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THE-“79

2 0014

This is to certify that the
dissertation entitled

RELATIONSHIPS BETWEEN GLYPHOSATE-RESISTANT SOYBEAN
[Glycine max (L.) MERR.] MANAGEMENT PRACTICES AND
Sclerotinia sclerotiorum (LIB.) DE BARY INFECTION

presented by

Chad David Lee

has been accepted towards fulfillment
of the requirements for

Ph.D. . Crop and Soil Sciences
degree in

 

 

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MW

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RELATIONSHIPS BETWEEN GLYPHOSATE-RESISTANT SOYBEAN
[Glycine max (L.) MERR.] MANAGEMENT PRACTICES AND Sclerotinia
sclerotiorum (LIB.) DE BARY INFECTION
By

Chad David Lee

A DISSERTATION

Submitted to
Michigan State University
In partial fulﬁllment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Crop and Soil Sciences

2002

ABSTRACT
RELATIONSHIPS BETWEEN GLYPHOSATE-RESISTANT SOYBEAN
[Glycine max (L.) MERR.] MANAGEMENT PRACTICES AND Sclerotinia
sclerotiorum (LIB.) DE BARY INFECTION
By

Chad David Lee

Field and greenhouse studies were conducted to investigate the effect of
glyphosate herbicide, adjuvants, shading, cultivar, row spacing, and plant population on
infection of soybean [Glycine max L. (Merr.)] by Sclerotinia sclerotiorum (Lib.) de Bary,
the fungus that causes Sclerotinia stem rot (SSR, also known as white mold). Two near-
isolines, Great Lakes ‘GL2415’ (glyphosate-sensitive) and ‘GL26OORR’ (glyphosate-
resistant), were compared for susceptibility to SSR. The isopropylamine salt of
glyphosate at 840, 1680, and 2520 g ae ha'l, the glyphosate formulation blank at 0.4%
v/v, organosilicone at 0.3% v/v, a crop oil concentrate at 1.0% v/v, and a nonionic
surfactant at 0.5% v/v were evaluated for their inﬂuence on V5 soybean susceptibility to
SSR in the ﬁeld. No differences were detected in SSR severity or yield between either
cultivar or among any herbicide/adjuvant treatment. Mycelia growth of S. sclerotiorum
was inhibited by high concentrations of formulated glyphosate, the glyphosate
formulation blank, and by the three adjuvants but not by glyphosate formulated without
adjuvants. Shikimate accumulation was detected 1, 4, and 7 days after glyphosate
treatment in GL2415 but not in GL26OORR. Soybean extracts containing glyceollin
inhibited S. sclerotiorum mycelia in a rate-dependent manner. Glyphosate had no impact
on glyceollin production in glyphosate-resistant soybeans, while lactofen induced

glyceollin synthesis in both GL2415 and GL26OORR. Shade levels of 60% did not inhibit

the induction of glyceollin synthesis. Three glyphosate-resistant cultivars, Pioneer
‘92B7l ’, Asgrow ‘AG2701’, and Asgrow ‘AGZ702’, were evaluated in row spacing by
target population combinations of 76 cm, 430 000 seeds ha"; 19 cm, 430 000 seeds ha";
and 19 cm, 560 000 seeds ha'1. Cultivar 92B7l was the most tolerant to SSR and the
highest yielding cultivar followed by A6270] and then AG2702. Disease severity
indexes were signiﬁcantly lower and yield was signiﬁcantly higher when target
population was reduced from 560 000 to 430 000 seeds ha’1 in l9-cm rows. Neither the
glyphosate-resistant trait nor the application of glyphosate and/or adjuvants inﬂuenced
soybean susceptibility to S. sclerotiorum. Planting soybeans in narrow rows at low

populations may be just as effective as planting soybeans in 30-cm rows for managing

SSR.

AKNOWLEDGEMENTS

Someone once said that obtaining a Ph.D. is not a matter of intelligence, but a
matter of perseverance. While that may be true, obtaining a Ph.D. is also a matter of
guidance and help from many others along the way. My advisor, Dr. Donald Penner, and
committee members, Drs. Karen Renner, Ray Hammerschmidt, and James Kelly, kept
their doors open and their watches in their pockets when questions were asked and
guidance was needed. Gary Powell, weed science technician, was instrumental in
establishing ﬁeld plots and providing a work crew. Many graduate students helped take
measurements, discussed statistics, and challenged my knowledge of weed science. The
most inﬂuential of those students was Kelly Nelson, not only for his labor, but for his
friendship. Undergraduate students helped water plants in the greenhouse and do
extractions in the laboratory. Of those students, Paula Crouse devoted the most time and
effort to my project. My parents, David and Carolyn Lee, were the ﬁrst to introduce me
to agriculture on a small farm in Ohio. Their concern for the land, for the food supply,
and for people helped shape my interests in agriculture. My wife, Verona, has been my
best friend and my cheering section throughout the trials of graduate school. Life is more
enjoyable due to her presence. Finally, my everlasting gratitude and praise belongs to

God for all the obvious reasons.

iv

TABLE OF CONTENTS

LIST OF TABLES ........................................................................................................... vi

LIST OF FIGURES ....................................................................................................... viii

INTRODUCTION .............................................................................................................. 1

CHAPTER 1

SCLEROTINIA STEM ROT IN SOYBEAN LITERATURE REVIEW .................... 2
HOST PENETRATION ..................................................................................... 3
RESISTANCE ................................................................................................. 13
EVALUATION OF SCLEROTINIA AND GLYPHOSATE ......................... 28
LITERATURE CITED .................................................................................... 36
TABLE I .......................................................................................................... 44

CHAPTER 2

INFLUENCE OF FORMULATED GLYPHOSATE AND ACTIVATOR
ADJ UVANTS ON Sclerotinia sclerotiorum IN GLYPHOSATE-RESISTANT AND —

SUSCEPTIBLE SOYBEAN (Glycine max) ................................................................... 45
ABSTRACT ..................................................................................................... 45
INTRODUCTION ........................................................................................... 47
MATERIALS AND METHODS ..................................................................... 49
RESULTS AND CONCLUSIONS .................................................................. 53
LIST OF RESOURCES ................................................................................... 56
ACKNOWLEDGEMENTS ............................................................................. 57
LITERATURE CITED .................................................................................... 58
TABLE 1 .......................................................................................................... 59
TABLE 2 .......................................................................................................... 60
TABLE 3 .......................................................................................................... 61
FIGURE 1 ........................................................................................................ 62
FIGURE 2 ........................................................................................................ 63
FIGURE 3 ........................................................................................................ 64

CHAPTER 3

GLYPHOSATE DOES NOT INHIBIT INDUCTION OF THE DEFENSE
RESPONSE OF GLYPHOSATE-RESISTANT SOYBEAN TO Sclerotinia

sclerotiorum ....................................................................................................................... 65
ABSTRACT ..................................................................................................... 65
INTRODUCTION ........................................................................................... 67
MATERIALS AND METHODS ..................................................................... 70
RESULTS AND CONCLUSIONS .................................................................. 75
SOURCES OF MATERIALS .......................................................................... 78
LITERATURE CITED .................................................................................... 80

TABLE 1 .......................................................................................................... 83

TABLE 2 .......................................................................................................... 84

FIGURE 1 ........................................................................................................ 85

FIGURE 2 ........................................................................................................ 86
CHAPTER 4

ROLE OF CULTIVAR, ROW SPACING, AND SEED POPULATION IN THE
INCIDENCE OF SCLEROTINIA STEM ROT IN GLYPHOSATE-RESISTANT

SOYBEAN ........................................................................................................................ 87
ABSTRACT ..................................................................................................... 87
INTRODUCTION ........................................................................................... 88
MATERIALS AND METHODS ..................................................................... 92
RESULTS AND CONCLUSIONS .................................................................. 99
SUMMARY ................................................................................................... 104
ACKNOWLEDGEMENTS ........................................................................... 106
LITERATURE CITED .................................................................................. 107
TABLE 1 ........................................................................................................ 110
TABLE 2 ........................................................................................................ l l 1
TABLE 3 ........................................................................................................ 112
TABLE 4 ........................................................................................................ 113
FIGURE 1 ...................................................................................................... 114
FIGURE 2 ...................................................................................................... 1 15
FIGURE 3 ...................................................................................................... 1 l6

SUNIMARY .................................................................................................................... 117

EXTENSION SUMMARY ............................................................................................ 119
FIGURE 1 ...................................................................................................... 122
FIGURE 2 ...................................................................................................... 123

vi

LIST OF TABLES

CHAPTER 1
LITERATURE REVIEW OF SCLEROTINIA STEM ROT IN SOYBEAN ........... 2
Table 1: Lesion diameters resulting from Sclerotinia sclerotiorum inoculation..44

CHAPTER 2

INFLUENCE OF FORMULATED GLYPHOSATE AND ACTIVATOR

ADJUV AN TS ON Sclerotinia sclerotiorum IN GLYPHOSATE-RESISTANT

AND —SUSCEPTIBLE SOYBEAN (Glycine max) ........................................................ 45
Table I .' Lesion diameters resulting from Sclerotinia sclerotiorum inoculation
of excised GL2415 and GL26OORR Glycine max leaﬂets (fourth trifoliate) at 1, 2,
3, 4, 5, 6, and 7 DAT. Lesion diameters were averaged over cultivar and
inoculation timings. Soybeans were grown in the ﬁeld and treated at the V5

growth stage. Means were averaged over inoculation timings and were analyzed
for differences with LSD (p = 0.05). ..................................................................... 59

Table 2: The SSR severity index (DSI) and yield from ﬁeld study of Glycine
max cultivars GL2415 and GL26OORR treated with adjuvants and formulated
glyphosate. Means were separated with LSD (p=0.05) ....................................... 60

Table 3: Lesion diameters measured 48 h after Sclerotinia sclerotiorum mycelial
inoculation of ‘GL26OORR’ Glycine max leaﬂets excised from plants 1, and 7
DAT. Soybean plants were grown under greenhouse conditions and chemical
treatments were made at the V4 growth stage. Means were separated by LSD

(p = 0.05) ................................................................................................................ 61

CHAPTER 3

GLYPHOSATE DOES NOT INHIBIT INDUCTION OF THE DEFENSE

RESPONSE OF GLYPHOSATE-RESISTANT SOYBEAN TO Sclerotinia

sclerotiorum ....................................................................................................................... 65
Table I. Glyphosate and lactofen effect on Sclerotinia sclerotiorum growth on
detached soybean leaﬂets and glyceollin synthesis. Relative glyceollin levels
determined by thin-layer chromatography separation using a Cladosporium
cucumerinum assay ................................................................................................ 83

Table 2. Inﬂuence of shade on lesion diameters on greenhouse-grown soybean
leaﬂets resulting from Sclerotinia sclerotiorum hyphae growth. Larger lesion size
indicates less glyceollin production (Dann et al. 1999). No signiﬁcant differences
between lesion diameters occurred among leaﬂet position. .................................. 84

vii

CHAPTER 4

ROLE OF CULTIVAR, ROW SPACING, AND SEED POPULATION IN THE
INCIDENCE OF SCLEROTINIA STEM ROT IN GLYPHOSATE-RESISTAN T
SOYBEAN ....................................................................................................................... 85

Table 1. Targeted and actual plant populations ................................................... 103

Table 2. Disease severity index (DSI) and yield for main plots of cultivar and
subplots of row spacing by seed population combined over years. Letters
following least squared means indicate signiﬁcant differences at p = 0.05 ......... 104

Table 3. Lesion diameters resulting from Sclerotinia sclerotiorum growth on
detached leaves from 92B7l and AG2702 soybean grown in the ﬁeld at different
row spacings and populations. Leaﬂets were harvested from R1 soybean. Letters
following least squared means represents signiﬁcant differences at p = 0.05. ....105

Table 4. Lesion diameters resulting from Sclerotinia sclerotiorum growth on
detached leaves of 92B7l soybean grown under various levels of shade in the
greenhouse. Soybean was at the V5 grth stage at the time of leaf harvest.

Letters following least squared means represent signiﬁcant differences at p =
0.05 ....................................................................................................................... 106

viii

LIST OF FIGURES

CHAPTER 2

INFLUENCE OF FORMULATED GLYPHOSATE AND ACTIVATOR

ADJUVANTS ON Sclerotinia sclerotiorum IN GLYPHOSATE-RESISTAN T AND —

SUSCEPTIBLE SOYBEAN (Glycine max) ................................................................... 44
Figure I: Lesion diameters averaged over GL2415 and GL26OORR Glycine max
leaﬂets inoculated with Sclerotinia sclerotiorum 15 min after being dipped into
various treatments of formulated glyphosate and adjuvants. Treatments included
the formulation blank of the isopropylamine salt of glyphosate (RUFB),
organosilicone (OSI), crop oil concentrate (COC), nonionic surfactant (NIS), and
formulated isopropylamine salt of glyphosate (RU). Rates for each treatment are
based on % v/v and included in parenthesis. Bars with different letters represent
signiﬁcant differences according to LSD (p=0.05) ................................................ 62

Figure 2. Mycelia] growth of Sclerotinia sclerotiorum on potato dextrose agar
with 100 and 1000 mM ae glyphosate as the formulation without adjuvants (AC),
formulation with an adjuvant (R0), with proprietary adjuvants (RU), and the
formulation blank with proprietary adjuvants (RUFB). Bars with different letters
represent signiﬁcant differences according to LSD (p=0.05). ............................... 63

Figure 3. Mycelia] growth of Sclerotinia sclerotiorum on potato dextrose agar
treated with l % v/v of organosilicone (OSI), crop oil concentrate (COC), or
nonionic surfactant (NIS). Bars with different letters represent signiﬁcant
differences according to LSD (p=0.05). ................................................................ 64

CHAPTER 3
GLYPHOSATE DOES NOT INHIBIT INDUCTION OF THE DEFENSE
RESPONSE OF GLYPHOSATE-RESISTANT SOYBEAN TO Sclerotinia

sclerotiorum ....................................................................................................................... 65
Figure 1. Inhibition of Sclerotinia sclerotiorum hyphae on potato
dextrose agar by extracts containing glyceollin from soybean leaﬂets ............. 85

Figure 2. Shikimate accumulation following glyphosate application to GL2415
(glyphosate-susceptible) and GL26OORR(glyphosate-resistant) soybean. T-bars
represent LSD at p=0.05 .................................................................. 86

ix

CHAPTER 4

ROLE OF CULTIVAR, ROW SPACING, AND SEED POPULATION IN THE

INCIDENCE OF SCLEROTINIA STEM ROT IN GLYPHOSATE-RESISTANT

SOYBEAN ....................................................................................................................... 87
Figure 1. Leaf area index in 1999 for the three cultivars (A) and the three row
spacings by populations (B) during the growing season. Differences of the least
squared means were detected with the PDIFF option in SAS at p= 0. 05. WL =
wide row, low population: 76 cm, 430 000 seeds ha; NL = narrow row, low
population: 19 cm], 430 000 seeds ha" ,=NH narrow row, high population: 19 cm,
560 000 seeds ha’l ................................................................................................ 114

Figure 2. Leaf area index in 2000 for the three cultivars (A) and the three row
spacings by populations (B) during the growing season. Differences of the least
squared means were detected with the PDIFF option in SAS at p= 0. 05. WL =
wide row, low population: 76 cm, 430 000 seeds ha; NL = narrow row, low
population: 19 cm], 430 000 seeds ha"; NH= narrow row, high population: 19 cm,
560 000 seeds ha ................................................................................................ 115

Figure 3. Differences in average ﬂower number per plant for the three cultivars
(A) and the three row spacings by populations (B) during the growing season.
Values are averaged over the subplots of row spacing by population and
combined over years. Letters above bars represent signiﬁcant differences between
least squared means at p= 0. 05. WL= wide row, low population. 76 cm, 430 000
seeds ha; NL= narrow row, low population: 19 cm, 430 000 seeds ha ,-NH ‘-
narrow row, high population. 19 cm, 560 000 seeds ha ............................ 116

EXTENSION SUMMARY ............................................................................................ 110
Figure 1 . Roundup Ready soybean variety effect on white mold disease levels (A)
and yield (B). White mold disease levels are determined by a disease severity
index (DSI) where 0 = no disease detected, and 100 = complete pod damage or
plant death from white mold ................................................................................ 116

Figure 2. Row spacing and population effect on white mold disease severity (A)
and soybean yield (B). White mold disease levels are determined by a disease
severity index (DSI) where 0 = no disease detected, and 100 = complete pod
damage or plant death from white mold .............................................................. 117

INTRODUCTION

Sclerotinia stem rot (SSR, also known as white mold) caused by the ﬁmgal
pathogen Sclerotinia sclerotiorum (Lib.) de Bary is a prevalent disease of soybean grown
in the Great Lakes region. Glyphosate-resistant soybean currently comprises about 70%
of the soybean acreage in the Great Lakes region. Sublethal doses of glyphosate to
glyphosate-susceptible soybean resulted in reduced soybean tolerance to pathogens.
Farmers in Michigan reported high levels of SSR in glyphosate-resistant soybean. This
research was initiated to determine if a glyphosate-resistant soybean cultivar was more
susceptible to S. sclerotiorum than a glyphosate-susceptible near-isoline and if
applications of formulated glyphosate and/or adjuvants increased plant susceptibility to S.
sclerotiorum. Another objective of this research was to determine if glyphosate or
shading reduced defense response of soybean to S. sclerotiorum. Since no comparisons of
the inﬂuence of different soybean populations within a row spacing on SSR and no
comparisons of the inﬂuence of a single population at different row spacings on SSR had
been reported, the ﬁnal objective of this research was to determine the impact of soybean
cultivar, row spacing, and population on SSR incidence in a glyphosate-resistant soybean

system.

CHAPTER 1
LITERATURE REVIEW OF SCLEROTINIA STEM ROT IN SOYBEAN

Sclerotinia sclerotiorum (Lib.) de Bary causes Sclerotinia stem rot (SSR, also
known as white mold disease) of soybean [Glycine max (L.) Merr] and is prevalent in the
Great Lakes region. Sclerotinia sclerotiorum has a wide range of hosts including 408
species and 75 families (Boland and Hall 1994). Broadleaf species that are hosts for S.
sclerotiorum include sunﬂower (Purdy 1979), soybean, common bean (Phaseolus
vulgaris L.) (Purdy 1979), canola (Brassica napus L.) (Kharbanda and Tewari 1996),
peas (Lathyrus sp.), clovers (T rifolium sp.), tomato (Lycopersicon esculentum Mill.)
(Boland and Hall 1994), Irish potato (Solanum tuberosum L.) (Purdy 1979), and pear
(Pyrus communis L.) (Spotts and Cervantes 1996). Grass species that are hosts for S.
sclerotiorum include oats (Avena sp.), barley (Hordeum vulgare L.), rye (Secale cereale
L.), sorghum [Sorghum bicolor (L.) Moench], wheat (T riticum aestivum L.), and maize
(Zea sp.) (Boland and Hall 1994). Some weed hosts of S. sclerotiorum include common
ragweed (Ambrosia artimisiifolia L.), various thistles (Cirsium sp.), chicory (Chicorum
intybus L.), mustards (Brassica sp.), velvetleaf (A butilon theophrasti Medicus), and
jimsonweed (Datura stramonium L.) (Boland and Hall 1994).

The general life and disease cycle of S. sclerotiorum was described by Purdy
(1979) and Li et al. (1999). Ascospores of S. sclerotiorum are released from apothecia
and germinate on nonliving or senescent plant parts. Flower petals are also a site of
ascospore germination (Sutton and Deverall 1983). Germ tubes from the ascospore
penetrate the plant tissue via either appressorium or direct penetration (Jaumax et al.

1995; Tariq and Jeffries 1984). Secretion of oxalic acid that alters plasma membranes

may play a role in penetration (Tu 1989). The invading mycelia will colonize plant tissue
and form white cottony masses on the plant (Purdy 1979). The mycelia deve10p sclerotia
in stem or fruit piths and the sclerotia over winter in the soil. In the mid-summer,
sclerotia produce apothecia in which ascospores are produced. Ascospores are released
from the apothecia, land and infect sensecing ﬂower petals, completing the cycle.

Agrios (1997) deﬁnes disease development as the interaction of the virulent
pathogen, susceptible host and the environment. Factors related to host penetration, plant
defenses and host susceptibility are reviewed.

HOST PENETRATION

Penetration of the host is a critical step in the disease and life cycle of S
sclerotiorum. In many cases, appressoria are a key process in the penetration of a host.
Formation of appressorium involves multiple steps (Kim et al. 2000) and occurs
relatively soon after inoculation (Buhr and Dickman 1997; Leandro et al. 2001; Tariq and
Jeffries 1984). Factors such as surface of host (Chaky et al. 2001; Jaumax et al. 1995;
Kim et al. 2000b Leandro et al. 2001; Tariq and J effries 1984), spore concentration (Buhr
and Dickman 1997; Kim et al. 2000b), nutrients (Chaky et al. 2001; Magalhaes et al.
1991), pH (Stumpf et al. 1991), and host genes (Heo et al. 1999; Reymond-Cotton et al.
1996; Warwar et al. 2000) regulate the development of appressoria. Plant inhibitors of
appressoria also play a role in appressoria development (Buhr and Dickman 1997; Choi
et al. 1998; Kim et al. 2000b).

Appressorium Formation

Stages of Development. Many fungi penetrate host tissue with the aid of

appresoria. Eight stages were detailed from conidia to appressorium development in

Colletotrichum gloeosporioides (Kim et al. 2000b). Stage 1 occurrs when phallodin-FITC
signals and PI signals co-localize in the nucleus at the center of the cells, while stage 2
occurrs when F-actin de-localizes from the nucleus (within 30 min). Stage 3 is the
gradual dispersal of PI throughout the cell and stage 4 occurrs when PI signals begin to
congregate at both ends of the cells, indicating nuclear division and F-actin associated
preferentially with one of the nuclei. In addition phalloidin-FITC signals become more
prevalent at the cell ends (about 60 min of incubation on hard surface). Stage 5 is the
completion of cytokenesis (within 2 to 4 h of incubation on the hard surface) and stage 6
occurrs when the germ tube begins to emerge speciﬁcally from the daughter cell (about 4
to 6 h of incubation). Stage 7 is the formation of F -actin-rich appressoria at the end of
germ tubes (6 to 10 h of incubation) and stage 8 is the melanization of the appressoria (8
to 16 h of incubation). Kim et al. (2000b) observed that the rate of appressorium
development was much quicker in low population densities than in higher population
densities.

Appressoria generally form within several hours after spore germination. S.
sclerotiorum spores on canola (Brassica napus) germinated within 3 h after inoculation
and collapse of epidermal cells in the host occurred by 7 h aﬁer inoculation (Jamaux et al.
1995). Appressorium development of Colletotrichurn trzfolii occurred with 1 h after germ
tube morphogenesis in all media except 4% yeast extract, which delayed appressorium
development to 2 h (Buhr and Dickman 1997). Leandro et al. (2001) reported that
Colletotrichum acutatum conidia began to germinate on strawberry leaves and cover slips
within 3 h after inoculation. Germination tubes adhered to the leaf surfaces by 3 h after

inoculation (Leandro et al. 2001). Appressoria developed on the distal ends of the

elongated germinated tubes. Leandro et al. (2001) ﬁrst observed appressorial pores as
melanization began. About 60 % of the appressoria developed appressorial pores by 48 to
168 h after inoculation.

Host Surface Effect on Appressorium Development. Surface rigidity, surface
hydrophobicity, and compatible reactions all affect conidia germination, formation of
appressorium, type of appressorium, and melanization of appressorium. A rigid host
surface resulted in more complex appressoria (Tariq and Jefﬁies 1984) and higher
frequencies of appressoria (Chaky et al. 2001). Sclerotinia sclerotiorum spores on canola
petals germinated and penetrated the petal surface without forming appressoria (J aumax
et al. 1995). Following germination of the spore, host epidermal cells collapsed,
indicating that enzymes may be excreted by the pathogen to compromise the host cell
wall (Jaumax et al. 1995). Sclerotinia sclerotiorum formed more complex appressoria on
surfaces that were more difficult to penetrate. Terminal S. sclerotiorum appressoria
formed on dichotomous branches of a single aerial hypha on biotic surfaces. Simple
appressoria were observed on leaves of common bean (Phaseolus vulgaris L.), canola
(Brassica napus ssp. oleifera), and lettuce (Lactuca sativa L.) (Tariq and Jeffries 1984).
Compound appressoria occurred on leaves and epicotyls of Phaseolus coccineus and on
leaves of Chrysanthemum (Chrysanthemum spp.) (Tariq and Jeffries 1984). The
compound appressoria developed as a result of dichotomous branching of single
subtending hypha so that a small pad of broad, multi-septate, short-celled hyphae was
formed. The apices of the short-celled hyphae were oriented perpendicularly to the biotic
surface (Tariq and Jeffries 1984). Well-developed S. sclerotiorum appressoria occurred

on leaves of P. coccineus and Chrysanthemum. The well-developed appressoria consisted

of numerous hyphal branches with the apices in close contact with the plant cuticle (Tariq
and Jeffries 1984). The largest and most complex appressoria were observed on
aluminum foil, glass coverslips, and the sides of plastic petri dishes. No appressoria were
observed on Whatman ﬁlter paper or millipore ﬁlters (Tariq and Jefﬁies 1984). These
observations indicated that leaves of P. coccineus and Chrysanthemum were more
difficult to penetrate than surfaces of common bean, canola, and lettuce (Tariq and
Jeffries 1984).

Colletotrichum graminicola spore germination and appressorial induction was
more prevalent on a rigid surface (plastic cover slip) than on a soft surface (silicone
grease), while germinated spores on silicone grease never developed appressoria (Chaky
et al. 2001). Hard surface contact with Colletotrichum gloeosporioides conidia induced
dispersal of F -actin and nuclear division (Kim et al. 2000b). A mitogen-activated protein
kinase (MAPK) signaling appeared to be necessary for polarized cell division (Kim et al.
2000b), resulting in conidia germination.

In their review of plant topography, Kolattukudy et al. (1995) reported that
microfabricated ridges of 0.5 pm on silicon wafers were optimal for differentiation into
appressorium for Uromyces appendiculatus (bean fungus). Researchers suggested that
mechanosensitive chemicals cause an inﬂux of Ca 2+ ions, which triggered the
differentiation process.

Biotic versus Abiotic Surfaces. Melanization of appressoria was dependent on
host surface. Fewer Colletotrichum acutatum appressoria became melanized on cover
slips than on the strawberry leaf surface (Leandro et al. 2001). Some appressoria on

strawberry leaves and cover slips became melanized and developed basal pores while

others that did not become melanized formed germ tubes. At the tips of these germ tubes
appressoria formed and became melanized (Leandro et al. 2001). Primary conidia that did
not develop germination tubes developed phialides which produced secondary conidia.

Surface hydrophobicity was positively related to Colletotrichum graminicola
spore germination rate (Chaky et al. 2001). As wettability increased, spore release
generally increased (Chaky et al. 2001). Spore germination was reduced as wettability
increased on UV-treated polystyrene sheets with different wettability characteristics.
Appressorial formation decreased as wettability increased (Chaky et al. 2001).

Host compatibility affects the type of appressorial formation. Ultrastructure of
compatible interactions between sorghum [Sorghum bicolor (L.) Moench] and
Colletotrichum sublineolum included the formation of infection pegs into globose
infection vesicles 42 h aﬁer inoculation (Wharton et al. 2001). The infection vesicles of
C. sublineolum were slightly smaller than appressoria. Primary hyphae were exclusively
intercellular. The C. sublineolum hyphae appeared to be in direct contact with the host
cell cytoplasm. Penetration of primary hyphae through additional cell walls resulted in
constriction of the primary hyphae (Wharton et al. 2001). A septum with Woronin bodies
was usually associated with cell wall penetration. By 24 h after inoculation, infected cells
began to degenerate once hyphae spread to additional cells (Wharton et al. 2001). The
cytoplasm of each infected cell was completely disrupted by 66 h after inoculation
(Wharton et al. 2001). The cell walls of primary hyphae in dead host cells were thicker
than those in living host cells (Wharton et al. 2001). At 66 h after inoculation, secondary
hyphae were observed in dead epidermal cells. Secondary hyphae proliferated throughout

the epidermis, mesophyll, and vascular tissue by 90 h and numerous acervuli had formed

by 114 h (Wharton et al. 2001). Acervuli resulted in conidiophores and setae that
occurred between the cell wall and the cuticle. The conidiophores and setae ruptured the
cuticle (Wharton et al. 2001). Ultrastructure of incompatible interactions between S.
bicolor and C. sublineolum included appressoria and penetration pegs that had penetrated
epidermal cells for form infection vesicles and hyphae. Infected host cells appeared dead
with cytoplasmic disruption by 42 h. At 66 b all hyphae appeared dead and did not
advance past the primary penetration cell (Wharton et al. 2001).

Sclerotinia sclerotiorum appressoria responded differently to resistant and
susceptible sunﬂower plants. Achbani et al. (1996) observed mucilage around ascospores
on susceptible plants and no mucilage on resistant plants.

Conidia Concentration and Germination

Conidia concentration impacts germination and appressorium formation. Lower
C. trifolli spore concentrations in suspension cultures resulted in increased conidia
germination and synchronization of conidia germination (Buhr and Dickman 1997). Kim
et al. (2000) observed that the rate of C. gloeosporioides appressorium development was
much quicker in low population densities than in higher population densities.

Nutrients and pH Effects on Appressoria

Ca”, carbon, and yeast extracts containing nitrogen and carbon either enhance or
are necessary for appressorium development. Zoophthora radicans required external Ca2+
for appressorium formation but not for conidia germination (Magalhaes et al. 1991).
More appressoria formed at higher Ca2+ concentrations compared to lower Ca2+
concentrations (Magalhaes et al. 1991). Carbon sources including yeast extract, glucose,

sucrose, and maltose increased spore germination (Chaky et al. 2001). Nitrogen had no

impact on appressorium development; however, yeast extract containing both nitrogen
and carbon resulted in the greatest increase of spore germination of Colletotrichum
graminicola (Chaky et al. 2001). Neither external K+ nor external Cl' were needed for
appressoria germination (Magalhaes et al. 1991).

Uromyces appendiculatus appressorium development was optimal at pH range of
5.5 to 6.5, while appressorium formed much less efﬁciently at a pH range of 7.0 to 8.0
(Stumpf et al. 1991). Appressorium development was inhibited by nonbuffered KCl in
distilled water (Stumpf et al. 1991).

Genes and Gene Expression for Appressoria

Expression of genes in both the host and the pathogen related to Ca2+ ﬂux
appeared to be required for appressorium formation. Two divergent soybean calmodulin
(SCaM) isoforrns (SCaM-4 and SCaM-S) acted as both a signal receptor and transmitter
of the pathogen-induced Ca 2+ signal (Heo et al. 1999). Both F usarium solani and
Phytophthora parasitica var. nicotianae resulted in increased mRN A production of
SCaM-4 and -5, but not in SCaM-l, -2, or -3 (Heo et al. 1999). Expression of the
calmodulin gene of C. trifolii was highest during spore germination and appressorial
development (Warwar et al. 2000). C. trifolii with an inducible promoter driving the
calmodulin gene in the antisense direction resulted in no appressorial formation (W arwar
et al. 2000).

SCaM-4 and -5 proteins reached maximum concentrations at 2 h and slowly
declined after 12 h (Heo et al. 1999), indicating a transient response of the host to the
fungi. Expression of SCaM-4 and -5 appears to be speciﬁc to pathogens and dependent

on Ca 2+ inﬂux (Heo et al. 1999). A Ca 2+ chelator abolished induction of SCaM-4 and -5

(Heo et al. 1999). The Ca 2+-ionophore A23187 was sufﬁcient to induce SCaM-4 and
SCaM-S expression as effective as the fungal elicitor (Heo et al. 1999), indicating that the
fungal elicitor contains Ca”. Exogenous salicylic acid (SA), hydrogen peroxide (H202),
an HZOZ-generating system, superoxide-generating system, jasmonic acid (JA) and
abscisic acid (ABA) all failed to induce SCaM-4 and -5 gene expression (Heo et al.
1999)

Transformed tobacco with either SCaM-4 or -5 expressed lesions indicating a
hypersensitive response (HR) on mature leaves, while no such lesions were observed on
un-transformed or empty construct plants (Heo et al. 1999). Based on protein production,
the HR lesions were likely a result of normal SCaM-4 or -5 protein levels similar to
activation by a pathogen attack (Heo et al. 1999). SCaM-4 and SCaM-5 transgenic plants
expressed high levels of resistance to Phytophthora parasitica var. nicotianae (black
shank). The transgenic plants also displayed resistance to Pseudomonas syringae pv.
tabaci (Heo et al. 1999).

Calmodulin genes appeared to be highly conserved between ﬁmgal species. The
predicted amino acid sequence of the C. trifolii calmodulin gene was 99% identical to
calmodulin genes in Neurospora crassa and Aspergillus nidulans (Warwar et al. 2000).
Within 160 bp upstream from the translation point, consensus sequences of a TATA box,
three CIT-rich regions, three A/T-rich regions, and four possible CAAT boxes were
identiﬁed (Reymond-Cotton et al. 1996). A sequence within pg] may correspond to the
D-glucose-mediated carbon metabolite repression by the DNA-binding repressor protein

CREA (Reymond—Cotton et al. 1996). Identiﬁcation of the CREA-binding sites suggested

10

that carbon-catabolite-repression regulatory system similar to CREA may regulate pgl
transcription (Reymond-Cotton et al. 1996).

PR Genes. Expression of SCaM-4 and -5 in transgenic plants was independent of
a pathogen and occurred throughout the growth and development of the transgenic plants
(Heo et al. 1999). Expression of PR genes in the transgenic plants was independent of
lesion formation, indicating that cell death was not required for the expression of PR
genes in these transgenic plants (Heo et a1. 1999).

SA did not appear to be involved in the induction of PR gene expression mediated
by SCaM-4 or -5 (Heo et al. 1999).

pgl. Expression of pgl at high levels appeared to occur during plant infection
(Reymond-Cotton et al. 1996). Total RNA of about 1150 bp from S. sclerotiorum
accumulated only from cultures grown on sunﬂower cotyledons as opposed to cultures
grown on carbon sources of glucose or polygalacturonate (Reymond-Cotton et al. 1996),
indicating that the sunﬂower cotyledons contained elicitors of pgl expression.

MEK. Mitogen-activated kinase kinase (MPAK kinase, MEK) played a role in
cell division, germination of conidia, and differentiation of the germ tube into
appressorium (Kim et al. 2000). CgMEKl protein was needed for germ tube
differentiation into appressorium (Kim et al. 2000).

Inhibitors of Appressoria

Various compounds have acted as inhibitors of appressoria. Yeast extracts and
cutin media delayed germination of conidia and appressorium development (Buhr and
Dickman 1997). Yeast extract delayed germ tube morphogenesis of C. trifolii (Buhr and

Dickman 1997) but enhanced appressorium development in Colletotrichum graminicola

11

(Chaky et al. 2001). Germ tube preinfection morphogenesis was affected by the balance
of self-germination inhibitors and available nutrients or cutin in the environment (Buhr
and Dickman 1997). Germination of conidia decreased in the presence of cutin or cutin-
like monomers (Buhr and Dickman 1997). Washing of C. trifolii conidia removed
germination inhibitors and increased synchronicity of conidial germination (Buhr and
Dickman 1997). Buhr and Dickman (1997) concluded that cutin constituents may act as a
plant signal molecules that bypass or inactive the germination inhibitors.

Treating wild-type conidia at the low population density with PD98059, an
inhibitor of mitogen-activated kinase kinase (MAPK kinase or MEK), inhibited
cytokinesis, germination of conidia, and appressorium development (Kim et al. 2000).
Ethylene overcame PD98059 effects on cytokinesis and germination of conidia, but
appressorium development was still inhibited (Kim et al. 2000). A CgMEKI mutant, in
which the CgMEKl protein was not produced, did not result in development of septum or
appressorium (Kim et al. 2000).

The polyamines sperrnidine, sperrnine, and putrescine inhibited appressorium
formation, while they had no effect on conidial or mycelial growth (Choi et al. 1998).
Cyclic-AMP reversed the inhibitory effects of the polyamines on appressorium formation
(Choi et al. 1998). Fresh conidia of Magnaporthe grisea had high levels of polyamines
and the polyamine levels decreased with conidial germination. The major polyamine was
sperrnidine followed by sperrnine and putrescine (Choi et al. 1998).

Con A, a lectin that interferes with attachment of C. graminicola to hydrophobic
surfaces, effectively reduced spore attachment to cover slips but did not interfere with

spore germination (Chaky et al. 2001). Con-A at 1 mg/ml reduced appressorial formation;

12

however, Con-A at lesser concentrations had no effect. Neither D-a-mannoside nor
glucose improved either spore attachment or appressorial formation (Chaky et al. 2001).

Antagonists of Ca2+ resulted in no appressorium formation, while calmodulin
inhibitors prevented the formation of appressoria but did not prevent spore germination
(Magalhaes et al. 1991).

RESISTANCE

Genetic Controls of Resistance

Heredity. Sunﬂower resistance to S. sclerotiorum was additive and male effects
were greater than female effects with the male parent correlation coefﬁcient of r =.99
(Castaﬁo et al. 1992). Speciﬁc combining ability indicated that interactions between
parental types are involved in the determination of resistance levels in offspring. There
was a speciﬁc correlation between level of resistance and heterosis (Castaﬂo et al. 1992).

Sources of Resistance. Interspeciﬁc crosses of common sunﬂower and other
Helianthus species were evaluated for genetic variability in response to S. sclerotiorum
(Kohler and Friedt. 1999). Three commercial sunﬂower hybrids and 29 progenies of
interspeciﬁc crosses were evaluated. The interspeciﬁc progenies expressed increased
variability in response to S. sclerotiorum, expanding resources for integrating resistance
into sunﬂower (Kohler and Friedt. 1999). Cultivated sunﬂower, perennial Helianthus
species, and the interspeciﬁc cross gave distinct patterns in the AP-PCR analysis (Kohler
and Friedt. 1999), indicating that the wild Helianthus species were sources of resistance
to S. sclerotiorum (Kbhler and Friedt. 1999).

Brassicus napus seeds from ‘Linetta’ were immersed in 0.03% v/v ethyl methane-

sulphonate (EMS) to cause mutagenesis to produce the M1 lines (Mullins et al. 1999). At

13

least two M2 mutants were more resistant to S. sclerotiorum than the susceptible Linetta
cultivar, while other mutants were more susceptible than Linetta to S. sclerotiorum
(Mullins et al. 1999). The M2 populations exhibited greater variability than the Linetta
populations in response to S. sclerotiorum, with narrow sense heritabilities of 0.75-0.83
(Mullins et al. 1999).

Genetic Analysis -— QTL’s Linked to Resistance. Park et al. (2001) attempted to
identify random ampliﬁed polymorphic DNA (RAPD) markers for linked to quantitative
trait loci (QTL) in common bean for partial physiological resistance, partial ﬁeld
resistance to SSR, porosity over the furrow, and plant height in a linkage map using
recombinant inbred lines from the cross of the resistant PC-50 by susceptible XAN-159
(Park et al. 2001). Quantitative trait loci candidates were identiﬁed for partial
physiological resistance, partial ﬁeld resistance, and porosity over the furrow in common
bean (Park et al. 2001). Nine QTL candidates were observed for partial physiological
resistance in common bean against S. sclerotiorum isolate 152 and three of those QTLs
were reported on linkage groups B4, B7, and B8 (Park et al. 2001). For partial
physiological resistance against isolate 279, strong evidence for QTL linked to the C
locus for seedcoat was identiﬁed (Park et al. 2001). Seven QTLs for partial ﬁeld
resistance were observed and six of the seven QTLs were in the same location as QTLs
for partial physiological resistance (Park et al. 2001). Two of the seven QTL for partial
ﬁeld resistance were also linked with porosity over the furrow, which may contribute to
disease avoidance (Park et al. 2001).

Both physiological and avoidance mechanisms contributed to ﬁeld resistance of

common bean to S. sclerotiorum have been identiﬁed with QTL’s (Miklas et al. 2001). A

14

QTL on linkage group B7 near the phaseolin seed protein (Phs) locus that explained 38%
of the phenotypic variation to S. sclerotiorum in the greenhouse (Miklas et al. 2001). The
same QTL on B7 and one on the B1 locus near the ﬁn gene for determinate growth habit
explained the majority of the phenotypic variation of common bean to S. sclerotiorum in
the ﬁeld (Miklas et al. 2001). Another QTL for canopy porosity was mapped to the ﬁn
locus (Miklas et al. 2001).

Soybean phenotypic variation explained by the signiﬁcant markers rangedfrom 4
to 10% (Arahana et al. 2001). Arahana et al. (2001) speculated that some susceptible
genotypes may contain resistance alleles. Linkage Groups C2 and M were associated
with escape mechanisms (Arahana et al. 2001). Linkage Group A2 and Sattl91 on
Linkage Group G were associated in SSR-tolerant cultivar S 19-90 (Arahana et al. 2001).
No known major genes have been reported in soybean for resistance to S. sclerotiorum
(Arahana et al. 2001).

Nutrients and Host Susceptibility

Nutrients supplied to the plant can affect host susceptibility to S. sclerotiorum.
Necrosis resulting from S. sclerotiorum 96 h after inoculation in pumpkin (Curcurbita
pepo) declined as Ca2+ applications increased from 0.005 to 0.05 and 0.5 mol m'3;
however, necrotic lesions were similar for all treatments at 48 and 120 h after inoculation
(Chrominski et al. 1987). Necrosis resulting from S. sclerotiorum 96 h after inoculation in
sunﬂower declined as Ca2+ increased from 0.005 to 0.05 mol m'3 (Chrominski et al.

1987 ). In both pumpkin and sunﬂower, necrotic lesions from S. sclerotiorum were always

among the largest in treatments with no calcium applied (Chrominski et al. 1987). Foliar

15

applications of CuSO4 and ZnSO4 suppressed Sclerotinia blight symptoms in peanuts
(Hallock and Porter 1981).

Phytochemicals that Inhibit Pathogens

Plant resistance to pathogens can be induced by biotic and abiotic factors. A
variety of plant compounds involved in induced resistance inhibit S. sclerotiorum and
other fungal pathogens. Some of these plant compounds include substilin-like inhibitors
(Konerav et al. 1999), phenols (Castaflo et al. 1992), oxalic acid (Toal and Jones 1999),
salicylic acid (De Meyer and Hofte 1997; Gorlach et al. 1996; Tenhaken et al. 2001) and
phytoalexins (Bailey 1974; Bailey and Burden 1993; Dann et al. 1999; Hammerschmidt
1999; Levene et al. 1998; Onoe et al. 1987; Stossel 1983; Yoshikawa et al. 1978).
Salicylic acid (SA) will induce systemic acquired resistance (SAR) within a host
(Tenhaken et al. 2001) and SA can be induced by biotic factors (De Meyer and H6er
1997). Other compounds can imitate SA action within a host (Gorlach et al. 1996).
Phytoalexin production is dependent on abiotic and/or biotic elicitors (Hammerschmidt
1999), environmental factors (Stab and Ebel 1987; Stossel 1982), and cultivar
competency (Abbasi et al. 2001).

Induced Resistance. Biotic organisms and synthetic compounds have induced
resistance of a host to the pathogen. The induction of resistance has been associated with
the hypersensitive response (HR) and with phytoalexins. Pseudomonas aeruginosa
7NSK2 grown on iron-deﬁcient media induced systemic resistance of bean to Botrytis
cinerea (De Meyer and Hofte 1997). Growth of Drechslera teres was inhibited on barley
previously inoculated with either nonbarley pathogen Bipolaris maydis or Septoria

nodorum (Jergensen et al. 1998). D. teres infection was inhibited by ﬂuorescent papillae

16

in induced cells (Jergensen et al. 1998). The formation of papillae appeared to be part of
the HR (Jergensen et al. 1998). Oxalic acid at concentrations as low as 0.5 mM induced
systemic resistance of canola to S. sclerotiorum (Toal and Jones 1999).

SSR disease severity in ﬁeld-grown soybean was signiﬁcantly reduced in two of
three years when bean were treated with INA (2,6-dichloroisonicotinic acid) and one of
two years when treated with BTH (benzo[1,2,3]thiadiazole-7-carbothioic acid S-methyl
ester) (Dann et a1. 1998). Applications of INA and BTH in the greenhouse reduced lesion
growth locally on treated leaves and systemically on other leaves. Dann et al. (1998)
proposed that treatments of INA and BTH stimulate inherent defense mechanisms
allowing the plant to respond more quickly to disease infections. The INA and BTH
treatments were especially effective in Williams 82 and Elgin 87 cultivars which are
more susceptible to white mold than Corsoy 79 and NKS 19-90 (Dann et al. 1998). Dann
et al. (1998) postulate that the INA and BTH treatments activated resistance mechanisms
more strongly in Williams 82 and Elgin 87, which contributed to enhanced resistance.

Substilin-Like Inhibitors. The pattern of inhibition of S. sclerotiorum in
sunﬂower was similar to inhibition of substilin (Konerav et a1. 1999). Sunﬂower leaves,
seeds, and heads contain substilin-like inhibitors (Konerav et al. 1999). Trypsin inhibitors
were isolated from three lines of sunﬂower and divided into three broad groups. Group 1
was included high pI (~ 85-10), which did not inhibit substilin and corresponded to
trypsin inhibitors. Others were present only in seeds with low pI (~ 6.5-8), which was
Group 2, while Group 3 were present in seeds and vegetative tissues and inhibited both

trypsin and substilin (Konerav et al. 1999).

17

Glucosinolates. Glucosinolates produced locally and systemically within canola
were not induced by inoculations of S. sclerotiorum and total glucosinolate levels did not
correspond with susceptibility or resistance to S. sclerotiorum (Li et al. 1999). However,
one European canola cultivar resistant to S. sclerotiorum and one Chinese line tolerant to
S. sclerotiorum did have increased production of aliphatic glucosinolates (Li et al. 1999).

Phenols. In uninfected leaves, six phenolic peaks separated SD*PAC1 from the
four sunﬂower hybrids (Castaﬁo et al. 1992). Five peaks occur at twice the concentration
in SD*PAC1 than in the other hybrids and peak 9 was considered as the most important
for resistance (Castai’io et al. 1992). No new phytoalexin compounds were identiﬁed in
the infected leaves (Castaﬁo et al. 1992).

Oxalic Acid. Oxalic acid at 20 mM caused induced resistance in canola for at
least 5 w after treatment and the highest level of resistance was at 5 w after treatment
(Toal and Jones 1999). Leaf sections furthest from the oxalate application expressed the
highest levels of resistance (Toal and Jones 1999).

Salicylic Acid. Tenhaken et al. (2001) postulated that salicylic acid (SA)
activation of genes in the soybean host and pathogen-derived signals from avrA
responsible for avirulence were both necessary for hypersensitive response (HR) cell
death in soybean. Salicylic acid activated both programmed cell death and expression of
DD-CA9, which was believed to encode a lectin that binds to a chitin monomer and may
be involved in plant defenses (Tenhaken et al. 2001). Cantharidin, a phosphatase
inhibitor, stabilized phosphorylated proteins and had a similar effect on augmentation of
DD-CA9 as did the avirulent pathogen, further supporting the role of protein kinases in

plant resistance (Tenhaken et al. 2001). SA and functional analogs of SA accelerated

18

 

programmed cell death to half the normal time (Tenhaken et al. 2001). The effect of SA
and functional analogs of SA was strictly dependent on the gene-for-gene interaction of
the soybean resistance gene Rng and the Pseudomonas avirulence gene avrA (Tenhaken
et al. 2001). Bacteria lacking the avrA gene did not cause cell death, indicating that
functional analogs of SA cannot bypass the pathway activated by the avrA gene
(Tenhaken et al. 2001). Functional analogs of SA that accelerated programmed cell death
included the NSAID’s ﬂufenamate and indomethacin, the synthetic PPAR ligan WY-
14643, leukotriene D4-antagonists LY-17l,883 and MK-0571, the anti-hypolipodemic
drug cloﬁbrate (activates PPAR transcription in vertebrates), the anti-diabetic drug
tolbutamide (activates PPAR transcription in vertebrates), and ETYA (a weak ligand of
PPAR transcription factors) (Tenhaken et al. 2001). Compounds that did not accelerate
programmed cell death included jasmonic acid and plant phytohorrnones structurally
similar to prostaglandins (Tenhaken et al. 2001). FAS and SA induced expression of DD-
CA9 (Tenhaken et al. 2001). SA alone was a weak inducer of DD-CA9 compared to the
SA-accelerated HR, indicating that a second signal is required for high gene expression
of DD-CA9 (Tenhaken et al. 2001). Pseudomonas syringae pv. glycinea (avr) increased
expression of DD-CA9 in the presence of F AD’s (Tenhaken et a1. 2001). Phosphorylation
increased expression of DD-CA9 while nitrous oxide did not augment DD-CA9
expression (Tenhaken et al. 2001).

SA is a key molecule in plant defenses and acted on two independent pathways,
which are activated by distinct classes of chemicals with little “cross-talk” between each
other (Tenhaken et al. 2001). The compounds SA, 2,6-dichloroisonicotinic acid (INA),

and benzothiadiazole (Bion) induced systemic acquired resistance (SAR) in plants

19

 

(Gorlach et al. 1996). SA, Bion, and FAS compounds WY-14463 and tolbutamide
induced expression of a B-l,3-glucanase gene (a PR gene) in soybean (Tenhaken et al.
2001). SAR compounds induced expression of the Arabidopsis SAR gene PR-l and
cucumber SAR class-III-chitnase gene but FAS compounds did not (Tenhaken et a1.
2001). Bion and INA accelerated programmed cell death in soybean (Tenhaken et al.
2001)

Biotic organisms that produce SA can reduce the growth of other biotic
organisms. De Meyer and Hofte (1997) concluded that Pseudomonas aeruginosa 7NSK2
grown on iron-deﬁcient media induced systemic resistance of bean to Botrytis cinerea.
SA-deﬁcient Pseudomonas aeruginosa mutants did not induce systemic resistance.
Pyochelin, of which SA is a precursor, may also be involved in induced systemic
resistance (De Meyer and Hoﬁe 1997).

Phytoalexins. Phytoalexins are described as low molecular weight, antimicrobial
compounds that are induced by biotic or abiotic factors (Hammershmidt 1999). Three
criteria are required to determine the role of phytoalexins on plant resistance to pathogen.
Those criteria are: 1) In race-speciﬁc resistance phytoalexin production must be
associated with the restriction of pathogen development conditioned by host-resistance
genes in race- or parasite-speciﬁc resistance or in nonhost resistance; 2) In all types of
resistance, phytoalexins must accumulate at antimicrobial levels at the infection site in
resistant plants at a sufﬁcient concentration to inhibit the pathogen at the time pathogen
development is stopped; and 3) Evidence must be present that the phytoalexins are
directly involved in defense, and that this defensive role has a measurable beneﬁt to the

plant.

20

 

Glyceollin. Glyceollin is a phytoalexin in soybean produced in the Shikimate acid
pathway (Figure 1). Increased levels of glyceollin within the soybean plant have been
associated with decreased growth of S. sclerotiorum hyphae (Dann et al. 1999), and other
pathogens including Phytophthora capsici (Onoe et al. 1987), Phytophthora megasperma
f. sp. glycinea (Stossel 1983; Yoshikawa et al. 1978) and soybean cyst nematode
(Heterodera glycines Ichinohe) (Levene et al. 1998). Glyceollin inhibited P. capsici by
distorting the plasma membrane and causing leakage (Onoe et al. 1987). Glyceollin was
also detrimental to plant membranes by causing membrane leakage in red beet vesicles

and vacuoles (Giannini et al. 1991).

Phytoalexins in other species have activity similar to that of glyceollin. The
phytoalexins phaseollin, phaseollidin, phaseollinisofalvin, and keivitone accumulated in
common bean infected with Colletotrichum lindemuthianum (Bailey 1974) or with
tobacco mosaic virus (Bailey and Burden 1973). Each of these compounds at low
concentrations inhibited spore germination and germ tube growth of Colletotrichum
lagenarium, C. lindemuthianum, and Uromycesfabae (Bailey and Burden 1973). Higher
concentrations of these compounds corresponded with smaller lesions resulting from
infection (Bailey 1974).

Glyceollin synthesis can be induced by various pathogens, various chemicals, and
herbicides. Pathogens reported to induce glyceollin included Macrophomina phaselina
(Chakraborty and Purkayastha 1987) and Pseudomonas pisi (Ingham et al. 1981),
Phytophthora megasperma f. sp. glycinea race 4 (Keen et al., 1971; Stbssel, 1982),
Phytophthora sojae (Abbasi et a1. 2001), Rhizoctonia solani Kuhn (Scharff et al.1997),

and S. sclerotiorum (Sutton and Deverall 1984). Ayers et al. (1976) reported that

21

 

 

Phytophthora megasperma f. sp. glycinea (ng) contained compounds within the

pathogen cell walls that induced glyceollin synthesis.

Glyceollin accumulation was greater with treatments of silver nitrate (AgNO3) or
copper chloride (CuClz) than with inoculation by zoospores of ng race 4 (Stossel,
1982). Glyceollin also accumulated after treatments of sodium iodoacetate (Ingham et al.,
1981) and sodium azide (Chakraborty and Purkayastha 1987). The inorganic salts
Ni(N03)2, ZnSO4, Pb(N03)2, and Hng induced synthesis of small amounts of glyceollin
(Stossel 1982). Glyceollin synthesis can also be induced by organic compounds such as
Triton X-100 (Stossel 1982), lactofen (Dann et a1. 1999), aciﬂuorfen, bentazon, crop oil
concentrate, and nonionic surfactant (Levene et al. 1998). Glyceollin was detected in
ﬁeld-grown soybean leaves treated with lactofen but not in soybean treated with water
(Dann et al. 1999), indicating that lactofen induced glyceollin production in soybean
leaves. Aciﬂuorfen plus a nonionic surfactant, bentazon plus a crop oil concentrate, a
crop oil concentrate, and a nonionic surfactant increased glyceollin levels in soybean
roots, while the lactofen plus crop oil concentrate did not increase glyceollin levels
(Levene et al. 1998).

Other plant compounds related to the defense response have been induced by
abiotic factors. Induction of coumestrol in common bean has occurred with ozone, sulfur
dioxide, and the herbicides endothall and oxadiazon (Rubin et al. 1983).

Glyceollin accumulation tends to occur in close proximity to the site of infection
or plant injury. Glyceollin was the most abundant isoﬂavonoid compound in necrotic
lesions of soybean leaves resulting from incompatible reactions with ng (Morris et al.

1991). Glyceollin levels were most abundant in cells within 2 mm of the incompatible

22

infection site and declined in cells ﬁrrther from the infection front (Graham et al. 1990).
Glyceollin levels were higher in the border zone between the lesion and the healthy tissue
than in the necrotic lesion 36 h after inoculation (Morris et al. 1991). Glyceollin
accumulation within soybean was highest when tissue browning was most pronounced
(Stossel 1982). Glyceollin accumulated at levels higher than the EDgo value in 0.25-mm-
thick tissue layers of soybean cultivar ‘Harosy 63 ’ that contained advancing hyphae of
ng (Yoshikawa et a1. 1978). Site of glyceollin accumulation can depend on the type of
elicitor. Soybean leaves yielded no glyceollin while hypocotyls contained glyceollin 24 h
after inoculation with S. sclerotiorum ascospores (Sutton and Deverall 1984). Soybean
leaves contained glyceollin while the hypocotyls contained no detectable levels following

infection by S. sclerotiorum mycelia (Sutton and Deverall 1984).

Although these studies found glyceollin to be a localized response, Nelson (2001)
found elevated glyceollin in new soybean leaves at 26 d after treatment with lactofen.
One reason for the difference in observations between these studies could be due to the
plant part examined, growing conditions, and age of the soybean plants. Stdssel (1982)
and Yoshikawa et al. (1978) tested hypocotyls incubated in growth chambers, while
Morris et al. (1991) tested soybean leaves of 14- and 21-day-old plants grown in a
controlled environment. Nelson (2000) examined ﬁeld-grown soybean that were
approximately 42 days old at the time glyceollin synthesis was induced by lactofen
herbicide. Glyceollin levels in untreated soybean roots increased over time (Scharff et al.
1997), which may also explain why Nelson (2000) found elevated glyceollin levels in

older soybean.

23

The response of glyceollin synthesis to an inducer appeared to be both rapid and
transient as glyceollin accumulated at inhibitory levels within 8 hours after inoculation
(Yoshikawa et al., 1978), and concentrations declined by 3 and 9 days after inoculation
(Stossel 1982; Yoshikawa et al. 1978). These studies were done on soybean hypocotyls
under a controlled environment and the transient occurrence of glyceollin in the
hypocotyls was not observed in ﬁeld-grown soybean. Nelson (2000) found elevated
concentrations of glyceollin in leaves of ﬁeld-grown soybean 26 DAT after induction by
lactofen.

Daidzein and genistein. The isoﬂavonoid compounds, daidzein and genistein
(Figure 1), appear to play a role in glyceollin accumulation. Malonylated conjugates of
daidzein and genistein occurr at high constitutive levels within all soybean tissues
examined (Graham et al. 1990). The daidzein conjugates are present at levels in excess of
that needed for accumulation of glyceollin to EDso levels (Graham et al. 1990).
Constitutive levels of conjugated daidzein and genistein occurred in cultivars with and
without the Rps genes for resistance to ng (Graham et al. 1990). Following inoculation
with Phytophthora megasperma, levels of conjugated daidzein and genistein decreased
while levels of free daidzein and genistein increased (Graham et a1. 1990). The increased
levels of daidzein corresponded with increased levels of glyceollin following inoculation
with P. megasperma (Graham et al. 1990), which indicated that constituents of the
daidzein conjugates may have played a role in glyceollin synthesis. Genistein conjugate
levels also declined and corresponded with the increase in glyceollin levels; however, the

direct link between genistein and glyceollin was not ascertained (Graham et al. 1990).

24

Light effect on glyceollin. Stossel (1982) investigated the effect of light on
glyceollin synthesis in soybean. Soybean seedlings were grown in the dark for 2 days or
received continuous light for the last two days. Glyceollin was induced by silver nitrate.
Seedlings were then incubated in the dark, in a 15-h light photoperiod, or in continuous
light. For seedlings initially grown in the dark, glyceollin accumulation was similar for
all light treatments following phytoalexin induction. Soybean treated to continuous light
48 h prior to and following treatment produced signiﬁcantly less glyceollin. Pennypacker
and Risius (1999) observed that shade increased SSR development in some soybean
cultivars (Pennypacker and Risius 1999). Exposure of subepidennal cells to light
triggered a net and highly selective accumulation of the malonylglucosyl conjugate of
genistein (Graham and Graham 1996). Graham et al. (1990) reported less glyceollin
accumulation in induced soybean cotyledons subjected to continuous darkness compared
to 14-h photoperiod of light. The increased level of disease on shaded soybean could
indicate reduced production of glyceollin. This observation is contradictory to the report
of Stossel (1982), where continuous light (no shade) resulted in reduced glyceollin
production.

Nutrient effect on glyceollin. The Ca2+ ionophore, A23187, enhanced
phytoalexin biosynthetic enzyme activities and accumulation of the phytoalexin
glyceollin in a dose-dependent manner (Stab and Ebel 1987).

Cultivar Effect on Glyceollin. Soybean cultivars differed in competency of
glyceollin elicitation by P. sojae (Abbasi et al. 2001). Harosoy and Bragg cultivars were
at least partially competent, meaning that HR or wounding was not required for

elicitation of glyceollin production by P. sojae. Williams cultivar required wounding or

25

 

the HR response to induced glyceollin production and was considered incompetent
(Abassi et al. 2001). Williams only produced glyceollin in response to P. sojae elicitors
when the soybean tissue was either wounded or treated with a speciﬁc chemical elicitor
prior to exposure to the P. sojae elicitors (Abassi et al. 2001). Cultivars of common bean
differed in coumestrol production when induced by ozone, sulpher dioxide and some
herbicides (Rubin et a1. 1983).

Induction and Extraction of Glyceollin. Several methods have been developed
to examine glyceollin production in soybean. One method for examining changes in
glyceollin production is the snapped-cotyledon assay. Eight-d-old cotyledons grown in
vermiculite with a 14 h photoperiod at 500 IE tn2 3'1 were used for the assay (Graham
and Graham 1991). Cotyledons were cut and dipped in 5% water agar treated with 12 pl
of elicitor. The cut cotyledons were incubated for 48 h and the upper 0.5 mm (closest to
the cut) was removed. Sections of 0.5 mm were made, layers were pooled and weighed,
and extraction occurred immediately or the tissue was stored intact at -—80 C (Graham and
Graham 1996). A wall glucan elicitor preparation (PWG) from Phytopthora sojae was
added to stimulate one-half maximum glyceollin response. PWG is a wall glucan elicitor
that elicits a defense response closely parallel to all aspects of infected tissues. The
cotyledons were kept at 25 C in continuous light and analyzed with HPLC or thioglycolic
acid assay (Graham and Graham 1996). The assay did not damage tissue at great levels
(0.89 :t 0.04%), indicating that the tissue was healthy and producing valid results
(Graham and Graham 1996). Extraction was facilitated by homogenizing 0.1 g tissue plus
400 111 of 80% ethanol with a plastic pestil in a microfuge tube at 600 rpm. Solid particles

were separated from the solution by centrifugation at 18,000 g for 3 min. The supernatant

26

was analyzed with HPLC (Graham 1991). Total extraction time was approximately 4 min
(Graham 1991). The extract was eluted by HPLC on a C18 reverse phase column at 1.5
ml min'1 with 0 to 55% linear gradient of acetonitrile in water (pH 3) for 25 min.
Compounds were analyzed with an ultra violet detector at 236 nm (Graham 1991).

Thirty cotyledons per treatment were examined. The cut surface was treated with
30 111 water or 30 pl of 50 mg ml-l elicitor(1/2-maximum glyceollin production) and
incubated at 25 C and 100 [E m'2 s". The centers were harvested with a number 1 cork
borer. The cylinder was cut in two cross sections of 0.6 and 2.5 mm (Graham and
Graham 1991). Sections for 10 replicate cotyledons were pooled, weighed, and kept
frozen at —80 C in microfuge tubes until HPLC analysis (Graham and Graham 1994).

The method of applying the water control and elicitor to cut cotyledons was
modiﬁed. To obtain “donor exudate” cotyledons were cut and placed on ﬁlter paper.
Aﬂer 5 min, 20 [.11 sterile water was placed on the surface and removed from the leaf four
times. One to ﬁve donors were used. 20 ul of donor exudate was applied to cotyledon
assay and 10 ml of water or elicitor was applied (Graham and Graham 1994).

Glyceollidin II (precursor to glyceollin) can be elicited in soybean leaves treated
with sodium iodoacetate or Pseudomonas syringa bv. pisi (Ingham et al. 1981 in Graham
and Graham 1996). PWG alone does not elicit glyceollin in snapped cotyledons but PWG
+ wound exudate from donor cut cotyledons increased glyceollin (Graham and Graham
1996). PWG stimulates the accumulation of conjugates of the 5-deoxy-isoﬂavone
daidzen, the ﬁrst committed precursor of glyceollin (Graham and Graham 1994).

In another study to investigate glyceollin, leaves on soybean plants that were 10 to

14 d-old were inﬁltrated with either Pseudomonas syringa bv. pisi or 1 mM sodium

27

 

iodoacetate. The soybean were allowed to grow for another 2 to 5 d. The leaves (100-500
g) were harvested and extracted with 40% ethanol. The solution was ﬁltered, and reduced
in vacuo at 45 C to half the original volume. This volume was shaken with diethyl ether
and the aqueous phase was discarded (Ingham et al. 1981). The extracted compounds
were separated on silica gel (Si gel) thin-layer chromatography (TLC) with chloroform:
acetone: concentrated ammonia hydroxide (50:50:1 vzvzv) resulting in O-
methylgyceofuran (Rf 0.71), isoformononetin (Rf 0.56), glyceollins I-III (Rf 0.50), and
glyceofuran and glyceocarpin (Rf 0.25) (Ingham et al. 1981). These four bands on the
TLC plates were not associated with comparable extracts of water-treated controls
(Ingham et al. 1981). The four bands were eluted with methanol prior to high pressure
liquid chromatography (HPLC) or TLC puriﬁcation. The glyceollins were separated on
HPLC using a Partisil (10 mg Whatrnan) column (0.94 x 50 cm) eluted with hexanes *-
iso-PrOH (50:4, ﬂow rate 5 ml/min). The eluate was monitored using a UV (245 nm) and
refractive index detectors (Ingham et al. 1981). Elution times for glyceollins I, II, and III
were approximately 15, 16 and 17 min, respectively. Leaf extracts typically contained the
glyceollins I, II, and III in a1:3:6 ratio (Ingham et al. 1981). The other compounds were
typically puriﬁed with TLC (Ingham et al. 1981).
EVALUATION OF SCLEROTINIA AND GLYPHOSATE

Sclerotinia Inoculation Studies

Carrot roots approximately 3 mm thick x 10 mm diameter were cut into 6
triangular pieces, autoclaved for 15 min at 121 C and placed around the growing edge of
advancing white mold mycelia on potato dextrose agar (PDA). The carrot was incubated

for 15 to 18 h at 21 C (Pennypacker and Risius 1999). Colonization was conﬁrmed by

28

microscopic evaluation before use (Pennypacker and Risius 1999). Soybean at V2 or V3
grth stage was inoculated with mycelia of white mold and were kept wet during the
infection period. Carrots infected with mycelia were placed in the axile of the oldest
trifoliate and pressed to the stern. Inoculum was removed after 48 h. Plants were rated for
disease 6 (1 later (Pennypacker and Risius 1999). A rating scale was developed to
evaluate whole plant symptoms to a limited-tenn-inoculation method. Individual plants
were rated on a scale of 1-6 for disease symptoms, where 1 = no symptoms, 2 = < 1 cm
lesion on petiole or main stem, 3 = petiole collapse and < 1 cm lesion on main stem, 4 =
lesion on main stem > 1 cm, 5 = apical portion of stem collapsed, 6 = plant dead
(Pennypacker and Risius 1999).

Determining Disease Severity

Disease incidence or disease severity index (DSI) may indicate yield of soybean
and dry bean. Yield was correlated with disease incidence (12 = -0.94) when averages of
16 soybean cultivars were examined (Chun et al. 1987). Correlation coefﬁcients (r) for
yield and D81 were —0.61 and 10.74 for 1979 and 1981, respectively (Grau and Radke
1984). When DSI versus yield for soybean cultivars were averaged over row spacings of
38 and 76 cm, correlation coefﬁcients (r) ranged from —0.74 to —0.97 (Grau and Radke
1984)

Ratings such as disease incidence and disease severity index (DSI) have been
used to estimate the level of SSR within a ﬁeld. Disease incidence is usually determined
by the percent of plants exhibiting above-ground infection (Boland and Hall 1988;

Schwartz et al. 1978).

29

 

Disease severity index (DSI) has been calculated in two different ways. DSI is
calculated as DSI = [sum[(severity class x number of plants in class)]/total number of
plants] x 100, where severity classes are 0 = no incidence, 1 = lesions less than 5 cm, 2 =
expanding lesions on stems and/or branches, 3 = up to V2 of branches/stems colonized, 4
= more than ‘/2 stems/branches colonized (Boland and Hall 1988). Disease severity index
(DSI) has also been calculated as DSI = [sum(class x number plants in class x 100)]/(total
number of plants x 3), with severity classes of 0 = no symptoms, 1 = lesions on lateral
branches, 2 = lesions on main stem (no effect on pod ﬁll), 3 = lesions on main stem (plant
death or poor pod ﬁll) (Grau et al. 1982; Kim et al. 1999). The method used by Boland
and Hall (1988) estimates the amount of the host colonized by the fungus, while the
method used by Grau et al. (1982) and Kim et al. (1999) attempts to estimate SSR impact
on plant yield by accounting for pod ﬁll.

Tests for Resistance

The leaf tests of sunﬂower correlated with a root test for resistance (Castafio et al.
1992). Based on this correlation, Castaﬁo et al. (1992) concluded that the leaf test was
nondestructive and should be used before ﬂowering, so lines could be eliminated prior to
crossing made. By using the leaf test, sunﬂower could be bred for S. sclerotiorum
resistance and the leaf test could be applied at the same time that other resistance tests
were being conducted on the same plant (Castaﬁo et al. 1992).

Oxalic acid is a determining factor in pathogenicity of S. sclerotiorum (Godoy et
al. 1990). Phaseolus vulgaris cultivars exhibited degrees of tolerance to oxalate in the
greenhouse (Kolkman and Kelly 2000). Cultivars tolerant of oxalic acid have greater

membrane stability than cultivars susceptible to oxalic acid (Tu 1989). Higher levels of

30

tolerance to oxalate in the greenhouse were positively correlated with lower severity of
SSR in the ﬁeld and negatively correlated with yield in common bean (Kolkman and
Kelly 2000).

Soybean Cultivar Sensitivity to Sclerotinia Stem Rot

Soybean cultivars vary in sensitivity to SSR development (Buzzell et al. 1993;
Grau and Radke 1984, Kim et al. 1999; Kurle et al. 2001). Several cultivars are
consistently more tolerant than others to SSR (Kim et al. 1999). Grau and Radke (1984)
found that differences in cultivar sensitivity to SSR are common across row widths. Some
research indicates that differences in disease development in cultivars are due in part to
maturity. Lower SSR incidence was found in earlier-maturing cultivars (Buzzell et al.
1993; Kim et al. 2000; Yang 1999) while other researchers found no correlation between
cultivar maturity and disease incidence (Chun et al. 1987; Grau et al. 1982; and Kim et al.
1999). These conﬂicts in ﬁndings on the correlations between cultivar maturity and
disease incidence indicate that differences in SSR development are likely due to escape
mechanisms rather than the factor of maturity, itself. Upright soybean cultivars are
associated with less disease incidence in several studies (Boland and Hall 1987b; and
Kim et al. 1999), which is consistent with correlations between upright canopies and SSR
development in dry bean (Coyne et al. 1974). Upright cultivars are speculated to have
more canopy air movement, less moisture, and less disease than lodged genotypes (Kim
et al. 1999). Cultivar sensitivity to S. sclerotiorum in soybean is also associated with
pedigree, and those genotypes tracing to either cultivar Williams or A3127, are more
sensitive to S. sclerotiorum (Kim et al. 1999). Glyphosate-resistant soybean compared to

glyphosate-susceptible near isolines are equally susceptible to SSR (Lee et al. 2000;

31

 

Nelson, 2000), indicating that the glyphosate-resistant trait does not impact cultivar
susceptibility to SSR.

Environmental and Cultural Factors

Environmental and cultural factors such as soybean row width (Grau and Radke
1984), soil moisture (Boland and Hall 1988a), timing of ﬂower development in
relationship to spore release (Boland and Hall 1988a), and shade (Pennypacker and
Risius 2000) impact SSR incidence. Narrow row widths allow the soybean crop to reach
canopy closure quickly to reduce weed competition (Legere and Schrieber 1989) and
increase yield potential (Nelson and Renner 1999). However, planting soybean in narrow
rows increases the risk of disease development (Grau and Radke 1984). Wide rows
decrease SSR severity in some studies (Grau and Radke 1984), while other studies have
shown no impact ﬁ'om row widths on SSR severity (Buzzell et al. 1993). Canopy
development as a result of row widths likely impacts SSR incidence. Sclerotinia stern rot
was ﬁrst observed after canopy closure in both white bean (Boland and Hall 1987b) and
soybean (Boland and Hall 1988a), and dense canopies were associated with increased
disease incidence in dry bean (Coyne et al. 1974; Schwartz and Steadman 1978). Narrow
rows promote a denser canopy which would foster the humid microclimate necessary for
SSR development (Boland and Hall 1988a). Narrow rows may also increase the level of
shade beneath the crop canopy and shade increases SSR development in some soybean
cultivars (Pennypacker and Risius 1999). Glyphosate resistant soybean is a system that
promotes use of narrow rows for weed control (Nelson and Renner 1999). Glyphosate

also has no impact on soybean canopy development (Nelson and Renner, 2001).

32

Extended periods of soil moisture and plant wetness are associated with disease
development (Boland and Hall 1998a). Soil manic potentials are generally -0.05 kPa
before disease epidemics start in white bean (Boland and Hall 19873). Disease incidence
is higher in ﬁelds receiving irrigation on a 5-d schedule compared to a lO-d schedule
(Weiss et al. 1980). Moisture appeared to have a direct effect on S. sclerotiorum
apothecia, the source of ascopsores. Schwartz and Steadman (1978) concluded that
irrigation on a 5-d schedule increased apothecium production compared to irrigation on a
10—d schedule. This impact of irrigation on apothecia production is important because
apothecia are directly correlated with disease incidence (Boland and Hall 1988b) to the
extent that SSR spatial variability correlates with apothecia spatial variability (Boland
and Hall 1988c). In addition to the factor of soil wetness, different frequencies of soil
wetting and drying explained differences in SSR development (Grau and Radke 1984).

Shade increased SSR development in some soybean cultivars (Pennypacker and
Risius 1999). Sunﬂower cultivars under longer periods of light were more tolerant than
cultivars grown under shorter photoperiods to Sclerotinia stalk rot, caused by S.
sclerotiorum (Orellana 1975).

Soybean Flowers and Sclerotinia Stem Rot

Signs of the SSR typically appear after ﬂowering has begun (Boland and Hall
1988a; Grau et a1. 1982), indicating that ﬂower development plays a critical role in
disease development. Kim et al. (2000) determined a positive correlation between later
ﬂowering and greater disease development. In canola, SSR never developed until a crop
was in mid-ﬂowering stage (Morrall et al. 1982). Primary infection of canola by S.

sclerotiorum is through ﬂower petals that land on leaves or leaf axils (Kharbanda and

33

Tewari 1996). Direct links between ascospores and soybean ﬂowers have also been
identiﬁed. When suspensions of ascospores were sprayed onto ﬂowering soybean plants,
initial symptoms of S. sclerotiorum infection on soybean were water-soaked petals
followed by lesions on the stem extending from the point of ﬂower attachment (Cline and
Jacobsen, 1983). On both dry bean and soybean, ascospores colonized to form white
cottony mycelia on ﬂowers and ﬂower parts but did not colonize on leaves or pods,
unless ﬂowers and ﬂower parts were present (Sutton and Deverall 1983).

Glyphosate and Sclerotinia

Glyphosate inhibits the conversion of Shikimate to chorismate (Amrhein et al.
1980) by directly inhibiting the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS)
enzyme (Steinrr’icken and Amrhein 1984) in the Shikimate pathway. Sublethal doses of
glyphosate on glyphosate-susceptible soybean reduced levels of the isoﬂavonoid
phytoalexin glyceollin in soybean leaves when induced by sodium iodoacetate or
aminooxyacetic acid (Holliday and Keen 1982), and reduced plant resistance to ng
(Keen et al. 1982). Sublethal doses of glyphosate to common bean reduced total
phytoalexins and host resistance to C. lindemuthianum (Johal and Rahe 1990).

Glyphosate-Resistant Soybean

Glyphosate-resistant soybean was developed by inserting a glyphosate-insensitive
bacterial EPSPS gene from Agrobacterium sp. strain CP4 into soybean (Padgette et al.
1995). Hypothetically, glyphosate applied to glyphosate-resistant soybean should not
inhibit the Shikimate pathway. Therefore, glyphosate applied to glyphosate-resistant
soybean should not reduce plant defenses against pathogens. A similar hypothesis

relating to sudden death syndrome was recently conﬁrmed. Glyphosate applied to ﬁeld-

34

 

grown glyphosate-resistant soybean did not reduce plant defenses against F usarium

solani f. sp. glycines, the causal agent of sudden death syndrome (Sanogo et al. 2001).

35

 

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39

 

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42

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43

0 0H 0
| l! j I]
0 OH OH 0 OH

Daidzein Genistein
Glyceollin I Glyceollin II

Glyceollin III

Figure 1. Chemical structures of daidzein, genistein, and glyceollin I, II, and III (Ingham
1982)

44

CHAPTER 2
INFLUENCE OF FORMULATED GLYPHOSATE AND ACTIVATOR
ADJUV ANTS ON Sclerotinia sclerotiorum IN GLYPHOSATE-RESISTAN T AND —
SUSCEPTIBLE SOYBEAN (Glycine max)
Abstract: Two Glycine max near-isolines, GL2415 (glyphosate-sensitive) and
GL26OORR (glyphosate-resistant), were compared for susceptibility to Sclerotinia
sclerotiorum, the fungus that causes Sclerotinia stem rot (SSR). Treatments of a
formulation of the isopropylamine salt of glyphosate (RU) at 840, 1680, and 2520 g ae
ha'l, the RU formulation blank which contained adjuvants (RUFB) at 0.4% v/v,
organosilicone at 0.3% v/v, a crop oil concentrate at 1.0% v/v, and a nonionic surfactant
at 0.5% v/v were evaluated for their inﬂuence on V5 soybean susceptibility to S.
sclerotiorum in the ﬁeld. To investigate the effects of the RU, RUFB, and adjuvants on
soybean susceptibility near the time of treatment, soybean leaﬂets were harvested 1, 2, 3,
4, 5, 6, and 7 d aﬁer treatment (DAT) and inoculated with S. sclerotiorum mycelial plugs.
Lesion diameters averaged over the inoculation timings and both years were similar in
respect to treatments of RU, RUFB and the adjuvants. The disease severity index ratings
and yields averaged over both years were similar for both cultivars and across all
herbicide and adjuvant treatments. To provide a less variable environment, treatments of
RU at 840, 1680, and 2520 g ae ha", a formulated isopropylamine salt of glyphosate
lacking adjuvants (AC) at 2520 g ae ha'1 and a formulated trimethylsulfonium salt of
glyphosate (TD) at 2520 g ae ha"l and the RUFB and three adjuvants at the rates
described earlier were applied to GL26OORR soybean in the greenhouse. Lesion

diameters were similar for all treatments when soybean leaﬂets were inoculated 1 and 7

DAT. Leaﬂets from both cultivars were dipped into and immediately removed from

45

solutions of the RU at 4, 7, and 10 % v/v, and the RUFB and adjuvants at the rates
described earlier. The leaﬂets were excised and inoculated 15 min after dipping. Lesion
development was similar across both cultivars. The organosilicone, crop oil concentrate,
and nonionic surfactant encouraged lesion development, while the RUFB and RU
inhibited lesion development. S. sclerotiorum mycelia growing on potato dextrose agar
were inhibited by high concentrations of RU, RUFB at 100 mM glyphosate ac and by the
three adjuvants at 1% v/v. AC did not inhibit mycelial growth on PDA. The glyphosate-
resistance trait did not appear to be associated with the susceptibility of soybean to S.
sclerotiorum. Neither the glyphosate-resistant trait nor the applications of glyphosate and

adjuvants inﬂuenced soybean susceptibility to S. sclerotiorum.

Nomenclature: Glyphosate, N—(phosphonomethyl)glycine; Glycine max (L.) Merr.,

soybean; ‘GL2415’, ‘GL2600RR’.

Key Words: Formulation, transgenic crops, plant disease.

46

INTRODUCTION

Sclerotinia sclerotiorum (Lib.) de Bary is the pathogen that causes SSR of
soybean and is prevalent in the Great Lakes region. Many environmental and cultural
factors promote or deter the incidence of SSR during the disease cycle. Application of the
diphenyl ether herbicide, lactofen ((i)-2-ethoxy-1 -methyl-2-oxoethyl 5-[2-chloro-4-
(triﬂuoromethyl)phenoxy]-2-nitrobenzoate), reduced SSR severity in the ﬁeld when
untreated disease severity was 40 to 60% (Dann et al. 1999). Lactofen reduced lesion
development resulting ﬁ'om S. sclerotiorum in detached leaﬂet studies (Dann et al. 1999).
Herbicides may adversely affect the viability of mycelia and reproductive structures of S.
sclerotiorum. The herbicides, diuron (N ’-(3,4-dichlorophenyl)-N,N-dimethylurea) at 50
ug ml'1 and atrazine (6-chloro-N-methyl-N’—(l-methylethyl)-1,3,5-triazine-2,4-diamine),
simazine (6-chloro-N,N ’-diethyl-l,3,5-t1iazine-2,4-diamine), and metribuzin (4-amino-6-
(1,1-diemthylethyl)-3-(methylthio)-1,2,4-triazin-5(4H)-one) at 500 ug ml", inhibited
mycelial development on 1.5% Bacto agar (Casale and Hart 1986). Carpogenic
germination of sclerotia in soil containing 0.5 to 10.0 mg g'1 soil of atrazine and simazine
was similar to the sclerotia in untreated soil; however, sclerotia in the treated soils did not
produce apothecia (Casale and Hart 1986). Sclerotia failed to germinate in soil containing
0.5 and 1.0 mg g'1 soil of metribuzin (Casale and Hart 1986). Effects were different in
soils treated with much lower doses of atrazine and metribuzin. Number of stipe initials
from sclerotia were higher in soils treated with metribuzin at 0.5 and 1.0 and atrazine at
0.5 pg g", but morphology of stipe initials and apothecia was altered by atrazine (Radke

and Grau 1986).

47

While lactofen and the triazines appeared to have a direct effect on the viability of
S. sclerotiorum, other herbicides and some adjuvants may impact the ability of plants to
defend themselves against pathogens. Glyphosate reduced the ability of soybean to
produce chemicals associated with plant defense (Keen et al. 1982). Sublethal doses of
glyphosate to glyphosate—susceptible (conventional) soybean hypocotyls lowered the
production of phytoalexins when challenged with Phytophthora megasperma f. sp.
glycinea (Keen et al. 1982) or Pseudomonas syringae f. sp. glycinea (Holliday and Keen
1982). Applications of two adjuvants, a crop oil concentrate1 and a nonionic surfactantz,
to the V3 grth stage soybean reduced J2 population densities of soybean cyst
nematodes in the soil and increased phytoalexin levels in soybean roots (Levene et al.
1998)

Glyphosate-resistant soybean was developed by inserting a bacterial
enolpyruvylshikimate-3-phosphate synthase gene from Agrobacterium sp. strain CP4 into
soybean (Padgette et al. 1995). Public concerns exist over the impact of genetically-
engineered crops on the environment. When glyphosate-resistant soybean cultivars were
ﬁrst introduced in Michigan, area farmers reported higher levels of S. sclerotiorum in
glyphosate-resistant soybean cultivars than in conventional soybean cultivars. These
reports could be due to the reduction in plant defenses resulting in greater plant
susceptibility to S. sclerotiorum. This reduction in plant defenses may have resulted from
either the gene insertion or from the application of glyphosate and/or adjuvants. The role
of adjuvants in a glyphosate-resistant soybean system and their possible impact on SSR
will gain new importance since glyphosate is now being marketed through several

companies. This investigation was initiated to determine if a glyphosate-resistant soybean

48

cultivar was more susceptible to S. sclerotiorum than a conventional near-isoline soybean
cultivar and if applications of formulated glyphosate and/or adjuvants increased plant
susceptibility to S. sclerotiorum.
MATERIALS AND METHODS
Cultivars, Adjuvants and Herbicides Evaluated. Two soybean near-isolines,
the conventional GL24153 cultivar and the glyphosate-resistant GL26OORR3 cultivar,
were evaluated in a ﬁeld study. Adjuvants included a nonionic surfactant", an

5, a crop oil concentrate6, and the formulation blank of the isopropylamine

organosilicone
salt of glyphosate7 (RUFB) that contained proprietary adjuvants (Feng 1998). Herbicides
included the formulated isopropylamine salt of glyphosate that included an adjuvants, a
formulated isopropylamine salt of glyphosate that included proprietary adjuvants9 (RU),
the isopropylamine salt of glyphosate that lacked adjuvantslo (AC), and a formulated
trimethylsulfonium salt of glyphosatell (TD). Data were analyzed using SAS software”.
Field Study. The two soybean near-isolines were evaluated in a ﬁeld study in
Ingham County, Michigan. S. sclerotiorum sclerotia in Phaseolus vulgaris L. plant
material was applied to the test site with a fertilizer spreader to maintain a stable disease
inoculum. Daily irrigation of the ﬁeld began on July 5, 1998 and July 3, 1999 and ended
at after soybean ﬂowering had ceased. Both cultivars were planted at 494,000 seeds ha'1
in 38 cm rows. Plot sizes were 2.3 x 4.3 m in 1998 and 1999. When soybean plants were
at the V5 growth stage (Herman 1997), both cultivars were treated with the herbicides
and adjuvants. For the GL2415 cultivar, treatments included the untreated control, the

nonionic surfactant at 0.5 %v/v, the organosilicone at 0.3% v/v, the crop oil concentrate

at 1.0 % v/v, and the RUFB at 0.4% v/v. Treatments for theGL2600RR cultivar included

49

the untreated control, the RUFB at 0.4% v/v and the RU at 840, 1680, and 2520 g ae ha".
All treatments were applied with a compressed CO2 backpack sprayer traveling 6.6 km/h
and using size 8002 ﬂat fan nozzlesl3 at a delivery rate of 178 L ha"1 and pressure of 131
kPa.

Detached Leaf Assay. A detached leaﬂet assay modiﬁed from Dann et al. (1999)
was used to evaluate the inﬂuence of the adjuvants and formulated glyphosate on the
susceptibility of soybean to S. sclerotiorum. Each day for seven consecutive days after
herbicide and adjuvant treatments, two trifoliate leaves most likely to intercept the
greatest amount of spray (typically the fourth trifoliate leaf) from separate plants were
harvested from each plot. A side leaﬂet from each trifoliate was placed into a 150 x 15
mm petri dish containing moistened Whatman #1 ﬁlter paper. Each leaﬂet was inoculated
with a 2.4 mm diam plug of S. sclerotiorum mycelia (Dann et al. 1999). The mycelia
plugs were selected from the outer margin of actively growing mycelia propagated on
potato dextrose agar (PDA) in 100 x 15 mm petri dishes. Inoculated soybean leaﬂets
were maintained for 48 h at 21 C. Each lesion was measured three times 48 h after
inoculation and the average of those three measurements was used in data analysis. The
study was replicated three times in 1998 and four in 1999. The study was analyzed as a
factorial randomized complete block with cultivar and herbicide/adjuvant combinations
as one factor, timing (1 through 7 DAT) as the second factor and year as the third factor.
Main effects and interactions were evaluated using a general linear models procedure
(PROC GLM), means were averaged over timings and were separated with LSD

=0.05).

50

Field Evaluations. A disease severity index (DSI) was used to score disease
severity within the plots by rating 15 consecutive plants from the two center rows of each
plot (Kim et al. 1999). Yields were taken at the end of each growing season. The center
two rows were harvested October 9, 1998 and October 6, 1999 with a plot combine and
yields were adjusted to 13% moisture. The study was a randomized complete block
design with three replications in 1998 and four replications in 1999. Main effects and
interactions were evaluated using a general linear models procedure (PROC GLM).
Yields were subjected to F-Max tests to test for homogeneity of variance among years
(Kuehl 1994) and data found to be homogenous was pooled. Means of each cultivar by
herbicide/adjuvant combination were separated using LSD (p=0.05).

Greenhouse Study. To reduce some of the potential environmental variability
that accompanies ﬁeld research, an evaluation of the impact of herbicides and adjuvants
on soybean susceptibility to S. sclerotiorum was conducted in the greenhouse. Two
soybean seeds each were planted in BACCTOl4 potting soil in 946-ml plastic pots and
thinned to one plant per pot after germination. Environmental data in the greenhouse
included a 16 h photoperiod with supplemental sodium-vapor lights providing a total of
approximately 1000 11E m'2 s". Day/night temperatures were set at 27 /20 C. When
soybean reached the V4 growth stage (Herman 1997), GL26OORR plants were treated
with the adjuvants, RUFB, and RU rates as listed previously. Plants ﬁ‘om GL26OORR
were also treated with AC at 2560 g ae ha’1 and a TD at 2560 g ae ha". Treatments were
delivered via a compressed air, continuous link belt sprayer ﬁtted with a size 8001B ﬂat
fan nozzlelo traveling at 1.53 km/h and delivering 187 L/ha at 165 kPa. The trifoliate leaf

from each plant most likely to intercept most of the spray (the third trifoliate leaf) was

51

harvested at 1 and 7 d after treatment (DAT). The excised leaﬂets were inoculated with
actively growing S. sclerotiorum mycelia and lesion diameters were measured as stated
previously. The study was a factorial randomized complete block design with four
replications and was repeated twice. Factors included the treatments and each
experiment. Main effects were separated with PROC GLM and means were separated
using LSD (p=0.05).

Leaﬂet Dip Study. Lesion development that resulted from the inoculation of S.
sclerotiorum on excised leaves was examined in both the ﬁeld and greenhouse studies.
The shortest time interval between treatment with RU or an adjuvant and inoculation was
1 DAT. To examine what effects the RU and/or adjuvants may have on lesion
development when inoculation occurs at a much shorter time interval, a leaﬂet dipping
study was initiated. Plants from GL2415 and GL26OORR were grown in the greenhouse
as described earlier and were treated at the V3 stage (Herman 1997). Leaﬂets from the
second trifoliate leaves were dipped into solutions of the organosilicone at 0.3 %v/v, the
crop oil concentrate at 1.0 %v/v, the nonionic surfactant at 0.5 % v/v, the RUFB at 0.4 %
v/v and RU at 4, 7, and 10 %v/v for 15 sec. The RU rates used for this leaﬂet dipping
study were equivalent to spray tank concentrations used in the ﬁeld study. The treated
leaﬂets were excised 15 min after dipping and inoculated with S. sclerotiorum mycelia
plugs as described previously. The lesion measurements 48 h after inoculation were
conducted as described previously. The study was conducted twice and analyzed as a
factorial randomized complete block design with four blocks. Factors included the
cultivars, the RU, RUFB or adjuvant, and each experiment. Average lesions were

subjected to F -Max tests to test for homogeneity of variance among runs (Kuehl 1994)

52

and data found to be homogenous was pooled. Means were separated with an LSD (p =
0.05).

Pure Culture Assay. The previous studies investigated the effect of herbicides
and adjuvants on plant susceptibility to S. sclerotiorum. Toxicity of various formulations
of glyphosate and adjuvants to S. sclerotiorum were analyzed on PDA to investigate the
effects of the herbicides and adjuvants directly on the S. sclerotiorum. Doses of RO, RU
and AC were added to molten PDA to give ﬁnal concentrations of 100 and 1000 mM ae
of glyphosate. (In the ﬁeld study, a spray tank on a sprayer set up to apply RU at 2520 g
ae ha”1 of RU and at a rate of 187 L ha ’1 contains 79 mM ae glyphosate.) RUFB was
added to molten PDA at rates equivalent to the RU. The organosilicone, nonionic
surfactant, and crop oil concentrate were added to molten PDA to give ﬁnal concentration
of 1.0 % v/v. The molten PDA was poured into 100x15 cm petri dishes and allowed to
cool. Cores of mycelia (5.25 mm in diam) from the leading edge of S. sclerotiorum
cultured on PDA were placed in the center of the petri dishes containing the herbicide
and adjuvant treatments. The plates were incubated at 22 C for 48 h. Aﬁer 48 h of
incubation, the mycelial leading edge diam was measured with digital calipers. Each
experiment consisted of ﬁve replications and was conducted twice.

RESULTS AND CONCLUSIONS

Field Study. Detached Leaf Assay. Since inoculation timing did not inﬂuence
lesion diameters (data not shown), S. sclerotiorum lesion diameters were averaged over
all inoculation timings. For the control treatments, lesion diameters were similar across
both of the cultivars (Table 1), indicating that the glyphosate-resistant trait did not

inﬂuence soybean susceptibility to S. sclerotiorum. Lesion diameters were similar across

53

the cultivar by herbicide/adjuvant treatments (Table 1), indicating that the chemical
treatments do not inﬂuence plant susceptibility to S. sclerotiorum.

Field Evaluations. The disease severity index (DSI) ratings for GL2415 control
and GL26OORR control were similar to each other (Table 2), indicating that the
glyphosate-resistant trait does not inﬂuence SSR severity in the ﬁeld. The RU and
adjuvant treatments did not affect the ﬁeld DSI ratings or yield either year (Table 2),
indicating that the RU, RUFB, and adjuvants do not inﬂuence SSR severity or yield.

Greenhouse Study. In a detached leaﬂet assay on soybean grown in the
greenhouse, lesion diameters were similar across all treatments when leaﬂets were
inoculated at 1 and 7 DAT (Table 3). These results support the results from the detached
leaﬂet assay conducted on plants grown in the ﬁeld. Results from both of the studies
indicate that applications of the organosilicone, crop oil concentrate, nonionic surfactant,
RUFB, and RU do not affect soybean susceptibility to S. sclerotiorum.

Leaﬂet Dip Study. The lesion diameters were averaged over both cultivars, since
the two cultivars displayed similar susceptibility to S. sclerotiorum lesion development
(data not shown). Lesion development increased when leaﬂets were dipped into the
organosilicone, crop oil concentrate, and nonionic surfactant and decreased when dipped
into the RUFB and RU (Figure 1). These results indicate that a direct interaction of the
RU and RUF B would deter lesion development while the organosilicone, crop oil
concentrate, and nonionic surfactant would encourage lesion development from S.
sclerotiorum, if such a scenario were plausible in the ﬁeld. However, this scenario is not
likely in the ﬁeld due to the difference in timing between herbicide application and

disease infection that occurs in the ﬁeld.

54

The two cultivars, GL2415 and GL26OORR displayed similar levels of
susceptibility to S. sclerotiorum in the ﬁeld. Applications of RU and adjuvants did
increase plant susceptibility to S. sclerotiorum. If the observations of increased SSR
levels in glyphosate-resistant soybean were accurate, factors other than the glyphosate-
resistant trait, the RU, RUFB, or the activator adjuvants were the cause.

Pure Culture Assay. S. sclerotiorum mycelia were inhibited by doses of 1000
mM ae of glyphosate of RO, RU, and RUFB; however, mycelia were not inhibited by AC
(Figure 2). Mycelia] growth was similar to the control for all glyphosate formulations at
the 100 mM rate (Figure 2). These results indicate that the adjuvants within R0 and RU
but not the glyphosate were inhibiting mycelial development in the PDA and also in the
leaﬂet dip study. Crop oil concentrate, nonionic surfactant, and organosilicone inhibited
mycelial grth on PDA at concentrations of 1.0 % v/v (Figure 3). These results indicate
that 48 h exposure of S. sclerotiorum to the activator adjuvants resulted in inhibition of
the mycelia.

Formulated glyphosate and activator adjuvants did not increase soybean
susceptibility to infection from S. sclerotiorum. The two cultivars displayed similar levels
of sensitivity to S. sclerotiorum, indicating that the glyphosate-resistant trait does not
cause an increase in plant susceptibility to SSR. Factors other than those examined in this
study are responsible for the farmers’ observation of increased SSR in glyphosate-
resistant soybean. Perhaps the architecture of the glyphosate-resistant soybean plants or
the trend to plant glyphosate-resistant soybean in narrow rows contributed to the

observed increase of SSR. Another possible explanation could be that the overall genetic

55

background of some glyphosate-resistant cultivars is prone to S. sclerotiorum
susceptibility.
LIST OF RESOURCES
l Prime Oil, Riverside/Terra Corp., Sioux City, IA 51101.

2 X-77, Valent U.S.A. Corp., 1333 North California Boulevard, Walnut Creek, CA

94596.

3Great Lakes Hybrids. 9915 West M-21, Ovid, MI, 48866.

4 Activator 90®, Alkylpolyoxyethylene ethers and free fatty acids, Loveland
Industries, Inc., PO. Box 1289, Greeley, CO 80632-1289.

5 Silwett® L-77®, Silicone-polyether copolymer 100%, Loveland Industries, Inc., PO.
Box 1289, Greeley, CO 80632-1289.

6Herbimax, 83% petroleum oil, 17% surfactant, Loveland Industries, Inc., PO. Box
1289, Greeley, CO 80632-1289.

7 Roundup Ultra® formulation blank, Monsanto Co., 800 N. Lindbergh Blvd., St.
Louis, MO, 63167.

8 Roundup Original®, Monsanto Co., 800 N. Lindbergh Blvd., St. Louis, MO, 63167.

9 Roundup Ultra®, Monsanto Co., 800 N. Lindbergh Blvd., St. Louis, MO, 63167.

‘0 Accord®, Monsanto Co., 800 N. Lindbergh Blvd., St. Louis, MO, 63167.

“ Touchdown 5®, ZENECA Inc., 1200 South 47th Street, Richmond, CA 94804.

‘2 SAS 6.12, SAS Institute, Box 8000, Cary, NC 27572.

'3 Teejet ﬂat fan nozzles, Spraying Systems Co., North Avenue and Schmale Road,
Wheaton, IL 60532.

‘4 Michigan Peat Co., PO. Box 98129, Houston, TX 77098.

56

ACKNOWLEDGEMENTS
The authors would like to thank the Michigan Soybean Promotion Committee for

funding this research.

57

LITERATURE CITED

Casale, W. L., and L. P. Hart. 1986. Inﬂuence of four herbicides on carpogenic and
apothecium development of Sclerotinia sclerotiorum. Physiol. Biochem. 76: 980-984.

Dann, E. K., B. W. Diers, and R. Hammerschmidt. 1999. Suppression of sclerotinia stem
rot of soybean by lactofen herbicide treatment. Phytopathol. 89: 598-602.

Feng, P. C. C., Jan S. Ryserse, and R. Douglas Sammons. 1998. Correlation of leaf
damage with uptake and translocation of glyphosate in velvetleaf (Abutilon theophrasti).
Weed Technol. 12: 300-307.

Herman, J. C. 1997. How a soybean plant develops. Iowa State University, Ames, IA.

Holliday, M. J ., and N. T. Keen. 1982. The role of phytoalexins in the resistance of
soybean leaves to bacteria: effect of glycophosphate on glyceollin accumulation.
Phytopathol. 72: 1470-1474.

Keen, N. T., M. J. Holliday, and M. Yoshikawa. 1982. Effects of glyphosate on
glyceollin production and the expression of resistance to Phytophthora megasperma f.sp.
glycinea in soybean. Phytopathol. 72: 1467-1470.

Kim, H. S., C. H. Sneller, and B. W. Diers. 1999. Evaluation of soybean cultivars for
resistance to Sclerotinia stem rot in ﬁeld environments. Crop Sci. 39: 64-68.

Kuehl, R. O. 1994. Statistical Principles of Research and Design Analysis. Wadsworth,
Inc., Belmont, CA.

Levene, B. C., M. D. K. Owen, and G. L. Tylka. 1998. Response of soybean cyst
nematodes and soybean (Glycine max) to herbicides. Weed Sci. 46: 264-270.

Padgette, S. R., K. H. Kolacz, X. Delannay, D. B. Re, B. J. LaVallee, C. N. Tinius, W. K.
Rhodes, Y. I. Otero, G. F. Barry, D. A. Eichholtz, V. M. Peschke, D. L. Nida, N. B.
Taylor, and G. M. Kishore. 1995. Development, identiﬁcation, and characterization of a
glyphosate-tolerant soybean line. Crop Sci. 35: 1451-1461.

Radke, V. L. and C. R. Grau. 1986. Effects of herbicides on carpogenic germination of
Sclerotinia sclerotiorum. Plant Disease 79: 19-23.

58

Table I. Lesion diameters resulting from Sclerotinia sclerotiorum inoculation of excised
GL2415 and GL26OORR Glycine max leaﬂets (fourth trifoliate) at l, 2, 3, 4, 5, 6, and 7
DAT. Lesion diameters were averaged over cultivar and inoculation timings. Soybean
were grown in the ﬁeld and treated at the V5 grth stage. Means were averaged over
inoculation timings and were analyzed for differences with LSD (p = 0.05).

 

 

 

Cultivar Treatmenta Rate Lesion Diameter

mm

GL2415 Untreated ---- 14.91

RUFB 0.4 % v/v 14.93

Organosilicone 0.3 % v/v 13.96

Cr0p oil concentrate 1 % v/v 13.89

Nonionic surfactant 0.5 % v/v 14.08

GL26OORR Untreated ---- 15.59

RUFB 0.4 % v/v 13.91

RU 840 g ae ha" 13.91

RU 1680 g ae ha'1 14.61

RU 2520 g ae ha'l 13.48
LSD (0.05) NS

 

“ All treatments included 2% v/v ammonium sulfate; treatment abbreviations: RUFB,
Roundup Ultra formulation blank; RU, Roundup Ultra formulation of glyphosate.

59

Table 2. The SSR severity index (DSI) and yield from ﬁeld study of Glycine max
cultivars GL2415 and GL26OORR treated with adjuvants and formulated glyphosate.
Means were separated with LSD (E005).

 

 

 

Cultivar Treatmentat Rate DSIb Yield

kg ha'I

GL2415 Untreated ---- 3 1 4006
GL2415 RUFB 0.4 % v/v 46 4247
GL2415 Organosilicone 0.3 % v/v 39 3842
GL2415 Crop oil concentrate l % v/v 40 4155
GL2415 Nonionic surfactant 0.5 % v/v 44 4075
GL26OORR Untreated ---- 49 3960
GL26OORR RUFB 0.4 % WV 50 4049
GL26OORR RU 840 g ae ha'1 50 3736
GL26OORR RU 1680 g ae ha" 43 4148
GL2600RR RU 2520 g ae ha'1 44 4024

LSD (0.05) NS NS

 

a All treatments included 2%v/v ammonium sulfate; treatment abbreviations: RUFB,
Roundup Ultra formulation blank; OSI, organosilicone; COC, petroleum crop oil
concentrate; NIS, nonionic surfactant; RU, Roundup Ultra;.

b DSI, disease severityindex, was calculated according to Kim et al. (1999) and averaged
over both years. 0 = no SSR and 100 = complete plant infestation of SSR.

60

Table 3. Lesion diameters measured 48 h after Sclerotinia sclerotiorum mycelial
inoculation of ‘GL26OORR’ Glycine max leaﬂets excised from plants 1, and 7 DAT.
Soybean plants were grown under greenhouse conditions and chemical treatments were
made at the V4 growth stage. Means were separated by LSD (p = 0.05).

 

 

 

 

Treatment8| Rate Lesion Diameter
1DATB 7DAT
mm
Untreated Control ---- 6.32 9.63
RUFB 0.4 % v/v 8.79 8.30
Organosilicone 0.3 % v/v 5.41 11.58
Crop oil concentrate 1 % v/v 5.93 9.73
Nonionic surfactant 0.5 % v/v 7.50 6.85
RU 840 g ae ha" 8.28 8.94
RU 1680 g ae ha'l 6.21 8.09
RU 2520 g ae ha" 6.33 7.47
AC 2520 g ae ha'I 6.82 6.69
TD 2520 g ae ha'l 5.91 7.12
LSD (0.05) NS NS

 

a Treatments included a formulation blank of the isopropylamine salt of glyphosate
(RUFB), nonionic surfactant (NTS), crop oil concentrate (COC) and organosilicone (OSI)
and formulated isopropylamine salt of glyphosate with adjuvants (RU), formulated
isopropylamine salt of glyphosate without adjuvants (AC), and formulated
trimethylsulfonium salt of glyphosate with adjuvants (TD).

b DAT: d after treatment

61

35.00

 

c LSD = 3.09
30.00 -

25.00 d
20.00 -
15.00 -
10.00 -

Lesion Diameter (mm)

5.00 -

 

 

 

0.00 -

Control RUFB 031 000 N13 RU (4) RU (7) RU (10)
(0.4) (0.3) (1) (0.5)

Figure I. Lesion diameters averaged over GL2415 and GL26OORR Glycine max leaﬂets
inoculated with Sclerotinia sclerotiorum 15 min after being dipped into various
treatments of formulated glyphosate and adjuvants. Treatments included the formulation
blank of the isopropylamine salt of glyphosate (RUFB), organosilicone (OSI), crop oil
concentrate (COC), nonionic surfactant (N18), and formulated isopropylamine salt of
glyphosate (RU). Rates for each treatment are based on % v/v and included in
parenthesis. Bars with different letters represent signiﬁcant differences according to LSD

(p=0.05).

62

 

 

 

 

 

 

 

 

 

 

 

120
100-
300- DAC
'8 .
no
93 60‘ Inn
2 IRIIB
4.0-4
2”
o

 

 

 

Figure 2. Mycelia] growth of Sclerotinia sclerotiorum on potato dextrose agar with 100
and 1000 mM ae glyphosate as the formulation without adjuvants (AC), formulation
with an adjuvant (RO), with proprietary adjuvants (RU), and the formulation blank with
proprietary adjuvants (RUFB). Bars with different letters represent signiﬁcant differences
according to LSD (p=0.05).

63

 

90' LSD=1

Myoeial Growth (°/o of Control)

 

 

 

Concentration (1% v/v)

Figure 3. Mycelial growth of Sclerotinia sclerotiorum on potato dextrose agar treated
with 1 % v/v of organosilicone (OSI), crop oil concentrate (COC), or nonionic surfactant
(NIS). Bars with different letters represent signiﬁcant differences according to LSD
(p=0.05).

CHAPTER 3
GLYPHOSATE DOES NOT INHIBIT INDUCTION OF THE DEFENSE
RESPONSE OF GLYPHOSATE-RESISTANT SOYBEAN TO Sclerotinia
sclerotiorum
Abstract: Farmers in Michigan reported higher levels of Sclerotinia stem rot (caused by
Sclerotinia sclerotiorum) in ﬁelds of glyphosate-resistant soybean [Glycine max (L.)
Merr]. Studies were conducted to determine if glyphosate or shading reduced the defense
response of glyphosate-resistant soybean to S. sclerotiorum. Shikimate accumulation was
detected 1, 4, and 7 days after treatment in the glyphosate-susceptible cultivar ‘G12415’
but not in the glyphosate-resistant cultivar ‘GL2600RR’ following glyphosate
application. The levels of Shikimate detected in both GL2415 and GL26OORR indicated
that these cultivars were responding as expected to glyphosate applications. Glyphosate
did not inhibit the Shikimate acid pathway in glyphosate-resistant soybean. Soybean
extracts containing the plant defense compound glyceollin were placed on potato
dextrose agar at varying concentrations. Sclerotinia sclerotiorum hyphae were inhibited
by the glyceollin extracts in a rate-dependant manner, indicating that glyceollin directly
inhibits S. sclerotiorum. Glyphosate had no impact on either baseline or induced levels of
glyceollin in glyphosate-resistant soybean, indicating that glyphosate did not impair plant
defense responses to S. sclerotiorum. Lactofen induced glyceollin synthesis equally in
both GL2415 and GL26OORR cultivars. Shade levels of 60% did not inhibit the induction
of glyceollin synthesis.
Nomenclature: Glyphosate; soybean, Glycine max (L.) Merr. ‘GL2415’,

‘GL2600RR’, and ‘92B71’; Sclerotinia stem rot (white mold), Sclerotinia sclerotiorum

(Lib.) de Bary.

65

Key Words: Glyphosate, glyphosate-resistant soybean, Sclerotinia stern rot,

glyceollin.

66

INTRODUCTION

Glyphosate inhibits the conversion of Shikimate to chorismate (Amrhein et al.,
1980) by directly inhibiting the 5-enolpyruvylshikimate—3-phosphate synthase (EPSPS)
enzyme (Steinri'lcken and Amrhein, 1984) in the Shikimate pathway. Sublethal doses of
glyphosate on glyphosate-susceptible soybean reduced levels of the isoﬂavonoid
phytoalexin glyceollin in soybean leaves when induced by sodium iodoacetate or
aminooxyacetic acid (Holliday and Keen, 1982), and reduced plant resistance to
Phytopthera megasperma f. sp. glycinea (Keen et al., 1982).

Glyphosate-resistant soybean was developed by inserting a glyphosate-insensitive
bacterial EPSPS gene from Agrobacterium sp. strain CP4 into soybean (Padgette et a1.
1995). Hypothetically, glyphosate applied to glyphosate-resistant soybean should not
inhibit the Shikimate pathway. Therefore, glyphosate applied to glyphosate-resistant
soybean should not reduce plant defenses against pathogens.

Sclerotinia sclerotiorum (Lib.) de Bary, the pathogen that causes Sclerotinia stem
rot (SSR) (also known as white mold) of soybean, is prevalent in the Great Lakes region.
Development of SSR in soybean depends on the interaction of the susceptible host,
virulent pathogen, and environmental conditions (Agrios, 1997; Boland and Hall, 1998).

Increased levels of the glyceollin within the soybean plant have been associated
with decreased grth of S. sclerotiorum hyphae (Dann et al., 1999), and other pathogens
including soybean cyst nematode (Heterodera glycines Ichinohe) (Levene et al., 1998)
and P. megasperma f. sp. glycinea (Stbssel, 1983; Yoshikawa et al., 1978).

Glyceollin synthesis can be induced by various pathogens, various chemicals, and

herbicides. Pathogens reported to induce glyceollin included Pseudomonas pisi (Ingham,

67

Keen et al., 1981), P. megasperma f. sp. glycinea race 4 (Keen et al., 1971; Stéssel,
1982), Rhizoctonia solani Kiihn (Scharff et al., 1997), and S. sclerotiorum (Sutton and
Deverall, 1984). Ayers et al. (1976) reported that P. megasperma f. sp. glycinea
contained compounds within the pathogen cell walls that induced glyceollin synthesis.
Certain chemicals have proven to be effective inducers of glyceollin synthesis. Glyceollin
accumulation was greater with treatments of silver nitrate (AgNO3) or copper chloride
(CuCl2) than with inoculation by zoospores of P. megasperma f. sp. glycinea race 4
(Stossel, 1982). Glyceollin also accumulated aﬁer treatments of sodium iodoacetate
(Ingham et al., 1981). The inorganic salts Ni(NO3)2, ZnSOa, Pb(NO3)2, and HgCl2
induced synthesis of small amounts of glyceollin (Stbssel, 1982). Glyceollin synthesis
can also be induced by organic compounds such as Triton X-100 (Stossel, 1982), lactofen
(Dann et al. 1999), aciﬂuorfen, bentazon, crop oil concentrate, and nonionic surfactant
(Levene et al., 1998).

Glyceollin accumulation tends to occur in close proximity to the site of infection
or plant injury. The most abundant isoﬂavonoid compound in necrotic lesions of soybean
leaves resulting from incompatible reactions with P. megasperma f. sp. glycinea was
glyceollin (Morris et al., 1991). The border zone between the lesion and the healthy tissue
contained higher concentrations of glyceollin than in the necrotic lesion 36 h after
inoculation (Morris et al., 1991). Glyceollin accumulation within soybean was highest
when tissue browning was most pronounced (Stdssel, 1982). Soybean cultivar ‘Harosoy
63’ tissue layers 0.25-mm thick that contained advancing hyphae of P. megasperma f. sp.
glycinea accumulated glyceollin at levels higher than the EDoo value (Yoshikawa et al.,

1978). Although these studies found glyceollin to be a localized response, Nelson (2001)

68

found elevated glyceollin in new soybean leaves at 26 d after treatment (DAT). The
difference in observations between these studies could be due to the plant part examined,
the growing conditions, and the age of the soybean plants. Stbssel (1982) and Yoshikawa
et al. (1978) tested hypocotyls incubated in growth chambers, while Morris et al. (1991)
tested soybean leaves of 14- and 21-d-old plants grown in a controlled environment.
Nelson (2001) examined ﬁeld-grown soybean that were approximately 42 days old at the
time glyceollin synthesis was induced.

The response of glyceollin synthesis to an inducer appears to be both rapid and
transient as glyceollin accumulated at inhibitory levels within 8 h after inoculation
(Yoshikawa et al., 1978), and concentrations declined by 3 and 9 d after inoculation
(Stossel, 1982; Yoshikawa et al., 1978). These studies were done on soybean hypocotyls
under a controlled environment and the transient occurrence of glyceollin in the
hypocotyls was not observed in ﬁeld-grown soybean. Nelson (2001) found elevated
concentrations of glyceollin in leaves of ﬁeld-grown soybean 26 DAT after induction by
lactofen.

Keen et al. ( 1971) investigated the effect of the ethyl acetate extracted fraction
from soybean on growth of P. megasperma f. sp. glycinea; however, no ethyl acetate
control was recorded in that research. In addition, there were no known reports of testing
the extracts from soybean on S. sclerotiorum.

Stossel (1982) investigated the effect of light on glyceollin synthesis in soybean.
Soybean seedlings were grown in the dark for 2 days or received continuous light for the
last two days. Glyceollin was induced by silver nitrate. Seedlings were then incubated in

the dark, in a 15/9 h light/dark regime, or in continuous light. For seedlings initially

69

grown in the dark, glyceollin accumulation was similar for all light treatments following
phytoalexin induction. Soybean treated to continuous light 48 h prior to and following
treatment produced signiﬁcantly less glyceollin. (Pennypacker and Risius, 1999)
observed that shade increased SSR development in some soybean cultivars (Pennypacker
and Risius, 1999). The increased level of disease on shaded soybean could indicate
reduced production of glyceollin. This observation is contradictory to the report of
Stossel (1982), where continuous light (no shade) resulted in reduced glyceollin
production.

Farmers in Michigan reported higher levels of SSR in glyphosate-resistant
soybean than in glyphosate-susceptible soybean ﬁelds. Field and greenhouse studies
found no signiﬁcant differences in SSR levels between near-isolines of glyphosate-
resistant and —susceptible cultivars (Lee et al., 2000; Nelson, 2000). Since ﬁeld
observations are dependent on crop growth, soil moisture, air temperature, relative
humidity, level of disease inoculum, timing of disease inoculum with soybean stage, and
inherent sensitivity of the cultivar (Boland and Hall, 1987; Boland and Hall; 1988; Kim
et al., 1999), the role of glyphosate on glyphosate-resistant cultivar response to pathogen
infection was investigated in the greenhouse and laboratory. This research was conducted
to determine if glyphosate or shading reduced the defense response of soybean to S.
sclerotiorum.

MATERIALS AND METHODS
Glyphosate Effect on Shikimate Accumulation
To determine if glyphosate caused an accumulation of Shikimate in glyphosate-

resistant soybean, glyphosate was applied to glyphosate-susceptible and glyphosate-

70

resistant cultivars. The soybean cultivars evaluated in this study were GL26OORRl
(glyphosate-resistant), GL24152 (glyphosate-susceptible), and 92B713 (glyphosate-
resistant).

A single seed of each cultivar was planted in BACCTO potting soil4 contained in
a 946-ml plastic pot. Environmental conditions in the greenhouse included a 16 h
photoperiod supplemented by sodium-vapor lights that provided a total photon ﬂux
density of approximately 1000 11E m’2 5". Minimum day/night temperatures were set at
27/20 C. Glyphosate, formulated as Accord“, at 1680 g a.e. ha'l was delivered in
distilled water at a spray volume of 93.5 L ha'l via a compressed air, continuous cable
belt sprayer ﬁtted with a size 8001B ﬂat fan nozzle6 traveling at 3.22 km h'1 at 172.5 kPa
to soybean at the V4 growth stage (Herman, 1997). At 1, 4, and 7 DAT, the apical
meristems, including 1- to 2-cm-long leaﬂets, of ﬁve soybean in each treatment were
harvested, weighed, and frozen until extraction. Following extraction, the optical density
was measured immediately at 380 nm7, and Shikimate levels were determined according
to Singh and Shaner (1998). This study was a completely randomized design with four
replications and repeated. The main effects of experiment, cultivar by herbicide, and day
were subjected to PROC GLM in SASs. Data found to be homogenous were pooled over
main effects. Means for Shikimate accumulation were separated with Fisher’s protected
LSD (p=0.05).

To determine if glyceollin inhibits mycelial growth of S. sclerotiorum, hyphae of
S. sclerotiorum were challenged with glyceollin extracted from soybean on potato
dextrose agar. Glyphosate-resistant cultivar ‘92B71 ’ was seeded and grown in the

greenhouse as described earlier. Lactofen9 at 105.1 g ai ha'l was applied to V4 soybeans

71

(Herman, 1997) to induce the synthesis of glyceollin (Dann et al., 1999; Nelson, 2000).
The trifoliolate leaves of soybean were harvested 4 DAT and glyceollin was extracted
along with other isoﬂavonoids according to (Ingham et al., 1981). Ethyl acetate
suspensions of the glyceollin extracted at a total volume of 200 111 and equivalent to 0.25,
0.5, l, 2, and 4 g ﬂesh wt were placed on ﬁrm potato dextrose agar. The ethyl acetate
extract was allowed to evaporate for 10 min. Controls included potato dextrose agar with
no ethyl acetate or glyceollin extract and potato dextrose agar with 200 pl ethyl acetate
but no glyceollin extract. A 2.5-mm plug of advancing S. sclerotiorum hyphae was placed
on top of the surface of the agar with the extract containing glyceollin and incubated at
room temperature. The diameter of the advancing hyphae was measured 48 h after
inoculation of the potato dextrose agar. This study was conducted as a completely
randomized design and repeated. Main effects of experiment and glyceollin were
subjected to PROC GLM in SAS. Data found to be homogenous were pooled over main
effects. Means for mycelia diameter were separated with Fisher’s protected LSD
=0.05).

Glyphosate Effect on Soybean Defenses

To determine if glyphosate inhibited the induction of phytoalexin synthesis in
soybean, glyphosate-susceptible and —resistant cultivars were treated with glyphosate or
lactofen and subsequently induced to synthesize glyceollin. Cultivars ‘GL2415’ and
‘GL2600RR’ were seeded and grown in the greenhouse as described earlier. When
soybean reached the V4 growth stage (Herman, 1997), herbicide treatments of
glyphosate, formulated as Accord, at 421 g a.e. ha'1 and lactofen at 105.1 g ai ha'I were

applied with the compressed air, continuous cable belt sprayer as described earlier. One

72

day after the herbicide treatment, silver nitrate at 10 mM plus an organosilicone adjuvant
at 0.3% v:v was applied with the compressed air, continuous cable belt sprayer as
described earlier to induce glyceollin synthesis (Stossel, 1982). Controls included
soybean treated with lactofen followed by silver nitrate, soybean treated with silver
nitrate alone, and untreated soybean. One day after the silver nitrate treatment, a side
soybean leaﬂet ﬂom the third trifoliolate was harvested and placed into a 150 x 15 mm
petri dish containing moistened Whatman #1 ﬁlter paper. The leaﬂet was inoculated by
placing a 2.4 mm diam plug of S. sclerotiorum mycelia near the center of the leaﬂet,
away ﬂom the leaf veins (Dann et al., 1999). The inoculated leaﬂet was maintained for
48 h at approximately 21 C on a laboratory bench. Mycelia spreading ﬂom the plug to the
leaf tissue caused the development of water-soaked lesions. The vertical, horizontal, and
diagonal diameters of the lesion were measured 48 h after inoculation and the average of
the three measurements was used in the analysis.

The center leaﬂet ﬂom the third trifoliolate was subjected to a glyceollin
extraction according to (Ingham et al., 1981). The extracted compounds were separated
on silica gel (Si gel) thin-layer chromatography (TLC) plates10 with chloroform: acetone:
concentrated ammonia hydroxide (50:50:l v:v:v) resulting in O-methylgyceoﬁlran (Rf
0.71), isoforrnononetin (Rf 0.56), glyceollins I, II, and 1H (Rf0.50), and glyceofuran and
glyceocarpin (Rf 0.25) (Ingham et al. 1981). To determine the relative amount of
glyceollin produced, a TLC phytoalexin bioassay was conducted. Cladosporium
cucumerinum spores suspended in a 100 ml solution 0.7 g KH2P04, 0.4 g KNO3, 0.3 g
Na2HP04, 0.1 g MgSOa, 0.1 g NaCl, and 5.0 g D-glucose that was adjusted to pH 6.5

(Allen, E.H. and J. Ku 1968). The suspended spores were sprayed on the TLC plate

73

surface and the plate was incubated at 100% relative humidity at approximately 21 C for
48 to 72 h. The C. cucumerinum hyphae spread across the TLC plate, except where
antimicrobial compounds were present, resulting in inhibitory sites appearing as white
zones on a dark background (Keen et al., 1971). Bands corresponding to glyceollin I, II,
and III (Rf 0.50) (Ingham et al. 1981) were measured for relative size.

This study was conducted as a factorial with soybean cultivar, herbicide
treatment, and silver nitrate as the factors. The study consisted of four replications and
was repeated once. Main effects of experiment, cultivar, herbicide, and silver nitrate were
analyzed with PROC GLM in SAS. Data were pooled over main effects where
homogenous. Means of lesion diameter and TLC band size were separated with Fisher’s
protected LSD (p=0.05).

Shade Effect on Soybean Defenses

To determine if shading reduced induction of plant defenses against S.
sclerotiorum, soybean cultivar ‘92B71 ’ was examined under shade regimes of 40 and
90% in the greenhouse. Soybean grown in no shade comprised the negative control.
Soybean plants at the V4 grth stage (Herman, 1997) were sprayed with silver nitrate at
10 mM and an organosilicone at 0.3% v:v with a compressed air, continuous cable belt
sprayer as described earlier. To mimic a ﬁeld scenario, the third and fourth trifoliolates
were placed above the shade cloth, while the cotyledons and ﬁrst and second trifoliolates
were covered by shade. Leaﬂets ﬂom the second and third trifoliolates were harvested 9
days after shade treatment was initiated. The leaﬂets were placed in petri dishes,
inoculated with S. sclerotiorum, and the resulting lesions were examined as described

earlier. This study was conducted as a factorial arranged in a randomized complete block

74

design with cultivar, shade, and silver nitrate as factors. The study contained four
replications and was repeated. Main effects of experiment, cultivar, shade, and silver
nitrate were analyzed in PROC GLM of SAS. Data found to be homogenous over main
effects were pooled and means of lesion diameters were separated by Fisher’s protected
LSD (p=0.05).
RESULTS AND CONCLUSIONS

Glyphosate Effect on Shikimate Accumulation

The interactions of cultivar by herbicide and cultivar by harvest date were
signiﬁcant (P<0.0001), so the cultivar by glyphosate effect was analyzed for each harvest
date. The cultivar by herbicide interaction was signiﬁcant (P<0.0001) for each harvest
date. Signiﬁcant Shikimate accumulation was detected 1, 4, and 7 DAT in cultivar
‘GL2415’ following treatment with glyphosate (Figure 1). Shikimate accumulation was
signiﬁcantly lower 1, 4, and 7 DAT in untreated GL2415, untreated GL26OORR, and h
GL26OORR treated with glyphosate compared to GL2415 treated with glyphosate (Figure
1). Glyphosate caused Shikimate to accumulate in GL2415 but not in GL26OORR,
indicating that both GL2415 and GL26OORR were responding as expected to glyphosate
(Singh and Shaner, 1998).

Glyceollin Effect on Sclerotinia sclerotiorum

As glyceollin increased, the grth of S. sclerotiorum hyphae decreased, such
that the plant extract corresponding to 0.25 g ﬂesh wt (12.5 111) resulted in a 13%
reduction of hyphae growth while plant extract corresponding to 4 g ﬂesh wt (200 111)
resulted in 80% inhibition (Figure 2). The plant extract contained glyceollin and other

isoﬂavonoids (Ingham et al., 1981). The plant extracts containing glyceollin had direct

75

inhibiting action against S. sclerotiorum, which further supports the link between
glyceollin activity and reduced S. sclerotiorum invasion of soybean tissue.

Glyphosate Effect on Soybean Defenses

Lesion diameters resulting ﬂom advancing S. sclerotiorum hyphae were reduced
by 51 and 63% when GL2415 and GL26OORR, respectively, were sprayed with lactofen
compared to the untreated controls (Table 1). Sclerotinia sclerotiorum lesion diameters
following applications of glyphosate to GL26OORR were not reduced compared to the
untreated control (Table 1). Lesion diameters following silver nitrate were reduced by 38
and 57% for GL2415 and GL26OORR, respectively, compared to the untreated controls
(Table l). Glyphosate followed by silver nitrate on GL2415 resulted in 38% smaller
lesion diameters compared with lesion diameters following silver nitrate (Table 1).
Therefore, glyphosate did not impair soybean response to a phytoalexin inducer.

Extracts ﬂom soybean sprayed with several of the herbicide by silver nitrate
combinations yielded antifungal compounds that corresponded to Rivalues previously
reported for a mixture of glyceollin I, II, and III (Rf 0.5) (Ingham et al. 1981). Extracts
ﬂom soybean sprayed with lactofen and lactofen followed by silver nitrate yielded 27 to
32 and 75 to 76% more glyceollin than extracts ﬂom soybean treated with silver nitrate
for GL2415 and GL26OORR, respectively (Table 1). Extracts ﬂom soybean sprayed with
glyphosate followed by silver nitrate resulted in 76 and 50% more glyceollin than
extracts ﬂom soybean sprayed with glyphosate for GL2415 and GL26OORR, respectively
(Table 1). These observations indicate that glyphosate was not inhibiting the induction of

glyceollin by silver nitrate.

76

Observed lactofen induction of glyceollin synthesis was consistent with previous
reports (Dann et al., 1999), but the observation that glyphosate did not decrease
glyceollin synthesis in glyphosate-susceptible soybean contradicted previous reports
(Keen et al., 1982; Ingham et al., 1981). The observation that glyphosate did not affect
glyceollin synthesis in glyphosate-resistant soybean is consistent with previous reports on
glyphosate and Sclerotinia stem rot (Lee et al., 2000; Nelson, 2000). Silver nitrate
induced glyceollin synthesis in soybean in this study consistent with previous reports
(Stdssel, 1982). Glyphosate application to glyphosate-susceptible and glyphosate-
resistant soybean did not inhibit induction of glyceollin by silver nitrate. This is an
important observation in that glyphosate applications to glyphosate-resistant soybean did
not appear to impair plant defenses against S. sclerotiorum. Since glyceollin has been
considered as a defense against several pathogens, this observation could be expanded to
those pathogens as well.

Shade Effect on Soybean Defenses

The soybean leaﬂets harvested from each shade regime in the greenhouse assay
exhibited lesions resulting ﬂom S. sclerotiorum similar in size to each other (Table 2).
Silver nitrate applied to soybean under no shade and soybean under 90% shade did not
inﬂuence lesion diameters; however, silver nitrate applied to soybean under 60% shade
resulted in a 55% reduction in lesion diameter (Table 2). For soybean grown in the
absence of shade, the similarity between S. sclerotiorum lesion diameters on soybean
sprayed with silver nitrate and untreated soybean contradicted the observations in the
study that investigated glyphosate effect on plant defenses. In that study soybean leaﬂets

sprayed with silver nitrate were harvested 1 DAT resulting in reduced S. sclerotiorum

77

lesion diameters and detection of glyceollin (Table 1); whereas, in this shade study
soybean were harvested at 9 DAT. The difference in time between induction of glyceollin
synthesis by silver nitrate and harvesting of the leaﬂet may be a factor in the
contradictory results and may support previous observations about glyceollin
accumulation being transient (Stdssel, 1982; Yoshikawa et al., 1978).

When treated with glyphosate, the glyphosate-resistant cultivar ‘GL2600RR’ did
not accumulate Shikimate acid as did the glyphosate-susceptible cultivar ‘GL2415’.
These results are consistent with previous research (Singh and Shaner, 1998). Soybean
plant extracts containing glyceollin inhibited mycelial grth in a dose-dependent
manner, suggesting that these compounds inhibited S. sclerotiorum mycelia growth on
soybean leaﬂets. Glyphosate applications to glyphosate-resistant soybean did not inhibit
the induction of glyceollin synthesis when the plant was challenged with an inducer.
Glyphosate applied to glyphosate-resistant soybean did not reduce the plant defense
system response within the soybean. Synthesis of glyceollin was related to inhibited
hyphae growth. Shade at 60% did not inhibit plant defenses against S. sclerotiorum.

SOURCES OF MATERIALS
' GL2415 soybean, Great Lakes Hybrids, 9915 West M-21, Ovid, MI 48866.
2 GL26OORR soybean, Great Lakes Hybrid, 9915 West M-21, Ovid, MI 48866.
3 92B71 soybean, Pioneer Hybrid International, 400 Locust Street, Suite 800, PO. Box
14453, Des Moines, IA 50306-3453.
4 BACCTO potting soil, Michigan Peat Co., P.O. Box 98129, Houston, TX 77098.

5 Accord®, Monsanto Co., 800 N. Lindbergh Blvd., St. Louis, MO 63167.

78

6 8001B TeeJet Flat Fan Nozzle, Spraying Systems Co., North PO. Box 7900 Wheaton,
IL 60189-7900.

7 Spectronic® GenesysTM 5 Spectrophotometer, ThermoSpectroni, 820 Linden Ave,
Rochester, NY 14625.

8 SAS Institute, Inc., SAS Campus Drive Cary, NC 27513-2414.

9 Cobra®, Valent U.S.A. Corporation, PO. Box 8025, Walnut Creek, CA 94596-8025.

'0 UniplateTM TLC Plate, Analtech, Inc. PO. Box 7558, Newark, DE 19714.

79

LITERATURE CITED

Allen, E.H. and J. Kuc.1968. a-Solanine and a-chaconine as ﬁrngitoxic compounds in
extracts of Irish potato tubers. Phytopathology 58: 776-781.

Amrhein, N., B. Deus, P. Gehrke, and H. C. Steinriicken. 1980. The site of glyphosate
with chorismate formation in vivo and in vitro. Plant Physiol. 66: 830-834.

Ayers, A. R., J. Ebel, B. Valent, P. Albersheim. 1976. Fractionation and biological
activity of an elicitor isolated ﬂom the mycellial walls of Phytophthora megasperma var.
sojae. Plant Physiol. 57: 760-765.

Bhattacharyya, M. K. and E. W. B. Ward. 1987. Biosynthesis and metabolism of
glyceollin I in soybean hypocotyls following wounding or inoculation with phytophthora
megasprem f.sp. glycinia. Physiol. Mol. Plant Pathol. 31: 387-405.

Boland, G. J. and R. Hall. 1987. Epidemiology of white mold of white bean in Ontario.
Can. J. Plant Pathol. 9: 218-224.

Boland, G. J. and R. Hall. 1988. Epidemiology of Sclerotinia stem rot of soybean in
Ontario. Phytopathol. 78: 1241-1245.

Dann, E. K., B. W. Diers, and R. Hammerschmidt. 1999. Suppression of Sclerotinia stem
rot of soybean by lactofen herbicide treatment. Phytopathol 89: 598-602.

Herman, J. C. 1997. How a soybean plant develops. Spec. Report. No. 5 3. Ames, IA,
Iowa State University. pp.3-16.

Holliday, M. J. and N. T. Keen. 1982. The role of phytoalexins in the resistance of

soybean leaves to bacteria: effect of glyphosate on glyceollin accumulation. Phytopathol.
72: 1470-1474.

Ingham, J. L., N. T. Keen, L. J. Mulheirn, and R. L. Lyne. 1981. Inducibly-forrned
isoﬂavonoids ﬂom leaves of soybean. Phytochem. 20: 795-798.

Keen, N. T. 1978. Phytoalexins: efficient extraction ﬂom leaves by a facilitated diffusion
technique. Phytopathol. 68: 1237-1239.

Keen, N. T., M. J. Holliday, and M. Yoshikawa. 1982. Effects of glyphosate on
glyceollin production and the expression of resistance to Phytophthora megasperma f.sp.
glycinea in soybean. Phytopathol. 72: 1467-1470.

Keen, N. T., J. J. Sims, D. C. Erwin, E. Rice, and J. E. Partridge. 1971. 6a-

Hydroxyphaseollin: an antifungal chemical induced in soybean hypocotyls by
Phytopathora megasperma var. sojae. Phytopathol. 61: 1084-1089.

80

Kim, H. S., C. H. Sneller, and B. W. Diers. 1999. Evaluation of soybean cultivars for
resistance to Sclerotinia stem rot in ﬁeld environments. Crop Sci. 39: 64-68.

Lee, C. D., D. Penner, and R. Hammerschmidt. 2000. Inﬂuence of formulated glyphosate
and activator adjuvants on Sclerotinia sclerotiorum in glyphosate-resistant and -
susceptible Glycine max. Weed Sci. 48: 710-715.

Levene, B. C., M. D. K. Owen, and G. L. Tylka. 1998. Response of soybean cyst
nematodes and soybean (Glycine max) to herbicides. Weed Sci. 46: 264-270.

Morris, P. F ., M. E. Savard, and E. W. B. Ward. 1991. Identiﬁcation and accumulation of
isoﬂavonoids and isoﬂavone glucosides in soybean leaves and hypocotyls in resistance

responses to Phytophthora megasperma f. sp. glycinea. Physiol. Mol. Plant Pathol. 39:
229-244.

Nelson, K. A. 2000. Soybean [Glycine max (L.) Merr] Growth and Development, White
Mold [Sclerotinia sclerotiorum (Lib.) de Bary] Incidence, and Yellow Nutsedge (Cyperus
esculentus L.) Control as Affected by Glyphosate and Other Herbicides. Ph.D.
Dissertation, Michigan State University, East Lansing, MI. pp.73-114.

Padgette, S. R., K.H. Kolacz, X. Delannay, D.B. Re, B.J. LaVallee, C.N. Tinius, W.K.
Rhodes, Y.I. Otero, G.F. Barry, D.A. Eichholtz, V.M. Peschke, D.L. Nida, N.B. Taylor,
and GM. Kishore. 1995. Development, identiﬁcation, and characterization of a
glyphosate-tolerant soybean line. Crop Sci. 35: 1451-1461.

Pennypacker, B. W. and M. L. Risius. 1999. Environmental sensitivity of soybean
cultivar response to Sclerotinia sclerotiorum. Phytopathol. 89: 618-622.

Scharff, A. M., I. Jakobsen, and L. Resendahl. 1997. The effect of symbiotic
microorganisms on phytoalexin contents of soybean roots. J. Plant Physiol. 151: 716-723.

Schopfer, C. R. and J. Ebel. 1998. Identiﬁcation of elicitor-induced cytochrome P450s of

soybean (Glycine max L.) using differential display of mRNA. Mol. Gen. Genetics. 258:
315-322.

Singh, B. K. and D. L. Shaner. 1998. Rapid determination of glyphosate injury to plants
and identiﬁcation of glyphosate-resistant plants. Weed Technol. 12: 527-530.

Stab, M. R. and J. Ebel. 1987. Effects of Ca2+ on phytoalexin induction by fungal elicitor
in soybean cells. Arch. Biochem. Biophysics. 257: 416-423.

Steinrilcken, HO and N. Amrhein. 1984. 5-Enolpyruvylshikimate-3-phosphate of
Klebsiella pneumoniae: 2. Inhibition by glyphosate [N-(phosphonomethyl)glycine)]. Eur.
J. Biochem. 143: 351-357.

Stbssel, P. 1982. Glyceollin production in soybean. Phtyopathol. Z. 105: 109-119.

81

Stossel, P. 1983. In vitro effects of glyceollin on Phytophthora megasperma f.sp.
glycinea. Phytopathol. 73: 1603-1607.

Sutton, D. C. and B. J. Deverall. 1984. Phytoalexin accumulation during infection of bean
and soybean by ascospores and mycelium of Sclerotinia sclerotiorum. Plant Pathol. 33:
377-3 83.

Yoshikawa, M., K. Yamauchi, and H. Masago. 1978. Glyceollin: its role in restricting
fungal growth in resistant soybean hypocotyls infected with Phytophthora megasperma
var. sojae. Physiol. Plant Pathol. 12: 73-82.

82

Table I. Glyphosate and lactofen effect on Sclerotinia sclerotiorum growth on detached
soybean leaﬂets and glyceollin synthesis. Relative glyceollin levels determined by thin-
layer chromatography separation using a Cladosporium cucumerinum assay.

 

 

Cultivar Treatment Rate AgNO3 (10 mM) AgNO3 (10 mM)
- + - +
Lesion diametera Glyceollin areab
(g ha'I) ----- mm ---------- cm2 -----
GL2415 Glyphosate 421 a.e.c 10.5 5.4 0.34 1.4
Lactofen 105 a.i. 6.4 3.1 1.80 1.9
Untreated -- 13.0 8.0 0.2 1.3
GL26OORR Glyphosate 421 a.e. 12.5 5.3 0.6 1.2
Lactofen 105 a.i. 4.8 4.4 1.6 1.7
Untreated -- 13.0 5.6 0.05 0.4
LSD (p =0.05) 1.7 0.33

 

' Combined over four experiments with four replications per experiment.
b Combined over two experiments with four replications per experiment.
° a.e. = acid equivalent; a.i. = active ingredient.

83

Table 2. Inﬂuence of shade on lesion diameters on greenhouse-grown soybean leaﬂets
resulting ﬂom Sclerotinia sclerotiorum hyphae growth.

 

Leaﬂet position

 

Below shade Above shade

 

Shadea Chemical Lesion diameter
(%) ---------- mm ----------
O Untreated 13.1 12.4
0 Silver nitrate (10 mM) 10.9 14.8
60a Untreated 1 1 .9 14.1
60 Silver nitrate (10 mM) 5.3 18.7
90b Untreated 16.4 16.7
90 Silver nitrate (10 mM) 17.9 17.1
LSD (H.051 6.3 6.0

 

’ Photosynthetic photon ﬂux density ranged ﬂom 550 t0128, 220 to 50, and 20 to 10

umol m"2 s'1 for 0, 60, and 90% shade respectively.

84

90.0 i
80.0 4‘

l-

N
.0
o

60.0 -
50.0 n
40.0 7
30.0 ‘
20.0 ‘

 

Dlam of Mycelia Growth (mm)

10.0 ‘

 

 

0.0 I I I 1
0 1 2 3 4

Glyceollin Extract Equivalent to Soybean Leaﬂet Fresh Wt. (9)

Figure 1. Inhibition of Sclerotinia sclerotiorum hyphae on potato dextrose agar by
extracts containing glyceollin ﬂom soybean leaﬂets.

85

 

 
 
 
 

 

 

 

 

 

9
-0-GL2415-Untreated
8 ~ -I-GL2415-Glyphoute
-a-GL2600RR-Untruhd
'1‘ 7 - +6L2600RR-Glyphoute
'01
to
.9 6 n
o
E
2 5-
.9.
E. 4 .
3
to
.§ 3 -
a .7
0’ 2+
12:——
1 i
o r r
1 4 7

Time After Treatment (days)

Figure 2. Shikimate accumulation following glyphosate application to GL2415
(glyphosate-susceptible) and GL26OORR (glyphosate-resistant) soybean. T-bars represent
LSD at p=0.05.

86

CHAPTER 4
ROLE OF CULTIVAR, ROW SPACING, AND SEED POPULATION IN THE
INCIDENCE OF SCLEROTINIA STEM ROT IN GLYPHOSATE-RESISTANT
SOYBEAN

Abstract. This research investigated the impact of glyphosate-resistant soybean [Glycine
max (L.) Merr.] cultivar, row spacing, and population in an irrigated system on the
incidence of Sclerotinia stem rot (SSR, also known as white mold), caused by the fungal
pathogen Sclerotinia sclerotiorum (Lib.) de Bary. Three glyphosate-resistant cultivars,
Pioneer ‘92B71’, Asgrow ‘AG2701’, and Asgrow ‘AG2702’, were evaluated in different
row spacing by target population combinations of 76 cm, 430 000 seeds ha"; 19 cm, 430
000 seeds ha"; and 19 cm, 560 000 seeds ha". Soybean canopy development, ﬂower
number, soil moisture, SSR severity, and soybean yield were evaluated. Cultivar ‘92B71 ’
had 43% lower DSI and 39% greater yield than AG2702. Cultivar ‘AG2701 ’ had 17%
lower D81 and 20% greater yield than AG2702. Actual average population of 92871 was
9 and 20% lower than actual average populations of AG2701 and AG2702, respectively.
Disease severity indexes were signiﬁcantly lower and yield was signiﬁcantly higher
when population was reduced ﬂom 560 000 seeds ha'l to 430 000 seeds ha'1 in 19-cm
rows. When averaged over the entire study, population was positively correlated with
DSI (r2 = 0.33 (p>0.0001)) and negatively correlated with yield (r2 = -013 (p=0.0140)).
Reduction of soybean population was more important than increasing row spacing to
manage SSR in an irrigated system. Average actual spacing between plants within a row
was 18 and 4 cm for 19- and 76-cm rows, respectively, at a target population of 430 000
seeds ha", which may have contributed to plant-to-plant transfer of S. sclerotiorum in the

76-cm rows.

87

INTRODUCTION

Sclerotinia sclerotiorum (Lib.) de Bary, the pathogen that causes Sclerotinia stem
rot (SSR) (also known as white mold) of soybean, is prevalent in the Great Lakes region.
The general life and disease cycle of S. sclerotiorum was described by Purdy (1979) and
Li et al. (1999). Ascospores of S. sclerotiorum are released ﬂom apothecia and germinate
on nonliving or senescent plant parts. Flower petals are also a site of ascospore
germination (Sutton and Deverall, 1983). Germ tubes ﬂom the ascospore penetrate the
plant tissue and colonize the plant tissue to form white cottony masses (Jaumax et al.,
1995; Purdy 1979; Tariq and Jeffries, 1984). The mycelia develop sclerotia in stem or
fruit piths and the sclerotia over winter in the soil. In the mid-summer, sclerotia produce
apothecia in which ascospores are produced. Ascospores are released from the apothecia,
land and infect sensecing ﬂower petals, completing the cycle. As with any disease,
development of SSR depends on the interaction of the susceptible host, virulent pathogen,
and environmental conditions (Agrios, 1997; Boland and Hall, 1988).

Soybean cultivars vary in sensitivity to SSR development (Buzzell et al., 1993;
Kim et al., 1999; Kurle et al., 2001). Differences in disease development in cultivars may
partially be due to maturity differences. Early-maturing cultivars developed less SSR in
some research (Buzzell et al., 1993; Kim and Diers, 2000; Yang, 1999), while no
correlation between cultivar maturity and disease incidence occurred in other research
(Chun et al., 1987; Grau et al., 1982; Kim et al., 1999). These discrepancies on the
correlations between cultivar maturity and disease incidence indicate that differences in
SSR development probably are due to escape mechanisms rather than the factor of

maturity alone.

88

Upright soybean cultivars are associated with less disease incidence (Boland and
Hall, 1987b; Kim et al., 1999), which is consistent with correlations between upright
canopies and white mold development in dry bean (Phaseolus vulgaris L.) (Coyne et al.,
1974; Kolkrnan and Kelly, 2002). Canopy air movement, less moisture, and less disease
has been speculated to occur more in upright soybean genotypes than in lodged
genotypes (Kim et al., 1999).

Cultivar sensitivity to S. sclerotiorum is associated with pedigree and is
independent of cultural practices such as row spacing. Soybean genotypes tracing back to
either cultivar ‘Williams’ or Asgrow ‘A3127’ are more sensitive to S. sclerotiorum (Kim
et al., 1999). Near isolines of glyphosate-resistant and glyphosate-susceptible cultivars
are equally susceptible to SSR (Lee et al., 2000; Nelson, 2000), indicating that the
glyphosate-resistant trait does not affect susceptibility of cultivar to SSR.

Environmental and cultural factors, such as soybean row spacing (Grau and
Radke, 1984), soil moisture (Boland and Hall, 1988), and timing of ﬂower development
in relationship to ascospore release (Boland and Hall, 1988) may inﬂuence SSR
incidence. Narrow row spacings allow the soybean crop to reach full canopy closure
quickly to reduce weed competition (Legere and Schrieber, 1989) and increase yield
potential (Nelson and Renner, 1999). However, planting soybean in narrow rows
increases the risk of disease development (Grau and Radke, 1984). Wide rows have
decreased SSR severity (Grau and Radke, 1984) or have had no impact on SSR severity
(Buzzell et al., 1993). In both studies conducted by Grau and Radke (1884) and Buzzell

et al. (1993), plant population varied across row spacings. Neither study investigated

89

similar plant populations across different row spacings or different plant populations
within a row spacing.

The ﬁrst observation of SSR occurrs aﬂer canopy closure in both white bean (P.
vulgaris L.) (Boland and Hall, 1987a) and soybean (Boland and Hall, 1988). Dense
canopies are associated with increased disease incidence in dry bean (Coyne et al., 1974;
Schwartz et al., 1978). Narrow rows promote a denser canopy that fosters a humid
microclimate necessary for SSR development (Boland and Hall, 1988). Narrow rows also
increase the level of shade beneath the crop canopy. Some soybean cultivars grown under
high levels of shade are more susceptible to SSR than the same cultivars grown under
little or no shade (Pennypacker and Risius, 1999).

Narrow rows are preferred in glyphosate-resistant soybean treated with
glyphosate herbicide to enhance weed control by increasing crop competition with weeds
and reducing the time to crop canopy closure (Nelson and Renner, 1999). Glyphosate
herbicide has no impact on soybean canopy development (Nelson and Renner, 2001), and
the glyphosate and adjuvants used with glyphosate have no impact on SSR severity in
glyphosate-resistant soybean (Lee et al., 2000). Because glyphosate does not injure
glyphosate-resistant soybean, ﬁlll canopy development is quicker in glyphosate-resistant
soybean treated with glyphosate herbicide than in conventional soybean ﬂoated with
conventional herbicides (Nelson, 2000). The glyphosate-resistant soybean system is an
excellent model for investigating the effect of cultural practices on SSR severity because
confounding factors such as reduced soybean canopy or increased phytoalexin production

ﬂom herbicide applications are not present in the glyphosate-resistant soybean system.

90

Glyphosate-resistant soybean comprised nearly 70 % of the United States soybean
acres in 2001. Even though the glyphosate-resistant trait and glyphosate herbicide have
no impact on SSR (Lee et al., 2000; Nelson, 2000), farmers in Michigan reported
increased SSR in glyphosate-resistant soybean cultivars compared to non-transgenic
soybean. The three hypotheses of this study were that glyphosate-resistant soybean
cultivars differed in susceptibility to SSR, wide row spacings would reduce SSR
compared with narrow row spacings, and lower plant populations would reduce SSR
compared with higher populations. Furthermore, shaded portions of the soybean canopy
should be more susceptible to SSR than non-shaded portions, ﬂower number should be
proportional to SSR, and SSR severity should be inversely proportional to yield. The
objectives of this research were to test the forementioned hypothesis.

Three studies were conducted to investigate the effect of soybean cultivar,
population, and row spacing on SSR. The ﬁrst study was conducted in the greenhouse to
determine cultivar susceptibility to S. sclerotiorum. The second study was conducted in
the ﬁeld to determine the effect of cultivar, row spacing, and plant population on S.
sclerotiorum. This study included evaluations of ﬂower number, soil moisture, and
soybean canopy development. The ﬁnal study was conducted in the greenhouse to
determine the effect of shading on the physiological susceptibility of soybean to S.
sclerotiorum. Since the third study supports data ﬂom the ﬁeld, the third study will be

discussed with the ﬁeld study in the section on soybean canopy development.

91

MATERIALS AND METHODS

The glyphosate-resistant cultivars Pioneer 92371, Asgrow AG2701, and Asgrow
AG2702 in the 2.7 maturity group were evaluated in three separate studies. AG2701 and
AGZ702 have bushy architectures, while 92871 is more upright. Although AG2701 and
AG2702 had similar architectures, AG2702 was marketed as having greater tolerance to

SSR (R. Stephenson, Monsanto Co., personal communication).

Cultivar Susceptibility to S. sclerotiorum

Three cultivars were evaluated in the spring in the greenhouse for physiological
susceptibility to S. sclerotiorum. A single seed of each cultivar was planted in BACCTO
(Michigan Peat Co., Houston, TX) potting soil contained in a 946-ml plastic pot. The
total number of pots with seeds was 20 % greater than the actual number of plants
needed. By initially planting extra seeds, uniform plants were selected for investigation.
Environmental conditions in the greenhouse included a 16-h photoperiod supplemented
by sodium-vapor lights that provided a total photosynthetic photon ﬂux density of
approximately 1000 umol m'2 8". Minimum day/night temperatures were set at 27/200 C.
At the V3 stage of grth (Fehr and Caviness, 1977), the second trifoliolate was
harvested for a detached leaﬂet S. sclerotiorum bioassay. Each leaﬂet was inoculated by
placing a 2.4-mm diam. plug of S. sclerotiorum mycelia near the center of the leaf but
away from leaﬂet veins (Dann et al., 1999) and maintained for 48 h at approximately 210
C on a laboratory bench. Mycelia spreading ﬂom the plug to the leaf tissue caused the

development of water-soaked lesions. The vertical, horizontal, and diagonal diameters of

92

each lesion were measured 48 h after inoculation and the average of the three

measurements used in analysis.

Cultivar, Row spacing, and Population Impact on SSR

Field research was conducted in Ingham County, MI (42.7° N lat, 84.5° W long)
in 1999 and 2000. Soil was a Capac loam (ﬁne-loamy, mixed mesic Aeric Ochraqualfs)
with 1.8% organic matter and a pH of 6.5. The year previous to the study, the study site
was planted to corn and no atrazine was used. In the fall after corn, dry bean thrash
infected with S. sclerotiorum sclerotia and mycelia was applied to the test site with a
fertilizer spreader to maintain consistent levels of disease inoculum. The inoculum was
worked into the soil with a chisel plow in the fall. A soil ﬁnisher completed soil
preparation for planting in the spring. This ﬁeld site was irrigated each evening
approximately 2 h before sunset with approximately 1 cm of water beginning when
soybean was at the V4 growth stage and ending when when soybean was at the R3
reproductive stage (Fehr and Caviness, 1977). The water was applied with Buckner 8600
nozzles (Buckner by Strom, Fresno, CA) spaced on 18-m centers. Water propelled ﬂom
the heads at 80 to 100 psi double-overlapped for complete coverage. The daily irrigation
combined with dew resulted in dampened foliage and moistened soil surface until
approximately 9:00 am. the following day.

Cultivars 92B71, AG2701, and AGZ7OZ were planted in row spacing and target
seed population combinations of 76 cm, 430 000 seeds ha"; 19 cm, 430 000 seeds ha";
and 19 cm, 560 000 seeds ha'1 in ﬁeld plots of 3 x 6.3 m. Planting 430 000 and 560 000

seeds ha'l was representative of common planting rates for 76- and 19-cm rows,

93

respectively, in Michigan. Row spacing affect on SSR was determined by comparing

soybean planted in 76- or 19-cm at 430 000 seeds ha". Plant population affect on SSR
was determined by comparing soybean planted in l9-cm rows at 430 000 and 560 000
seeds ha'l. Actual plant populations were counted when soybean plants were at the V2

growth stage (Fehr and Caviness, 1977) and those values are presented in Table 1.

Glyphosate herbicide (formulated as Roundup Ultra, Monsanto Co. St. Louis,
M0) at 480 g a.e. ha’l plus ammonium sulfate [(NH4)2SOa] at 2% w/v was applied to
soybean at V5 grth stage (Fehr and Caviness, 1977) for weed control. The spray
solution was applied with a compressed CO2 backpack sprayer (R & D Sprayers,
Opelousas, LA) traveling 6.6 km h’1 and using size 8002 ﬂat fan tips (Spraying Systems

Co., Wheaton, IL) at a delivery rate of 178 L ha'l at 131 kPa.

A disease severity index (DSI) was recorded at the R6 to R7 soybean growth
stage (Fehr and Caviness, 1977) by rating 15 consecutive plants ﬂom each of the center
two rows of each plot on a scale of 0 to 3, where 0 = no symptoms, 1 = lesions on lateral
branches only, 2 = lesions on the main stem but little or no effect on pod ﬁll and 3 =
lesions on main stem resulting in plant death or poor pod ﬁll (Kim et al. 1999). The
formula used to calculate DSI was: DSI = [Z R/(Nx3)]x100 where R = rating for each

plant and N = number of plants.

Soybean seeds were harvested with a Massey 10 plot harvester (Kincaid
Equipment Manufacturing, Haven, KS) ﬂom the center 1.5 m of each plot and yields

were calculated at 13 % moisture.

94

Canopy Development

Canopy development was monitored at the time of S. sclerotiorum apothecia
(spore cup) development by measuring leaf area index (LAI) obtained by a non-
destructive SunScan Canopy Analysis System (Delta-T Devices Ltd, Cambridge,
England). The Sunscan Canopy Analysis System is comprised of a sensor placed above
the crop canopy and a l-m wand with 64 sensors that record photosynthetically active
radiation beneath the crop canopy. Three measurements were randomly taken in each plot
by laying the l-m probe level at the soil surface and at a 450 angle to the soybean row.
This angle was chosen to minimize damage to the soybean stems ﬂom the wand.
Transmitted, diffused, and incident light was captured by the wand and was compared
with simultaneous readings above the canopy. These readings ﬂom above and below the
soybean canopy were used to estimate LAI (Nelson and Renner, 2001). Measurements
were taken only between 1100 and 1300 h when the zenith angle was near 900.
Measurements were taken at 4, 7, 10, 11, 13, and 16 wk after planting in 1999. Due to
cloudy weather and equipment failure in 2000, LA] measurements were recorded at 4, 8,

and 13 wk after planting.

The inﬂuence of shade on soybean physiological susceptibility to S. sclerotiorum
was examined in the greenhouse and the ﬁeld. In the ﬁeld, levels of shade for cultivars
92B7l and AG2701 were determined in each row spacing and population system. The
third trifoliolates (below canopy) and sixth trifoliolates (above canopy) were harvested
ﬂom three plants in each subplot for both cultivars at the R2 growth stage (Fehr and
Caviness, 1977). The trifoliolates were inoculated with S. sclerotiorum mycelia,

incubated, and evaluated as described earlier.

95

Soybean cultivar 92B7] was examined under three shade scenarios (0, 60, and
90%) in the greenhouse during the winter. Greenhouse conditions were similar to those
described earlier, except that maximum photosynthetic photon ﬂux density ranged ﬂom
550 to 128 mol tn2 8'1 depending upon winter cloud cover. To mimic a ﬁeld scenario,
the third and fourth trifoliolates were placed above the black shade cloth, while the
cotyledons and ﬁrst and second trifoliolates were covered by the shade cloth.
Photosynthetic photon ﬂux density ranged ﬂom 550 to 128, 200 to 50, and 20 to 10 mol
m'2 s'1 for 0, 60, and 90% shade, respectively. The second and third trifoliolates were
harvested 9 d after shade treatment was initiated. The trifoliolates were inoculated with S.

sclerotiorum mycelia, incubated, and evaluated as described earlier.

Flower Number and Soil Moisture

Flower number per plant was counted and soil moisture was monitored once each
week in each plot beginning at 7 and ending near 13 wk after planting. Three plants per
subplot were randomly selected for each ﬂower count in 1999. Six plants per subplot
were randomly selected and marked for all ﬂower counts in 2000. Soil matric potential
was measured throughout the growing season with a Theta Meter Soil Moisture Probe

(Delta-T Devices Ltd, Cambridge, England) at three random locations per subplot.

Statistical Analysis
The cultivar susceptibility study was arranged in the greenhouse in a randomized

complete block design, with four replications per treatment, and repeated. Cultivar was

96

treated as the ﬁxed effect while block and experiment were treated as random effects.
Main effects were analyzed with the GLM procedure in SAS system (SAS, 2000). Means
were calculated with the LSMEANS statement and signiﬁcant differences (p = 0.05)
were separated with the PDIFF option.

The ﬁeld study was a split-plot design, with cultivar as the main plot and row
spacing by population combinations as subplots. The main plots and subplots were
arranged in randomized complete blocks and the study was conducted in 1999 and 2000.
The main effects were analyzed with the MIXED procedure in SAS system (SAS, 2000)
using Satterthwaite's procedure to determine appropriate error degrees of ﬂeedom (Littell
et al., 1996). The MIXED procedure was selected to analyze the ﬁeld study because it
incorporates the appropriate calculations for standard errors for main effects' and simple-
effects' means and comparisons in split-plot designs compared to the GLM procedure
(Littell et al., 1996). Means were calculated with the LSMEANS statement and
signiﬁcant differences (p = 0.05) were separated with the PDIFF option. Cultivar and the
combination of row spacing by plant population were ﬁxed effects. Year and block were
random effects. Disease severity index and yield data were analyzed over both years.
Canopy data was analyzed separately for each year. No interactions occurred for ﬂower
counts or soil moisture and those data were combined over years.

The greenhouse shade study was a split-plot design with soybean plant as the
main plot and trifoliolate position as the subplot. The soybean plants were arranged in a
randomized complete block design with four replications and the study was repeated.

Main effects were analyzed with the GLM procedure in SAS system (SAS, 2000). Means

97

were calculated with the LSMEAN S statement and signiﬁcant differences (p = 0.05)

were separated with the PDIFF option.

98

RESULTS AND DISCUSSION

Cultivar Susceptibility to S. sclerotiorum

Leaﬂets of the three cultivars had similar levels of susceptibility to advancing S.
sclerotiorum mycelia (data not shown). These similarities in susceptibility indicate that
any difference detected in SSR development were likely due ﬂower numbers, canopy

development, microclimate, or combinations thereof.

Cultivar, Row spacing, and Population Impact on SSR

Soybean cultivars differed in susceptibility to SSR in the ﬁeld. Cultivar 92B7]
had the lowest SSR levels and the highest yields at all row spacings and plant
populations. Disease severity index values combined over row spacing and plant
populations were 17 and 43 % lower in AG2701 and 92B71, respectively, compared to
AG2702 (Table 2). Seed yield combined over row spacing and populations was highest in
92371 at 3570 kg ha'l whereas yield was 20 and 39% lower in AGZ701 and AG2702,

respectively (Table 2).

The differences in SSR between cultivars may be inﬂuenced by the population of
each cultivar. Cultivar 92B7] had an average population of 129 373 plants ha", which
was 9% and 20% less than the average populations of A6270] and AGZ7OZ, respectively
(Table 1). Although 92B7] appeared more tolerant to SSR than the other cultivars,

92B7] may have had lower disease severity due the effects of a decreased population.

The severity of SSR was 22% lower in 19-cm rows when population was reduced

ﬂom target populations of 560 000 to 430 000 seeds ha’l (Table 2). Although a difference

99

in DSI was observed between plant populations, no differences in yield between target
populations in 19-cm rows was determined (Table 2). While yields between two target
populations at 19-cm rows were similar, increased actual plant population for the entire
study was positively correlated with DSI (r2 = 0.33 (p<0.0001)) and negatively correlated

with yield (r2 = -013 (p=0.0140)).

Soybean in wide row spacings resulted in similar levels of SSR compared with
soybean in narrow row spacings. Although soybean planted in 19 and 76 cm rows at a
target population of 430 000 seeds ha'l had the similar levels of SSR, soybean in 19 cm

rows had a 13% greater yield than soybean in 76 cm rows (Table 2).

Canopy Development

Canopy development in 1999 was similar for each of the three cultivars ﬂom 4
wk after planting until 7 wk after planting (Figure 1). Cultivar 92871 had a smaller
canopy than AG2702 at 8 and 10 wk after planting in 1999; however, no differences in
canopy development were detected by 11 wk after planting (Figure 1). No differences in
cultivar canopy development were detected in 2000 (Figure 2). The reduced canopy in
92871 ﬂom 4 to 7 wk after planting could have been due to a combination of its upright
architecture and to the reduced plant population compared the other cultivars. The similar
canopies among the three cultivars by 11 wk after planting in 1999 and throughout 2000
could have been due to the expected increase in plant biomass due to daily irrigation
(Ball et a1. 2000). In a non-irrigated system, which is typical of Michigan soybean

production, the differences in canopy between the upright and bushy cultivars likely

100

would be of a greater magnitude. The differences in canopy development would likely
have increased differences in soil moisture (Grau and Radke, 1984).

Leaf area index was signiﬁcantly lower for soybean planted in 76-cm rows at 430
000 seeds ha’l than the other two planting conﬁgurations at 7, 8, and 10 wk after planting
in 1999; however LAI was similar for all three planting conﬁgurations by 11 wk after
planting (Figure 18). No differences in canopy development for all three planting
conﬁgurations were detected at 8 and 13 wk after planting in 2000. Decreased canopy
development is typically associated with decreased SSR levels (Grau and Radke, 1984).
However, lower SSR severity was not associated with decreased canopy development in
this study.

Shade caused by differences in canopy development did not explain SSR
development in the ﬁeld. Leaﬂets ﬂom soybean grown in the ﬁeld under different row
spacings and population showed no signiﬁcant differences in lesion development caused
by advancing S. sclerotiorum mycelia (Table 3), nor were differences detected between
cultivars (Table 3). Shade on the lower portion of the soybean plant, resulting ﬂom the
soybean canopy, did not increase shaded leaﬂet susceptibility to S. sclerotiorum in the
ﬁeld (Table 3). In contrast, leaﬂets beneath shade in the greenhouse were more
susceptible to S. sclerotiorum (Table 4). Lesion diameters were larger on leaﬂets grown
under 90% shade than on leaﬂets grown at the 0 or 60% shade levels (Table 4).

Shade leaf response in the ﬁeld and greenhouse did not concur. Chun et al.
(1987), Pennypacker and Risius (1999), and Nelson et al. (1991) observed similar
discrepancies between the greenhouse and ﬁeld. Differences in soybean response to S.

sclerotiorum in the greenhouse and ﬁeld may be due in part to differences in

101

photosynthetic photon ﬂux densities (Nelson et al. 1991). On a clear, bright day,
photosynthetic photon ﬂux densities can be as high as 4600 umol rn'2 s'l (Fitter and Hay,
1987), while maximum photosynthetic photon ﬂux densities in the greenhouse was
recorded near 1000 and 550 mol m'2 8'1 during the summer and winter, respectively.
Photosynthetic photon ﬂux density in the ﬁeld was likely too high in the shaded
conditions to result in reduced photosynthesis. Reduced photosynthetic photon ﬂux
densities could result in reduced respiration and a reduced photosynthetic rate (Fitter and
Hay, 1987). Pennypacker and Risius (1999) speculated that a reduced photosynthetic rate
could decrease the physiological response of soybean to a pathogen. That hypothesis
agrees with the observation that reduced photoperiods caused sunﬂower cultivars to be
susceptible to SSR while increased photoperiods resulted in tolerance to SSR (Orellana,

1975)

Flower Numbers and Soil Moisture
Flower numbers for each cultivar were statistically similar at 9 wk after planting

when apothecia were ﬁrst observed. By 11 wk after planting, ﬂower numbers were
signﬁcantly highest in 92871 (Figure 3). Higher ﬂower counts on 92871 at 2 wk
following apothecia development appear to contradict the results of Kim and Diers
(2000) where the timing of ﬂower development in relation to ascospore release ﬂom
apothecia was critical to SSR development. However, ﬂower counts could have been
higher on AG2701 and AG2702 than 92871 in weeks that ﬂowers were not counted.

Flower numbers were similar for all row spacing and population subplots ﬂom 8 to 12

102

wk after planting (Figure 3). Flower numbers do not explain differences in SSR
development between cultivars or row spacing and population systems.

Soil moisture did not explain the differences in SSR development. Differences in
soil moisture were detected sporadically in this study with the wide row system having
slightly drier soils at times (data not shown). The increased ﬂequency of wetting and
drying of soil that accompanies wide row spacings (Grau and Radke, 1984) was negated
in this study by daily irrigation, and may explain why 76-cm rows had greater SSR levels
than 19-cm rows (Table 2). The greater SSR levels in 76-cm rows may not occur in non-
irrigated systems where soils may be drier in wider rows.

Since canopy development, ﬂower number, and soil moisture were not associated
with SSR development in this irrigated study, arrangement of soybean plants within the
ﬁeld may explain some of the differences in SSR. Average actual spacing between plants
within a row was 18 and 4 cm for 19- and 76-cm rows, respectively, at a target
population of 430 000 seeds ha". The reduced spacing between plants within the 76-cm
row may have increased plant-to—plant transfer of S. sclerotiorum mycelia (Boland and

Hall, 1988).

103

SUMMARY

Differences in glyphosate-resistant cultivar susceptibility to SSR were found in
this study. Although farmers may choose glyphosate-resistant soybean for weed control
purposes, farmers in areas of high risk to SSR need to select soybean cultivars that are
tolerant to SSR for effective management of SSR (Kurle et al., 2001). The lower D81 and
higher yield in the l9-cm rows at 430 000 seeds ha'l were not explained by the canopy
development, shading, ﬂower number, or soil moisture, but may be attributed to three
other factors. The ﬁrst factor is that glyphosate herbicide applied to glyphosate-resistant
soybean results in quicker canopy development compared to other herbicides applied to
glyphosate-susceptible soybean. The second factor could be that daily irrigation of the
ﬁeld reduced differences in soil drying that normally occur between wide and narrow
rows. The third factor could be related to the arrangement of soybean plants within a
given area. In this study, soybean plants within 19-cm rows at a target population of 430
000 seeds ha'l had an average stand of 342 000 plants ha'1 and an average of l8-cm
between plants while soybeans in 76-cm rows at an average stand of 310 000 plants ha'l
had an average of 4-cm between plants. With differences in canopy and soil moisture
minimized, DSI in the 76-cm rows may be a result of plant-to-plant transfer of S.

sclerotiorum.

Soybean population within a row spacing appears to be an important
consideration for the management of SSR. Reducing soybean population ﬂom a target
population of 560 000 to 430 000 seeds ha‘1 in 19-cm rows decreased SSR, possibly
because of increasing within-row plant spacing from 4 to 18 cm. Managing SSR in

glyphosate-resistant soybean should rely on choosing SSR tolerant cultivars, and planting

104

those SSR tolerant cultivars at 430 000 seeds ha”1 in 19-cm rows. Further research should
determine if actual stand counts of reductions of soybean population in l9-cm rows
reduce the incidence of SSR; however, establishing a stand with uniform plant spacing
within the row at these low populations with a drill may be difﬁcult. Reducing soybean
population within 19-cm rows may be as effective as increasing row spacing to 76-cm

rows for managing SSR.

105

ACKNOWLEDGEIVIENTS

The authors would like to thank the Michigan Soybean Promotion Committee for
sponsoring this research, Pioneer Hi-Bred for donating seed, Monsanto Company for
donating seed and herbicide. The authors would also like to thank Gary Powell and John

Boyse for their help in this project.

106

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109

Table 1. Targeted and actual plant populations.

 

 

Row Target Actual Percent of
Cultivar Spacing Population Population Target
cm Plants ha'I
92871 76 430 000 300 000 70
19 430 000 260 000 60
19 560 000 400 000 72
AG2701 76 430 000 364 000 85
19 430 000 297 000 69
19 560 000 433 000 78
AGZ702 76 430 000 363 000 84
19 430 000 373 000 87
19 560 000 501 000 90

 

110

Table 2. Disease severity index (DSI) and yield for main plots of cultivar and subplots of
row spacing by seed population combined over years. Letters following least squared
means indicate signiﬁcant differences at p = 0.05.

 

 

DSI Yield
Cultivar ' (kg ha")
92871 34 c 3570 a
AGZ701 50 b 2890 b
AGZ702 59 a 2220 c
Row spacing; Seed Population
(cm; seeds ha")
76; 430 000 50 ab 2690 b
19; 430 000 42 b 3100 a

19; 560000 53 a 2800 ab

 

111

Table 3. Lesion diameters resulting ﬂom Sclerotinia sclerotiorum growth on detached
leaves ﬂom 92871 and AGZ702 soybean grown in the ﬁeld at different row spacings and
populations. Leaﬂets were harvested ﬂom R1 soybean. Letters following least squared

means represents signiﬁcant differences at p = 0.05.

 

 

Cultivar Row spacing; Trifoliolate above Trifoliolate below

stand population canopy canopy
(6th trifoliolate) (3" trifoliolate)
Lesion diameter

(cm; plants ha")

92871 76; 299 110 10.5 a 8.3 a **"
19; 259 301 10.9 a 9.2 a
19; 400 246 10.5 a 8.7 a

A6270] 76; 362 589 9.5 a 8.5 a
19; 373 348 11.2 a 9.2 a
19; 501 385 10.4 a 9.2 a

 

“ Signiﬁcant difference (p=0.05) between S. sclerotiorum lesion diameters on 3rd and 6“1

trifoliolates within a row width and plant population. A blank indicates no signiﬁcant

differences.

112

Table 4. Lesion diameters resulting ﬂom Sclerotinia sclerotiorum growth on detached
leaves of 92871 soybean grown under various levels of shade in the greenhouse. Soybean
was at the V5 grth stage at the time of leaf harvest. Letters following least squared
means represent signiﬁcant differences at p = 0.05.

Shade‘ Trifoliolate above shade Trifoliolate below shade
(3rd trifoliolate) (2"d trifoliolate)

Lesion diameter

 

% (mm)

0 13.9 a 15.1 b

60 16.7 a 13.9 c

90 16.4 a 18.6 a "b

 

a Maximum photosynthetic photon ﬂux density ranged between 550 and 128 umol m’2 s".
b Signiﬁcant difference (p=0.05) between S. sclerotiorum lesion diamters between 2"d
and 3rd trifoliolates within a shade regime. A blank indicates no signiﬁcant differences.

113

Leaf Area Index (LA!)

Leaf Area Index (LA!)

10'
9.
3.
7a
5.
5']
4a
3.
2.
1.

 

Differences in LAI Between Cultivars in 1999

+AGZ701
-I-AGZ702
+ 92371

  

 

1B

4 7 8 10 11 13 16
Week: After Planting

Differences in LAI Between Row Widths by Populations in 1999

 

 
 
    

 

1° 7 +111".
9 l -l-NL
8 ‘ +1!!!
7 i
6 -i
5 4
4 q
3 ‘ us
2 d
1 2 First Observation
0 «We
4 7 8 10 11 13 16
Week: After Planting

Figure I . Leaf area index in 1999 for the three cultivars (A) and the three row spacings
by populations (8) during the growing season. Differences of the least squared means
were detected with the PDIFF option in SAS at p = 0.05. WL = wide row, low
population: 76 cm, 430 000 seeds ha"; NL = narrow row, low population: 19 cm, 430
000 seeds ha"; NH = narrow row, high population: 19 cm, 560 000 seeds ha".

114

Differences in LAI Between Cultivars in 2000

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Differences in LAI Between Row Widths by Populations in 2000

 

 

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Figure 2. Leaf area index in 2000 for the three cultivars (A) and the three row spacings
by populations (8) during the growing season. Differences of the least squared means
were detected with the PDIFF option in SAS at p = 0.05. WL = wide row, low
population: 76 cm, 430 000 seeds ha"; NL = narrow row, low population: 19 cm, 430
000 seeds ha"; NH = narrow row, high population: 19 cm, 560 000 seeds ha".

115

3A
Flower Differences Between Cultivars

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Flowers Differences Between Row Widths by Populations

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Figure 3. Differences in average ﬂower number per plant for the three cultivars (A) and
the three row spacings by populations (8) during the growing season. Values are
averaged over the subplots of row spacing by population and combined over years.
Letters above bars represent signiﬁcant differences between least squared means at p =
0.05. WL = wide row, low population: 76 cm, 430 000 seeds ha"; NL = narrow row, low
population: 19 cm, 430 000 seeds ha"; NH = narrow row, high population: 19 cm, 560
000 seeds ha".

116

SUMMARY

Formulated glyphosate and activator adjuvants did not increase soybean
susceptibility to infection ﬂom S. sclerotiorum. The two cultivars displayed similar levels
of sensitivity to S. sclerotiorum, indicating that the glyphosate-resistant trait does not
cause an increase in plant susceptibility to Sclerotinia stem rot (SSR). When treated with
glyphosate, the glyphosate-resistant cultivar ‘GL2600RR’ did not accumulate Shikimate
acid as did the glyphosate-susceptible cultivar ‘GL2415’. Soybean plant extracts
containing glyceollin inhibited mycelial grth in a dose-dependent manner, suggesting
that these compounds inhibited S. sclerotiorum mycelia growth on soybean leaﬂets.
Glyphosate applied to glyphosate-resistant soybean did not reduce the plant defense
system response within the soybean plant. Synthesis of glyceollin was related to inhibited
hyphae growth. Shade at 60% did not inhibit plant defenses against S. sclerotiorum.
Differences in glyphosate-resistant cultivar susceptibility to SSR were found in this
study. Flower numbers were similar among cultivars when apothecia were present. The
lower DSI and higher yield in the 19-cm rows at 430 000 seeds ha'l were not explained
by the canopy development, shading, or soil moisture, but may be attributed to three
factors. The ﬁrst factor is that glyphosate herbicide applied to glyphosate-resistant
soybean results in quicker canopy closure compared to other herbicides applied to
glyphosate-susceptible soybean. The second factor could be that daily irrigation of the
ﬁeld reduced differences in soil drying that normally occur between wide and narrow
rows. The third factor could be related to placement of soybean plants within a given
area. Soybean planted at 430 000 seeds ha'I in 19-cm rows have more spacing between

each plant (18 cm) than do soybean planted at the same population but in 76-cm rows (4

117

 

cm). With differences in canopy and soil moisture minimized, the increased DSI in the
76-cm rows may be a result of plant-to-plant transfer of S. sclerotiorum and an increase

in ﬂower number per meter row because of an increase in plant number per meter row.

118

EXTENSION SUMMARY

White mold disease in soybean is prevalent in Michigan and surrounding states.
White mold disease is caused by the fungus Sclerotinia sclerotiorum, which can infect
many crops including soybean, dry bean, snap bean and sugarbeets. Roundup Ready®
soybean comprises approximately 60 to 70% of the soybean acreage in Michigan. When
Roundup Ready soybean was ﬁrst introduced into the state, farmers reported higher
levels of white mold in their Roundup Ready soybean ﬁelds. Research was conducted to
determine the possible cause for these observations.

A Roundup Ready soybean, GL26OORR, and its conventional near-isoline,
GL2415, were found to be equally susceptible to white mold. Glyphosate herbicide,
formulated as Roundup Ultram, did not increase white mold disease development in
soybean. Crop oil concentrate, nonionic surfactant, and organosilicone adjuvants also had
no effect on white mold disease in soybean. In the laboratory high doses of the adjuvants
contained in Roundup Ultra actually reduced growth of the fungus, Sclerotinia
sclerotiorum. Crop oil concentrate, nonionic surfactant, and organosilicone adjuvants also
reduced growth of the fungus. Glyphosate did not impair soybean defenses against white
mold in laboratory investigations.

Three Roundup Ready soybean varieties were planted at three row spacings and
plant populations to investigate susceptibility to white mold. The three soybean varieties
were Pioneer ‘92871’, Asgrow ‘2701 ’ and Asgrow ‘2702’. All three varieties were
planted in 30-inch rows at 175,000 seeds/A, 7.5-inch (drilled) rows at 175,000 seeds/A,
and 7.5-inch rows at 225,000 seeds/A. Soybean in Michigan are typically planted in 30-

inch rows at about 175,000 seeds/A and in drilled rows at about 225,000 seeds/A.

119

Soybean variety 92871 was more tolerant to white mold than AG2701 or AG2702 at all
three row spacings and populations (Figure 1A). Flowers numbers were similar on all
varieties at the time of white mold development. 92871 was also the highest yielding
variety in the presence of white mold (Figure 18).

Less white mold developed in soybean planted in 7.5-inch rows at 175,000
seeds/A than in soybean in 7.5-inch rows at 225,000 seeds/A or in 30-inch rows at
175,000 seeds/A (Figure 2A). Soybean drilled at 175,000 seeds/A produced the highest
yield in the presence of white mold (Figure 28). The observation of lower white mold
levels in the drilled rows at 175,000 seeds/A than in the 30-inch rows at 175,000 seeds/A
may be due to three factors. The ﬁrst factor is that glyphosate herbicide applied to
Roundup Ready soybean results in quicker soybean canopy development compared to
other herbicides applied to conventional soybean. The second factor could be due to daily
irrigation of the ﬁeld. This research was irrigated daily to provide a suitable microclimate
for disease development. Daily irrigation likely reduced differences in soil drying that
normally occur between wide and narrow rows. Daily irrigation also likely reduced
differences in soybean canopy architecture. Avoidance mechanisms would have been
minimized. The third factor could be related to placement of soybean plants within a
given area. Soybean planted at 175,000 seeds/A in drilled rows have more spacing
between each plant within a row (7.1 inches) than soybean planted at the same population
30-inch rows (1.5 inches). The increased white mold in the 30-inch rows may be a result
of plant-to-plant transfer of S. sclerotiorum.

Applying glyphosate to Roundup Ready soybean had no impact on white mold

disease development. Selecting a white mold tolerant soybean variety was more

120

important than soybean row spacing or plant population for management of white mold
disease. When farmers are selecting soybean varieties based on weed management
decisions, they should also consider disease tolerance before deciding what row spacing
and plant population to use. Drilling white mold tolerant varieties at lower populations
(175,000 seeds/A instead of 225,000 seeds/A) may be as effective as planting soybean in
wide rows for management of white mold, but further research needs to be conducted in
this area.

Reports ﬂom university white mold tolerance trials are excellent resources for
ﬁnding white mold tolerant varieties. Most reports include a white mold disease severity
rating and yield for each variety. Yield under the pressure of white mold is typically the
best indicator of variety tolerance to white mold. If at all possible, compare variety

tolerance to white mold over several sites and at least two years.

121

A Variety Effect on White Mold Disease Severity as Measured by the D51*

 

 

 

 

 

 

 

 

 

   

 

   

 

 

 

 

 

 

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Figure I. Roundup Ready soybean variety effect on white mold disease levels (A) and
yield (8). White mold disease levels are determined by a disease severity index (DSI)
where 0 = no disease detected, and 100 = complete pod damage or plant death ﬂom white
mold.

122

A Row Width by Population Effect on White Mold Disease Severity as
Measured by the 051*

 

 

 

 

 

 

    

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3 Row Width by Population Effect on Soybean Yield in the Presence of
White Mold*
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*Combined over varieties and years.

Figure 2. Row spacing and population effect on white mold disease severity (A) and
soybean yield (8). White mold disease levels are determined by a disease severity index
(DSI) where 0 = no disease detected, and 100 = complete pod damage or plant death ﬂom
white mold.

123

      
 

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