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
By
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!
iv
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.
v
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
vi
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
ix
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
x
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
xi
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
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
xiv
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).
1
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
2
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
3
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,
4
and in many cases decreased yields (Broders et al., 2009; Campa et al., 2010; MurilloWilliams & 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.
5
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
6
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
7
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
8
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.
9
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.
10
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.
11
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).
12
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).
13
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.
14
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
15
(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
16
(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
17
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
18
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
19
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.
23
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
24
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.
27
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
Aug 1-1-3
Aug 1-1-5
Craw 1-2-3
Craw 1-1-13
Darke 3-3-8
Erie 2-4-2
Hen 1-2-8
Fay 1-1-3
Mad 2-6-7
Miami 1-3-14
Miami 1-1-5
Miami 1-1-8
Miami 1-3-7
Pick 1-3-5
Sand 1-3-13
Will 1-6-7
Wyan 1-1-9
Species
Pythium ultimum var. ultimum
Pythium ultimum var. ultimum
Pythium ultimum var. ultimum
Pythium ultimum var. ultimum
Pythium ultimum var. ultimum
Pythium ultimum v.
sporangiiferum
Pythium ultimum var. ultimum
Pythium ultimum v.
sporangiiferum
Pythium ultimum var. ultimum
Pythium ultimum var. ultimum
Pythium ultimum v.
sporangiiferum
Pythium ultimum var. ultimum
Pythium ultimum var. ultimum
Pythium ultimum var. ultimum
Pythium ultimum var. ultimum
Pythium ultimum v.
sporangiiferum
Pythium ultimum var. ultimum
28
County
Auglaize
Auglaize
Crawford
Crawford
Darke
Erie
Henry
Fayette
Madison
Miami
Miami
Miami
Miami
Pickaway
Sandusky
Williams
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).
29
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
Weight
% Control Root
Weight
Pr > F
F Value
Pr > F
F Value
Pr > F
F Value
Pr > F
Pr > F
22.63
<.0001
128.26
<.0001
0.0049
F
Value
0.28
Pr > F
0.0207
F
Value
8.11
Pr > F
5.4
F
Value
107.06
<.0001
30
Source
DF
exp
1
F
Value
106.46
isol
2
547.8
<.0001
1846.96
<.0001
1528.75
<.0001
1320.03
<.0001
1651.1
3
<.0001
709.8
5
<.0001
982.03
<.0001
exp*iso
l
2
25.55
<.0001
38.84
<.0001
41.3
<.0001
31.97
<.0001
35.98
<.0001
16.81
<.0001
21.62
<.0001
8
10.62
<.0001
10.37
<.0001
8.11
<.0001
15.09
<.0001
25
<.0001
12.48
<.0001
33.59
<.0001
47
6.61
<.0001
3.79
<.0001
11.12
<.0001
14.78
<.0001
14.49
<.0001
3.18
<.0001
1.86
0.0019
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
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
*isol)
line
exp*lin
e
<.0001
0.5989
exp(rep
*line)
Continued
30
Table 2 continued
Stand (%Stand)
Root Score
Pr > F
F Value
0.0007
0.837
Source
DF
line*iso
l
94
F
Value
1.63
exp*lin
e*isol
94
0.85
Mean Height
Plant Weight
Root Weight
% Control Plant
Weight
% Control Root
Weight
Pr > F
F Value
Pr > F
F Value
Pr > F
Pr > F
1.68
0.0004
1.58
0.0015
<.0001
0.8555
F
Value
0.61
Pr > F
0.0351
F
Value
0.77
Pr > F
1.33
F
Value
2.69
0.83
0.8633
0.79
0.9159
0.56
0.9995
0.75
0.9517
1.18
0.2245
1.02
0.4429
31
31
0.9758
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
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
Pr > F
Pr > F
0.0033
F
Value
14.35
Pr > F
0.0049
F
Value
39.46
0.0438
% Control
Plant Weight
F
Pr > F
Value
11.8
0.0264
% Control
Root Weight
F
Pr > F
Value
52.43 0.0019
Pr > F
0.0369
F
Value
8.46
Pr > F
0.0193
F
Value
9.49
33
Source
DF
exp
1
F
Value
31.69
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
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
33
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
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
0.0274
% Control
Plant Weight
F
Pr > F
Value
0
0.9918
% Control Root
Weight
F
Pr > F
Value
1.74
0.2577
Pr > F
Pr > F
0.0447
F
Value
11.54
Pr > F
0.2515
F
Value
8.33
Pr > F
0.0823
F
Value
1.79
Pr > F
0.6219
F
Value
5.32
Source
DF
Exp
1
F
Value
0.28
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
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
exp(rep*line)
35
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
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
Pr > F
Pr > F
Pr > F
Pr > F
Pr > F
37
Source
DF
Exp
1
F
Value
266.72
<.0001
F
Value
49.05
<.0001
F
Value
321.62
<.0001
F
Value
220.8
<.0001
F
Value
134.95
Isol
1
124.37
<.0001
21.82
<.0001
211.97
<.0001
103.93
<.0001
52.4
<.0001
exp*isol
1
124.37
<.0001
19.18
<.0001
125.51
<.0001
92.97
<.0001
41.95
<.0001
<.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
37
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
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
Pr > F
Pr > F
Pr > F
DF
Plant Weigtht
Root Weight
Pr > F
Pr > F
39
Source
DF
exp
2
F
Value
0.05
0.9555
F
Value
1.78
0.1695
F
Value
10.44
<.0001
2
F
Value
37.05
<.0001
F
Value
126.96
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
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
39
<.0001
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
Cultivar
M1-37
2.5
BC
2.0
C
nt
P1-3-5
Hutchison
M1-314
2.3
BC
1.8
BC
nt
Jack
nt
Kottman
Pythium ultimum var. sporangiiferum
Mean
3.7
BC
2.5
C
nt
W1-19
5.0
A
2.8
C
nt
nt
nt
nt
nt
2.2
BC
1. 7
C
2.3
BC
2.7
BC
4.7
AB
2.8
C
5.0
A
4.2
B
3.5
2.0
BC
2.3
BC
2.5
B
3. 7
A
nt
2.4
BC
2.2
BC
2.8
B
3.8
A
nt
3.7
BC
4.2
AB
4.5
AB
5.0
A
nt
4.7
AB
5.0
A
5.0
A
5.0
A
nt
2.5
Overall
Mean
2.3
2.6
3.9
LSD
0.76
0.74
1.29
Burlison
Dennison
Ohio FG1
Prohio
Williams82
Williams
Wooster
Wyandot
E2.4.
2
nt
F1.1.
3
nt
M.1.1.
5
nt
W.1.6.
7
nt
Mean
1.7
C
1.7
C
5.0
A
2.8
B
2.0
BC
2.2
D
3.7
B
5.0
A
3.5
BC
3.0
BC
2.0
D
2.2
CD
5.0
A
3.7
B
2.7
BCD
1.5
C
2.2
BC
3.8
A
2.5
B
2.2
BC
1.8
2.8
CD
nt
3.2
BC
nt
2.3
BC
nt
2.7
3.4
2.3
BC
nt
3.7
nt
nt
nt
nt
nt
3.5
5.0
A
3.0
BC
3.5
4.8
A
3.5
B
3.4
3. 7
A
2.2
BC
2.5
4.6
4.6
5.0
A
2. 7
B
2.9
0.79
0.89
0.81
1.08
0.95
3.4
2.3
nt
2.8
nt
40
nt
2.4
4.7
3.1
2.5
nt
2.8
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
Table 8. P. ultimum v. ultimum NAM population Means of lines evaluated.
Level of
genotype
4J10534
5M20252
C0J09546
C0J17368
Dennison
HS63976
Kottman
LD003309
LD015907
LD024485
LD029050
LG003372
LG032979
LG033191
LG044717
LG046000
LG054292
LG054317
LG054464
LG054832
LG902550
LG921255
LG941128
LG941906
LG977012
LG981605
Magellan
Percent Mean
Stand
Height
(mm)
93.80
84.30
87.50
80.80
79.20
64.00
81.30
73.00
85.40
89.50
87.50
68.50
95.80
72.70
72.90
63.30
66.70
61.20
54.20
65.00
81.30
68.90
70.80
82.10
83.30
70.40
75.00
64.50
77.10
93.20
56.30
47.90
89.60
76.50
77.10
55.80
75.00
71.40
70.80
54.60
81.30
71.10
68.80
56.20
70.80
61.70
58.30
59.60
77.10
81.50
58.30
52.60
79.20
80.20
Plant
Weight
(g)
10.20
10.10
8.20
9.20
11.40
10.40
10.90
6.70
5.10
4.80
8.80
7.50
7.70
8.50
7.30
5.40
8.90
7.30
9.50
7.30
7.90
6.20
5.20
5.40
8.40
4.60
7.80
Root
Weight
(g)
3.30
2.80
2.60
2.90
4.20
3.20
3.60
2.10
1.20
1.40
2.80
2.10
2.30
2.60
1.90
1.50
2.90
2.30
2.80
2.40
2.40
1.60
1.50
1.40
2.20
1.40
2.40
42
Root
Score
2.00
1.50
2.00
2.50
2.50
2.20
1.50
2.20
2.80
3.00
2.30
2.70
2.80
2.80
3.00
3.20
2.00
2.50
2.20
2.30
2.20
2.60
3.00
3.30
2.30
2.30
2.70
Continued
Table 8 continued
Level of
genotype
Percent Mean
Stand
Height
(mm)
Maverick 64.60
68.40
NE3001
81.30
82.60
OhioFG1 85.40
77.20
P404188A 87.50
78.80
P437169B 72.90
58.20
P507681B 39.60
55.50
PI398881 93.80
81.70
PI427136 75.00
69.00
PI518751 85.40
80.10
PI561370 60.40
70.40
PI574486 87.50
71.80
Prohio
80.70
87.90
S0613640 93.80
84.10
Skylla
64.60
71.00
Streeter
81.30
77.20
Summit
92.50
85.90
U3100612 81.30
68.60
Wooster
35.40
21.20
Wyandot
81.30
74.80
conrad
70.80
68.00
sloan
85.00
74.10
76.08
70.54
Overall
Mean
28.19
19.30
LSD
Plant
Weight
(g)
7.00
7.80
11.30
9.30
5.90
3.40
10.90
8.80
9.20
5.60
9.10
8.20
11.50
5.90
9.50
8.60
6.60
2.00
9.80
5.80
10.00
7.81
Root
Weight
(g)
2.10
2.40
3.50
2.70
1.50
1.00
3.40
2.40
2.90
1.30
2.20
2.50
3.90
1.60
3.10
3.20
2.10
0.40
3.40
1.80
3.20
2.36
Root
Score
3.52
1.34
0.97
43
2.60
2.50
2.30
2.70
2.50
2.80
2.70
2.30
2.50
3.00
2.70
2.40
2.00
3.00
2.50
2.30
3.00
3.80
2.30
2.80
2.00
2.52
Table 9. NAM population means of lines evaluated with P. ultimum v. sporangiiferum.
Level of
genotype
4J10534
5M20252
C0J09546
C0J17368
Dennison
HS63976
Kottman
LD003309
LD015907
LD024485
LD029050
LG003372
LG032979
LG033191
LG044717
LG046000
LG054292
LG054317
LG054464
LG054832
LG902550
LG921255
LG941128
LG941906
LG977012
LG981605
Magellan
Maverick
NE3001
Percent Mean
Stand
Height
(mm)
58.33
3.27
29.17
2.05
27.08
1.35
41.67
2.18
70.83
4.38
68.75
4.53
39.58
2.45
58.33
2.80
18.75
1.83
17.50
0.78
27.08
1.38
56.25
3.08
37.50
2.00
54.17
3.00
58.33
2.62
35.42
1.70
52.08
2.93
35.42
1.45
41.67
2.57
85.42
5.28
31.25
1.95
39.58
2.10
8.33
0.32
12.50
0.55
35.42
1.85
29.17
1.37
35.42
2.20
37.50
1.82
37.50
1.97
Plant
Weight
(g)
0.53
0.23
0.18
0.32
0.83
0.70
0.47
0.50
0.63
0.14
0.20
0.40
0.32
0.47
0.35
0.22
0.48
0.17
0.47
0.90
0.28
0.30
0.05
0.05
0.28
0.20
0.33
0.27
0.30
Root
Weight
(g)
3.83
4.00
4.17
3.67
3.83
3.67
3.67
3.83
4.67
4.20
4.00
4.00
4.00
4.00
4.00
4.33
3.67
3.67
3.67
3.33
4.00
4.17
4.67
4.83
4.00
4.00
4.00
3.83
4.17
44
Root
Score
30.86
21.42
19.38
33.74
45.52
31.17
24.90
25.05
15.43
16.46
17.27
32.72
16.47
28.68
39.58
16.28
30.67
16.68
30.68
33.33
21.08
19.95
4.95
3.45
23.38
14.62
20.95
20.78
26.12
Continued
Table 9 continued
Level of
Percent Mean
genotype Stand
Height
(mm)
OhioFG1 77.08
6.17
P404188A 31.25
1.97
P437169B 37.50
1.97
P507681B 18.75
0.73
PI398881 41.67
2.67
PI427136 39.58
2.63
PI518751 39.58
2.17
PI561370 25.00
1.40
PI574486 35.42
2.30
Prohio
60.42
3.24
S0613640 72.92
4.80
Skylla
16.67
0.82
Streeter
57.50
3.24
Summit
58.33
2.92
U3100612 39.58
2.00
Wooster
0.00
0.00
Wyandot
56.25
3.57
conrad
27.08
1.13
sloan
41.67
2.38
41.18
24.41
Overall
Mean
28.13
1.78
LSD
Plant
Weight
(g)
1.13
0.32
0.30
0.08
0.42
0.43
0.28
0.13
0.27
0.63
0.75
0.12
0.44
0.38
0.28
0.00
0.53
0.17
0.38
2.35
Root
Weight
(g)
3.50
4.00
4.17
4.33
3.67
3.33
4.00
4.33
3.83
4.00
3.83
4.17
4.00
4.00
3.83
5.00
3.67
4.17
3.67
0.37
Root
Score
0.41
0.67
15.31
45
41.62
22.57
23.70
12.45
23.40
29.28
23.38
14.17
27.17
39.07
34.50
15.70
29.68
28.17
28.23
0.00
40.45
21.22
22.77
3.99
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
genotype
Conrad
Dunbar
Forrest
Kottman
Magellan
Maverick
OhioFG1
Miss001
Miss002
Miss003
Miss004
Miss005
Miss006
Miss007
Miss008
Miss009
Miss010
Miss011
Miss012
Miss013
Pana
Prohio
Miss014
Miss015
Miss016
Percent Mean
Stand
Height
(mm)
75.00
61.67
6.25
17.50
2.08
10.83
58.33
67.89
42.50
64.75
27.08
42.67
62.50
68.56
8.33
19.83
58.33
48.22
64.58
52.67
54.17
53.17
22.92
56.00
72.92
80.17
52.08
44.22
50.00
52.08
64.58
65.47
14.58
33.17
10.42
10.78
12.50
28.50
52.08
60.31
8.33
24.17
56.25
70.03
54.17
58.61
62.50
72.53
56.25
58.33
Plant
Weight
(g)
6.12
0.28
0.12
5.95
3.40
1.98
6.38
0.37
1.94
3.50
2.47
2.15
6.48
2.95
3.72
5.52
0.90
0.40
0.58
2.97
0.65
4.52
3.72
5.22
3.70
46
Root
Weight
(g)
1.52
0.07
0.02
1.43
0.86
0.47
1.33
0.07
0.44
0.77
0.38
0.50
1.63
0.77
0.95
1.62
0.30
0.10
0.12
0.68
0.17
1.07
0.83
1.50
0.97
Root
Score
3.67
4.50
4.83
3.67
3.60
4.00
3.67
4.50
3.33
4.00
3.67
4.00
3.17
3.83
3.33
3.50
4.33
4.67
4.50
3.50
4.50
3.17
3.33
3.17
3.67
Continued
Table 10 continued
Level of
Percent Mean
genotype Stand
Height
(mm)
37.50
39.69
Miss017
18.75
24.19
Miss018
Will82
22.92
29.83
Wyandot 54.17
60.03
Dennison 89.58
81.33
Streeter
66.67
71.78
Summit
64.58
68.78
Wooster
0.00
0.00
42.51
48.42
Overall
Mean
23.5
24.34
LSD
Plant
Weight
(g)
3.02
1.42
1.75
5.00
9.37
6.20
5.22
0.00
3.27
Root
Weight
(g)
0.80
0.20
0.40
1.23
2.57
1.38
1.12
0.00
0.80
Root
Score
2.22
0.72
0.70
47
3.50
4.33
4.17
3.50
3.33
3.17
3.50
5.00
3.84
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
genotype
Conrad
Dunbar
Forrest
Kottman
Magellan
Maverick
OhioFG1
Miss001
Miss002
Miss003
Miss004
Miss005
Miss006
Miss007
Miss008
Miss009
Miss010
Miss011
Miss012
Miss013
Pana
Prohio
Miss014
Miss015
Miss016
Miss017
Miss018
Will82
Percent Mean
Stand
Height
(mm)
91.67
85.72
50.00
64.94
25.00
49.50
89.58
81.39
83.33
88.67
87.50
70.39
89.58
93.11
20.83
33.83
81.25
69.67
83.33
64.33
85.42
67.00
45.83
71.33
93.75
75.56
85.42
80.39
77.08
77.39
93.75
85.00
66.67
83.50
39.58
57.39
58.33
64.67
68.75
74.67
62.50
78.44
87.50
93.00
75.00
74.39
79.17
88.28
95.83
87.78
79.17
76.39
42.50
55.10
81.25
79.89
Plant
Weight
(g)
9.75
3.85
1.30
9.78
7.28
8.12
13.40
1.10
3.40
5.37
5.77
3.60
9.35
6.00
7.32
9.20
4.57
2.30
3.77
6.07
4.30
8.82
5.90
7.83
8.38
6.92
4.10
8.75
Root
Weight
(g)
3.25
0.92
0.18
2.83
1.98
2.50
4.07
0.25
0.85
1.58
1.32
0.82
2.95
1.83
2.37
3.00
1.48
0.67
0.98
1.75
1.28
2.55
1.48
2.53
2.58
2.10
0.68
2.63
48
Root
Score
2.50
3.00
3.67
2.17
2.67
2.67
2.17
3.83
2.67
3.00
2.67
3.33
3.00
2.83
2.33
2.17
2.17
3.00
3.17
2.50
2.67
2.67
2.67
2.83
2.67
2.83
3.00
2.17
Continued
Table 11 continued
Level of
Percent Mean
genotype Stand
Height
(mm)
Wyandot 64.58
81.72
dennison 89.58
102.06
streeter
97.92
95.00
summit
87.50
92.67
wooster
41.67
43.56
72.91
75.46
Overall
Mean
15.10
14.50
LSD
Plant
Weight
(g)
7.60
12.27
10.65
9.58
2.83
6.64
Root
Weight
(g)
2.25
4.58
2.92
2.78
0.68
1.96
Root
Score
2.00
0.87
0.70
49
2.33
1.83
2.33
2.50
3.17
2.70
Table 12. Overall means of Virginia Tech population inoculated with P. ultimum v.
ultimum.
Level of
genotype
Percent
Stand
Archer
VT001
Dennison
Essex
Hutchinson
VT002
VT003
VT004
VT005
VT006
VT007
VT008
VT009
VT010
VT011
VT012
VT013
VT014
VT015
VT016
VT017
VT018
VT019
VT020
VT021
VT022
VT023
VT024
VT025
VT026
VT027
83.33
91.67
91.67
91.67
91.67
89.58
47.92
91.67
89.58
79.17
97.92
93.75
89.58
85.42
83.33
93.75
91.67
92.71
89.58
95.83
93.75
87.50
89.58
95.83
87.50
87.50
93.75
91.67
87.50
87.50
89.58
Mean
Height
(mm)
127.39
111.33
137.28
101.28
114.28
112.44
79.17
155.11
146.61
137.06
107.94
104.50
140.17
102.56
112.17
117.89
145.06
107.08
137.56
119.17
132.56
119.20
93.83
142.61
112.83
115.67
90.06
146.39
114.07
127.00
113.00
Plant
Weight
(g)
11.58
12.07
12.82
9.50
10.93
10.83
3.13
18.33
16.15
9.48
9.15
15.05
15.47
5.32
9.50
10.80
15.03
13.40
11.92
15.38
15.53
12.05
10.32
11.87
9.05
13.18
6.72
10.20
8.82
9.62
8.57
Root
Weight
(g)
3.28
5.05
5.15
3.13
4.12
3.90
0.72
4.97
4.62
3.03
3.17
5.50
4.98
1.92
3.00
3.47
4.33
4.73
3.48
4.55
4.28
3.38
4.02
3.43
2.93
4.38
2.87
3.63
3.15
3.20
3.08
Root
Score
2.50
2.67
2.17
2.17
2.50
2.17
3.17
2.00
2.17
2.83
2.50
2.17
1.83
2.83
2.50
2.83
2.50
2.00
2.83
2.67
2.83
2.83
2.83
2.33
2.67
2.33
2.83
2.67
2.17
2.67
2.83
Continued
50
Table 12 continued
Level of
genotype
Percent
Stand
Plant
Weight
(g)
10.58
8.40
14.25
6.70
13.00
12.63
11.92
5.47
2.72
10.48
10.13
10.12
13.32
10.22
12.00
12.13
10.82
12.12
11.27
9.82
5.08
11.48
10.9
Root
Weight
(g)
3.88
2.52
4.73
2.57
4.67
3.77
3.88
2.22
0.53
2.75
3.75
2.75
4.57
4.22
3.97
4.42
3.97
4.22
3.97
3.22
1.53
4.03
3.60
Root
Score
91.67
85.42
89.58
89.58
91.67
95.83
93.75
83.33
31.25
81.25
97.92
89.58
97.92
81.25
89.58
79.17
91.67
91.67
87.50
87.50
64.58
87.50
87.5
Mean
Height
(mm)
117.50
107.11
113.61
125.06
129.56
132.78
134.06
86.50
56.33
111.50
98.44
107.67
107.83
104.61
105.11
114.00
121.44
116.56
118.11
117.67
74.22
109.33
115.54
VT028
VT029
VT030
VT031
VT032
VT033
VT034
VT035
VT036
VT037
VT038
VT039
Williams
conrad
kottman
ohiofg1
prohio
sloan
streeter
summit
wooster
wyandot
Overall
Mean
LSD
17.57
21.75
3.33
1.67
0.68
51
2.83
2.67
2.83
2.67
2.17
2.50
2.67
2.50
3.67
2.33
2.67
2.67
1.83
2.33
1.83
2.33
2.17
2.17
2.00
2.50
3.00
1.83
2.5
Table 13. Overall means of Virginia Tech population evaluated with P. ultimum v.
sporangiiferum.
Level of
genotype
Percent
Stand
Mean
Height
(mm)
Plant
Weight
(g)
Root
Weight
(g)
Root
Score
Archer
VT001
Dennison
Essex
Hutchinson
VT002
VT003
VT004
VT005
VT006
VT007
VT008
VT009
VT010
VT011
VT012
VT013
VT014
VT015
VT016
VT017
VT018
VT019
VT020
VT021
VT022
VT023
VT024
VT025
VT026
VT027
68.75
64.58
93.75
93.75
87.50
87.50
2.08
83.33
83.33
85.42
60.42
62.50
79.17
81.25
66.67
93.75
87.50
69.79
91.67
87.50
87.50
91.67
95.83
83.33
77.08
79.17
93.75
89.58
95.83
66.67
79.17
3.17
3.33
2.67
2.83
2.33
2.83
4.67
2.33
2.33
3.17
3.50
2.67
2.17
3.17
3.00
3.00
3.00
2.92
3.00
2.33
2.50
2.33
3.00
3.17
3.00
3.33
3.33
2.67
2.50
3.33
3.17
70.28
62.19
79.56
72.67
76.33
75.67
6.00
112.00
122.28
104.83
73.86
68.11
123.39
72.89
86.06
89.40
116.72
74.19
90.72
95.39
103.94
97.72
78.94
102.33
91.94
82.22
73.61
126.28
94.44
87.06
81.50
6.60
5.82
9.78
7.52
8.45
8.90
0.08
15.87
14.20
8.13
3.92
7.32
10.60
3.50
6.02
9.70
11.76
7.14
9.78
11.82
12.62
12.15
8.92
7.95
7.10
8.65
5.10
8.23
8.55
5.85
5.85
1.62
1.67
3.35
2.18
2.85
2.63
0.02
4.38
3.65
2.28
1.05
2.07
3.10
0.95
1.43
2.62
2.86
1.91
2.60
3.02
3.42
3.10
2.98
2.10
1.95
2.15
1.68
2.55
2.60
1.70
1.72
Continued
52
Table 13 continued
Level of
genotype
Percent
Stand
Plant
Weight
(g)
109.00
84.87
91.17
88.78
85.61
92.28
107.61
70.61
15.72
69.11
71.22
58.78
52.72
67.39
76.33
87.17
68.47
52.78
93.94
63.50
8.40
84.56
81.11
Root
Weight
(g)
9.25
7.50
10.58
4.33
11.72
7.23
9.33
3.83
0.85
9.38
6.88
4.88
4.97
5.82
6.63
9.38
6.33
5.17
9.83
6.08
0.12
7.90
7.6
Root
Score
89.58
91.67
81.25
79.17
89.58
66.67
91.67
70.83
8.33
83.33
87.50
56.25
68.75
75.00
70.83
81.25
87.50
60.42
91.67
72.92
2.50
72.92
76.50
Mean
Height
(mm)
2.67
3.00
2.67
3.00
3.00
3.17
3.00
3.17
4.50
2.50
3.33
3.33
3.67
3.33
2.83
3.00
3.17
3.50
2.83
3.17
4.40
3.17
3.00
VT028
VT029
VT030
VT031
VT032
VT033
VT034
VT035
VT036
VT037
VT038
VT039
Williams
conrad
kottman
ohiofg1
prohio
sloan
streeter
summit
wooster
wyandot
Overall
Mean
LSD
18.42
0.89
24.05
2.81
1.26
53
3.22
1.93
3.22
1.28
3.12
1.77
2.45
1.33
0.18
2.32
1.88
1.08
1.05
1.65
1.75
2.55
1.58
1.32
2.90
1.62
0.02
2.33
2.12
Table 14. Overall means of Genetic Gain population inoculated with Pythium ultimum v.
ultimum.
Level of
genotype
Percent Mean
Stand
Height
(mm)
Plant
Root
Root
Weight Weight (g) Score
(g)
Burlison
Corsoy79
Dwight
IA2021
IA2022
IA2038
IA2050
IA2052
IA2065
IA2068
IA2094
Rango
Richland
Amcor
Amsoy71
Amsoy
Beeson80
Beeson
Century84
Century
Conrad
Corsoy
Dennison
Elgin87
Elgin
Harosoy63
Harcor
Harosoy
45.83
14.58
12.50
4.17
27.08
4.17
25.00
27.08
12.50
2.08
8.33
14.58
31.25
25.00
41.67
35.42
0.00
12.50
33.33
27.08
18.75
16.67
85.42
29.17
10.42
25.00
27.08
25.00
3.88
1.10
0.75
0.25
1.68
0.28
1.65
2.05
0.80
0.10
0.67
0.90
2.28
1.68
3.18
2.17
0.00
0.85
2.38
2.00
1.62
1.50
10.70
1.33
0.58
1.87
1.87
1.83
47.25
29.07
29.78
6.42
29.17
16.33
27.08
41.00
17.80
7.00
11.33
19.28
32.07
34.67
42.39
33.37
0.00
20.17
31.63
43.83
18.17
26.33
77.66
21.38
14.63
35.27
34.50
29.53
1.08
0.33
0.22
0.50
0.42
0.05
0.42
0.60
0.18
0.02
0.17
0.22
0.63
0.50
1.05
0.48
0.00
0.22
0.62
0.55
0.52
0.48
3.77
0.30
0.13
0.60
0.57
0.55
54
3.17
4.50
3.83
4.83
3.83
4.67
4.17
3.67
4.67
4.83
4.83
4.50
4.17
4.17
3.33
4.17
5.00
4.67
4.17
4.17
4.50
4.50
2.67
4.17
4.67
4.00
4.00
4.33
Continued
Table 14 continued
Level of
genotype
Percent Mean
Stand
Height
(mm)
Hawkeye63 29.17
26.72
Hawkeye
18.75
23.22
Jack
22.92
31.22
Korean
0.00
0.00
Kottman
70.83
63.43
Kenwood
10.42
23.08
Lindarin
20.83
30.33
Loda
4.17
6.42
Mukden
18.75
23.22
OhioFG1
75.00
63.50
Preston
4.17
15.50
Private-210 33.33
27.85
Private-211 31.25
23.77
Private-212 35.42
24.60
Private-213 20.83
29.23
Private-214 41.67
29.88
Private-215 6.25
5.67
Private-216 18.75
17.62
Private-217 10.42
11.28
Private-218 14.58
26.08
Private-219 22.92
20.67
Private-220 27.08
21.35
Private-21
35.42
29.35
Private-22
27.08
20.33
Private-23
27.08
29.22
Private-24
22.92
22.38
Private-25
20.83
22.12
Private-26
22.92
27.55
Private-27
16.67
26.75
Private-28
20.83
22.72
Private-29
37.50
30.38
Plant
Weight
(g)
2.08
1.10
1.00
0.00
8.42
0.62
1.57
0.30
0.78
10.57
0.25
1.83
1.65
2.48
1.58
3.33
0.25
1.10
0.68
0.80
1.05
1.72
2.18
1.87
1.42
1.47
1.23
1.12
1.22
1.38
2.55
Root
Root
Weight (g) Score
0.52
0.18
0.22
0.00
2.83
0.18
0.43
0.08
0.13
3.65
0.07
0.50
0.37
0.70
0.55
0.98
0.07
0.28
0.17
0.17
0.22
0.47
0.52
0.47
0.33
0.45
0.28
0.28
0.32
0.37
0.78
55
4.33
4.50
4.17
5.00
2.83
4.50
4.33
4.67
4.00
2.67
4.67
4.50
4.50
4.17
4.33
4.00
4.83
4.67
4.83
4.50
4.33
4.33
4.50
4.33
4.00
4.50
4.50
4.50
4.17
4.33
4.17
Continued
Table 14 continued
Level of
genotype
Prohio
Savoy
Sloan
Streeter
Summit
Vickery
Wells II
Wells
Wooster
Wyandot
Overall
Mean
LSD
Percent Mean
Stand
Height
(mm)
54.17
56.45
29.17
37.22
56.25
56.43
81.25
69.33
66.67
59.39
27.08
29.22
16.67
24.58
41.67
40.99
0.00
0.00
75.00
61.45
26.93
28.81
Plant
Weight
(g)
5.48
2.58
6.25
9.58
5.92
1.93
0.95
2.97
0.00
7.98
2.19
Root
Root
Weight (g) Score
1.75
0.73
1.82
3.27
1.55
0.65
0.22
0.67
0.00
2.50
0.65
3.33
3.67
3.17
2.67
3.00
4.00
4.33
3.83
5.00
3.00
4.17
32.35
2.52
0.82
0.87
29.48
56
Table 15. Overall means of Genetic Gain population inoculated with P. ultimum v.
sporangiiferum.
Level of
genotype
Burlison
Corsoy79
Dwight
IA2021
IA2022
IA2038
IA2050
IA2052
IA2065
IA2068
IA2094
Rango
Richland
Amcor
Amsoy71
Amsoy
Beeson80
Beeson
Century84
Century
Conrad
Corsoy
Dennison
Elgin87
Elgin
Harosoy63
Harcor
Harosoy
Percent Mean
Stand
Height
(mm)
10.42
9.83
12.50
19.33
2.08
3.83
0.00
0.00
2.08
9.00
0.00
0.00
2.08
2.67
0.00
0.00
2.08
2.67
0.00
0.00
0.00
0.00
0.00
0.00
33.33
31.73
4.17
4.92
10.42
11.17
6.25
8.33
2.08
3.67
0.00
0.00
8.33
12.22
0.00
0.00
6.25
6.12
8.33
25.17
95.83
66.62
2.08
4.17
0.00
0.00
8.33
19.33
2.08
7.50
4.17
6.83
Plant
Weight
(g)
0.53
0.75
0.08
0.00
0.18
0.00
0.08
0.00
0.23
0.00
0.00
0.00
1.87
0.28
0.48
0.50
0.08
0.00
0.45
0.00
0.33
0.53
12.45
0.13
0.00
0.38
0.12
0.25
Root
Weight
(g)
0.10
0.23
0.02
0.00
0.05
0.00
0.02
0.00
0.05
0.00
0.00
0.00
0.45
0.12
0.10
0.17
0.02
0.00
0.07
0.00
0.07
0.18
5.58
0.02
0.00
0.12
0.02
0.07
57
Root Score
4.67
4.17
4.67
5.00
4.83
5.00
4.83
5.00
4.83
5.00
5.00
5.00
3.50
4.67
4.33
4.67
4.67
5.00
4.50
5.00
4.83
3.33
2.33
4.83
5.00
4.00
4.83
4.33
Continued
Table 15 continued
Level of
genotype
Percent Mean
Stand
Height
(mm)
Hawkeye63 2.08
4.17
Hawkeye
2.08
2.50
Jack
0.00
0.00
Korean
0.00
0.00
Kottman
31.25
31.18
Kenwood
4.17
16.17
Lindarin
10.42
8.95
Loda
0.00
0.00
Mukden
0.00
0.00
OhioFG1
85.42
54.06
Preston
0.00
0.00
Private-210 8.33
13.67
Private-211 4.17
8.33
Private-212 2.08
5.33
Private-213 6.25
15.08
Private-214 12.50
25.50
Private-215 0.00
0.00
Private-216 2.08
2.67
Private-217 16.67
22.25
Private-218 0.00
0.00
Private-219 2.08
3.67
Private-220 0.00
0.00
Private-21
14.58
14.30
Private-22
8.33
21.75
Private-23
4.17
2.83
Private-24
14.58
13.72
Private-25
2.08
8.83
Private-26
0.00
0.00
Private-27
8.33
17.33
Private-28
8.33
20.50
Private-29
6.25
2.33
Plant
Weight
(g)
0.13
0.12
0.00
0.00
2.50
0.15
0.60
0.00
0.00
11.20
0.00
0.33
0.17
0.08
0.37
0.67
0.00
0.08
0.85
0.00
0.07
0.00
0.60
0.45
0.22
1.00
0.10
0.00
0.43
0.70
0.25
Root
Weight
(g)
0.03
0.03
0.00
0.00
0.77
0.03
0.18
0.00
0.00
3.87
0.00
0.08
0.03
0.02
0.08
0.15
0.00
0.03
0.18
0.00
0.02
0.00
0.10
0.13
0.03
0.30
0.02
0.00
0.10
0.20
0.02
58
Root Score
4.67
4.83
5.00
5.00
3.17
4.67
4.67
5.00
5.00
2.50
5.00
4.33
4.33
4.83
4.67
3.50
5.00
4.83
3.50
5.00
4.83
5.00
4.33
3.67
4.83
4.50
4.83
5.00
4.17
3.83
4.83
Continued
Table 15 continued
Level of
genotype
Prohio
Savoy
Sloan
Streeter
Summer
Vicker
Wells II
Wells
Wooster
Wyandot
Overall
Mean
LSD
Percent Mean
Stand
Height
(mm)
72.92
50.98
8.33
8.92
47.92
21.06
83.33
54.66
83.33
56.73
6.25
11.25
0.00
0.00
16.67
23.25
0.00
0.00
81.25
52.67
12.74
12.29
Plant
Weight
(g)
7.28
0.50
4.33
8.78
8.12
0.38
0.00
1.27
0.00
7.30
1.14
Root
Weight
(g)
2.62
0.12
1.28
3.22
2.67
0.10
0.00
0.40
0.00
2.23
0.38
Root Score
13.26
1.14
0.45
0.80
15.41
59
2.50
4.50
3.50
2.50
2.67
4.50
5.00
4.00
5.00
2.83
4.41
Table 16. Overall means of The Ohio State University population inoculated with P.
ultimum v. ultimum.
Level of
genotype
OSU001
OSU002
OSU003
OSU004
OSU005
OSU006
OSU007
OSU008
OSU009
OSU010
OSU011
OSU012
Archer
Clermont
Conrad
Danbkong
Dennison
Dilworth
Flint
OSU013
OSU014
OSU015
OSU016
OSU017
OSU018
OSU019
OSU020
OSU021
Percent Mean
Stand
Height
(mm)
54.2
40.4
50
50
54.2
64.7
45.8
46
66.7
69.7
83.3
55.7
41.7
34.9
75
58.9
83.3
66.3
91.7
82
54.2
70.1
83.3
67.4
62.5
66.5
25
25.8
33.3
45.3
83.3
67.7
58.3
61.7
79.2
78.9
70.8
89.3
70.8
67.8
83.3
64.8
20.8
45
58.3
69.9
50
47.6
50
43.8
58.3
60.9
37.5
37.1
66.7
48.2
Plant
Weight
(g)
8.9
8.9
6.7
8.2
9.8
5.2
6.7
5.9
4.8
14.4
6.7
5.1
7.9
1
2.6
6.7
6.1
6.7
7.5
5.6
7.3
3.7
4.5
3.2
3.7
4.9
4.3
5.3
Root
Weight
(g)
3.2
3.4
2.3
2.4
3.6
2.2
2.1
2.2
1.9
5.5
2.6
1.8
2.5
0.2
0.9
1.9
2
2.1
2.5
1.7
1.8
1.1
1.3
0.8
1
1.4
0.9
1.6
60
Root
Score
3.3
4
3
4
2.7
3.3
3.7
3
2.7
2.7
2.3
3.3
3.3
4
3.3
2
2.7
2.7
2.7
3
2.7
3.3
3
3.7
3.7
2.7
3.7
3.3
Continued
Table 16 continued
Level of
genotype
Percent Mean
Stand
Height
(mm)
58.3
49.8
OSU022
83.3
56.7
OSU023
83.3
81.8
OSU024
50
55.2
OSU025
41.7
51.9
OSU026
37.5
49.1
OSU027
58.3
63.9
OSU028
66.7
63.4
OSU029
66.7
60.6
OSU030
35.4
54.3
OSU031
58.3
66.6
OSU032
95.8
59
OSU033
66.7
68.6
OSU034
70.8
65.3
OSU035
75
84.3
OSU036
29.2
54.5
OSU037
37.5
44.2
OSU038
25
39.7
OSU039
75
53.7
OSU040
Hutchinson 75
53.1
Jack
33.3
37.2
Kottman
41.7
28.1
45.8
75.2
OSU041
70.8
63.9
OSU042
25
48.8
OSU043
37.5
24.2
OSU044
45.8
43
OSU045
54.2
57.7
OSU046
50
53.6
OSU047
75
70.9
OSU048
OhioFG1
91.7
64
Plant
Weight
(g)
4.7
6.7
7.3
3.8
2.4
2.5
6.1
5.2
4.8
3.4
4.3
8.5
5.1
5.7
7.1
2.5
5.5
2.2
5
6.7
4.5
3.3
4.2
5.5
4.7
4.5
5.2
4.5
3.5
8.6
10.3
Root
Weight
(g)
1.5
2.2
2.4
1
0.6
0.5
2.1
1.6
1.6
0.9
1.2
2.7
1.5
2.1
2.2
0.7
1.5
0.5
1.8
1.9
1.3
0.8
0.9
1.9
1.7
1.5
2
1.5
1.1
3.1
2.9
61
Root
Score
3
2.7
2.7
3
3
3.3
3
3
3.3
3.7
3.7
2.3
3
2.3
2.7
3.3
4
3.3
2.7
3.3
4
3.3
3.7
3
3.3
3.3
4
3.3
3.3
3
2.7
Continued
Table 16 continued
Level of
genotype
OhioFG5
PI408105A
PI408225A
PI424234B
PI427105B
PI567301B
PI567321A
PI567336A
PI567352B
PI567516C
PI243540
PI291327
PI398233
PI398841
PI399073
PI407985
PI416783
PI417142
PI417459
PI423885
PI427106
PI567324
PI567343
PI594599
Prohio
OSU049
OSU050
Resnik
Ripley
OSU052
Sloan
Percent Mean
Stand
Height
(mm)
50
49.8
62.5
70.3
45.8
61.2
58.3
50
79.2
64.8
87.5
65
41.7
59.7
87.5
65.3
91.7
68.5
12.5
30
79.2
67.1
37.5
27.1
33.3
57.4
45.8
71.7
58.3
51.9
37.5
38.9
16.7
49.7
20.8
38.6
41.7
56
45.8
56.4
41.7
53.3
20.8
38
41.7
49.3
37.5
38.9
66.7
97.9
50
69
45.8
54.7
62.5
81.7
16.7
33.3
66.7
55.4
25
42.7
Plant
Weight
(g)
4.6
5.1
3.2
4.2
5.4
4.4
3.5
4
4.4
0.5
9.1
2
2.6
4
5.2
4.7
2.2
1.6
4.9
5.3
4.1
0.6
2.5
1.7
7
3.4
5.7
6.6
2.6
3.9
4
Root
Weight
(g)
1.2
1.7
1
1
1.6
1.7
1.3
1.6
1.7
0.1
2.4
0.4
0.6
0.9
1.5
1.7
0.4
0.5
1.4
1.4
1.1
0.2
0.8
0.5
2.2
0.9
1.4
2.1
0.9
1.2
1.1
62
Root
Score
3
3.7
3.3
3.7
2.3
2.7
2.7
2.7
3
4.3
3
4.3
3.7
2.7
3.3
3.7
3.7
4.3
3
3.3
3.3
3.3
4
3
2.7
3.7
3.3
2.3
4
3.3
3.7
Continued
Table 16 continued
Level of
genotype
Stout
Streeter
Stressla
Strong
Summit
W82
Williams
Wooster
Wyandot
OSU052
OSU053
Overall
Mean
LSD
Percent Mean
Stand
Height
(mm)
33.3
36.8
29.2
37.3
54.2
82.9
54.2
75.3
33.3
52.1
54.2
78.8
66.7
80.2
33.3
75.2
41.7
52.2
45.8
34.9
81.3
63.5
54.06
56.89
Plant
Weight
(g)
2.1
5.1
4.8
6.1
2.8
4.6
6
3.4
4
5.2
5.9
4.99
Root
Weight
(g)
0.5
1.7
1.5
1.9
0.9
0.9
1.5
1.2
1.3
1.8
2.2
1.56
Root
Score
43.24
4.59
1.72
1.32
39.32
63
4
3.7
3
2.7
3.7
3.3
3
3
3.7
3.3
2.5
3.2
Table 17. Overall means of The Ohio State University population inoculated with P.
ultimum v. sporangiiferum.
Level of
genotype
OSU001
OSU002
OSU003
OSU004
OSU005
OSU006
OSU007
OSU008
OSU009
OSU010
OSU011
OSU012
Archer
Clermont
Conrad
Danbkong
Dennison
Dilworth
Flint
OSU013
OSU014
OSU015
OSU016
OSU017
OSU018
OSU019
OSU020
Percent Mean
Stand
Height
(mm)
87.50
57.80
66.67
70.57
91.67
78.20
91.67
66.57
70.83
68.53
95.83
64.33
70.83
67.87
75.00
66.23
91.67
69.90
87.50
82.90
91.67
74.80
91.67
70.53
83.33
69.43
79.17
57.10
45.83
27.23
70.83
38.90
95.83
65.90
87.50
65.00
83.33
54.10
75.00
61.57
79.17
58.20
62.50
58.63
58.33
40.23
79.17
52.80
37.50
35.83
91.67
53.47
70.83
63.77
Plant
Weight
(g)
6.70
6.87
8.43
9.17
8.10
5.50
6.97
4.87
4.53
12.93
9.40
6.10
10.93
6.63
3.00
4.43
9.93
7.07
7.90
4.87
5.33
5.33
4.95
5.13
2.20
7.10
7.23
Root
Weight
(g)
2.00
1.90
2.47
2.37
1.93
1.93
1.77
1.50
1.47
4.17
3.00
2.20
3.40
1.93
0.53
0.83
3.60
1.90
2.43
1.20
1.27
1.33
1.10
1.17
0.47
1.70
2.13
64
Root Score
2.67
2.33
2.67
2.67
3.00
2.67
2.67
3.00
3.00
2.00
2.33
2.67
2.33
2.33
4.00
3.00
2.67
3.00
3.00
2.33
2.67
1.33
3.00
2.33
3.33
2.33
2.33
Continued
Table 17 continued
Level of
Percent Mean
genotype
Stand
Height
(mm)
OSU021
70.83
48.77
75.00
67.00
OSU022
79.17
57.33
OSU023
75.00
67.77
OSU024
75.00
54.20
OSU025
45.83
38.27
OSU026
95.83
47.67
OSU027
75.00
59.00
OSU028
58.33
42.13
OSU029
75.00
38.67
OSU030
72.92
60.32
OSU031
75.00
39.80
OSU032
87.50
44.47
OSU033
66.67
44.10
OSU034
75.00
46.33
OSU035
70.83
45.33
OSU036
87.50
57.53
OSU037
100.00 70.65
OSU038
83.33
64.33
OSU039
79.17
44.10
OSU040
Hutchinson 79.17
59.43
Jack
70.83
65.13
Kottman
91.67
64.87
75.00
66.67
OSU041
66.67
45.20
OSU042
79.17
57.43
OSU043
45.83
35.00
OSU044
45.83
35.87
OSU045
58.33
67.43
OSU046
54.17
31.43
OSU047
79.17
58.33
OSU048
Plant
Weight
(g)
4.53
5.50
5.70
5.00
5.50
2.87
7.00
6.00
3.80
4.97
6.15
4.45
6.33
4.43
4.90
5.10
6.70
11.30
9.77
5.03
6.43
6.80
9.37
7.20
4.00
7.40
2.80
2.70
3.73
2.70
6.77
Root
Weight
(g)
1.10
1.47
1.50
1.17
1.57
0.73
2.10
1.83
0.93
1.43
1.83
1.05
1.47
1.13
1.23
1.23
1.90
3.50
2.77
1.37
1.77
2.00
2.97
1.93
0.97
2.33
0.63
0.70
0.80
0.63
2.20
65
Root Score
2.67
2.67
2.33
3.00
2.67
3.00
2.00
2.00
2.67
3.00
2.67
3.00
3.00
2.67
3.00
2.67
3.00
2.00
2.33
2.67
2.67
2.67
2.00
3.00
2.67
2.33
2.67
3.00
3.00
3.67
3.33
Continued
Table 17 continued
Level of
genotype
OhioFG1
OhioFG5
PI408105A
PI408225A
PI424234B
PI427105B
PI567301B
PI567321A
PI567336A
PI567352B
PI567516C
PI243540
PI291327
PI398233
PI398841
PI399073
PI407985
PI416783
PI417142
PI417459
PI423885
PI427106
PI567324
PI567343
PI594599
Prohio
OSU049
OSU050
Resnik
Ripley
OSU051
Percent Mean
Stand
Height
(mm)
70.83
58.23
83.33
54.23
58.33
70.00
58.33
58.27
29.17
39.10
37.50
46.77
62.50
44.67
70.83
52.57
83.33
55.57
95.83
72.10
62.50
61.53
70.83
76.20
33.33
43.77
41.67
54.20
37.50
31.43
54.17
58.07
54.17
71.90
16.67
25.23
25.00
36.10
33.33
44.00
50.00
46.67
37.50
45.90
41.67
40.10
75.00
70.67
66.67
62.33
95.83
73.67
87.50
54.13
33.33
52.67
79.17
66.13
41.67
42.00
41.67
38.10
Plant
Weight
(g)
8.47
7.97
4.50
1.93
2.10
1.67
2.03
4.00
2.73
4.40
3.40
8.60
3.00
3.20
3.95
5.20
4.40
1.50
2.40
4.40
3.87
3.37
2.40
5.13
4.13
7.67
7.27
2.97
6.50
5.30
8.13
Root
Weight
(g)
2.70
2.57
1.07
0.67
0.47
0.37
0.63
0.90
0.83
1.63
0.97
2.23
0.80
0.70
0.85
1.33
1.17
0.25
0.70
1.30
0.83
0.73
0.75
1.70
1.23
2.23
2.60
0.93
1.77
1.30
3.17
66
Root Score
2.33
2.67
3.33
2.00
3.33
2.67
3.33
3.33
3.33
2.67
2.33
2.67
3.00
3.00
3.67
3.00
2.33
4.00
3.33
3.00
3.33
2.67
4.00
2.33
2.67
2.67
2.00
2.33
3.33
4.00
2.67
Continued
Table 17 continued
Level of
Percent Mean
genotype
Stand
Height
(mm)
Sloan
37.50
32.50
Stout
95.83
47.67
Streeter
75.00
53.30
Stressla
91.67
63.57
Strong
87.50
66.57
Summit
91.67
61.87
W82
70.83
64.90
Williams
70.83
46.53
Wooster
75.00
64.53
Wyandot
70.83
49.87
OSU052
58.33
47.60
OSU053
50.00
47.13
69.10
55.02
Overall
Mean
30.37
24.76
LSD
Plant
Weight
(g)
2.77
6.60
7.33
8.03
8.80
8.53
6.93
7.17
5.87
5.50
3.07
4.40
5.67
Root
Weight
(g)
0.53
1.87
2.13
2.77
2.37
2.47
2.10
1.73
1.70
1.27
0.73
1.20
1.60
Root Score
3.71
1.57
1.28
67
3.33
2.67
2.33
2.67
2.33
2.33
3.00
3.00
3.00
3.00
3.33
3.00
2.8
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
Genotype Root
Root
Weight
Score
Kottman
0.47
3.67
Prohio
0.63
4.00
Streeter
0.44
4.00
Summit
0.38
4.00
Wooster
0.00
5.00
Wyandot 0.53
3.67
Conrad
0.17
4.17
Sloan
0.38
3.67
OhioFG1 1.13
3.50
Dennison 0.83
3.83
0.50
3.95
Overall
0.41
0.67
LSD
P. ultimum v. ult
Root
Root
Weight
Score
3.60
1.50
2.50
2.40
3.10
2.50
3.20
2.30
0.40
3.80
3.40
2.30
1.80
2.80
3.20
2.00
3.50
2.30
4.20
2.50
2.89
2.44
1.34
0.97
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.
P. ultimum v. spor
Genotype Root
Root
Weight
Score
Kottman
2.83
2.17
Prohio
2.55
2.67
Streeter
2.92
2.33
Summit
2.78
2.50
Wooster
0.68
3.17
Wyandot 2.25
2.33
Conrad
3.25
2.50
Sloan
NA
NA
OhioFG1 4.07
2.17
Dennison 4.58
1.83
2.88
2.41
Overall
0.87
0.70
LSD
P. ultimum v. ult
Root
Root
Weight
Score
1.43
3.67
1.07
3.17
1.38
3.17
1.12
3.50
0.00
5.00
1.23
3.50
1.52
3.67
NA
NA
1.33
3.67
2.57
3.33
1.29
3.63
0.72
0.70
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.
P. ultimum v. spor
Genotype Root
Root
Weight
Score
Kottman
1.75
2.83
Prohio
1.58
3.17
Streeter
2.90
2.83
Summit
1.62
3.17
Wooster
0.02
4.4
Wyandot 2.33
3.17
Conrad
1.65
3.33
Sloan
1.32
3.50
OhioFG1 2.55
3.00
Dennison 3.35
2.67
6.20
1.91
Overall
1.26
0.89
LSD
P. ultimum v. ult
Root
Root
Weight
Score
3.97
2.17
3.97
2.17
3.97
2.00
3.22
2.50
1.53
3.00
4.03
1.83
4.22
2.33
4.22
2.17
4.42
2.33
5.15
2.17
3.87
2.27
1.11
0.67
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.
P. ultimum v. spor
Genotype Root
Root
Weight
Score
Kottman
0.77
3.17
Prohio
2.62
2.50
Streeter
3.22
2.50
Summit
2.67
2.67
Wooster
0.00
5.00
Wyandot 2.23
2.83
Conrad
0.07
4.83
Sloan
1.28
3.50
OhioFG1 3.87
2.50
Dennison 5.58
2.33
2.23
3.18
Overall
0.47
0.83
LSD
P. ultimum v. ult
Root
Root
Weight
Score
2.83
2.83
1.75
3.33
3.27
2.67
1.55
3.00
0.00
5.00
2.50
3.00
0.52
4.50
1.82
3.17
3.65
2.67
3.77
2.67
2.17
3.28
0.84
0.91
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.
P. ultimum v. spor
Genotype Root
Root
Weight
Score
Kottman
2.97
2.00
Prohio
2.23
2.67
Streeter
2.13
2.33
Summit
2.47
2.33
Wooster
1.70
3.00
Wyandot 1.27
3.00
Conrad
0.53
4.00
Sloan
0.53
3.33
OhioFG1 2.70
2.33
Dennison 3.60
2.67
2.01
2.77
Overall
1.57
1.28
LSD
P. ultimum v. ult
Root
Root
Weight
Score
0.80
3.30
2.20
2.70
1.70
3.70
0.90
3.70
1.20
3.00
1.30
3.70
0.90
3.30
1.10
3.70
2.90
2.70
2.00
2.70
1.50
3.25
1.72
1.32
72
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
74
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 +
75
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
76
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 codominant 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 nonamended 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.
79
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
80
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
81
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.
82
Pythium irregulare
80
Number of Isolates
70
60
50
40
30
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.
83
Pythium sylvaticum
8
Number of Isolates
7
6
5
4
3
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.
84
Number of Isolates
Pythium torulosum
4.5
4
3.5
3
2.5
2
1.5
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.
85
Pythium dissotocum
Number of Isolates
1.2
1
0.8
0.6
0.4
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.
86
Number of Isolates
Pythium attrantheridium
4.5
4
3.5
3
2.5
2
1.5
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.
87
Pythium ultimum var. ultimum
14
Number of Isolates
12
10
8
6
4
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;
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
Pythium ultimum var. sporangiiferum
Number of Isolates
6
5
4
3
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 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
Number of Isolates
P. aphanidermatum
4.5
4
3.5
3
2.5
2
1.5
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.
90
Pythium inflatum
3.5
Number of Isolates
3
2.5
2
1.5
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 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.
91
Phytophthora sojae
35
Number of Isolates
30
25
20
15
10
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 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
2/2
15/37
1/3
1/2
3/4
5/9
4/9
1/1
1/1
2/2
1/4
2/3
3/3
4/6
1/4
4/5
3/8
1/3
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
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.
<http://www.frac.info/>.
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.
94
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-2013-0422-01RS.
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.
96
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.
97
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
98
Appendix A: ANOVA for checks & Metalaxyl and Pyraclostrobin Table
99
Table 23. ANOVA for root score of checks. Excluding Conrad and the experiments for
Virginia Tech.
Source
DF
loc
rep(loc)
isol
line
isol*line
loc*isol
loc*line
loc*isol*line
3
8
1
8
8
3
23
22
F
Value
2.44
3.84
2.85
14.57
0.41
58
2.36
1.05
Pr > F
0.0645
0.0003
0.0927
< .0001
0.9152
<.0001
0.0006
0.4047
Table 24. ANOVA for root weight of checks. Excluding Conrad and the experiments for
Virginia Tech.
Source
DF
loc
rep(loc)
isol
line
isol*line
loc*isol
loc*line
loc*isol*line
3
8
1
8
8
3
23
22
F
Value
9.08
5.69
0.02
26.12
1.35
62.68
2.54
2.3
Pr > F
<.0001
<.0001
0.8974
<.0001
0.219
<.0001
0.0002
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
Def 11
Y
NWB 1-w
NWB 3e
OH.12142.06.03
OH12001.10.02
OH12103.01.04
OH12110.08.01
OH12121.02.01
OH-12135. 02.
04
OH12135.05.02
OH12137.05.03
OH12138.10.02
OH12139.04.04
OH12139.06.03
OH12142.03.02
OH12142.03.05
OH12142.05.02
OH-
Y
Y
Y
Phytophthora
sansomeana
Phytophthora sojae
Phytophthora sojae
Phytophthora sojae
Phytophthora sojae
Metalaxyl Pyraclo
Rating
1
Year
2013
3
1
1
1
2013
2013
2012
2012
Phytophthora sojae
1
2012
Phytophthora sojae
1
2012
Phytophthora sojae
1
2012
Phytophthora sojae
1
2012
Phytophthora sojae
1
2012
Phytophthora sojae
2012
Phytophthora sojae
2.5
2012
Y
Phytophthora sojae
1
2012
Y
Phytophthora sojae
1
2012
Y
Phytophthora sojae
1
2012
Y
Phytophthora sojae
1
2012
Y
Phytophthora sojae
1
2012
Y
Phytophthora sojae
1
2012
101
12142.08.05
OH12144.09.02
OH-12147.
05.04
OH12147.08.01
OH12148.01.07
OH12150.01.03
OH-1215003.02
OH12154.07.03
OH12164.05.01
OH12164.07.01
OH12164.08.01
OH12164.10.03
OH12167.05.01
OH12168.01.02
OH12170.10.01
OH12172.01.04
OH12180.04.02
P2-106 P1-3
P2-103 P2-2
Y
Phytophthora sojae
Phytophthora sojae
2.5
1
2012
2012
Y
Phytophthora sojae
1
2012
Y
Phytophthora sojae
1
2012
Y
Phytophthora sojae
1
2012
Y
Phytophthora sojae
1
2012
Y
Phytophthora sojae
1
2012
Phytophthora sojae
1
2012
Phytophthora sojae
1
2012
Phytophthora sojae
1
2012
Phytophthora sojae
2.5
2012
Phytophthora sojae
1
2012
Phytophthora sojae
1
2012
Phytophthora sojae
1
2012
Phytophthora sojae
1
2012
Phytophthora sojae
1
2012
Pythium
Pythium
aphanadermatum
Pythium
aphanadermatum
Pythium
aphanadermatum
Pythium
aphanadermatum
Pythium
aphanadermatum
1
4
2010
2010
Parsely
Y
pepper
Y
squash
Y
Sweet Pepper
Y
102
0.7
P
2012
0.2
P
2012
3
0
2012
P
2012
Tomato
18 Def 8c exp1
Br 209 pb2
D10 407-6r
H3-511 P5-1
Shelby 1-1-3
Y
Pythium
aphanadermatum
Pythium attrantheridium
Pythium attrantheridium
Pythium attrantheridium
Pythium attrantheridium
Pythium cryptoirregulare
Pythium dissotocum
Pythium dissotocum
1
2012
0
1
2010
Aug 3-3-3
Pythium
heterothallicum
Pythium
heterothallicum
Pythium inflatum
2010
2010
2010
2010
2006/
2007
2010
2006/
2007
2010
Erie 1-1-1 A1
Pythium inflatum
0.7
Erie 1-2-11 C3
Pythium inflatum
5
Erie 1-6-5 H6
Pythium inflatum
1
Erie 1-6-6 A7
Pythium inflatum
3
Pythium irregulare
Pythium irregulare
Pythium irregulare
Pythium irregulare
Pythium irregulare
Pythium irregulare
Pythium irregulare
2
1
1
1
2
1
2
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2010
2010
2010
2010
2010
2010
2010
Pythium irregulare
2
2010
Y
Pythium irregulare
2
2010
Y
Pythium irregulare
3
2010
Y
Y
Pythium irregulare
Pythium irregulare
Pythium irregulare
103
2
1
1
2010
2010
2010
Y
Y
Y
18 Def 2A e1
Erie 1-1-9 A2
H3-506 P1-1
P2-103 P1-2
18 Def 12AE1
18 Pike 2A e1
18 Pike 2B e1
18 Pike 3A e1
18Adams2Be2
18Brown 2Ae1
18HIGH11A
EXP 1
18HIGH 1B
EXP1
18HIGH 1C
EXP 1
18HIGH 2A
EXP2
18Pike4c e1
24 Brown 1B e1
24 Brown 2A e1
Y
Y
Y
Y
Y
Y
Y
1
1
1
1
2
P
P*
P*
4
1.3
3
P*
24 High 5B e1
24adams 3A-1
e2
24Adams 4A1
e2
24Brown1A e1
24Brown3A e1
24High 5A e1
Ash 1-1-9
Y
Y
Pythium irregulare
Pythium irregulare
1
1
2010
2010
Y
Pythium irregulare
1
2010
Y
Y
Y
Y
Pythium irregulare
Pythium irregulare
Pythium irregulare
Pythium irregulare
2
2
1
1
Ash 1-6-3
Y
Pythium irregulare
0
Ash 2-1-13
Pythium irregulare
2
Ash 2-1-14
Pythium irregulare
2
P*
P*
2010
2010
2010
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
Ash 2-1-17
Y
Pythium irregulare
0
Ash 2-2-10
Y
Pythium irregulare
2
Ash 2-2-6
Y
Pythium irregulare
2
P*
Ash 2-4-2
Y
Pythium irregulare
1
P*
Ash 2-6-2
Y
Pythium irregulare
2
Pythium irregulare
1
Aug 3-2-1
BR 2-3-5
Y
Pythium irregulare
1
Br 2-5-14
Y
Pythium irregulare
0
Br 2-5-3
Y
Pythium irregulare
1.5
Cham 2-3-4
Y
Pythium irregulare
2
Clark 1-4-13
Y
Pythium irregulare
2
Clark1-4-16
Y
Pythium irregulare
1.7
Cle 1-1-12
Y
Pythium irregulare
2
Cler 1-1-12
Y
Pythium irregulare
104
2
P*
P*
P*
Cler 1-4-1
Y
Pythium irregulare
2
P*
Cler 1-6-11
Y
Pythium irregulare
2
P*
Cler 1-6-6
Pythium irregulare
1
P*
Craw 1-1-8
Pythium irregulare
1
Craw 1-2-9
Pythium irregulare
0
Darke 3-1-8
Y
Pythium irregulare
1.3
Def 2-4-14
Y
Pythium irregulare
0
Def 2-5-22
Y
Pythium irregulare
1
Def 2-5-22
Y
Pythium irregulare
1.7
Pythium irregulare
1.7
Def 2-6-15 (G2)
Erie 2-5-5
Y
Pythium irregulare
1
P*
Erie 2-6-1
Y
Pythium irregulare
0
P*
Fay 2-3-2
Y
Pythium irregulare
1.5
P
Fay 2-4-17
Y
Pythium irregulare
0.5
Fay 2-4-3
Y
Pythium irregulare
1.5
Pythium irregulare
5
Pythium irregulare
1.3
Pythium irregulare
1
P*
P*
Ful 2-2-8
(check)
Ful 2-6-4
Y
Green 1-5-4
Green 2-3-3
Y
Pythium irregulare
0
Green 2-4-7
Y
Pythium irregulare
1
Pythium irregulare
1.3
Hen 1-5-11
105
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
Hen 1-6-4
Y
Pythium irregulare
1
P*
High 2-5-8
Y
Pythium irregulare
1.7
Hur 1-4-11
Y
Pythium irregulare
2
Huron 1-1-13
Y
Pythium irregulare
cont
Logan 1-6-6
Y
Pythium irregulare
1.7
Logan 2-3-15
Pythium irregulare
0.7
Logan 2-3-4
Pythium irregulare
1.3
Logan 2-4-8
Pythium irregulare
1.3
P*
Logan 2-5-16
Pythium irregulare
0.7
P*
Logan 2-5-4
Pythium irregulare
1
P*
Logan 2-6-11
Pythium irregulare
1.3
P*
Logan 2-6-7
Pythium irregulare
0.7
P*
Logan 2-6-9
Pythium irregulare
0
P*
P*
P*
Lucas 2-1-1
Y
Pythium irregulare
1
P*
Mad 2-1-4
Y
Pythium irregulare
cont
P*
Pythium irregulare
1
Pythium irregulare
0
Pythium irregulare
1
Miami 1-2-8
Miami 1-4-8
Y
Mont 1-1-12
Mont 1-1-5
Y
Pythium irregulare
0
Mont 1-1-7
Y
Pythium irregulare
1.5
Mont 1-4-1
Y
Pythium irregulare
1
Pythium irregulare
0
Mor 2-2-5
106
P*
P*
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
Mor 2-3-11
Pythium irregulare
0
Pythium irregulare
0
P*
Put 1-1-5
Pythium irregulare
1
P*
Put 1-1-6
Pythium irregulare
1
P*
Put 1-3-2
Pythium irregulare
0
P*
Put 1-3-5
Pythium irregulare
0
Put 1-3-6
Pythium irregulare
0
Mor 2-4-8
Y
Put 1-5-7
Y
Pythium irregulare
0
Put 1-5-8
Y
Pythium irregulare
0.5
Put 1-6-5
Pythium irregulare
0
sand 1-1-10
Pythium irregulare
1
Sand 1-1-15
Pythium irregulare
1
Sand 1-1-3
Pythium irregulare
1
Sand 1-1-8
Pythium irregulare
0
Sand 1-2-1
Pythium irregulare
0
Sand 1-2-10
Pythium irregulare
1
Sand 1-2-13
Pythium irregulare
0
Sand 1-2-14
Pythium irregulare
0
Sand 1-2-16
Pythium irregulare
0
Sand 1-2-2
Pythium irregulare
0
Sand 1-2-20
Pythium irregulare
0.3
107
P*
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
Sand 1-2-21
Pythium irregulare
0
Sand 1-2-5
Pythium irregulare
1
Sand 1-2-6
Pythium irregulare
1
Sand 1-3-1
Pythium irregulare
0
Sand 1-3-10
Pythium irregulare
0.3
Sand 1-3-11
Pythium irregulare
0.3
Sand 1-3-4
Pythium irregulare
1
Sand 1-3-9
Pythium irregulare
1
Sand 1-4-10
Pythium irregulare
1
Sand 1-4-13
Pythium irregulare
1.3
P*
Sand 1-4-14
Pythium irregulare
1.3
P*
Sand 1-4-19
Pythium irregulare
1.3
P*
Sand 1-4-23
Pythium irregulare
1.3
P*
Pythium irregulare
2
P*
Sand 1-4-5
Pythium irregulare
1.7
Sand 1-4-9
Pythium irregulare
0.3
Sand 1-6-11
Pythium irregulare
1
Sand 1-6-12
Pythium irregulare
1.3
P*
Sand 1-6-14
Pythium irregulare
2
P*
Pythium irregulare
2.3
P*
Sand 1-6-16
Pythium irregulare
1.7
Sand 1-6-17
Pythium irregulare
1.7
Sand 1-4-25
Sand 1-6-15
Y
Y
108
P*
P*
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
Sand 1-6-18
Pythium irregulare
1.7
P*
Sand 1-6-25
Pythium irregulare
1.3
P*
Sand 1-6-4
Pythium irregulare
2
P*
Sand 1-6-9
Pythium irregulare
2
P*
Shelby 1-6-4
Pythium irregulare
0
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2013
2010
2010
2010
War 1-1-13
Y
Pythium irregulare
2
War 1-1-19
Y
Pythium irregulare
2
War 1-6-3
Y
Pythium irregulare
2
Wood 1-4-13
Y
Pythium irregulare
2
wood 1-5-15
Pythium irregulare
0
wood 1-5-2
Pythium irregulare
0
Pythium oopapillum
Pythium spp.
Pythium sylvaticum
Pythium sylvaticum
3
5
1
3
Pythium sylvaticum
3
2010
Pythium sylvaticum
2
2010
Pythium sylvaticum
3
2010
Pythium sylvaticum
2
2010
Pythium sylvaticum
3
2010
Pythium sylvaticum
3
2010
Pythium sylvaticum
2
2010
Pythium sylvaticum
109
3
2010
Def 9
H3-504 P1-1
18 Def 4A e1
18CLER 4A
EXP1
18DEF 11C
EXP1
18DEF 14A
EXP2
18DEF 15B
EXP2
18DEF 2A
EXP2
18DEF 3B
EXP2
18DEF 5B
EXP2
18DEF 6A
EXP2
Ad-1 pb2
Y
Y
P
P
Aug 3-1-5
Pythium sylvaticum
1
Br 213 Pb3
high3 p4-2
Huron 1-1-5
Y
Y
Pythium sylvaticum
Pythium sylvaticum
Pythium sylvaticum
3
2
1
P3-105 P2-1
Wood 2-5-17
Y
Y
Pythium sylvaticum
Pythium sylvaticum
2
2
18Cler 11A e1
18Def 11A e1
18DEF 1A
EXP1
18Def 1B e1
Erie 1-4-4 F4
Pythium torulosum
Pythium torulosum
Pythium torulosum
1
0
2
Pythium torulosum
Pythium torulosum
2.5
0
Erie 1-6-9 D7
Pythium torulsoum
2.7
P2-105 P2-1
ERIE 2-4-2
Y
1
0
P
Fay 1-1-3
Y
0.3
P
HEN 2-2-11
Y
Miami 1-1-5
Y
Miami 1-3-7
Y
Will 1-6-7
Y
Wood 2-6-1
Y
18Def 14B e1
Y
Pythium ultimum
Pythium ultimum
sporangiiferum
Pythium ultimum
sporangiiferum
Pythium ultimum
sporangiiferum
Pythium ultimum
sporangiiferum
Pythium ultimum
sporangiiferum
Pythium ultimum
sporangiiferum
Pythium ultimum
sporangiiferum
Pythium ultimum
ultimum
Pythium ultimum
ultimum
Pythium ultimum
ultimum
Pythium ultimum
ultimum
Pythium ultimum
Aug 1-1-11 B2
Aug 1-1-5
Aug 1-3-1 F4
Aug 1-6-3
Y
110
P*
1
0
P
0
P
2
P
1.2
0
0
0
0.7
0
P*
2006/
2007
2010
2010
2006/
2007
2010
2006/
2007
2010
2010
2010
2010
2006/
2007
2006/
2007
2010
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2010
2006/
2007
2006/
2007
2006/
2007
2006/
Craw 1-1-13
Y
Craw1-2-3
Y
Darke 3-3-8
Y
H2-508 P2-2
Hen 1-2-8
Y
Mad 2-6-7
Y
Miami 1-1-8
Y
Miami 1-3-14
Y
Pick 1-3-5
Y
sally radish
2013
Sand 1-3-13
Y
Y
Wyan 1-1-9
Y
(check)
Br 109 PB3
Y
Br 113 pb1
Y
Br 203 Pb2
Y
Br 305 Pb3
Y
0.3.01
18ADAMS5EEXP2
18Def 1c e1
Br 103 Pb2
Br 314 pb1
H2-510 P2-2
Logan 2-5-5
Rich 30ft
ultimum
Pythium ultimum
ultimum
Pythium ultimum
ultimum
Pythium ultimum
ultimum
Pythium ultimum
ultimum
Pythium ultimum
ultimum
Pythium ultimum
ultimum
Pythium ultimum
ultimum
Pythium ultimum
ultimum
Pythium ultimum
ultimum
Pythium ultimum
ultimum
Pythium ultimum
ultimum
Pythium ultimum
ultimum
Pythium vexans
Pythium vexans
Pythium vexans
Pythium vexans
unknown
unknown
unknown
unknown
unknown
unknown
unknown
unknown
111
1
0
0
P*
1
P*
2
1
1
P
1.5
P*
1
P
1
1
0
P
2
2
2
2
1
3
2.5
2
1
1
0
P*
cont.
P*
2007
2006/
2007
2006/
2007
2006/
2007
2010
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2006/
2007
2013
2006/
2007
2006/
2007
2010
2010
2010
2010
2010
2010
2010
2010
2010
2010
2006/
2007
2013
Field isolates from 2014 tested in Metalaxyl and Pyraclostrobin.
Unknowns from
field 2014
140005-1
140005-2
140005-5
140006-3
140003-5
140010-3
140004-7
140002-3
140004-2
140004-1
140003-6
140005-7
140005-7
140013-4
140013-3
140016-7
140016-3
140015-1
140015-3
140025-1
140012-1
140016-3
140016-7
140012-2
140015-2
140015-5
140012-3
140041-4
140041-3
140041-5
140041-6
140040-3
Metalaxyl Date
0.33
4.5
1.2
0
5
0
0
5
1.33
5
0
2.33
C
C
0.67
C
C
5
C
0
Pyro
6/4/2014
6/4/2014
6/4/2014
6/4/2014
6/4/2014
6/4/2014
6/4/2014
6/4/2014
6/4/2014
6/4/2014
6/4/2014
6/4/2014
6/23/2014
6/23/2014
6/23/2014
6/23/2014
6/23/2014
6/23/2014
6/23/2014
6/23/2014
7/3/2014
7/3/2014
7/3/2014
7/3/2014
7/3/2014
7/3/2014
7/3/2014
7/8/2014
7/8/2014
7/8/2014
7/8/2014
7/8/2014
112
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
140004-1
140005-5
140032-1
140006-3
140041-1
140041-7
140041-2
140037-1
140037-3
140035-2
140035-3
140034-3
140036-3
140040-4
140033-2
140033-1
140038-1
140020-1
140020-2
140021-1
140019-2
140026-2
140039-1
140038-2
140027-1
140027-2
140029-2
140031-5
1.33
0
C
C
C
C
C
C
C
5
C
5
C
0
C
4
C
4
0
4
3
C
C
4
C
C
4
5
7/8/2014
7/8/2014
7/8/2014
7/8/2014
7/8/2014
7/8/2014
7/8/2014
7/9/2014
7/9/2014
7/9/2014
7/9/2014
7/9/2014
7/9/2014
7/9/2014
7/9/2014
7/9/2014
7/9/2014
7/9/2014
7/9/2014
7/9/2014
7/9/2014
7/9/2014
7/9/2014
7/9/2014
7/9/2014
7/9/2014
7/9/2014
7/9/2014
113
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
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.
115
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.
116