rot that developed following inoculation with both Pythium species (Table 2-7). There were also differences for all other parameters- percent stand, mean height, root weight, and plant weight.

Checks

There were 10 genotypes used as controls across experiments. Conrad was excluded from the final analysis of checks due to problems with the seed. The Virginia Tech experiments were also excluded due to less disease in those 2 experiments. Once analyzed, there was not an isolate x genotype interaction (Root score P-value= 0.9152,

Root weight P-value= 0.2190, Appendix A).

Nested Association Mapping

There was a clear separation between resistance to Pythium ultimum var. ultimum and

Pythium ultimum var. sporangiiferum. More soybean genotypes within the NAM

population had low root rot scores (<3.0) and higher stand following inoculation with P.

ultimum var. ultimum compared to P. ultimum var. sporangiiferum (Figure 1), which all had root scores >3.0. All of the soybean genotypes were highly susceptible to P. ultimum var. sporangiiferum. For all measurements taken, the experiments were significantly different except for the percent of the control root weight, (P = 0.5989). Pythium is a root rotter, so this could indicate that there was not a difference between experiments. Overall, the isolates were highly significantly different for all measurements (P= <0.0001). The

isolate x genotype interaction was not significant for the percent of the control of plant

weight (P = 0.8555) and percent of the control root weight (P=0.9758) (Table 2).

For % control root weight there was a significant interaction between experiment and

genotype (P=0.0255). This indicates that the isolate could have been more aggressive in

one experiment over the other. However, there was not a significant interaction for

experiment x line x isolate (P= 0.4429, Table 2). Since there is not a significant

interaction between the 3 factors, the experiments were combined for final analysis.

University of Missouri

The genotypes from the University of Missouri also differed in resistance to the two

varieties of Pythium ultimum. Most genotypes had root rot scores ≤3, indicating

resistance to P. ultimum var. sporangiiferum (Figure 2). Whereas all of the root rot scores

were ≥3 for P. ultimum var. ultimum, indicating there was no resistance (Figure 2).

For isolates and experiments, there was a P-value < 0.05 for all measurements taken.

There was a significant difference between isolates and between experiments. There was

a significant interaction for isolate x line, except for percent of control root weight

(P=0.0865) (Table 3). There was not a significant interaction for experiment x line x isolate in percent of control root weight (P=0.1624,Table 3).

Virginia Tech Genotypes

There was a high level of partial resistance to both species with the genotypes selected by

Saghai Maroof of Virginia Tech (Figure 3). Forty-five lines were moderately resistant 20

(mean root score between 2-3) to Pythium ultimum var. ultimum compared to 30 lines

expressing moderate resistance towards Pythium ultimum var. sporangiiferum. Seven

genotypes had high levels of resistance (1.0-2.0) to Pythium ultimum var. ultimum while

24 genotypes were highly susceptible (3.9-5.0) towards Pythium ultimum var.

sporangiiferum (Figure 3). These numbers seem to be similar; however, there was a

significant interaction for all measurements (P-value ≤0.05) for isolate x line (Table 4).

Therefore, the isolates were responding differently to the genotypes within this population from each experiment. There was a significant difference in plant weight (P=

0.045) and root weight (P=0.027) for experiments (Table 4). However, percent control of these was not. There was a significant difference (P≤0.05) between isolates for all measurements. There was a significant interaction between experiment x line for all variables except percent stand (P= 0.4061) and root rot score (P=0.7618, Table 4).

Genetic Gain

This group of genotypes represents elite soybean cultivars and all responded similarly to both of the Pythium species. Burlison had the lowest root rot score (mean= 3.2,Table 14) for P. ultimum var. ultimum, and Corsoy (mean= 3.3) for Pythium ultimum var. sporangiiferum (Table 15). Over 50 lines were highly susceptible, with root rot scores

between 3.9-5.0, in response to inoculation with one or both of the Pythium species

(Figure 4). There was a significant difference for both experiment and isolates (P≤0.05).

There was a significant interaction for isolate x genotype (P ≤0.05) for all measurements

(Table 5). The Pythium ultimum variety had an effect on the genotype resistance 21

response. For plant weight (P=0.0127) and root weight (P=0.0001) there was a significant

interaction for experiment x genotype x isolate (Table 5).

Ohio State University Genotypes

There was variability among the OSU population of soybeans. Over 70 lines conveyed

moderate resistance to Pythium ultimum var. sporangiiferum, and over 40 lines to

Pythium ultimum var. ultimum. Hutchinson, PI424354, OSU038, OSU015, OSU028,

PI408225A, OSU049, and OSU027 all conferred high levels of resistance (0-1) to

Pythium ultimum var. sporangiiferum (Figure 5; Table 17). Danbaekkong and Williams

had high levels of resistance to Pythium ultimum var. ultimum (Figure 5; Table 16). There

was a significant difference between experiments for mean height, plant weight, and root

weight (P< 0.0001) (Table 6). There was a significant interaction for isolate x genotype

in the plant weight (P=0.0518) (Table 6).

Comparison of isolates and genotypes

To determine if there were differences or similarities in response to both varieties of P.

ultimum, the response of 11 varieties to 8 isolates were compared. There were 2

genotypes of each resistance category (resistant, moderately resistant, moderately

susceptible, and susceptible) for each P. ultimum variety evaluated (Table 7). In this

evaluation, Dennison, which was a check, had the highest level of resistance based on the

lowest mean root rot score of 2.3 to four isolates of Pythium ultimum var. ultimum, and

1.8 to four isolates of Pythium ultimum var. sporangiiferum (Table 7). Williams was the most susceptible variety for Pythium ultimum var. ultimum with a mean score of 3.7 22

(Table 7). Jack (mean score=4.7) and Wooster (mean score=4.6) were both susceptible to

Pythium ultimum var. sporangiiferum (Table 7). Wooster was susceptible to both P. ultimum varieties. For OhioFg1 there was a wide range of responses from low root rot scores (mean score=1.7) for Miami 1-3-14, to high root rot scores for Wyan 1-1-9 (mean score= 4.2) (Table 7). Isolate Wyan1-1-9 was the most aggressive among the Pythium ultimum var. ultimum isolates with an average root score of 4.6 (Table 7). Whereas

Miami 1-3-14 was a weak isolate. The most aggressive isolate for Pythium ultimum var. sporangiiferum was Fay 1-1-3.

Ultimately this is strong evidence that there are differences in the resistance response among soybean genotypes towards isolates of P. ultimum var. ultimum and P. ultimum var. sporangiiferum. This also means that there are differences in the level of pathogenicity among isolates of the two P. ultimum varieties. These lines tested would be good checks for future assays. However, the most aggressive isolate in the table should also be used.

There were different levels of disease between the isolates of the two varieties. There was strong evidence of an isolate x line interaction in the Virginia Tech experiments and the genetic gain experiments.

Discussion

Two varieties of the species P. ultimum var. ultimum & sporangiiferum were used to evaluate a large collection of soybean genotypes for resistance. These two varieties of P. ultimum have been scrutinized as to whether or not they are completely separate species or part of a species complex. Pythium ultimum var. ultimum is more prevalent, has a larger host range and has molecularly more base pairs than Pythium ultimum v. sporangiiferum (Pythium Genome Database, 2014). Both varieties of Pythium ultimum are aggressive towards corn and soybean seeds and seedlings. They are commonly found in many Ohio fields, which is why it was crucial to identify resistance against this pathogen (Broders et al., 2009).

The soybean genotypes evaluated in this study came from soybean breeders at University of Missouri, Virginia Tech, OSU, as well as North Central region for the NAM, and

Genetic Gain populations. Many of the genotypes selected for this study have resistance to other oomycetes pathogens or other soybean pests and pathogens. Within all of these different groups of genotypes, resistance was expressed towards Pythium ultimum in the soybean roots as reduction in the amount of root rot and increase in root weight following inoculation in a greenhouse seed and seedling assay.

The genotype Hutchinson expressed a high level of resistance to P. ultimum v. ultimum.

There was previous work done by Kirkpatrick et al. (2006) that concluded that

Hutchinson was not resistant to P. ultimum but did not define which variety. This line

should be evaluated with more isolates of Pythium ultimum var. ultimum prior to mapping

specific loci for resistance.

The genotypes from Missouri, which were later maturity group, had higher levels of resistance towards P. ultimum var. sporangiiferum, than for P. ultimum var. ultimum

(Figure 2), as did the genotypes from the OSU breeders (Figure 5). Soybean genotype maturity group is important to know because it correlates to when the soybean is supposed to flower. If the wrong maturity group is used in a region, it could lead to more disease and less yield. However, genotypes from Virginia Tech and the NAM population, which are later and earlier maturity groups, respectively, were more resistant to P. ultimum var. ultimum. The reaction can change for these “on the line” genotypes. They are the most variable with environmental conditions. Environmental conditions easily fluctuate in greenhouses, and especially in the field. Therefore, making it difficult to make a correlation between maturity group and resistance.

The genetic gain population demonstrated low levels of resistance. However, there were a lot of intermediate root rot scores. Since it is elite soybean germplasm, one explanation could be that its resistance is favored towards another pathogen other than P. ultimum.

With this conclusion, other management strategies would need to be integrated.

There were differences between experiments. All of these experiments, except for the last experiment testing for cultivar by isolate interactions, were completed in a greenhouse. A

25 greenhouse environment can be variable. Therefore, some conditions could have favored the pathogen, while others may have favored the soybean. This is also likely to occur in the field, which is why it’s important to examine resistance in all environments. In order to reduce variability, it is important to run experiments as close together in time as possible, monitor the temperature and humidity, and run all experiments in the same greenhouse space.

There was variability in the pathogenicity of the isolates. This outcome raises the question of genetic variability within species concerning resistance. The next part of this research will be to map these resistant genotypes and define which genes contain resistance towards these two Pythium species.

There were differences in the pathogenicity within both varieties of Pythium ultimum.

Overall most experiments had similar results. However, for P. ultimum var. sporangiiferum, the NAM and Genetic Gain had higher root scores than the other 3 populations, concluding that there was variation in the greenhouse studies.

When comparing the two varieties, P. ultimum var. sporangiiferum and P. ultimum var. ultimum, with the checks, between the experiments, there is not an interaction between isolate and genotype. Therefore, there no conclusions drawn in terms of these two varieties being two separate species. There was less disease in the Virginia Tech

26 experiments, and there were questions with seed quality of Conrad. Both of these were not used in the final analysis comparing the checks across experiments.

It is important to use integrated management strategies to limit the losses from these pathogens. Since most fields contain more than one Pythium species it is important to use a cultivar that confers resistance to multiple species. However, from the data compiled from these experiments, I would recommend using Dennison or Hutchinson seed with a seed treatment. OhioFG1 varies in its resistance, but is food grade seed. If food grade quality is needed, OhioFG1 is reasonable.

For future studies with Pythium ultimum, the Fay 1-1-3 should be used. Fay 1-1-3 displayed a clear separation between resistant and susceptible, therefore making it easier to differentiate between what’s susceptible and what’s resistant.

Table 1. Isolates of Pythium ultimum that originate in Ohio and were used to evaluate soybean genotypes for resistance in greenhouse cup experiments.

Isolate Code Species County Aug 1-1-3 Pythium ultimum var. ultimum Auglaize Aug 1-1-5 Pythium ultimum var. ultimum Auglaize Craw 1-2-3 Pythium ultimum var. ultimum Crawford Craw 1-1-13 Pythium ultimum var. ultimum Crawford Darke 3-3-8 Pythium ultimum var. ultimum Darke Erie 2-4-2 Pythium ultimum v. Erie sporangiiferum Hen 1-2-8 Pythium ultimum var. ultimum Henry Fay 1-1-3 Pythium ultimum v. Fayette sporangiiferum Mad 2-6-7 Pythium ultimum var. ultimum Madison Miami 1-3-14 Pythium ultimum var. ultimum Miami Miami 1-1-5 Pythium ultimum v. Miami sporangiiferum Miami 1-1-8 Pythium ultimum var. ultimum Miami Miami 1-3-7 Pythium ultimum var. ultimum Miami Pick 1-3-5 Pythium ultimum var. ultimum Pickaway Sand 1-3-13 Pythium ultimum var. ultimum Sandusky Will 1-6-7 Pythium ultimum v. Williams sporangiiferum Wyan 1-1-9 Pythium ultimum var. ultimum Wyandot

Figure 1. The number of soybean genotypes of the Nested Association Mapping parent population with (A) mean heights (mm) of 3 seedlings, (B) mean percent plant stand of 8 soybeans, (C) mean plant weight (g) of all seedlings, (D) mean root weight (g) of all seedlings, and (E) root rot score of overall roots: (R – 1.0-2.0), moderate resistance (MR

–2.0-3.0), moderate susceptibility (MS- 3.0-3.8), and susceptibility (S- 3.8-5.0) to

Pythium ultimum var. ultimum and Pythium ultimum var. sporangiiferum (N= 48).

Table 2. Analysis of variance (ANOVA) for Nested Association Mapping population evaluated for resistance to Will 1-6-7 (P.

ultimum var. sporangiiferum) and Miami 1-3-7 (P. ultimum var. ultimum) in greenhouse assays.

Stand (%Stand) Root Score Mean Height Plant Weight Root Weight % Control Plant % Control Root Weight Weight Source DF F Pr > F F Value Pr > F F Value Pr > F F Value Pr > F F Pr > F F Pr > F F Pr > F Value Value Value Value exp 1 106.46 <.0001 5.4 0.0207 22.63 <.0001 128.26 <.0001 107.06 <.0001 8.11 0.0049 0.28 0.5989 isol 2 547.8 <.0001 1846.96 <.0001 1528.75 <.0001 1320.03 <.0001 1651.1 <.0001 709.8 <.0001 982.03 <.0001 3 5

exp*iso 2 25.55 <.0001 38.84 <.0001 41.3 <.0001 31.97 <.0001 35.98 <.0001 16.81 <.0001 21.62 <.0001

30 l

8 10.62 <.0001 10.37 <.0001 8.11 <.0001 15.09 <.0001 25 <.0001 12.48 <.0001 33.59 <.0001 exp(rep *isol) line 47 6.61 <.0001 3.79 <.0001 11.12 <.0001 14.78 <.0001 14.49 <.0001 3.18 <.0001 1.86 0.0019

exp*lin 47 0.93 0.6151 1.4 0.0497 1.07 0.3617 0.81 0.8154 0.87 0.7204 1.48 0.0356 1.53 0.0255 e 188 0.97 0.5846 1.04 0.3663 0.95 0.6671 0.9 0.7982 0.96 0.6301 1.31 0.0307 1.58 0.0009 exp(rep *line)

Continued 30

Table 2 continued

Stand (%Stand) Root Score Mean Height Plant Weight Root Weight % Control Plant % Control Root Weight Weight Source DF F Pr > F F Value Pr > F F Value Pr > F F Value Pr > F F Pr > F F Pr > F F Pr > F Value Value Value Value line*iso 94 1.63 0.0007 1.33 0.0351 1.68 0.0004 1.58 0.0015 2.69 <.0001 0.77 0.8555 0.61 0.9758 l

exp*lin 94 0.85 0.837 0.83 0.8633 0.79 0.9159 0.56 0.9995 0.75 0.9517 1.18 0.2245 1.02 0.4429 e*isol

Figure 2. The number of soybean genotypes of the Missouri population with (A) mean

heights (mm) of 3 seedlings, (B) mean percent plant stand of 8 soybeans, (C) mean plant

weight (g) of all seedlings, (D) mean root weight (g) of all seedlings, and (E) root rot score of overall roots: (R – 1.0-2.0), moderate resistance (MR –2.0-3.0), moderate

susceptibility (MS- 3.0-3.8), and susceptibility (S- 3.8-5.0) to Pythium ultimum var.

ultimum and Pythium ultimum var. sporangiiferum (N= 48).

Table 3. Analysis of variance (ANOVA) for The University of Missouri population evaluated for resistance to Will 1-6-7 (P.

ultimum var. sporangiiferum) and Miami 1-3-7 (P. ultimum var. ultimum) in greenhouse assays.

Stand (%stand) Root Score Height Plant weight Root Weight % Control % Control Plant Weight Root Weight Source DF F Pr > F F Pr > F F Pr > F F Pr > F F Pr > F F Pr > F F Pr > F Value Value Value Value Value Value Value exp 1 31.69 0.0049 39.46 0.0033 14.35 0.0193 9.49 0.0369 8.46 0.0438 11.8 0.0264 52.43 0.0019 isol 1 286.38 <.0001 46.68 0.0024 106.88 0.0005 63.14 0.0014 47.59 0.0023 62.77 0.0014 40.97 0.0031

exp*isol 1 8.5 0.0434 0 0.9551 14.25 0.0195 2.14 0.2174 0.86 0.407 3.61 0.13 0.22 0.6655 exp(rep*isol) 4 1.15 0.3351 6.71 <.0001 2.34 0.0585 6.23 0.0001 6.77 <.0001 2.84 0.0271 4.48 0.002 33

line 32 17.9 <.0001 6.73 <.0001 11.28 <.0001 20.75 <.0001 13.88 <.0001 3.03 <.0001 1.38 0.1071

exp*line 32 1.42 0.0906 1 0.4769 0.96 0.5305 0.77 0.8023 0.64 0.9295 1.25 0.1915 1.09 0.3555 exp(rep*line) 128 1.16 0.1984 0.8 0.8979 1.13 0.2521 1.41 0.028 1.4 0.0301 1.25 0.1098 1.25 0.1026

line*isol 32 2.18 0.0012 1.65 0.0278 1.92 0.0058 2.21 0.001 2.55 0.0001 1.74 0.0171 1.43 0.0865

exp*line*isol 32 2.78 <.0001 1.35 0.1264 1.98 0.0041 2.14 0.0016 1.59 0.0367 1.4 0.0979 1.29 0.1624

Figure 3. The number of soybean genotypes of the Virginia Tech population with (A)

mean heights (mm) of 3 seedlings, (B) mean percent plant stand of 8 soybeans, (C) mean

plant weight (g) of all seedlings, (D) mean root weight (g) of all seedlings, and (E) root rot score of overall roots: (R – 1.0-2.0), moderate resistance (MR –2.0-3.0), moderate

susceptibility (MS- 3.0-3.8), and susceptibility (S- 3.8-5.0) to Pythium ultimum var.

ultimum and Pythium ultimum var. sporangiiferum (N= 54).

Table 4. Analysis of variance (ANOVA) Virginia Tech population evaluated for resistance to Will 1-6-7 (P. ultimum var.

sporangiiferum) and Miami 1-3-7 (P. ultimum var. ultimum) in greenhouse assays.

Stand (%Stand) Root Score Mean Height Plant Weight Root Weight % Control % Control Root Plant Weight Weight Source DF F Pr > F F Pr > F F Pr > F F Pr > F F Pr > F F Pr > F F Pr > F Value Value Value Value Value Value Value Exp 1 0.28 0.6219 5.32 0.0823 1.79 0.2515 8.33 0.0447 11.54 0.0274 0 0.9918 1.74 0.2577 Isol 1 52.27 0.0019 126.18 0.0004 48.81 0.0022 59.59 0.0015 30.27 0.0053 93.55 0.0006 27.85 0.0062

exp*isol 1 2.56 0.1852 12.11 0.0254 5.22 0.0844 5.13 0.0863 2.72 0.1742 3.28 0.1442 0.46 0.5361

35 4 1.86 0.1192 0.98 0.4195 12.75 <.0001 6 0.0001 13.98 <.0001 3.96 0.004 14.76 <.0001

exp(rep*isol) Line 52 10.21 <.0001 3.53 <.0001 13.49 <.0001 19.65 <.0001 9.92 <.0001 3.66 <.0001 1.79 0.0022

exp*line 52 1.04 0.4061 0.87 0.7618 1.41 0.0499 1.83 0.0015 1.91 0.0008 1.73 0.0038 1.58 0.0133 208 1.28 0.0363 1.24 0.0559 1.29 0.0326 1.19 0.0997 1.16 0.1434 1.86 <.0001 2.16 <.0001 exp(rep*line) line*isol 52 3.27 <.0001 1.83 0.0015 2.18 <.0001 2.45 <.0001 2.29 <.0001 2.92 <.0001 2.12 <.0001

exp*line*isol 52 1.49 0.027 1.36 0.0667 1.13 0.2733 1.44 0.0394 1.57 0.0135 1.36 0.0685 1.41 0.0471

Figure 4. The number of soybean genotypes of the Genetic Gain population with (A)

mean heights (mm) of 3 seedlings, (B) mean percent plant stand of 8 soybeans, (C) mean

plant weight (g) of all seedlings, (D) mean root weight (g) of all seedlings, and (E) root rot score of overall roots: (R – 1.0-2.0), moderate resistance (MR –2.0-3.0), moderate

susceptibility (MS- 3.0-3.8), and susceptibility (S- 3.8-5.0) to Pythium ultimum var.

ultimum and Pythium ultimum var. sporangiiferum (N= 71).

Table 5. Analysis of variance (ANOVA) for Genetic Gain population evaluated for resistance to Will 1-6-7 (P. ultimum var.

sporangiiferum) and Miami 1-3-7 (P. ultimum var. ultimum) in greenhouse assays.

Stand (%Stand) Root Score Mean Height Plant weight Root Weight

Source DF F Pr > F F Pr > F F Pr > F F Pr > F F Pr > F Value Value Value Value Value Exp 1 266.72 <.0001 49.05 <.0001 321.62 <.0001 220.8 <.0001 134.95 <.0001 Isol 1 124.37 <.0001 21.82 <.0001 211.97 <.0001 103.93 <.0001 52.4 <.0001 37

exp*isol 1 124.37 <.0001 19.18 <.0001 125.51 <.0001 92.97 <.0001 41.95 <.0001 4 2.92 0.0217 2.81 0.0258 6.35 <.0001 2.64 0.0342 2.07 0.0847 exp(rep*isol) Line 70 17.4 <.0001 9.72 <.0001 10.93 <.0001 37.33 <.0001 37.79 <.0001

exp*line 70 1.46 0.0182 1.34 0.0511 1.39 0.0352 1.56 0.0064 1.23 0.1262 268 0.96 0.6276 0.98 0.5588 0.99 0.5319 0.92 0.7613 0.94 0.7046 exp(rep*line) line*isol 70 1.66 0.0024 1.82 0.0004 1.44 0.0216 2.15 <.0001 2.84 <.0001

exp*line*isol 70 1.27 0.096 1.32 0.0628 1.13 0.2469 1.5 0.0127 1.91 0.0001

Figure 5. The number of soybean genotypes of the OSU population with (A) mean

heights (mm) of 3 seedlings, (B) mean percent plant stand of 8 soybeans, (C) mean plant

weight (g) of all seedlings, (D) mean root weight (g) of all seedlings, and (E) root rot score of overall roots: (R – 1.0-2.0), moderate resistance (MR –2.0-3.0), moderate

susceptibility (MS- 3.0-3.8), and susceptibility (S- 3.8-5.0) to Pythium ultimum var.

ultimum and Pythium ultimum var. sporangiiferum (N= 101).

Table 6. Analysis of variance (ANOVA) for the OSU population evaluated for resistance to Will 1-6-7 (P. ultimum var.

sporangiiferum) and Miami 1-3-7 (P. ultimum var. ultimum) in greenhouse assays.

Stand (%Stand) Root Score Mean Height Plant Weigtht Root Weight

Source DF F Pr > F F Pr > F F Pr > F DF F Pr > F F Pr > F Value Value Value Value Value exp 2 0.05 0.9555 1.78 0.1695 10.44 <.0001 2 37.05 <.0001 126.96 <.0001 isol 2 142.01 <.0001 367.71 <.0001 88.11 <.0001 2 250.57 <.0001 325.51 <.0001 exp*isol 3 12.4 <.0001 6.46 0.0003 9.39 <.0001 3 10.44 <.0001 26.68 <.0001

39 line 100 2.11 <.0001 0.76 0.9509 1.42 0.0095 100 3.58 <.0001 2.79 <.0001

line*isol 199 1.14 0.1362 0.8 0.9674 0.8 0.9629 197 1.22 0.0518 1.17 0.0935

Table 7. Comparison of the mean root score (1-5) for soybeans following inoculation with four isolates of both Pythium ultimum var. ultimum, and Pythium ultimum var. sporangiiferum, in a growth chamber assay.

Pythium ultimum var. ultimum Pythium ultimum var. sporangiiferum Cultivar M1-3- M1-3- P1-3-5 W1-1- Mean E2.4. F1.1. M.1.1. W.1.6. Mean 14 7 9 2 3 5 7 Burlison 2.3 2.5 3.7 5.0 3.4 nt nt nt nt nt BC BC BC A Dennison 1.8 2.0 2.5 2.8 2.3 1.7 2.2 2.0 1.5 1.8 BC C C C C D D C Hutchison nt nt nt nt nt 1.7 3.7 2.2 2.2 2.4 C B CD BC Jack nt nt nt nt nt 5.0 5.0 5.0 3.8 4.7 A A A A Kottman 2.2 2.3 4.7 5.0 3.5 2.8 3.5 3.7 2.5 3.1 BC BC AB A B BC B B Ohio FG1 1. 7 2.7 2.8 4.2 2.8 2.0 3.0 2.7 2.2 2.5 C BC C B BC BC BCD BC

Prohio 2.0 2.4 3.7 4.7 2.5 2.3 2.8 3.2 2.3 2.7 BC BC BC AB BC CD BC BC Williams82 2.3 2.2 4.2 5.0 3.4 nt nt nt nt nt BC BC AB A Williams 2.5 2.8 4.5 5.0 3.7 nt nt nt nt nt B B AB A Wooster 3. 7 3.8 5.0 5.0 3.5 5.0 5.0 4.8 3. 7 4.6 A A A A A A A A Wyandot nt nt nt nt nt 2. 7 3.0 3.5 2.2 2.8 B BC B BC Overall 2.3 2.6 3.9 4.6 2.9 3.5 3.4 2.5 Mean

LSD 0.76 0.74 1.29 0.79 0.89 0.81 1.08 0.95

Figure 6. Soybeans inoculated with Pythium spp. Root rot scores from the left to right:

1, 2, 3 and 4 where (R – 1.0-2.0), moderate resistance (MR – 2.0-3.0), moderate

susceptibility (MS- 3.0-3.8), and susceptibility (S- 3.8-5.0) to Pythium ultimum var.

ultimum and Pythium ultimum var. sporangiiferum.

Table 8. P. ultimum v. ultimum NAM population Means of lines evaluated.

Level of Percent Mean Plant Root Root genotype Stand Height Weight Weight Score (mm) (g) (g) 4J10534 93.80 84.30 10.20 3.30 2.00 5M20252 87.50 80.80 10.10 2.80 1.50 C0J09546 79.20 64.00 8.20 2.60 2.00 C0J17368 81.30 73.00 9.20 2.90 2.50 Dennison 85.40 89.50 11.40 4.20 2.50 HS63976 87.50 68.50 10.40 3.20 2.20 Kottman 95.80 72.70 10.90 3.60 1.50 LD003309 72.90 63.30 6.70 2.10 2.20 LD015907 66.70 61.20 5.10 1.20 2.80 LD024485 54.20 65.00 4.80 1.40 3.00 LD029050 81.30 68.90 8.80 2.80 2.30 LG003372 70.80 82.10 7.50 2.10 2.70 LG032979 83.30 70.40 7.70 2.30 2.80 LG033191 75.00 64.50 8.50 2.60 2.80 LG044717 77.10 93.20 7.30 1.90 3.00 LG046000 56.30 47.90 5.40 1.50 3.20 LG054292 89.60 76.50 8.90 2.90 2.00 LG054317 77.10 55.80 7.30 2.30 2.50 LG054464 75.00 71.40 9.50 2.80 2.20 LG054832 70.80 54.60 7.30 2.40 2.30 LG902550 81.30 71.10 7.90 2.40 2.20 LG921255 68.80 56.20 6.20 1.60 2.60 LG941128 70.80 61.70 5.20 1.50 3.00 LG941906 58.30 59.60 5.40 1.40 3.30 LG977012 77.10 81.50 8.40 2.20 2.30 LG981605 58.30 52.60 4.60 1.40 2.30 Magellan 79.20 80.20 7.80 2.40 2.70

42 Continued

Table 8 continued Level of Percent Mean Plant Root Root genotype Stand Height Weight Weight Score (mm) (g) (g) Maverick 64.60 68.40 7.00 2.10 2.60 NE3001 81.30 82.60 7.80 2.40 2.50 OhioFG1 85.40 77.20 11.30 3.50 2.30 P404188A 87.50 78.80 9.30 2.70 2.70 P437169B 72.90 58.20 5.90 1.50 2.50 P507681B 39.60 55.50 3.40 1.00 2.80 PI398881 93.80 81.70 10.90 3.40 2.70 PI427136 75.00 69.00 8.80 2.40 2.30 PI518751 85.40 80.10 9.20 2.90 2.50 PI561370 60.40 70.40 5.60 1.30 3.00 PI574486 87.50 71.80 9.10 2.20 2.70 Prohio 80.70 87.90 8.20 2.50 2.40 S0613640 93.80 84.10 11.50 3.90 2.00 Skylla 64.60 71.00 5.90 1.60 3.00 Streeter 81.30 77.20 9.50 3.10 2.50 Summit 92.50 85.90 8.60 3.20 2.30 U3100612 81.30 68.60 6.60 2.10 3.00 Wooster 35.40 21.20 2.00 0.40 3.80 Wyandot 81.30 74.80 9.80 3.40 2.30 conrad 70.80 68.00 5.80 1.80 2.80 sloan 85.00 74.10 10.00 3.20 2.00 Overall 76.08 70.54 7.81 2.36 2.52 Mean LSD 28.19 19.30 3.52 1.34 0.97

Table 9. NAM population means of lines evaluated with P. ultimum v. sporangiiferum.

Level of Percent Mean Plant Root Root genotype Stand Height Weight Weight Score (mm) (g) (g) 4J10534 58.33 3.27 0.53 3.83 30.86 5M20252 29.17 2.05 0.23 4.00 21.42 C0J09546 27.08 1.35 0.18 4.17 19.38 C0J17368 41.67 2.18 0.32 3.67 33.74 Dennison 70.83 4.38 0.83 3.83 45.52 HS63976 68.75 4.53 0.70 3.67 31.17 Kottman 39.58 2.45 0.47 3.67 24.90 LD003309 58.33 2.80 0.50 3.83 25.05 LD015907 18.75 1.83 0.63 4.67 15.43 LD024485 17.50 0.78 0.14 4.20 16.46 LD029050 27.08 1.38 0.20 4.00 17.27 LG003372 56.25 3.08 0.40 4.00 32.72 LG032979 37.50 2.00 0.32 4.00 16.47 LG033191 54.17 3.00 0.47 4.00 28.68 LG044717 58.33 2.62 0.35 4.00 39.58 LG046000 35.42 1.70 0.22 4.33 16.28 LG054292 52.08 2.93 0.48 3.67 30.67 LG054317 35.42 1.45 0.17 3.67 16.68 LG054464 41.67 2.57 0.47 3.67 30.68 LG054832 85.42 5.28 0.90 3.33 33.33 LG902550 31.25 1.95 0.28 4.00 21.08 LG921255 39.58 2.10 0.30 4.17 19.95 LG941128 8.33 0.32 0.05 4.67 4.95 LG941906 12.50 0.55 0.05 4.83 3.45 LG977012 35.42 1.85 0.28 4.00 23.38 LG981605 29.17 1.37 0.20 4.00 14.62 Magellan 35.42 2.20 0.33 4.00 20.95 Maverick 37.50 1.82 0.27 3.83 20.78 NE3001 37.50 1.97 0.30 4.17 26.12 44 Continued

Table 9 continued Level of Percent Mean Plant Root Root genotype Stand Height Weight Weight Score (mm) (g) (g) OhioFG1 77.08 6.17 1.13 3.50 41.62 P404188A 31.25 1.97 0.32 4.00 22.57 P437169B 37.50 1.97 0.30 4.17 23.70 P507681B 18.75 0.73 0.08 4.33 12.45 PI398881 41.67 2.67 0.42 3.67 23.40 PI427136 39.58 2.63 0.43 3.33 29.28 PI518751 39.58 2.17 0.28 4.00 23.38 PI561370 25.00 1.40 0.13 4.33 14.17 PI574486 35.42 2.30 0.27 3.83 27.17 Prohio 60.42 3.24 0.63 4.00 39.07 S0613640 72.92 4.80 0.75 3.83 34.50 Skylla 16.67 0.82 0.12 4.17 15.70 Streeter 57.50 3.24 0.44 4.00 29.68 Summit 58.33 2.92 0.38 4.00 28.17 U3100612 39.58 2.00 0.28 3.83 28.23 Wooster 0.00 0.00 0.00 5.00 0.00 Wyandot 56.25 3.57 0.53 3.67 40.45 conrad 27.08 1.13 0.17 4.17 21.22 sloan 41.67 2.38 0.38 3.67 22.77 Overall 41.18 24.41 2.35 0.37 3.99 Mean LSD 28.13 1.78 0.41 0.67 15.31

Table 10. University of Missouri population inoculated with P. ultimum v. ultimum

(There was poor seed quality with some lines in the non-inoculated controls. This table only includes lines that had growth in the non-inoculated control).

Level of Percent Mean Plant Root Root genotype Stand Height Weight Weight Score (mm) (g) (g) Conrad 75.00 61.67 6.12 1.52 3.67 Dunbar 6.25 17.50 0.28 0.07 4.50 Forrest 2.08 10.83 0.12 0.02 4.83 Kottman 58.33 67.89 5.95 1.43 3.67 Magellan 42.50 64.75 3.40 0.86 3.60 Maverick 27.08 42.67 1.98 0.47 4.00 OhioFG1 62.50 68.56 6.38 1.33 3.67 Miss001 8.33 19.83 0.37 0.07 4.50 Miss002 58.33 48.22 1.94 0.44 3.33 Miss003 64.58 52.67 3.50 0.77 4.00 Miss004 54.17 53.17 2.47 0.38 3.67 Miss005 22.92 56.00 2.15 0.50 4.00 Miss006 72.92 80.17 6.48 1.63 3.17 Miss007 52.08 44.22 2.95 0.77 3.83 Miss008 50.00 52.08 3.72 0.95 3.33 Miss009 64.58 65.47 5.52 1.62 3.50 Miss010 14.58 33.17 0.90 0.30 4.33 Miss011 10.42 10.78 0.40 0.10 4.67 Miss012 12.50 28.50 0.58 0.12 4.50 Miss013 52.08 60.31 2.97 0.68 3.50 Pana 8.33 24.17 0.65 0.17 4.50 Prohio 56.25 70.03 4.52 1.07 3.17 Miss014 54.17 58.61 3.72 0.83 3.33 Miss015 62.50 72.53 5.22 1.50 3.17 Miss016 56.25 58.33 3.70 0.97 3.67 Continued 46

Table 10 continued Level of Percent Mean Plant Root Root genotype Stand Height Weight Weight Score (mm) (g) (g) Miss017 37.50 39.69 3.02 0.80 3.50 Miss018 18.75 24.19 1.42 0.20 4.33 Will82 22.92 29.83 1.75 0.40 4.17 Wyandot 54.17 60.03 5.00 1.23 3.50 Dennison 89.58 81.33 9.37 2.57 3.33 Streeter 66.67 71.78 6.20 1.38 3.17 Summit 64.58 68.78 5.22 1.12 3.50 Wooster 0.00 0.00 0.00 0.00 5.00 Overall 42.51 48.42 3.27 0.80 3.84 Mean LSD 23.5 24.34 2.22 0.72 0.70

Table 11. University of Missouri population inoculated with P. ultimum v. sporangiiferum (There was poor seed quality with some lines in the non-inoculated controls. This table only includes lines that had growth in the non-inoculated control.).

Level of Percent Mean Plant Root Root genotype Stand Height Weight Weight Score (mm) (g) (g) Conrad 91.67 85.72 9.75 3.25 2.50 Dunbar 50.00 64.94 3.85 0.92 3.00 Forrest 25.00 49.50 1.30 0.18 3.67 Kottman 89.58 81.39 9.78 2.83 2.17 Magellan 83.33 88.67 7.28 1.98 2.67 Maverick 87.50 70.39 8.12 2.50 2.67 OhioFG1 89.58 93.11 13.40 4.07 2.17 Miss001 20.83 33.83 1.10 0.25 3.83 Miss002 81.25 69.67 3.40 0.85 2.67 Miss003 83.33 64.33 5.37 1.58 3.00 Miss004 85.42 67.00 5.77 1.32 2.67 Miss005 45.83 71.33 3.60 0.82 3.33 Miss006 93.75 75.56 9.35 2.95 3.00 Miss007 85.42 80.39 6.00 1.83 2.83 Miss008 77.08 77.39 7.32 2.37 2.33 Miss009 93.75 85.00 9.20 3.00 2.17 Miss010 66.67 83.50 4.57 1.48 2.17 Miss011 39.58 57.39 2.30 0.67 3.00 Miss012 58.33 64.67 3.77 0.98 3.17 Miss013 68.75 74.67 6.07 1.75 2.50 Pana 62.50 78.44 4.30 1.28 2.67 Prohio 87.50 93.00 8.82 2.55 2.67 Miss014 75.00 74.39 5.90 1.48 2.67 Miss015 79.17 88.28 7.83 2.53 2.83 Miss016 95.83 87.78 8.38 2.58 2.67 Miss017 79.17 76.39 6.92 2.10 2.83 Miss018 42.50 55.10 4.10 0.68 3.00 Will82 81.25 79.89 8.75 2.63 2.17 48 Continued

Table 11 continued Level of Percent Mean Plant Root Root genotype Stand Height Weight Weight Score (mm) (g) (g) Wyandot 64.58 81.72 7.60 2.25 2.33 dennison 89.58 102.06 12.27 4.58 1.83 streeter 97.92 95.00 10.65 2.92 2.33 summit 87.50 92.67 9.58 2.78 2.50 wooster 41.67 43.56 2.83 0.68 3.17 Overall 72.91 75.46 6.64 1.96 2.70 Mean LSD 15.10 14.50 2.00 0.87 0.70

Table 12. Overall means of Virginia Tech population inoculated with P. ultimum v. ultimum.

Level of Percent Mean Plant Root Root genotype Stand Height Weight Weight Score (mm) (g) (g) Archer 83.33 127.39 11.58 3.28 2.50 VT001 91.67 111.33 12.07 5.05 2.67 Dennison 91.67 137.28 12.82 5.15 2.17 Essex 91.67 101.28 9.50 3.13 2.17 Hutchinson 91.67 114.28 10.93 4.12 2.50 VT002 89.58 112.44 10.83 3.90 2.17 VT003 47.92 79.17 3.13 0.72 3.17 VT004 91.67 155.11 18.33 4.97 2.00 VT005 89.58 146.61 16.15 4.62 2.17 VT006 79.17 137.06 9.48 3.03 2.83 VT007 97.92 107.94 9.15 3.17 2.50 VT008 93.75 104.50 15.05 5.50 2.17 VT009 89.58 140.17 15.47 4.98 1.83 VT010 85.42 102.56 5.32 1.92 2.83 VT011 83.33 112.17 9.50 3.00 2.50 VT012 93.75 117.89 10.80 3.47 2.83 VT013 91.67 145.06 15.03 4.33 2.50 VT014 92.71 107.08 13.40 4.73 2.00 VT015 89.58 137.56 11.92 3.48 2.83 VT016 95.83 119.17 15.38 4.55 2.67 VT017 93.75 132.56 15.53 4.28 2.83 VT018 87.50 119.20 12.05 3.38 2.83 VT019 89.58 93.83 10.32 4.02 2.83 VT020 95.83 142.61 11.87 3.43 2.33 VT021 87.50 112.83 9.05 2.93 2.67 VT022 87.50 115.67 13.18 4.38 2.33 VT023 93.75 90.06 6.72 2.87 2.83 VT024 91.67 146.39 10.20 3.63 2.67 VT025 87.50 114.07 8.82 3.15 2.17 VT026 87.50 127.00 9.62 3.20 2.67 VT027 89.58 113.00 8.57 3.08 2.83 Continued 50

Table 12 continued

Level of Percent Mean Plant Root Root genotype Stand Height Weight Weight Score (mm) (g) (g) VT028 91.67 117.50 10.58 3.88 2.83 VT029 85.42 107.11 8.40 2.52 2.67 VT030 89.58 113.61 14.25 4.73 2.83 VT031 89.58 125.06 6.70 2.57 2.67 VT032 91.67 129.56 13.00 4.67 2.17 VT033 95.83 132.78 12.63 3.77 2.50 VT034 93.75 134.06 11.92 3.88 2.67 VT035 83.33 86.50 5.47 2.22 2.50 VT036 31.25 56.33 2.72 0.53 3.67 VT037 81.25 111.50 10.48 2.75 2.33 VT038 97.92 98.44 10.13 3.75 2.67 VT039 89.58 107.67 10.12 2.75 2.67 Williams 97.92 107.83 13.32 4.57 1.83 conrad 81.25 104.61 10.22 4.22 2.33 kottman 89.58 105.11 12.00 3.97 1.83 ohiofg1 79.17 114.00 12.13 4.42 2.33 prohio 91.67 121.44 10.82 3.97 2.17 sloan 91.67 116.56 12.12 4.22 2.17 streeter 87.50 118.11 11.27 3.97 2.00 summit 87.50 117.67 9.82 3.22 2.50 wooster 64.58 74.22 5.08 1.53 3.00 wyandot 87.50 109.33 11.48 4.03 1.83 Overall 87.5 115.54 10.9 3.60 2.5 Mean LSD 17.57 21.75 3.33 1.67 0.68

Table 13. Overall means of Virginia Tech population evaluated with P. ultimum v. sporangiiferum.

Level of Percent Mean Plant Root Root genotype Stand Height Weight Weight Score (mm) (g) (g)

Archer 68.75 3.17 70.28 6.60 1.62 VT001 64.58 3.33 62.19 5.82 1.67 Dennison 93.75 2.67 79.56 9.78 3.35 Essex 93.75 2.83 72.67 7.52 2.18 Hutchinson 87.50 2.33 76.33 8.45 2.85 VT002 87.50 2.83 75.67 8.90 2.63 VT003 2.08 4.67 6.00 0.08 0.02 VT004 83.33 2.33 112.00 15.87 4.38 VT005 83.33 2.33 122.28 14.20 3.65 VT006 85.42 3.17 104.83 8.13 2.28 VT007 60.42 3.50 73.86 3.92 1.05 VT008 62.50 2.67 68.11 7.32 2.07 VT009 79.17 2.17 123.39 10.60 3.10 VT010 81.25 3.17 72.89 3.50 0.95 VT011 66.67 3.00 86.06 6.02 1.43 VT012 93.75 3.00 89.40 9.70 2.62 VT013 87.50 3.00 116.72 11.76 2.86 VT014 69.79 2.92 74.19 7.14 1.91 VT015 91.67 3.00 90.72 9.78 2.60 VT016 87.50 2.33 95.39 11.82 3.02 VT017 87.50 2.50 103.94 12.62 3.42 VT018 91.67 2.33 97.72 12.15 3.10 VT019 95.83 3.00 78.94 8.92 2.98 VT020 83.33 3.17 102.33 7.95 2.10 VT021 77.08 3.00 91.94 7.10 1.95 VT022 79.17 3.33 82.22 8.65 2.15 VT023 93.75 3.33 73.61 5.10 1.68 VT024 89.58 2.67 126.28 8.23 2.55 VT025 95.83 2.50 94.44 8.55 2.60 VT026 66.67 3.33 87.06 5.85 1.70 VT027 79.17 3.17 81.50 5.85 1.72 Continued 52

Table 13 continued

Level of Percent Mean Plant Root Root genotype Stand Height Weight Weight Score (mm) (g) (g) VT028 89.58 2.67 109.00 9.25 3.22 VT029 91.67 3.00 84.87 7.50 1.93 VT030 81.25 2.67 91.17 10.58 3.22 VT031 79.17 3.00 88.78 4.33 1.28 VT032 89.58 3.00 85.61 11.72 3.12 VT033 66.67 3.17 92.28 7.23 1.77 VT034 91.67 3.00 107.61 9.33 2.45 VT035 70.83 3.17 70.61 3.83 1.33 VT036 8.33 4.50 15.72 0.85 0.18 VT037 83.33 2.50 69.11 9.38 2.32 VT038 87.50 3.33 71.22 6.88 1.88 VT039 56.25 3.33 58.78 4.88 1.08 Williams 68.75 3.67 52.72 4.97 1.05 conrad 75.00 3.33 67.39 5.82 1.65 kottman 70.83 2.83 76.33 6.63 1.75 ohiofg1 81.25 3.00 87.17 9.38 2.55 prohio 87.50 3.17 68.47 6.33 1.58 sloan 60.42 3.50 52.78 5.17 1.32 streeter 91.67 2.83 93.94 9.83 2.90 summit 72.92 3.17 63.50 6.08 1.62 wooster 2.50 4.40 8.40 0.12 0.02 wyandot 72.92 3.17 84.56 7.90 2.33 Overall 76.50 3.00 81.11 7.6 2.12 Mean LSD 18.42 0.89 24.05 2.81 1.26

Table 14. Overall means of Genetic Gain population inoculated with Pythium ultimum v. ultimum.

Level of Percent Mean Plant Root Root genotype Stand Height Weight Weight (g) Score (mm) (g)

Burlison 45.83 47.25 3.88 1.08 3.17 Corsoy79 14.58 29.07 1.10 0.33 4.50 Dwight 12.50 29.78 0.75 0.22 3.83 IA2021 4.17 6.42 0.25 0.50 4.83 IA2022 27.08 29.17 1.68 0.42 3.83 IA2038 4.17 16.33 0.28 0.05 4.67 IA2050 25.00 27.08 1.65 0.42 4.17 IA2052 27.08 41.00 2.05 0.60 3.67 IA2065 12.50 17.80 0.80 0.18 4.67 IA2068 2.08 7.00 0.10 0.02 4.83 IA2094 8.33 11.33 0.67 0.17 4.83 Rango 14.58 19.28 0.90 0.22 4.50 Richland 31.25 32.07 2.28 0.63 4.17 Amcor 25.00 34.67 1.68 0.50 4.17 Amsoy71 41.67 42.39 3.18 1.05 3.33 Amsoy 35.42 33.37 2.17 0.48 4.17 Beeson80 0.00 0.00 0.00 0.00 5.00 Beeson 12.50 20.17 0.85 0.22 4.67 Century84 33.33 31.63 2.38 0.62 4.17 Century 27.08 43.83 2.00 0.55 4.17 Conrad 18.75 18.17 1.62 0.52 4.50 Corsoy 16.67 26.33 1.50 0.48 4.50 Dennison 85.42 77.66 10.70 3.77 2.67 Elgin87 29.17 21.38 1.33 0.30 4.17 Elgin 10.42 14.63 0.58 0.13 4.67 Harosoy63 25.00 35.27 1.87 0.60 4.00 Harcor 27.08 34.50 1.87 0.57 4.00 Harosoy 25.00 29.53 1.83 0.55 4.33

54 Continued

Table 14 continued

Level of Percent Mean Plant Root Root genotype Stand Height Weight Weight (g) Score (mm) (g) Hawkeye63 29.17 26.72 2.08 0.52 4.33 Hawkeye 18.75 23.22 1.10 0.18 4.50 Jack 22.92 31.22 1.00 0.22 4.17 Korean 0.00 0.00 0.00 0.00 5.00 Kottman 70.83 63.43 8.42 2.83 2.83 Kenwood 10.42 23.08 0.62 0.18 4.50 Lindarin 20.83 30.33 1.57 0.43 4.33 Loda 4.17 6.42 0.30 0.08 4.67 Mukden 18.75 23.22 0.78 0.13 4.00 OhioFG1 75.00 63.50 10.57 3.65 2.67 Preston 4.17 15.50 0.25 0.07 4.67 Private-210 33.33 27.85 1.83 0.50 4.50 Private-211 31.25 23.77 1.65 0.37 4.50 Private-212 35.42 24.60 2.48 0.70 4.17 Private-213 20.83 29.23 1.58 0.55 4.33 Private-214 41.67 29.88 3.33 0.98 4.00 Private-215 6.25 5.67 0.25 0.07 4.83 Private-216 18.75 17.62 1.10 0.28 4.67 Private-217 10.42 11.28 0.68 0.17 4.83 Private-218 14.58 26.08 0.80 0.17 4.50 Private-219 22.92 20.67 1.05 0.22 4.33 Private-220 27.08 21.35 1.72 0.47 4.33 Private-21 35.42 29.35 2.18 0.52 4.50 Private-22 27.08 20.33 1.87 0.47 4.33 Private-23 27.08 29.22 1.42 0.33 4.00 Private-24 22.92 22.38 1.47 0.45 4.50 Private-25 20.83 22.12 1.23 0.28 4.50 Private-26 22.92 27.55 1.12 0.28 4.50 Private-27 16.67 26.75 1.22 0.32 4.17 Private-28 20.83 22.72 1.38 0.37 4.33 Private-29 37.50 30.38 2.55 0.78 4.17 55 Continued

Table 14 continued Level of Percent Mean Plant Root Root genotype Stand Height Weight Weight (g) Score (mm) (g) Prohio 54.17 56.45 5.48 1.75 3.33 Savoy 29.17 37.22 2.58 0.73 3.67 Sloan 56.25 56.43 6.25 1.82 3.17 Streeter 81.25 69.33 9.58 3.27 2.67 Summit 66.67 59.39 5.92 1.55 3.00 Vickery 27.08 29.22 1.93 0.65 4.00 Wells II 16.67 24.58 0.95 0.22 4.33 Wells 41.67 40.99 2.97 0.67 3.83 Wooster 0.00 0.00 0.00 0.00 5.00 Wyandot 75.00 61.45 7.98 2.50 3.00 Overall 26.93 28.81 2.19 0.65 4.17 Mean LSD 32.35 29.48 2.52 0.82 0.87

Table 15. Overall means of Genetic Gain population inoculated with P. ultimum v. sporangiiferum.

Level of Percent Mean Plant Root Root Score genotype Stand Height Weight Weight (mm) (g) (g) Burlison 10.42 9.83 0.53 0.10 4.67 Corsoy79 12.50 19.33 0.75 0.23 4.17 Dwight 2.08 3.83 0.08 0.02 4.67 IA2021 0.00 0.00 0.00 0.00 5.00 IA2022 2.08 9.00 0.18 0.05 4.83 IA2038 0.00 0.00 0.00 0.00 5.00 IA2050 2.08 2.67 0.08 0.02 4.83 IA2052 0.00 0.00 0.00 0.00 5.00 IA2065 2.08 2.67 0.23 0.05 4.83 IA2068 0.00 0.00 0.00 0.00 5.00 IA2094 0.00 0.00 0.00 0.00 5.00 Rango 0.00 0.00 0.00 0.00 5.00 Richland 33.33 31.73 1.87 0.45 3.50 Amcor 4.17 4.92 0.28 0.12 4.67 Amsoy71 10.42 11.17 0.48 0.10 4.33 Amsoy 6.25 8.33 0.50 0.17 4.67 Beeson80 2.08 3.67 0.08 0.02 4.67 Beeson 0.00 0.00 0.00 0.00 5.00 Century84 8.33 12.22 0.45 0.07 4.50 Century 0.00 0.00 0.00 0.00 5.00 Conrad 6.25 6.12 0.33 0.07 4.83 Corsoy 8.33 25.17 0.53 0.18 3.33 Dennison 95.83 66.62 12.45 5.58 2.33 Elgin87 2.08 4.17 0.13 0.02 4.83 Elgin 0.00 0.00 0.00 0.00 5.00 Harosoy63 8.33 19.33 0.38 0.12 4.00 Harcor 2.08 7.50 0.12 0.02 4.83 Harosoy 4.17 6.83 0.25 0.07 4.33 57 Continued

Table 15 continued

Level of Percent Mean Plant Root Root Score genotype Stand Height Weight Weight (mm) (g) (g) Hawkeye63 2.08 4.17 0.13 0.03 4.67 Hawkeye 2.08 2.50 0.12 0.03 4.83 Jack 0.00 0.00 0.00 0.00 5.00 Korean 0.00 0.00 0.00 0.00 5.00 Kottman 31.25 31.18 2.50 0.77 3.17 Kenwood 4.17 16.17 0.15 0.03 4.67 Lindarin 10.42 8.95 0.60 0.18 4.67 Loda 0.00 0.00 0.00 0.00 5.00 Mukden 0.00 0.00 0.00 0.00 5.00 OhioFG1 85.42 54.06 11.20 3.87 2.50 Preston 0.00 0.00 0.00 0.00 5.00 Private-210 8.33 13.67 0.33 0.08 4.33 Private-211 4.17 8.33 0.17 0.03 4.33 Private-212 2.08 5.33 0.08 0.02 4.83 Private-213 6.25 15.08 0.37 0.08 4.67 Private-214 12.50 25.50 0.67 0.15 3.50 Private-215 0.00 0.00 0.00 0.00 5.00 Private-216 2.08 2.67 0.08 0.03 4.83 Private-217 16.67 22.25 0.85 0.18 3.50 Private-218 0.00 0.00 0.00 0.00 5.00 Private-219 2.08 3.67 0.07 0.02 4.83 Private-220 0.00 0.00 0.00 0.00 5.00 Private-21 14.58 14.30 0.60 0.10 4.33 Private-22 8.33 21.75 0.45 0.13 3.67 Private-23 4.17 2.83 0.22 0.03 4.83 Private-24 14.58 13.72 1.00 0.30 4.50 Private-25 2.08 8.83 0.10 0.02 4.83 Private-26 0.00 0.00 0.00 0.00 5.00 Private-27 8.33 17.33 0.43 0.10 4.17 Private-28 8.33 20.50 0.70 0.20 3.83 Private-29 6.25 2.33 0.25 0.02 4.83 58 Continued

Table 15 continued

Level of Percent Mean Plant Root Root Score genotype Stand Height Weight Weight (mm) (g) (g) Prohio 72.92 50.98 7.28 2.62 2.50 Savoy 8.33 8.92 0.50 0.12 4.50 Sloan 47.92 21.06 4.33 1.28 3.50 Streeter 83.33 54.66 8.78 3.22 2.50 Summer 83.33 56.73 8.12 2.67 2.67 Vicker 6.25 11.25 0.38 0.10 4.50 Wells II 0.00 0.00 0.00 0.00 5.00 Wells 16.67 23.25 1.27 0.40 4.00 Wooster 0.00 0.00 0.00 0.00 5.00 Wyandot 81.25 52.67 7.30 2.23 2.83 Overall 12.74 12.29 1.14 0.38 4.41 Mean LSD 13.26 15.41 1.14 0.45 0.80

Table 16. Overall means of The Ohio State University population inoculated with P. ultimum v. ultimum.

Level of Percent Mean Plant Root Root genotype Stand Height Weight Weight Score (mm) (g) (g) OSU001 54.2 40.4 8.9 3.2 3.3 OSU002 50 50 8.9 3.4 4 OSU003 54.2 64.7 6.7 2.3 3 OSU004 45.8 46 8.2 2.4 4 OSU005 66.7 69.7 9.8 3.6 2.7 OSU006 83.3 55.7 5.2 2.2 3.3 OSU007 41.7 34.9 6.7 2.1 3.7 OSU008 75 58.9 5.9 2.2 3 OSU009 83.3 66.3 4.8 1.9 2.7 OSU010 91.7 82 14.4 5.5 2.7 OSU011 54.2 70.1 6.7 2.6 2.3 OSU012 83.3 67.4 5.1 1.8 3.3 Archer 62.5 66.5 7.9 2.5 3.3 Clermont 25 25.8 1 0.2 4 Conrad 33.3 45.3 2.6 0.9 3.3 Danbkong 83.3 67.7 6.7 1.9 2 Dennison 58.3 61.7 6.1 2 2.7 Dilworth 79.2 78.9 6.7 2.1 2.7 Flint 70.8 89.3 7.5 2.5 2.7 OSU013 70.8 67.8 5.6 1.7 3 OSU014 83.3 64.8 7.3 1.8 2.7 OSU015 20.8 45 3.7 1.1 3.3 OSU016 58.3 69.9 4.5 1.3 3 OSU017 50 47.6 3.2 0.8 3.7 OSU018 50 43.8 3.7 1 3.7 OSU019 58.3 60.9 4.9 1.4 2.7 OSU020 37.5 37.1 4.3 0.9 3.7 OSU021 66.7 48.2 5.3 1.6 3.3 60 Continued

Table 16 continued

Level of Percent Mean Plant Root Root genotype Stand Height Weight Weight Score (mm) (g) (g) OSU022 58.3 49.8 4.7 1.5 3 OSU023 83.3 56.7 6.7 2.2 2.7 OSU024 83.3 81.8 7.3 2.4 2.7 OSU025 50 55.2 3.8 1 3 OSU026 41.7 51.9 2.4 0.6 3 OSU027 37.5 49.1 2.5 0.5 3.3 OSU028 58.3 63.9 6.1 2.1 3 OSU029 66.7 63.4 5.2 1.6 3 OSU030 66.7 60.6 4.8 1.6 3.3 OSU031 35.4 54.3 3.4 0.9 3.7 OSU032 58.3 66.6 4.3 1.2 3.7 OSU033 95.8 59 8.5 2.7 2.3 OSU034 66.7 68.6 5.1 1.5 3 OSU035 70.8 65.3 5.7 2.1 2.3 OSU036 75 84.3 7.1 2.2 2.7 OSU037 29.2 54.5 2.5 0.7 3.3 OSU038 37.5 44.2 5.5 1.5 4 OSU039 25 39.7 2.2 0.5 3.3 OSU040 75 53.7 5 1.8 2.7 Hutchinson 75 53.1 6.7 1.9 3.3 Jack 33.3 37.2 4.5 1.3 4 Kottman 41.7 28.1 3.3 0.8 3.3 OSU041 45.8 75.2 4.2 0.9 3.7 OSU042 70.8 63.9 5.5 1.9 3 OSU043 25 48.8 4.7 1.7 3.3 OSU044 37.5 24.2 4.5 1.5 3.3 OSU045 45.8 43 5.2 2 4 OSU046 54.2 57.7 4.5 1.5 3.3 OSU047 50 53.6 3.5 1.1 3.3 OSU048 75 70.9 8.6 3.1 3 OhioFG1 91.7 64 10.3 2.9 2.7 61 Continued

Table 16 continued Level of Percent Mean Plant Root Root genotype Stand Height Weight Weight Score (mm) (g) (g) OhioFG5 50 49.8 4.6 1.2 3 PI408105A 62.5 70.3 5.1 1.7 3.7 PI408225A 45.8 61.2 3.2 1 3.3 PI424234B 58.3 50 4.2 1 3.7 PI427105B 79.2 64.8 5.4 1.6 2.3 PI567301B 87.5 65 4.4 1.7 2.7 PI567321A 41.7 59.7 3.5 1.3 2.7 PI567336A 87.5 65.3 4 1.6 2.7 PI567352B 91.7 68.5 4.4 1.7 3 PI567516C 12.5 30 0.5 0.1 4.3 PI243540 79.2 67.1 9.1 2.4 3 PI291327 37.5 27.1 2 0.4 4.3 PI398233 33.3 57.4 2.6 0.6 3.7 PI398841 45.8 71.7 4 0.9 2.7 PI399073 58.3 51.9 5.2 1.5 3.3 PI407985 37.5 38.9 4.7 1.7 3.7 PI416783 16.7 49.7 2.2 0.4 3.7 PI417142 20.8 38.6 1.6 0.5 4.3 PI417459 41.7 56 4.9 1.4 3 PI423885 45.8 56.4 5.3 1.4 3.3 PI427106 41.7 53.3 4.1 1.1 3.3 PI567324 20.8 38 0.6 0.2 3.3 PI567343 41.7 49.3 2.5 0.8 4 PI594599 37.5 38.9 1.7 0.5 3 Prohio 66.7 97.9 7 2.2 2.7 OSU049 50 69 3.4 0.9 3.7 OSU050 45.8 54.7 5.7 1.4 3.3 Resnik 62.5 81.7 6.6 2.1 2.3 Ripley 16.7 33.3 2.6 0.9 4 OSU052 66.7 55.4 3.9 1.2 3.3 Sloan 25 42.7 4 1.1 3.7 Continued 62

Table 16 continued Level of Percent Mean Plant Root Root genotype Stand Height Weight Weight Score (mm) (g) (g) Stout 33.3 36.8 2.1 0.5 4 Streeter 29.2 37.3 5.1 1.7 3.7 Stressla 54.2 82.9 4.8 1.5 3 Strong 54.2 75.3 6.1 1.9 2.7 Summit 33.3 52.1 2.8 0.9 3.7 W82 54.2 78.8 4.6 0.9 3.3 Williams 66.7 80.2 6 1.5 3 Wooster 33.3 75.2 3.4 1.2 3 Wyandot 41.7 52.2 4 1.3 3.7 OSU052 45.8 34.9 5.2 1.8 3.3 OSU053 81.3 63.5 5.9 2.2 2.5 Overall 54.06 56.89 4.99 1.56 3.2 Mean LSD 43.24 39.32 4.59 1.72 1.32

Table 17. Overall means of The Ohio State University population inoculated with P. ultimum v. sporangiiferum.

Level of Percent Mean Plant Root Root Score genotype Stand Height Weight Weight (mm) (g) (g) OSU001 87.50 57.80 6.70 2.00 2.67 OSU002 66.67 70.57 6.87 1.90 2.33 OSU003 91.67 78.20 8.43 2.47 2.67 OSU004 91.67 66.57 9.17 2.37 2.67 OSU005 70.83 68.53 8.10 1.93 3.00 OSU006 95.83 64.33 5.50 1.93 2.67 OSU007 70.83 67.87 6.97 1.77 2.67 OSU008 75.00 66.23 4.87 1.50 3.00 OSU009 91.67 69.90 4.53 1.47 3.00 OSU010 87.50 82.90 12.93 4.17 2.00 OSU011 91.67 74.80 9.40 3.00 2.33 OSU012 91.67 70.53 6.10 2.20 2.67 Archer 83.33 69.43 10.93 3.40 2.33 Clermont 79.17 57.10 6.63 1.93 2.33 Conrad 45.83 27.23 3.00 0.53 4.00 Danbkong 70.83 38.90 4.43 0.83 3.00 Dennison 95.83 65.90 9.93 3.60 2.67 Dilworth 87.50 65.00 7.07 1.90 3.00 Flint 83.33 54.10 7.90 2.43 3.00 OSU013 75.00 61.57 4.87 1.20 2.33 OSU014 79.17 58.20 5.33 1.27 2.67 OSU015 62.50 58.63 5.33 1.33 1.33 OSU016 58.33 40.23 4.95 1.10 3.00 OSU017 79.17 52.80 5.13 1.17 2.33 OSU018 37.50 35.83 2.20 0.47 3.33 OSU019 91.67 53.47 7.10 1.70 2.33 OSU020 70.83 63.77 7.23 2.13 2.33

64 Continued

Table 17 continued Level of Percent Mean Plant Root Root Score genotype Stand Height Weight Weight (mm) (g) (g) OSU021 70.83 48.77 4.53 1.10 2.67 OSU022 75.00 67.00 5.50 1.47 2.67 OSU023 79.17 57.33 5.70 1.50 2.33 OSU024 75.00 67.77 5.00 1.17 3.00 OSU025 75.00 54.20 5.50 1.57 2.67 OSU026 45.83 38.27 2.87 0.73 3.00 OSU027 95.83 47.67 7.00 2.10 2.00 OSU028 75.00 59.00 6.00 1.83 2.00 OSU029 58.33 42.13 3.80 0.93 2.67 OSU030 75.00 38.67 4.97 1.43 3.00 OSU031 72.92 60.32 6.15 1.83 2.67 OSU032 75.00 39.80 4.45 1.05 3.00 OSU033 87.50 44.47 6.33 1.47 3.00 OSU034 66.67 44.10 4.43 1.13 2.67 OSU035 75.00 46.33 4.90 1.23 3.00 OSU036 70.83 45.33 5.10 1.23 2.67 OSU037 87.50 57.53 6.70 1.90 3.00 OSU038 100.00 70.65 11.30 3.50 2.00 OSU039 83.33 64.33 9.77 2.77 2.33 OSU040 79.17 44.10 5.03 1.37 2.67 Hutchinson 79.17 59.43 6.43 1.77 2.67 Jack 70.83 65.13 6.80 2.00 2.67 Kottman 91.67 64.87 9.37 2.97 2.00 OSU041 75.00 66.67 7.20 1.93 3.00 OSU042 66.67 45.20 4.00 0.97 2.67 OSU043 79.17 57.43 7.40 2.33 2.33 OSU044 45.83 35.00 2.80 0.63 2.67 OSU045 45.83 35.87 2.70 0.70 3.00 OSU046 58.33 67.43 3.73 0.80 3.00 OSU047 54.17 31.43 2.70 0.63 3.67 OSU048 79.17 58.33 6.77 2.20 3.33 65 Continued

Table 17 continued Level of Percent Mean Plant Root Root Score genotype Stand Height Weight Weight (mm) (g) (g) OhioFG1 70.83 58.23 8.47 2.70 2.33 OhioFG5 83.33 54.23 7.97 2.57 2.67 PI408105A 58.33 70.00 4.50 1.07 3.33 PI408225A 58.33 58.27 1.93 0.67 2.00 PI424234B 29.17 39.10 2.10 0.47 3.33 PI427105B 37.50 46.77 1.67 0.37 2.67 PI567301B 62.50 44.67 2.03 0.63 3.33 PI567321A 70.83 52.57 4.00 0.90 3.33 PI567336A 83.33 55.57 2.73 0.83 3.33 PI567352B 95.83 72.10 4.40 1.63 2.67 PI567516C 62.50 61.53 3.40 0.97 2.33 PI243540 70.83 76.20 8.60 2.23 2.67 PI291327 33.33 43.77 3.00 0.80 3.00 PI398233 41.67 54.20 3.20 0.70 3.00 PI398841 37.50 31.43 3.95 0.85 3.67 PI399073 54.17 58.07 5.20 1.33 3.00 PI407985 54.17 71.90 4.40 1.17 2.33 PI416783 16.67 25.23 1.50 0.25 4.00 PI417142 25.00 36.10 2.40 0.70 3.33 PI417459 33.33 44.00 4.40 1.30 3.00 PI423885 50.00 46.67 3.87 0.83 3.33 PI427106 37.50 45.90 3.37 0.73 2.67 PI567324 41.67 40.10 2.40 0.75 4.00 PI567343 75.00 70.67 5.13 1.70 2.33 PI594599 66.67 62.33 4.13 1.23 2.67 Prohio 95.83 73.67 7.67 2.23 2.67 OSU049 87.50 54.13 7.27 2.60 2.00 OSU050 33.33 52.67 2.97 0.93 2.33 Resnik 79.17 66.13 6.50 1.77 3.33 Ripley 41.67 42.00 5.30 1.30 4.00 OSU051 41.67 38.10 8.13 3.17 2.67 66 Continued

Table 17 continued Level of Percent Mean Plant Root Root Score genotype Stand Height Weight Weight (mm) (g) (g) Sloan 37.50 32.50 2.77 0.53 3.33 Stout 95.83 47.67 6.60 1.87 2.67 Streeter 75.00 53.30 7.33 2.13 2.33 Stressla 91.67 63.57 8.03 2.77 2.67 Strong 87.50 66.57 8.80 2.37 2.33 Summit 91.67 61.87 8.53 2.47 2.33 W82 70.83 64.90 6.93 2.10 3.00 Williams 70.83 46.53 7.17 1.73 3.00 Wooster 75.00 64.53 5.87 1.70 3.00 Wyandot 70.83 49.87 5.50 1.27 3.00 OSU052 58.33 47.60 3.07 0.73 3.33 OSU053 50.00 47.13 4.40 1.20 3.00 Overall 69.10 55.02 5.67 1.60 2.8 Mean LSD 30.37 24.76 3.71 1.57 1.28

Table 18. Mean root weight and root rot score for the NAM experiments of both P. ultimum v. ultimum and P. ultimum v. sporangiiferum for genotype checks.

P. ultimum v. spor P. ultimum v. ult Genotype Root Root Root Root Weight Score Weight Score Kottman 0.47 3.67 3.60 1.50 Prohio 0.63 4.00 2.50 2.40 Streeter 0.44 4.00 3.10 2.50 Summit 0.38 4.00 3.20 2.30 Wooster 0.00 5.00 0.40 3.80 Wyandot 0.53 3.67 3.40 2.30 Conrad 0.17 4.17 1.80 2.80 Sloan 0.38 3.67 3.20 2.00 OhioFG1 1.13 3.50 3.50 2.30 Dennison 0.83 3.83 4.20 2.50 Overall 0.50 3.95 2.89 2.44 LSD 0.41 0.67 1.34 0.97

Table 19. Mean root weight and root rot score for the University of Missouri experiments of both P. ultimum v. ultimum and P. ultimum v. sporangiiferum for genotype checks.

P. ultimum v. spor P. ultimum v. ult Genotype Root Root Root Root Weight Score Weight Score Kottman 2.83 2.17 1.43 3.67 Prohio 2.55 2.67 1.07 3.17 Streeter 2.92 2.33 1.38 3.17 Summit 2.78 2.50 1.12 3.50 Wooster 0.68 3.17 0.00 5.00 Wyandot 2.25 2.33 1.23 3.50 Conrad 3.25 2.50 1.52 3.67 Sloan NA NA NA NA OhioFG1 4.07 2.17 1.33 3.67 Dennison 4.58 1.83 2.57 3.33 Overall 2.88 2.41 1.29 3.63 LSD 0.87 0.70 0.72 0.70

Table 20. Mean root weight and root rot score for the Virginia Tech experiments of both

P. ultimum v. ultimum and P. ultimum v. sporangiiferum for genotype checks.

P. ultimum v. spor P. ultimum v. ult Genotype Root Root Root Root Weight Score Weight Score Kottman 1.75 2.83 3.97 2.17 Prohio 1.58 3.17 3.97 2.17 Streeter 2.90 2.83 3.97 2.00 Summit 1.62 3.17 3.22 2.50 Wooster 0.02 4.4 1.53 3.00 Wyandot 2.33 3.17 4.03 1.83 Conrad 1.65 3.33 4.22 2.33 Sloan 1.32 3.50 4.22 2.17 OhioFG1 2.55 3.00 4.42 2.33 Dennison 3.35 2.67 5.15 2.17 Overall 6.20 1.91 3.87 2.27 LSD 1.26 0.89 1.11 0.67

Table 21. Mean root weight and root rot score for the Genetic Gain experiments of both

P. ultimum v. ultimum and P. ultimum v. sporangiiferum for genotype checks.

P. ultimum v. spor P. ultimum v. ult Genotype Root Root Root Root Weight Score Weight Score Kottman 0.77 3.17 2.83 2.83 Prohio 2.62 2.50 1.75 3.33 Streeter 3.22 2.50 3.27 2.67 Summit 2.67 2.67 1.55 3.00 Wooster 0.00 5.00 0.00 5.00 Wyandot 2.23 2.83 2.50 3.00 Conrad 0.07 4.83 0.52 4.50 Sloan 1.28 3.50 1.82 3.17 OhioFG1 3.87 2.50 3.65 2.67 Dennison 5.58 2.33 3.77 2.67 Overall 2.23 3.18 2.17 3.28 LSD 0.47 0.83 0.84 0.91

Table 22. Mean root weight and root rot score for the OSU experiments of both P. ultimum v. ultimum and P. ultimum v. sporangiiferum for genotype checks.

P. ultimum v. spor P. ultimum v. ult Genotype Root Root Root Root Weight Score Weight Score Kottman 2.97 2.00 0.80 3.30 Prohio 2.23 2.67 2.20 2.70 Streeter 2.13 2.33 1.70 3.70 Summit 2.47 2.33 0.90 3.70 Wooster 1.70 3.00 1.20 3.00 Wyandot 1.27 3.00 1.30 3.70 Conrad 0.53 4.00 0.90 3.30 Sloan 0.53 3.33 1.10 3.70 OhioFG1 2.70 2.33 2.90 2.70 Dennison 3.60 2.67 2.00 2.70 Overall 2.01 2.77 1.50 3.25 LSD 1.57 1.28 1.72 1.32

Chapter 3: Efficacy of Metalaxyl and Pyraclostrobin to Pythium species affecting

soybean and corn in Ohio

Introduction

Phytophthora sojae and Pythium species are two of the main causes of seedling diseases

in the United States (Wrather et al., 2010). In Ohio, as well as other Midwestern states,

oomycetes contribute substantially to crop loss every year, primarily through root rot and

damping-off of seedlings (Broders et al., 2009; Campa et al., 2010; Murillo-Williams and

Pedersen, 2008; Zhang and Yang, 2000). Seedling damping-off incurs additional

expenses for producers in the form of costs associated with replanting and reductions in

yield due to later planting dates. Therefore, it is important that effective management of

oomycetes is implemented each year to produce the highest soybean yield.

Fungicide seed treatments are a proven effective management strategy for many types of seedling diseases of soybean and corn (Bradley et al., 2008; Dorrance et al., 2004, 2009;

Esker & Conley, 2012). Metalaxyl and strobilurins are currently the primary seed applied fungicides for Pythium control. Metalaxyl, an acylalanine fungicide targeting oomycetes, has been used for a long time as a seed treatment application against Pythium species and Phytophthora sojae. The specific mode of action for metalaxyl is that it

73 inhibits ribosomal RNA synthesis (Cohen & Coffey 1986). The inhibition of RNA synthesis in the ribosome ultimately inhibits mycelial growth. As a result of the specific mode of action of metalaxyl, some Pythium species have been identified that are insensitive to this chemistry (Broders et al., 2007; Dorrance et al., 2004; Moorman et al.,

2004; Olson et al., 2013). For some of these species, it is not clear if they were always insensitive to metalaxyl or if they developed this insensitivity following repeated exposure. Due to the increasing number of reports of insensitivity to metalaxyl, other fungicides have been studied for their effectiveness against Pythium species.

Strobilurin fungicides, or Quinone outside Inhibitors (Qol), are in this category of other fungicides used to manage Pythium, as well as other fungal pathogens (Broders et al.,

2007; Ypema & Gold, 1999). Originally derived from Strobilurus tenacellus, a wood rotting fungus, strobilurins were eventually modified by scientists to be more stable in the environment (Vincelli, 2002; Ypema & Gold, 1999). They have activity towards a broad spectrum of pathogens, including oomycetes, but expanding to true fungi such as

Fusarium species, as well as rusts (Vincelli, 2002, Ypema & Gold, 1999). Even though strobilurins have activity towards a broad spectrum of pathogens, interestingly, this fungicide class also has one specific target site. This is the quinol oxidation site in the cytochrome bc1 complex (Vincelli, 2002, Ypema & Gold, 1999). Considering this group of fungicides is site specific, the oomycetes (in this case), need only one mutation at this site, and a resistant strain of the pathogen can develop (Vincelli, 2002). There have been numerous cases of resistance reported to strobilurin fungicides, one of which is Downy

Mildew (Gee et al., 2013; Ishii et al., 2001, Ypema & Gold, 1999). There are many different types of strobilurins, however, they all have the same site specific mode of action. Therefore, the fungi recognize these as the same fungicide, and may become resistant to one strobilurin, but in effect it will actually be resistant to all strobilurins

(Vincelli, 2002). This action is referred to as cross-resistance (Vincelli, 2002). It is important to test for resistance to any high risk fungicides every few years.

Recent reports from Ohio have identified a few isolates that are insensitive to one or both of these fungicides (Broders et al., 2007; Dorrance et al., 2004). Therefore, the objective of this study was to: determine the fungicide sensitivity of a population of isolates of

Pythium species within Ohio, to the high rate of metalaxyl (100 ppm) and the commercial rate of pyraclostrobin, and then make recommendations based on these results.

Materials & Methods

Phytophthora and Pythium isolates

The isolates for this study were collected during 2005-2014 in the state of Ohio. Isolates were collected from infected soybean tissue. To do so, the roots of infected soybean samples were washed, by hand, with detergent (Tide, Proctor & Gamble, Cincinatti,

Ohio). This step is important to remove dirt and saprophytes on the exterior layer of the soybean roots. Then the roots were disinfected with 75% ethanol for 30 seconds, and

rinsed twice with sterile deionized water, in two separate beakers, for 30 seconds. The

root tissue was placed in a petri dish under PIBNC agar (V-8 media +

pentachloronitrobenzene, iprodione, benlate, neomycin sulfate, and chloramphenicol,

Dorrance et al., 2008). Clean isolates were then transferred to potato carrot agar (PCA) vials for storage. All isolates were stored in a 15°C cold room until used.

Sensitivity to metalaxyl

An amended broth assay as described by Olson et al. (2013) was used to evaluate metalaxyl sensitivity. Prior to testing, each isolate was transferred to potato carrot agar.

After 3 days of growth, a 3 mm plug of each isolate was placed into 2 wells of a 24 well,

cell-culture cluster plate (Corning Inc., Corning, NY). In every other row (A & C) the

control well contained 2 ml of sterile potato-carrot (PC) broth, and the opposite rows (B

& D) contained 2 ml of sterile PC broth amended with 100 ppm of technical grade metalaxyl (Syngenta). The amended broth was made by first mixing 100 mg of metalaxyl

in 1 mL of dimethylsulfoxide (DMSO) to dissolve, before it was added to 1 L of PC

broth. Each cell-culture plate held 12 different isolates at a time. Each isolate was

evaluated three times along with the controls. There were two control isolates; one that

was insensitive to metalaxyl: Ful 2-2-8 (P. irregulare), and one that was completely

sensitive to metalaxyl: Wyan 1-1-9 (P. ultimum var. ultimum). Both of these isolates were from the study previously reported by Broders et al (2009). The two controls were used to ensure that the PC broth and metalaxyl amended PC broth were consistent for every experiment. The experimental cell-culture plates were covered with a plastic bag and placed in an incubator at 22°C, in the dark. The cultures were examined 24 and 48 hours after inoculation. The isolates were rated on a scale from 0-5 for sensitivity, as described

by Olson et al. (2013), using a compound microscope (10x power): 0= no growth; 1= hyphae only grew microscopically, sporadic hyphae grew from plug- No uniformity in hyphae growth; 2= uniform hyphae growth, seen only microscopically; 3= uniform mycelium visible macroscopically; 4= mycelium uniform, however, not full growth compared to it’s non-amended control- quantified by 85-95% of control; 5= mycelium visible, uniform, and equal to the PCB control (100% of control).

Statistical Analysis

The means of the isolates from all replicates were analyzed using a Fisher’s exact test. It is important to use the mean of each isolates ratings to get the central value of all rates taken. Since the rate of metalaxyl used was the highest rate in which Pythium spp. could still grow, a rating under 1.0 was deemed sensitive to metalaxyl. If there was any growth at this high rate, then the isolate was considered insensitive (Gisi & Cohen, 1996). At the high concentration of 100 ppm metalaxyl, the resistance is controlled by a single co-

dominant nuclear locus (Gisi & Cohen, 1996).

Sensitivity to pyraclostrobin

Potato carrot agar (PCA) amended with pyraclostrobin and salicylhydroxamic acid

(SHAM) at the rate of 234.38 ppm (0.6 fl oz/cwt), PCA amended with SHAM, and non- amended PCA were prepared to evaluate the sensitivity of Pythium spp. to pyraclostrobin. The SHAM was dissolved in dimethylsulfoxide (DMSO) and methanol for a ratio of 1:1 and final concentration 50 ppm (50 μg/ml). SHAM was added to inhibit

77 the alternative oxidase respiratory pathway. It is important to inhibit this pathway, for accurate readings of the sensitivity to pyraclostrobin.

After 3 days of growth on PCA, a 3 mm plug of each isolate was placed into the center of

Petri plate with PCA, PCA+SHAM, or PCA+ SHAM + pyraclostrobin. After 48 hours of growth, two diameter readings from two different areas of growth were taken from each of the plates. The growth of each isolate on the SHAM-amended media was compared with the pyraclostrobin + SHAM amended media in order to determine the percent growth of the control.

Results

For this study, 250 Pythium or Phytophthora isolates were evaluated for their sensitivity to metalaxyl at 100 ppm from isolates collected before 2014. Fifty isolates collected from

Ohio fields in 2014 were also tested in 100 ppm metalaxyl. One hundred and forty-four isolates were evaluated for sensitivity to pyraclostrobin at the current commercial rate of

234.38 ppm, 127 of these were confirmed Pythium or Phytophthora. Some isolates were collected during the 2014 field season, and do not yet have species identification (Table,

26 ). The metalaxyl sensitive control (Wyan 1-1-9) and the metalaxyl insensitive control

(Ful 2-2-8) responded as expected across all experiments. Overall, the Pythium species evaluated in this study from different locations in Ohio, had differences in sensitivity to metalaxyl. Among these isolates representing 5 Pythium species, 2.7 % of all isolates evaluated had growth equal to the non-amended control. All isolates were sensitive to

78 pyraclostrobin at the highest commercial rate of 234.38 ppm (0.6 fl oz/cwt) amended with SHAM.

Metalaxyl

Pythium ultimum var. ultimum (18 isolates) and Pythium ultimum var. sporangiiferum (7 isolates) were almost all sensitive to metalaxyl (Figures 12 &13). There were 4 isolates rated between 1.0 and 2.0 (Appendix A). Pythium ultimum was previously reported to be completely sensitive to metalaxyl (Broders et al., 2007). Reports of intermediate sensitivity to metalaxyl may be an indication that this species is developing insensitivity.

There were 131 Pythium irregulare isolates with more than 50 % (75 isolates) with ratings 0-1.0. Less than 50 percent of isolates had ratings that ranged from 1.01 to 5.0, indicating insensitivity, confirming the results of Broders et al. (2007). One isolate of

Pythium crypto-irregulare, a species within the P. irregulare species complex, was evaluated and rated 2.0.

There were 38 isolates of Phytophthora sojae evaluated. Four of which had a mean score of 2-3, which indicates insensitivity. However, the remaining 34 isolates were all sensitive (<1).

One isolate of Pythium dissotocum had intermediate growth with a rating 1.0-2.0 and one with a rating of 4.

There were a few isolates evaluated representing 4 species, Phytophthora sansomeana (1 isolate), Pythium oopapillum (1 isolate), Pythium vexans (4 isolates), and Pythium attrantheridium (4 isolates), which had intermediate growth in the metalaxyl, indicating that they were all insensitive (Appendix A). There was a mixed response of sensitive and insensitive among other four Pythium species including: P. sylvaticum (18 isolates), P. torulosum (7 isolates), P. aphanidermatum (6 isolates), and P. inflatum (6 isolates)

(Appendix A).

Pyraclostrobin

Among the 125 Pythium isolates for sensitivity to pyraclostrobin, none were insensitive at this rate. There was no growth on the PCA + pyraclostrobin + SHAM amended agar plates.

Both the plates amended with SHAM alone and control PCA, had growth from the isolates tested. SHAM reduced the growth of the isolates by 35 %.

Discussion

In a previous study by Broders et al. (2007), variability among Pythium species for sensitivity to mefenoxam, the active isomer of metalaxyl, was found (Sukul & Spiteller,

2000). For example, most P. sylvaticum isolates tested were sensitive to mefenoxam at the high rate (100 ppm); while, 2 isolates were insensitive. P. irregulare was also variable in its sensitivity/insensitivity to mefenoxam in this previous study. The results

found in this current study confirm these earlier findings. In fact, Pythium species that

had more than one isolate tested had a mixed response to metalaxyl, except for P. attrantheridium which were all sensitive. The two varieties of Pythium ultimum had intermediate growth at 100 ppm. This is in contrast to Broders et al. (2007) previous findings where Pythium ultimum isolates were sensitive to mefenoxam. We detected moderate growth (score 1.01-3.9) in 4 out of 24 isolates in the study. This indicated that this population is now shifting towards resistance to metalaxyl.

The same trend may be beginning for Phytophthora sojae where 4 isolates tested from the 2013 field season had intermediate growth in metalaxyl (score 1.01-3.9). These 4 isolates were all from different fields. This is a significant finding. Previous to this study, there has been no insensitivity found from P. sojae in Ohio. Nelson et al. (2008) did report insensitivity to metalaxyl in North Dakota. Many of the fungicides used in seed treatments applied to corn and soybeans included a rate of 0.375 oz metalaxyl per 100 wt of seed. One specific isolate, from northwest Ohio, is from a field where metalaxyl has been used every year. With continuous use in the same field, it is not surprising that insensitivity developed. A broader range of isolates of P. sojae should be tested for sensitivity towards metalaxyl to determine how widespread this may be.

A difference in isolate reaction to pyraclostrobin was expected. However, all 144 isolates in this study were sensitive to pyraclostrobin at the rate tested. Evaluation of isolate sensitivity of P. sojae to pyraclostrobin at this and lower rates should continue before any

conclusions can be made about its efficacy towards the Pythium and Phytophthora

population in Ohio. Previously, Broders et al. (2007) did not test at a rate higher than 100 ppm, which could be another explanation for different results.

Fields in Ohio have more than one species of Pythium. Some more pathogenic and prevalent species such as P. sylvaticum, P. irregulare and P. ultimum (especially if they are all in the same field), can cause massive amounts of loss, if they are insensitive to these fungicides. With the differing results from the metalaxyl assay and sensitive results from the pyraclostrobin assay, combinations of these two fungicides as seed treatments would provide the best protection for Pythium and Phytophthora that contribute to seed and seedling damping-off. These results also suggest that high rates of metalaxyl/mefenoxam may be needed for best protection. Although additional greenhouse studies with treated seed are required to confirm the high rates will protect seed towards isolates that have limited (scores of 1-2) growth in 100 ppm.

In summary, metalaxyl and mefenoxam were effective to more than 50 % of the isolates collected in Ohio for this study. There was a range of insensitivity both among and within the Pythium spp. that were tested. Thus no conclusions about a specific species as insensitive can be determined. Ultimately though, new fungicides are needed that can provide better protection in this vulnerable life stage of soybean and corn.

Pythium irregulare 80 70

60 50 40 30

Number of of Number Isolates 20 10 0 0.0-1.0 1.0-2.0 2.0-3.0 3.0-4.0 4.0-5.0 Mean score

Figure 7. The Mean score of 131 Pythium irregulare isolates tested in potato carrot broth amended with 100 ppm metalaxyl. Rated on a scale from 0-5: 0= no growth; 1= hyphae

only grew microscopically, sporadic hyphae grew from plug- No uniformity in hyphae

growth; 2= uniform hyphae growth, seen only microscopically; 3= uniform mycelium

visible macroscopically; 4= mycelium uniform, however, not full growth compared to it’s

non-amended control- quantified by 85-95% of control; 5= mycelium visible, uniform,

and equal to the PCB control (100% of control), after 24 and 48 hours of growth.

Pythium sylvaticum 8 7

6 5 4 3

Number of of Number Isolates 2 1 0 0.0-1.0 1.0-2.0 2.0-3.0 3.0-4.0 4.0-5.0 Mean Score

Figure 8. The Mean score of 18 Pythium sylvaticum isolates tested in potato carrot broth amended with 100 ppm metalaxyl. Rated on a scale from 0-5: 0= no growth; 1= hyphae

only grew microscopically, sporadic hyphae grew from plug- No uniformity in hyphae

growth; 2= uniform hyphae growth, seen only microscopically; 3= uniform mycelium

visible macroscopically; 4= mycelium uniform, however, not full growth compared to it’s

non-amended control- quantified by 85-95% of control; 5= mycelium visible, uniform,

and equal to the PCB control (100% of control), after 24 and 48 hours of growth.

Pythium torulosum 4.5 4 3.5 3 2.5 2 1.5

Number of of Number Isolates 1 0.5 0 0.0-1.0 1.0-2.0 2.0-3.0 3.0-4.0 4.0-5.0 Mean Score

Figure 9. The mean score of 7 Pythium torulosum isolates tested in potato carrot broth amended with 100 ppm metalaxyl. Rated on a scale from 0-5: 0= no growth; 1= hyphae only grew microscopically, sporadic hyphae grew from plug- No uniformity in hyphae growth; 2= uniform hyphae growth, seen only microscopically; 3= uniform mycelium visible macroscopically; 4= mycelium uniform, however, not full growth compared to it’s non-amended control- quantified by 85-95% of control; 5= mycelium visible, uniform, and equal to the PCB control (100% of control), after 24 and 48 hours of growth.

Pythium dissotocum 1.2

0.8

0.6

0.4

Number of of Number Isolates 0.2

0 0.0-1.0 1.0-2.0 2.0-3.0 3.0-4.0 4.0-5.0 Mean Score

Figure 10. The mean score of 2 Pythium dissotocum isolates tested in potato carrot broth amended with 100 ppm metalaxyl. Rated on a scale from 0-5: 0= no growth; 1= hyphae only grew microscopically, sporadic hyphae grew from plug- No uniformity in hyphae growth; 2= uniform hyphae growth, seen only microscopically; 3= uniform mycelium visible macroscopically; 4= mycelium uniform, however, not full growth compared to it’s non-amended control- quantified by 85-95% of control; 5= mycelium visible, uniform, and equal to the PCB control (100% of control), after 24 and 48 hours of growth.

Pythium attrantheridium 4.5 4 3.5 3 2.5 2 1.5

Number of of Number Isolates 1 0.5 0 0.0-1.0 1.0-2.0 2.0-3.0 3.0-4.0 4.0-5.0 Mean Score

Figure 11. The mean score of 4 Pythium attrantheridium isolates tested in potato carrot

broth amended with 100 ppm metalaxyl. Rated on a scale from 0-5: 0= no growth; 1=

hyphae only grew microscopically, sporadic hyphae grew from plug- No uniformity in hyphae growth; 2= uniform hyphae growth, seen only microscopically; 3= uniform mycelium visible macroscopically; 4= mycelium uniform, however, not full growth compared to its non-amended control- quantified by 85-95% of control; 5= mycelium visible, uniform, and equal to the PCB control (100% of control), after 24 and 48 hours of

growth.

Pythium ultimum var. ultimum 14 12

10 8 6 4 Number of of Number Isolates 2 0 0.0-1.0 1.0-2.0 2.0-3.0 3.0-4.0 4.0-5.0 Mean Score

Figure 12. The mean score of 16 Pythium ultimum var. ultimum isolates tested in potato

carrot broth amended with 100 ppm metalaxyl. Rated on a scale from 0-5: 0= no growth;

growth.

Pythium ultimum var. sporangiiferum 6

Number of of Number Isolates 1

0 0.0-1.0 1.0-2.0 2.0-3.0 3.0-4.0 4.0-5.0 Mean Score

Figure 13. The mean score of 7 Pythium ultimum v. sporangiiferum isolates tested in

potato carrot broth amended with 100 ppm metalaxyl. Rated on a scale from 0-5: 0= no

growth; 1= hyphae only grew microscopically, sporadic hyphae grew from plug- No uniformity in hyphae growth; 2= uniform hyphae growth, seen only microscopically; 3= uniform mycelium visible macroscopically; 4= mycelium uniform, however, not full growth compared to its non-amended control- quantified by 85-95% of control; 5= mycelium visible, uniform, and equal to the PCB control (100% of control), after 24 and

48 hours of growth.

P. aphanidermatum 4.5 4 3.5 3 2.5 2 1.5

Number of of Number Isolates 1 0.5 0 0.0-1.0 1.0-2.0 2.0-3.0 3.0-4.0 4.0-5.0 Mean Score

Figure 14. The mean score of 6 Pythium aphanidermatum isolates tested in potato carrot

broth amended with 100 ppm metalaxyl. Rated on a scale from 0-5: 0= no growth; 1=

hyphae only grew microscopically, sporadic hyphae grew from plug- No uniformity in hyphae growth; 2= uniform hyphae growth, seen only microscopically; 3= uniform mycelium visible macroscopically; 4= mycelium uniform, however, not full growth compared to it’s non-amended control- quantified by 85-95% of control; 5= mycelium visible, uniform, and equal to the PCB control (100% of control), after 24 and 48 hours of

growth.

Pythium inflatum 3.5 3

2.5 2 1.5 1 Number of of Number Isolates 0.5 0 0.0-1.0 1.0-2.0 2.0-3.0 3.0-4.0 4.0-5.0 Mean Score

Figure 15. The mean score of 6 Pythium inflatum isolates tested in potato carrot broth amended with 100 ppm metalaxyl. Rated on a scale from 0-5: 0= no growth; 1= hyphae only grew microscopically, sporadic hyphae grew from plug- No uniformity in hyphae growth; 2= uniform hyphae growth, seen only microscopically; 3= uniform mycelium visible macroscopically; 4= mycelium uniform, however, not full growth compared to it’s non-amended control- quantified by 85-95% of control; 5= mycelium visible, uniform, and equal to the PCB control (100% of control), after 24 and 48 hours of growth.

Phytophthora sojae 35 30

25 20 15 10 Number of of Number Isolates 5 0 0.0-1.0 1.0-2.0 2.0-3.0 3.0-4.0 4.0-5.0 Mean Score

2/2

15/37 3/4 1/2 1/3

5/9

4/9 1/1

1/1

2/2

1/4

2/3 3/3

4/6 4/5 1/4

3/8 1/3

References

Anonymous. "FRAC Code List ©*2013: Fungicides Sorted by Mode of Action (including FRAC Code Numbering)." Frac. Fungicide Resistance Action Committee, 2013. Web. 27 Mar. 2013. .

Bates, G. D., Rothrock, C. S., and Rupe, J. C. 2008. Resistance of the soybean cultivar Archer to Pythium damping-off caused by several Pythium spp. Plant Dis. 92:763-766.

Beuerlein, J., and A.E. Dorrance, 2005. Soybean Production. Ohio Agronomy Guide 14th Edition 14: 57-71.

Bowen, C., Melouk, H. A., Jackson, K. E., and Payton, M. E. 2000. Effect of a select group of seed protectant fungicides on growth of Sclerotinia minor in vitro and its recovery from infested peanut seed. Plant Dis. 84:1217-1220.

Bradley, C. A. 2008. Effect of Fungicide Seed Treatments on Stand Establishment, Seedling Disease, and Yield of Soybean in North Dakota. Plant Dis. 92: 120-125.

Broders, K. D., Wallhead, M. W., Austin, G. D., Lipps, P. E., Paul, P. A., Mullen, R. W., and Dorrance, A. E. 2009. Association of soil chemical and physical properties with Pythium species diversity, community composition, and disease incidence. Phytopathology 99:957-967.

Broders, K. D., Lipps, P. E., Ellis, M. L., and Dorrance, A. E. 2009. Pythium delawarii – a new species isolated from soybean in Ohio. Mycologia. 101:232-238.

Broders, K. D., Lipps, P. E., Paul, P. A., and Dorrance, A. E. 2007. Characterization of Pythium spp. associated with corn and soybean seed and seedling disease in Ohio. Plant Dis. 91:727-735.

Campa, A., Pérez-Vega, E., Pascual, A., and Ferreira, J. J. 2010. Genetic analysis and molecular mapping of quantitative train loci in common bean against Pythium ultimum. Phytopathology 100:1315-1320

Cook, P. J., Landschoot, P. J., and Schlossberg, M. J. 2009. Inhibition of Pythium spp. and suppression of Pythium blight of turfgrasses with phosphonate fungicides. Plant. Dis. 95:809-814.

Donaldson, S. P., and J. W. Deacon. 1993. Effects of amino acids and Sugars on Zoospore Taxis, Encystment and Cyst Germination in Pythium Aphanidermatum (Edson) Fitzp., P. Catenulatum Matthews and P. Dissotocum Drechs. New Phytologist. 123:289-95.

Dorrance, A. E., McClure, S. A., and deSilva, A. 2003. Pathogenic diversity of Phytophthora sojae in Ohio soybean fields. Plant Dis. 87:139-146.

Dorrance, A. E., S. A. Berry, P. Bowen, and P. E. Lipps. 2004 "Characteristics of Pythium Spp. from Three Ohio Fields for Pathogenicity on Corn and Soybean and Metalaxyl Sensitivity. Online. Plant Health Progress Doi: 10.1094/PHP-2004-0202-01-RS.

Dorrance, A. E., Berry, S. A., Anderson, T. R. and Meharg, C. 2008. Isolation, storage, pathotype characterization, and evaluation of resistance for Phytophthora sojae in soybean. Online. Plant Health Progress doi:10.1094/PHP-2008-0118-01-DG.

Dorrance, A.E., Robertson, A.E., Cianzo, S., Giesler, L.J., Grau, C.R., Draper, M.A., Tenuta, A.U., and Anderson, T.R. 2009. Integrated management strategies for Phytophthora sojae combining host resistance and seed treatment. Plant Dis. Vol. 93: 875-882.

Ellis, M. L., Paul, P. A., Dorrance, A. E., and Broders, K. D. 2012. Two new species of Pythium, P. schmitthenneri and P. selbyi pathogens of corn and soybean in Ohio. Mycologia. 104:477-487.

Ellis, M.L., McHale, L.K., Paul, P. A., St. Martin, S.K., and Dorrance, A. E. 2013. Soybean Germplasm Resistant to Pythium irregulare and Molecular Mapping of Resistance Quantitative Trait Loci derived from the Soybean Accession PI 424354. 53:1008-1021.

Esker, P. D., and S. P. Conley. 2012. Probability of Yield Response and Breaking Even for Soybean Seed Treatments. Crop Science. 52: 351-359.

Francis, D.M., and DA St. Clair. 1993. Outcrossing in the homothallic oomycete, Pythium ultimum detected with molecular markers. Current Genetics. 100-106.

Garzón, C. D., Molineros, J. E., Yánez, J. M., Flores, F. J., Jiménez-Gasco, M. M., and Moorman, G. W. 2011. Sublethal doses of mefenoxam enhance Pythium damping-off of geranium. Plant Dis. 95:1233-1238.

Gee, C. T., Chestnut, S., Duberow, E., Collins, A., and Shields, M. A. 2013. Downy mildew from Lake Erie vineyards is diverse for the G143A SNP conferring resistance to Quinone outside inhibitor fungicides. Online. Plant Health Progress doi:10.1094/PHP-2013-0422-01- RS.

Gisi, U., and Y. Cohen. 1996. Resistance to phenylamide fungicides: A case study with Phytophthora infestans involving mating type and race structure. Annual Rev. Phytopathology. 34:549-72.

Grau, Craig R., Anne E. Dorrance, Jason Bond, and John S. Russin. "Fungal Diseases."Soybeans: Improvement, Production, and Uses. Ed. H. Roger. Boerma and James Eugene. Specht. 3rd ed. Vol. 16. Madison, WI: American Society of Agronomy, Crop Science Society of America, Soil Science Society of America, 2004. 679-763. Print.

Griffin, G. J. 1990. Importance of Pythium ultimum in a disease syndrome of cv. Essex soybean. Can. J. Plant Pathol. 12:135-140. 95

Grove, G. G., Ellis, M. A., Madden, L. V., and Schmitthenner, A. F. 1985. Overwinter survival of Phytophthora cactorum in infected strawberry fruit. Plant Dis. 69:514-515.

Ishii, H., Fraaije, B. A., Sugiyama, T., Noguchi, K., Nishimura, K., Takeda, T., Amano, T., and Hollomon, D. W. 2001. Occurrence and molecular characterization of strobilurin resistance in cucumber powdery mildew and downy mildew. Phytopathology 91:1166-1171.

Kamoun, S. 2003. Molecular genetics of pathogenic oomycetes. Eukaryotic Cell. 2:191-199.

Kirkpatrick, M. T., Rothrock, C. S., and Rupe, J. C. 2006. The effect of Pythium ultimum and soil flooding on two soybean cultivars. Plant Dis. 90:597-602.

Lévesque, C. A. and de Cock, A. W. A. M. 2004. Molecular phylogeny and taxonomy of the genus Pythium. Mycol. Res. 108:1363-1383.

Martin, Frank. "Pythium Genetics." Oomycete Genetics and Genomics: Diversity, Interactions, and Research Tools. Ed. Kurt Lamour and Sophien Kamoun. Hoboken, NJ: Wiley-Blackwell, 2009. 213-39. Print.

Frank N. Martin & Joyce E. Loper.1999. Soilborne Plant Diseases Caused by Pythium spp.: Ecology,Epidemiology, and Prospects for Biological Control, Critical Reviews in Plant Sciences, 18:2, 111-181.

Mcgee, D. C. 1986. Treatment of Soybean Seeds.Seed Treatment. 2nd ed. Thornton Heath: British Crop Protection Council. 185-97. Print.

Moorman, G. W., and Kim, S. H. 2004. Species of Pythium from greenhouses in Pennsylvania exhibit resistance to propamocarb and mefenoxam. Plant Dis. 88:630-632.

Mueller, D. S., Hartman, G. L, and Pedersen, W. L. 1999. Development of sclerotia and apothecia of Sclerotinia sclerotiorum from infected soybean seed and its control by fungicide seed treatment. Plant Dis. 83:1113-1115.

Mueller, D. S., Nelson, R. L., Hartman, G. L., and Pederson, W. L. 2003. Response of commercially developed soybean cultivars and the ancestral soybean lines to Fusaium solani f. sp. glycines. Plant Dis. 87:827-831.

Murillo-Williams, A., and Pedersen, P. 2008. Early incidence of soybean seedling pathogens in Iowa. Agron. J. 100:1481-1487.

Nelson, B. D., Mallik, I., McEwen, D., and Christianson, T. 2008. Pathotypes, distribution, and metalaxyl sensitivity of Phytophthora sojae from North Dakota. Plant Dis. 92:1062-1066.

Olive, L. S. 1963. Genetics of homothallic fungi. . Mycologia. 55: 93-103.

Olson, H. A., Jeffers, S. N., Ivors, K. L., Steddom, K. C., Williams-Woodward, J. J., Mmbaga, M. T., Benson, D. M., and Hong, C. X. 2013. Diversity and mefenoxam sensitivity of Phytophthora spp. associated with the ornamental horticulture industry in the southeastern United States. Plant Dis. 97:86-92.

Porter, D. M., and Phipps, P. M. 1985. Effects of three fungicides on mycelial growth, sclerotium production, and development of fungicide-tolerant isolates of Sclerotinia minor. Plant Dis. 69:143-146.

Rebollar-Alviter, A., Madden, L. V., Jeffers, S. N., and Ellis, M. A. 2007. Baseline and differential sensitivity to two QoI fungicides among isolates of Phytophthora cactorum that cause leather rot and crown rot on strawberry. Plant Dis. 91:1625-1637.

Schmitthenner, A. F., Hobe, M., and Bhat, R. G. 1994. Phytophthora sojae races in Ohio over a 10-year interval. Plant Dis. 78:269-276.

Schroeder, K. L., Martin, F. N., de Cock, A. W. M., Lévesque, C. A., Spies, C. F. J., Okubara, P. A., and Paulitz, T. C. 2013. Molecular detection and quantification of Pythium species: evolving taxonomy, new tools, and challenges. Plant Dis. 97:4-20.

Stoskopf, N. C., Tomes, D. T., and Christie, B. R. 1993. Plant Breeding Theory and Practice. Westview Press, Boulder, Co.pp91.

Sukul, P., and Spiteller, M. 2000. Metalaxyl: persistence, degradation, metabolism, and analytical methods. Rev. Environ. Contam. Toxicol. 164: 1-26.

Taylor, R. J., Salas, B., Secor, G. A., Rivera, V., and Gudmestad, N. C. 2002. Sensitivity of North American isolates of Phytophthora erythroseptica and Pythium ultimum to mefenoxam (metalaxyl). Plant Dis. 86:797-802.

Tu, C. X. 1982. Effects of Some Pesticides on Rhizobium japonicum and on the seed germination and pathogens of soybean. Chemosphere. 11:1027-1033.

Varshney, R. K., Terauchi, R., and McCouch, S. R. 2014. Harvesting the Promising Fruits of Genomics: Applying Genome Sequencing Technologies to Crop Breeding. PLoS Biol 12(6): e1001883. doi:10.1371/journal.pbio.1001883.

Wilson, E. W., Rowntree, S.C., Suhre, J.J., Weidenbenner, N.H., Conley, S.P., Davis, V.M., Diers, B.W., Esker, P.D., Naeve, S.L., Specht, J.E., & Casteel, S.N. 2014. Genetic Gain x Management Interactions in Soybean: II Nitrogen Utilization. Crop Science. 54:340-348.

Wrather, A., Shannon, G., Balardin, R., Carregal, L., Escobar, R., Gupta, G. K., Ma, Z., Morel, W., Ploper, D., and Tenuta, A. 2010. Effect of diseases on soybean yield in the top eight producing countries in 2006. Online. Plant Health Progress doi:10.1094/PHP-2010-0125-01-RS.

Ypema, H.L., and Gold, R.E. 1999. Kresoxim-methyl: modification of a naturally occurring compound to produce a new fungicide. Plant Disease. 83: 4-19.

Zhang, B. Q., Chen, W. D., and Yang, X. B. 1998. Occurrence of Pythium species in long-term maize and soybean monoculture and maize/soybean rotation. Mycol. Res. 102:1450-1452.

Zhang, B. Q., and Yang, X. B. 2000. Pathogenicity of Pythium populations from corn-soybean rotation fields. Plant Dis. 84:94-99.

National Statistics for Soybeans. National Agricultural statistics service, USDA. Retrieved 25 July 2013 from http://www.nass.usda.gov/Statistics_by_Subject/index.php.

Pythium Genome Database: Pythium Ultimum Var. Ultimum. Michigan State University. Retrieved 13 June 2014, from: http://pythium.plantbiology.msu.edu/Pythium_ultimum_var_ultimum.shtml

Pythium Genome Database: Pythium Ultimum Var. sporangiiferum. Michigan State University. Retrieved 13 June 2014, from: http://pythium.plantbiology.msu.edu/Pythium_ultimum_var_sporangiiferum.shtml

Appendix A: ANOVA for checks & Metalaxyl and Pyraclostrobin Table

Table 23. ANOVA for root score of checks. Excluding Conrad and the experiments for

Virginia Tech.

Source DF F Pr > F Value loc 3 2.44 0.0645 rep(loc) 8 3.84 0.0003 isol 1 2.85 0.0927 line 8 14.57 < .0001 isol*line 8 0.41 0.9152 loc*isol 3 58 <.0001 loc*line 23 2.36 0.0006 loc*isol*line 22 1.05 0.4047

Table 24. ANOVA for root weight of checks. Excluding Conrad and the experiments for

Virginia Tech.

Source DF F Pr > F Value loc 3 9.08 <.0001 rep(loc) 8 5.69 <.0001 isol 1 0.02 0.8974 line 8 26.12 <.0001 isol*line 8 1.35 0.219 loc*isol 3 62.68 <.0001 loc*line 23 2.54 0.0002 loc*isol*line 22 2.3 0.001

100

Table of isolates tested in Metalaxyl and Pyraclostrobin. Including if they were sequenced, species id, metalaxyl rating, if tested in pyraclostrobin, and the year they were isolated.

Isolate Code Seq Species Metalaxyl Pyraclo Year Rating Def 11 Y Phytophthora 1 2013 sansomeana NWB 1-w Y Phytophthora sojae 3 2013 NWB 3e Y Phytophthora sojae 1 2013 OH.12142.06.03 Y Phytophthora sojae 1 2012 OH- Phytophthora sojae 1 2012 12001.10.02 OH- Phytophthora sojae 1 2012 12103.01.04 OH- Phytophthora sojae 1 2012 12110.08.01 OH- Phytophthora sojae 1 2012 12121.02.01 OH-12135. 02. Phytophthora sojae 1 2012 04 OH- Phytophthora sojae 1 2012 12135.05.02 OH- Phytophthora sojae 2012 12137.05.03 OH- Phytophthora sojae 2.5 2012 12138.10.02 OH- Y Phytophthora sojae 1 2012 12139.04.04 OH- Y Phytophthora sojae 1 2012 12139.06.03 OH- Y Phytophthora sojae 1 2012 12142.03.02 OH- Y Phytophthora sojae 1 2012 12142.03.05 OH- Y Phytophthora sojae 1 2012 12142.05.02 OH- Y Phytophthora sojae 1 2012

101

12142.08.05 OH12144.09.02 Phytophthora sojae 2.5 2012 OH-12147. Y Phytophthora sojae 1 2012 05.04 OH- Y Phytophthora sojae 1 2012 12147.08.01 OH- Y Phytophthora sojae 1 2012 12148.01.07 OH- Y Phytophthora sojae 1 2012 12150.01.03 OH-12150- Y Phytophthora sojae 1 2012 03.02 OH- Y Phytophthora sojae 1 2012 12154.07.03 OH- Phytophthora sojae 1 2012 12164.05.01 OH- Phytophthora sojae 1 2012 12164.07.01 OH- Phytophthora sojae 1 2012 12164.08.01 OH- Phytophthora sojae 2.5 2012 12164.10.03 OH- Phytophthora sojae 1 2012 12167.05.01 OH- Phytophthora sojae 1 2012 12168.01.02 OH- Phytophthora sojae 1 2012 12170.10.01 OH- Phytophthora sojae 1 2012 12172.01.04 OH- Phytophthora sojae 1 2012 12180.04.02 P2-106 P1-3 Pythium 1 2010 P2-103 P2-2 Pythium 4 2010 aphanadermatum Parsely Y Pythium 0.7 P 2012 aphanadermatum pepper Y Pythium 0.2 P 2012 aphanadermatum squash Y Pythium 3 2012 aphanadermatum Sweet Pepper Y Pythium 0 P 2012 aphanadermatum 102

Tomato Y Pythium 1 P 2012 aphanadermatum 18 Def 8c exp1 Pythium attrantheridium 1 2010 Br 209 pb2 Y Pythium attrantheridium 1 2010 D10 407-6r Pythium attrantheridium 1 P* 2010 H3-511 P5-1 Y Pythium attrantheridium 1 2010 Shelby 1-1-3 Y Pythium crypto- 2 P* 2006/ irregulare 2007 18 Def 2A e1 Pythium dissotocum 4 2010 Erie 1-1-9 A2 Pythium dissotocum 1.3 2006/ 2007 H3-506 P1-1 Pythium 0 2010 heterothallicum P2-103 P1-2 Pythium 1 2010 heterothallicum Aug 3-3-3 Pythium inflatum 3 P* 2006/ 2007 Erie 1-1-1 A1 Pythium inflatum 0.7 2006/ 2007 Erie 1-2-11 C3 Pythium inflatum 5 2006/ 2007 Erie 1-6-5 H6 Pythium inflatum 1 2006/ 2007 Erie 1-6-6 A7 Pythium inflatum 3 2006/ 2007 18 Def 12AE1 Y Pythium irregulare 2 2010 18 Pike 2A e1 Y Pythium irregulare 1 2010 18 Pike 2B e1 Y Pythium irregulare 1 2010 18 Pike 3A e1 Y Pythium irregulare 1 2010 18Adams2Be2 Y Pythium irregulare 2 2010 18Brown 2Ae1 Y Pythium irregulare 1 2010 18HIGH11A Y Pythium irregulare 2 2010 EXP 1 18HIGH 1B Pythium irregulare 2 2010 EXP1 18HIGH 1C Y Pythium irregulare 2 2010 EXP 1 18HIGH 2A Y Pythium irregulare 3 2010 EXP2 18Pike4c e1 Y Pythium irregulare 2 2010 24 Brown 1B e1 Y Pythium irregulare 1 2010 24 Brown 2A e1 Pythium irregulare 1 2010 103

24 High 5B e1 Y Pythium irregulare 1 2010 24adams 3A-1 Y Pythium irregulare 1 2010 e2 24Adams 4A1 Y Pythium irregulare 1 2010 e2 24Brown1A e1 Y Pythium irregulare 2 2010 24Brown3A e1 Y Pythium irregulare 2 2010 24High 5A e1 Y Pythium irregulare 1 2010 Ash 1-1-9 Y Pythium irregulare 1 2006/ 2007 Ash 1-6-3 Y Pythium irregulare 0 2006/ 2007 Ash 2-1-13 Pythium irregulare 2 2006/ 2007 Ash 2-1-14 Pythium irregulare 2 P* 2006/ 2007 Ash 2-1-17 Y Pythium irregulare 0 P* 2006/ 2007 Ash 2-2-10 Y Pythium irregulare 2 2006/ 2007 Ash 2-2-6 Y Pythium irregulare 2 P* 2006/ 2007 Ash 2-4-2 Y Pythium irregulare 1 P* 2006/ 2007 Ash 2-6-2 Y Pythium irregulare 2 2006/ 2007 Aug 3-2-1 Pythium irregulare 1 P* 2006/ 2007 BR 2-3-5 Y Pythium irregulare 1 2006/ 2007 Br 2-5-14 Y Pythium irregulare 0 2006/ 2007 Br 2-5-3 Y Pythium irregulare 1.5 2006/ 2007 Cham 2-3-4 Y Pythium irregulare 2 2006/ 2007 Clark 1-4-13 Y Pythium irregulare 2 P* 2006/ 2007 Clark1-4-16 Y Pythium irregulare 1.7 2006/ 2007 Cle 1-1-12 Y Pythium irregulare 2 P* 2006/ 2007 Cler 1-1-12 Y Pythium irregulare 2 2006/ 104

2007 Cler 1-4-1 Y Pythium irregulare 2 P* 2006/ 2007 Cler 1-6-11 Y Pythium irregulare 2 P* 2006/ 2007 Cler 1-6-6 Pythium irregulare 1 P* 2006/ 2007 Craw 1-1-8 Pythium irregulare 1 2006/ 2007 Craw 1-2-9 Pythium irregulare 0 2006/ 2007 Darke 3-1-8 Y Pythium irregulare 1.3 2006/ 2007 Def 2-4-14 Y Pythium irregulare 0 2006/ 2007 Def 2-5-22 Y Pythium irregulare 1 2006/ 2007 Def 2-5-22 Y Pythium irregulare 1.7 2006/ 2007 Def 2-6-15 (G2) Pythium irregulare 1.7 2006/ 2007 Erie 2-5-5 Y Pythium irregulare 1 P* 2006/ 2007 Erie 2-6-1 Y Pythium irregulare 0 P* 2006/ 2007 Fay 2-3-2 Y Pythium irregulare 1.5 P 2006/ 2007 Fay 2-4-17 Y Pythium irregulare 0.5 2006/ 2007 Fay 2-4-3 Y Pythium irregulare 1.5 2006/ 2007 Ful 2-2-8 Pythium irregulare 5 2006/ (check) 2007 Ful 2-6-4 Y Pythium irregulare 1.3 2006/ 2007 Green 1-5-4 Pythium irregulare 1 P* 2006/ 2007 Green 2-3-3 Y Pythium irregulare 0 P* 2006/ 2007 Green 2-4-7 Y Pythium irregulare 1 2006/ 2007 Hen 1-5-11 Pythium irregulare 1.3 2006/ 2007 105

Hen 1-6-4 Y Pythium irregulare 1 P* 2006/ 2007 High 2-5-8 Y Pythium irregulare 1.7 2006/ 2007 Hur 1-4-11 Y Pythium irregulare 2 2006/ 2007 Huron 1-1-13 Y Pythium irregulare cont P* 2006/ 2007 Logan 1-6-6 Y Pythium irregulare 1.7 2006/ 2007 Logan 2-3-15 Pythium irregulare 0.7 P* 2006/ 2007 Logan 2-3-4 Pythium irregulare 1.3 2006/ 2007 Logan 2-4-8 Pythium irregulare 1.3 P* 2006/ 2007 Logan 2-5-16 Pythium irregulare 0.7 P* 2006/ 2007 Logan 2-5-4 Pythium irregulare 1 P* 2006/ 2007 Logan 2-6-11 Pythium irregulare 1.3 P* 2006/ 2007 Logan 2-6-7 Pythium irregulare 0.7 P* 2006/ 2007 Logan 2-6-9 Pythium irregulare 0 P* 2006/ 2007 Lucas 2-1-1 Y Pythium irregulare 1 P* 2006/ 2007 Mad 2-1-4 Y Pythium irregulare cont P* 2006/ 2007 Miami 1-2-8 Pythium irregulare 1 2006/ 2007 Miami 1-4-8 Y Pythium irregulare 0 P* 2006/ 2007 Mont 1-1-12 Pythium irregulare 1 2006/ 2007 Mont 1-1-5 Y Pythium irregulare 0 P* 2006/ 2007 Mont 1-1-7 Y Pythium irregulare 1.5 2006/ 2007 Mont 1-4-1 Y Pythium irregulare 1 2006/ 2007 Mor 2-2-5 Pythium irregulare 0 2006/ 106

2007 Mor 2-3-11 Pythium irregulare 0 2006/ 2007 Mor 2-4-8 Y Pythium irregulare 0 P* 2006/ 2007 Put 1-1-5 Pythium irregulare 1 P* 2006/ 2007 Put 1-1-6 Pythium irregulare 1 P* 2006/ 2007 Put 1-3-2 Pythium irregulare 0 P* 2006/ 2007 Put 1-3-5 Pythium irregulare 0 2006/ 2007 Put 1-3-6 Pythium irregulare 0 2006/ 2007 Put 1-5-7 Y Pythium irregulare 0 2006/ 2007 Put 1-5-8 Y Pythium irregulare 0.5 P* 2006/ 2007 Put 1-6-5 Pythium irregulare 0 2006/ 2007 sand 1-1-10 Pythium irregulare 1 2006/ 2007 Sand 1-1-15 Pythium irregulare 1 2006/ 2007 Sand 1-1-3 Pythium irregulare 1 2006/ 2007 Sand 1-1-8 Pythium irregulare 0 2006/ 2007 Sand 1-2-1 Pythium irregulare 0 2006/ 2007 Sand 1-2-10 Pythium irregulare 1 2006/ 2007 Sand 1-2-13 Pythium irregulare 0 2006/ 2007 Sand 1-2-14 Pythium irregulare 0 2006/ 2007 Sand 1-2-16 Pythium irregulare 0 2006/ 2007 Sand 1-2-2 Pythium irregulare 0 2006/ 2007 Sand 1-2-20 Pythium irregulare 0.3 2006/ 2007 107

Sand 1-2-21 Pythium irregulare 0 2006/ 2007 Sand 1-2-5 Pythium irregulare 1 2006/ 2007 Sand 1-2-6 Pythium irregulare 1 2006/ 2007 Sand 1-3-1 Pythium irregulare 0 2006/ 2007 Sand 1-3-10 Pythium irregulare 0.3 2006/ 2007 Sand 1-3-11 Pythium irregulare 0.3 2006/ 2007 Sand 1-3-4 Pythium irregulare 1 2006/ 2007 Sand 1-3-9 Pythium irregulare 1 2006/ 2007 Sand 1-4-10 Pythium irregulare 1 2006/ 2007 Sand 1-4-13 Pythium irregulare 1.3 P* 2006/ 2007 Sand 1-4-14 Pythium irregulare 1.3 P* 2006/ 2007 Sand 1-4-19 Pythium irregulare 1.3 P* 2006/ 2007 Sand 1-4-23 Pythium irregulare 1.3 P* 2006/ 2007 Sand 1-4-25 Y Pythium irregulare 2 P* 2006/ 2007 Sand 1-4-5 Pythium irregulare 1.7 2006/ 2007 Sand 1-4-9 Pythium irregulare 0.3 P* 2006/ 2007 Sand 1-6-11 Pythium irregulare 1 2006/ 2007 Sand 1-6-12 Pythium irregulare 1.3 P* 2006/ 2007 Sand 1-6-14 Pythium irregulare 2 P* 2006/ 2007 Sand 1-6-15 Y Pythium irregulare 2.3 P* 2006/ 2007 Sand 1-6-16 Pythium irregulare 1.7 2006/ 2007 Sand 1-6-17 Pythium irregulare 1.7 P* 2006/ 108

2007 Sand 1-6-18 Pythium irregulare 1.7 P* 2006/ 2007 Sand 1-6-25 Pythium irregulare 1.3 P* 2006/ 2007 Sand 1-6-4 Pythium irregulare 2 P* 2006/ 2007 Sand 1-6-9 Pythium irregulare 2 P* 2006/ 2007 Shelby 1-6-4 Pythium irregulare 0 2006/ 2007 War 1-1-13 Y Pythium irregulare 2 2006/ 2007 War 1-1-19 Y Pythium irregulare 2 2006/ 2007 War 1-6-3 Y Pythium irregulare 2 2006/ 2007 Wood 1-4-13 Y Pythium irregulare 2 P 2006/ 2007 wood 1-5-15 Pythium irregulare 0 2006/ 2007 wood 1-5-2 Pythium irregulare 0 2006/ 2007 Def 9 Y Pythium oopapillum 3 2013 H3-504 P1-1 Pythium spp. 5 2010 18 Def 4A e1 Pythium sylvaticum 1 2010 18CLER 4A Pythium sylvaticum 3 P 2010 EXP1 18DEF 11C Pythium sylvaticum 3 2010 EXP1 18DEF 14A Pythium sylvaticum 2 2010 EXP2 18DEF 15B Pythium sylvaticum 3 2010 EXP2 18DEF 2A Pythium sylvaticum 2 2010 EXP2 18DEF 3B Pythium sylvaticum 3 2010 EXP2 18DEF 5B Pythium sylvaticum 3 2010 EXP2 18DEF 6A Pythium sylvaticum 2 2010 EXP2 Ad-1 pb2 Y Pythium sylvaticum 3 2010 109

Aug 3-1-5 Pythium sylvaticum 1 2006/ 2007 Br 213 Pb3 Y Pythium sylvaticum 3 2010 high3 p4-2 Y Pythium sylvaticum 2 P* 2010 Huron 1-1-5 Pythium sylvaticum 1 2006/ 2007 P3-105 P2-1 Y Pythium sylvaticum 2 2010 Wood 2-5-17 Y Pythium sylvaticum 2 2006/ 2007 18Cler 11A e1 Pythium torulosum 1 2010 18Def 11A e1 Pythium torulosum 0 2010 18DEF 1A Pythium torulosum 2 2010 EXP1 18Def 1B e1 Pythium torulosum 2.5 2010 Erie 1-4-4 F4 Pythium torulosum 0 2006/ 2007 Erie 1-6-9 D7 Pythium torulsoum 2.7 2006/ 2007 P2-105 P2-1 Pythium ultimum 1 2010 ERIE 2-4-2 Y Pythium ultimum 0 P 2006/ sporangiiferum 2007 Fay 1-1-3 Y Pythium ultimum 0.3 P 2006/ sporangiiferum 2007 HEN 2-2-11 Y Pythium ultimum 1 2006/ sporangiiferum 2007 Miami 1-1-5 Y Pythium ultimum 0 P 2006/ sporangiiferum 2007 Miami 1-3-7 Y Pythium ultimum 0 P 2006/ sporangiiferum 2007 Will 1-6-7 Y Pythium ultimum 2 P 2006/ sporangiiferum 2007 Wood 2-6-1 Y Pythium ultimum 1.2 2006/ sporangiiferum 2007 18Def 14B e1 Y Pythium ultimum 0 2010 ultimum Aug 1-1-11 B2 Pythium ultimum 0 P* 2006/ ultimum 2007 Aug 1-1-5 Y Pythium ultimum 0 2006/ ultimum 2007 Aug 1-3-1 F4 Pythium ultimum 0.7 2006/ ultimum 2007 Aug 1-6-3 Pythium ultimum 0 2006/

110

ultimum 2007 Craw 1-1-13 Y Pythium ultimum 1 2006/ ultimum 2007 Craw1-2-3 Y Pythium ultimum 0 2006/ ultimum 2007 Darke 3-3-8 Y Pythium ultimum 0 P* 2006/ ultimum 2007 H2-508 P2-2 Pythium ultimum 1 P* 2010 ultimum Hen 1-2-8 Y Pythium ultimum 2 2006/ ultimum 2007 Mad 2-6-7 Y Pythium ultimum 1 2006/ ultimum 2007 Miami 1-1-8 Y Pythium ultimum 1 P 2006/ ultimum 2007 Miami 1-3-14 Y Pythium ultimum 1.5 P* 2006/ ultimum 2007 Pick 1-3-5 Y Pythium ultimum 1 P 2006/ ultimum 2007 sally radish Y Pythium ultimum 1 2013 2013 ultimum Sand 1-3-13 Y Pythium ultimum 1 2006/ ultimum 2007 Wyan 1-1-9 Y Pythium ultimum 0 P 2006/ (check) ultimum 2007 Br 109 PB3 Y Pythium vexans 2 2010 Br 113 pb1 Y Pythium vexans 2 2010 Br 203 Pb2 Y Pythium vexans 2 2010 Br 305 Pb3 Y Pythium vexans 2 2010 0.3.01 unknown 1 2010 18ADAMS5EEXP2 unknown 3 2010 18Def 1c e1 unknown 2.5 2010 Br 103 Pb2 unknown 2 2010 Br 314 pb1 unknown 1 2010 H2-510 P2-2 unknown 1 2010 Logan 2-5-5 unknown 0 P* 2006/ 2007 Rich 30ft unknown cont. P* 2013

111

Field isolates from 2014 tested in Metalaxyl and Pyraclostrobin.

Unknowns from Metalaxyl Date Pyro field 2014 140005-1 6/4/2014 0 140005-2 6/4/2014 0 140005-5 6/4/2014 0 140006-3 6/4/2014 0 140003-5 6/4/2014 0 140010-3 6/4/2014 0 140004-7 6/4/2014 0 140002-3 6/4/2014 0 140004-2 6/4/2014 0 140004-1 6/4/2014 0 140003-6 6/4/2014 0 140005-7 6/4/2014 0 140005-7 0.33 6/23/2014 0 140013-4 4.5 6/23/2014 0 140013-3 1.2 6/23/2014 0 140016-7 0 6/23/2014 0 140016-3 5 6/23/2014 0 140015-1 0 6/23/2014 0 140015-3 0 6/23/2014 0 140025-1 5 6/23/2014 0 140012-1 1.33 7/3/2014 0 140016-3 5 7/3/2014 0 140016-7 0 7/3/2014 0 140012-2 2.33 7/3/2014 0 140015-2 C 7/3/2014 0 140015-5 C 7/3/2014 0 140012-3 0.67 7/3/2014 0 140041-4 C 7/8/2014 0 140041-3 C 7/8/2014 0 140041-5 5 7/8/2014 0 140041-6 C 7/8/2014 0 140040-3 0 7/8/2014 0

112

140004-1 1.33 7/8/2014 0 140005-5 0 7/8/2014 0 140032-1 C 7/8/2014 0 140006-3 C 7/8/2014 0 140041-1 C 7/8/2014 0 140041-7 C 7/8/2014 0 140041-2 C 7/8/2014 0 140037-1 C 7/9/2014 0 140037-3 C 7/9/2014 0 140035-2 5 7/9/2014 0 140035-3 C 7/9/2014 0 140034-3 5 7/9/2014 0 140036-3 C 7/9/2014 0 140040-4 0 7/9/2014 0 140033-2 C 7/9/2014 0 140033-1 4 7/9/2014 0 140038-1 C 7/9/2014 0 140020-1 4 7/9/2014 0 140020-2 0 7/9/2014 0 140021-1 4 7/9/2014 0 140019-2 3 7/9/2014 0 140026-2 C 7/9/2014 0 140039-1 C 7/9/2014 0 140038-2 4 7/9/2014 0 140027-1 C 7/9/2014 0 140027-2 C 7/9/2014 0 140029-2 4 7/9/2014 0 140031-5 5 7/9/2014 0

113

Appendix B: Protocols for Chapter 2 & 3

114

Chapter 2- Resistance Screening

Materials & Methods:

1. With clean isolates of species being tested, plate onto PCA for 3-4 day growth. 2. Add 950ml of sand, 50 ml of cornmeal, and 250 ml of deionized water to the myco-bag (Myco Supply; Pittsburgh PA) and mix. 3. Autoclave- 1 hour sterilization, 20 exhaust, 10 dry. Repeat after 24 hours. 4. After bags are cool, a day after autoclaving, inoculate. 5. Add eight plugs, at 10-mm diameter, of each isolate, into each bag. 6. Seal with a sealer-electrical impulse (Harbor Freight Tools; Calabasas, CA). Make sure to test which setting the sealer is on. It varies. If it’s too hot it will burn through the bag. 7. Mix bags every other day for ten days, to ensure even mycelia growth.

Greenhouse Studies.

1. Mix single myco-bag with 4-liters of fine vermiculite, for a 4:1 ratio. 2. Add 100 mL of coarse vermiculite to the bottom of each cup. 3. Place 300 ml of inoculum mixture into a 500ml cup. Make sure cups are saturated for 24 hours following. 4. After 24 hours, plant 8 seeds of each line directly on inoculum mixture and cover with 100ml of coarse vermiculite. 5. After planting, water cups twice daily to ensure saturation for Pythium growth.

Data collection.

1. After roughly 2 weeks, or V1 growth stage, wash soybean root for the removal of any vermiculite and debris. 2. Rate root rot on a scale from 1-5. Where- 1= all roots healthy, with no symptoms on root system; 2= 1-20% of root system has visible lesions on lateral roots; 3= 21-75% of roots showing visible symptoms, with symptoms beginning to show on tap root; 4= 76-100% of roots infected with symptoms on lateral roots and tap root; and 5= complete root rot, no germination of seeds. 3. Take stand count, height of three soybeans (from first root to crown), plant weight, and root weight.

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Chapter 3- Fungicide sensitivity.

Materials and Methods: Metalaxyl:

1. Make Potato Carrot broth and 100 ppm metalaxyl amended PC broth.

To make PC broth: autoclave 20 g potato, 20 g carrot in 1L of deionized water. Then filter through cheese cloth. Make sure to re-autoclave once in the container you will store it in. Store it in the refrigerator. To make the amended broth, add 100 mg of metalaxyl to the liter of broth. Make sure to dissolve metalaxyl in 1.5 mL of DMSO first. If want to make half liter just add 50 mg of metalaxyl.

2. Make clean PCA plates of isolates for testing. Let them grow for 3-4 days. 3. Add 2 ml of the 100 ppm metalaxyl PCB to rows B and D of a 24 well plate. Add Non-amended PCB in wells in the opposite rows, A and C. 4. Then add a 3mm diameter plug to both a control well and amended well, taken from newest growth of 3-4 day old culture. Twelve isolates per plate per rep – 3 reps per experiment. Meaning there will be 3 plates in each experiment with 12 different isolates. Each rep will need to be re-randomized each time. There should be one control that grows in the metalaxyl and one control that does not grow. 5. Cover the plates in a plastic bag and put into an incubator at 22°C in the dark. 6. After 24 hours and 48 hours rate the wells. Rate the wells on a scale from 0-5, to determine sensitivity: 0= no growth; 1= hyphae only growing microscopically, only a few hyphae growing from plug; 2= uniform hyphae growth, seen only microscopically; 3= uniform mycelium visible macroscopically; 4= mycelium uniform, not as much growth as in PCB wells; 5= mycelium visible and equal to the PCB control. The means of the isolates from both experiments will be analyzed.

Pyraclostrobin:

1. Make PCA, PCA+SHAM, & PCA + SHAM+ pyraclostrobin. Refer to media book on how to make PCA. Then for the SHAM amended plates add _ to 500 mL for a 50 ug/mL concentration. Do the same for the next type of media but add _ of pyraclostrobin to make a 234 ppm concentration. 2. Make sure to use large pertri plates. 3. After 48 hours in the incubator measure the diameter of each plate twice (2 different spots) and take the average. The growth of the pyraclostrobin plates will then be compared with the control.

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