STUDY OF CHEMICAL AND MICROBIAL-MEDIATED STRATEGIES FOR MANAGING
SCLEROTINIA STEM ROT OF SOYBEAN (GLYCINE MAX L.)
By
Carolina Escobar-Ochoa

A THESIS
Submitted to
Michigan State University
in partial fulfillment of requirements
for the degree of
Plant Pathology - Master of Science
2014

ABSTRACT
STUDY OF CHEMICAL AND MICROBIAL-MEDIATED STRATEGIES FOR MANAGING
SCLEROTINIA STEM ROT OF SOYBEAN (GLYCINE MAX L)
By
Carolina Escobar-Ochoa
The management of Sclerotinia stem rot of soybean caused by [Sclerotinia sclerotiorum
(Lib) de Bary] integrates several tactics that may include biological control. However, the
successful incorporation of biocontrol agents into the management program might be hindered
by agrochemical application. To determine the compatibility of potential biocontrol agents with
agrochemicals, growth of Trichoderma asperellum, T. gamsii and Coniothyrium minitans was
tested with selected chemicals on agar plates. Effects of selected agrochemicals on C. minitans
growth and biocontrol activity were also tested in soil and in water. On amended agar media,
Trichoderma spp. and C. minitans were insensitive to the herbicide lactofen and were inhibited
to varying degrees by some of the tested fungicides. The survival and the biocontrol activity of
C. minitans in the soil was not impaired by propamocarb HCl, tetraconazole or lactofen
treatments. When mixed in a water suspension, none of the selected the herbicides or a
biofertilizer affected C. minitans population in the suspension over a 4 h period, suggesting that
they can be applied in a tank mix. Under field conditions, Trichoderma spp. and C. minitans
reduced the disease severity and viable sclerotial population of S. sclerotiorum, but their efficacy
varied across years and location. Co-application with lactofen enhanced the efficacy of disease
suppression and yield in some cases but reduced the efficacy in others. The effect of the
biological control agents or chemicals evaluated on yield during three years of trials was
inconsistent.

To my mother Martha for showing me the value of hard work and discipline, to my father
Alfonso for teaching me to pursue my dreams with optimism, to my brother Mario for being my
confidence provider, to my husband Jose Luis for his love and unconditional support, and to
Jeronimo for being my motivation now and forever, I dedicated this thesis.

iii

ACKNOWLEDGEMENTS

I would like to thank my major advisor Dr. Ray Hammerschmidt for accepting me as one
of his student, with a contagious optimism. I would also like express my gratitude to Dr. Jianjun
Hao for his instruction, patience, friendship, dedication, and his confidence in me. I’ll always
appreciate the chance that Dr. Hao has given to my family and I to make our dreams come true. I
sincerely thank Drs. Linda Hanson and Martin Chilvers for serving on my graduate advisory
committee and for their excellent contribution to my scientific training as well as their warmth
and kindness to me.
I would want to express my gratitude to people in Hao’s lab. I am grateful to Michelle
Meng for her friendship and help during my graduate study; I especially appreciate Helen Jiang
for her active contribution to my research and her lovely sense of humor. Dr. Noah Rosenzweig
helped and provided me with his critical view on my research over these three years. I cherish
our friendship. Gary Zehr, Rob Shafer, Jerry Skeltis, and especially John Boyse and Randy
Laurenz unconditionally supported me in fieldwork. Samantha Adamovicz has committed
laboratory assistance. I appreciate Linzi Kaniszewski for her companionship and help with some
laboratory techniques and Juan Bosco for helping me with the statistical analysis. The Michigan
Soybean Promotion Committee (MSPC) and Michigan State University Project GREEEN
provided partial funding for research. I deeply appreciate the encouragement and support of my
lovely family, even being far from my home.

Carolina Escobar-Ochoa

iv

TABLE OF CONTENTS

LIST OF TABLES ........................................................................................................................ vii
LIST OF FIGURES ....................................................................................................................... ix
CHAPTER ONE: LITERATURE REVIEW .................................................................................. 1
SOYBEAN ...................................................................................................................................... 1
SCLEROTINIA SCLEROTIORUM ................................................................................................. 3
Biology and life cycle ................................................................................................................. 3
Host range ................................................................................................................................... 4
Molecular mechanisms of pathogenicity .................................................................................... 5
SCLEROTINIA STEM ROT .......................................................................................................... 6
Symptoms.................................................................................................................................... 6
Disease cycle ............................................................................................................................... 6
Epidemiology .............................................................................................................................. 8
DISEASE MANAGEMENT .......................................................................................................... 9
Tillage and crop rotation ............................................................................................................. 9
Resistant cultivars ....................................................................................................................... 9
Chemical control. ...................................................................................................................... 10
Resistance inducing chemicals .................................................................................................. 12
Biological control. ..................................................................................................................... 13
Integrated disease management ................................................................................................ 17
OBJECTIVES OF THIS STUDY ................................................................................................. 19
CHAPTER TWO: SENSITIVITY OF BIOCONTROL AGENTS TO PESTICIDES ................ 20
ABSTRACT .................................................................................................................................. 20
INTRODUCTION ........................................................................................................................ 21
MATERIALS AND METHODS .................................................................................................. 24
Sensitivities of biological control agents to pesticides in vitro ................................................. 24
Isolation of biological control agents from commercial products ........................................ 24
Preparation of inoculum of biological control agents and pesticides ................................... 25
Sensitivity of biological control agents to pesticides using a spiral gradient dilution
method .................................................................................................................................. 26
Survival of Coniothyrium minitans with herbicides and fertilizers under tank mix conditions 27
Inoculum preparation ............................................................................................................ 27
Herbicides and fertilizer tested ............................................................................................. 27
Evaluation of Coniothyrium minitans survival ..................................................................... 28
Effect of pesticides on Coniothyrium minitans survival and its biocontrol activity under
growth chamber conditions ....................................................................................................... 28
Pesticides and commercial biological control preparation ................................................... 28
Soil infestation with pesticides and Coniothyrium minitans ................................................. 29
v

Soil sample collection and processing .................................................................................. 29
Sclerotia viability .................................................................................................................. 30
Data Analysis ............................................................................................................................ 30
RESULTS ..................................................................................................................................... 31
Sensitivity of biological control agents to pesticides in vitro ................................................... 31
Survival of Coniothyrium minitans with herbicides and fertilizers under tank mix condition . 32
Effect of pesticides on Coniothyrium minitans survival and its biocontrol activity under
growth chamber conditions ....................................................................................................... 34
DISCUSSION ............................................................................................................................... 37
CHAPTER THREE: MANAGEMENT OF SCLEROTINIA STEM ROT BY INTEGRATION
OF BIOCONTROL AND CHEMICAL STRATEGIES UNDER FIELD CONDITION ............ 42
ABSTRACT .................................................................................................................................. 42
INTRODUCTION ........................................................................................................................ 43
MATERIALS AND METHODS .................................................................................................. 46
Field plots .................................................................................................................................. 46
Soil infestation with Sclerotinia sclerotiorum .......................................................................... 46
Soil and foliar treatments .......................................................................................................... 47
Viability of sclerotial population in soil ................................................................................... 48
Disease evaluation..................................................................................................................... 48
Soybean yield evaluation .......................................................................................................... 50
Number of sclerotia in harvested beans .................................................................................... 50
Data analysis ............................................................................................................................. 50
RESULTS ..................................................................................................................................... 51
Disease severity index (DSI) and density of sclerotia in beans ................................................ 51
Sclerotia viability in the soil ..................................................................................................... 56
Yield evaluation ........................................................................................................................ 58
DISCUSSION ............................................................................................................................... 63
APPENDIX ................................................................................................................................... 68
LITERATURE CITED ................................................................................................................. 78

vi

LIST OF TABLES

Table 2-1. Pesticides used in the soybean cropping system in the U.S. included for the evaluation
of commercial biological control agents sensitivity test. All chemicals are fungicides with
the exception of lactofen, a post emergence herbicide. .................................................... 25
Table 2-2. Description of treatments combinations included to evaluate the effect of two selected
fungicides and one herbicide on a commercial formulation of Coniothyrium minitans
(CON/M/91-08) survival and its biological control activity against Sclerotinia
sclerotiorum under 20°C, 14h light:10 h dark. ................................................................. 30
Table 2-3. Effective concentration for 50% reduction of mycelial growth (EC50) values for
fifteen fungicides and one herbicide. Data are for three biological control fungi as
determined by the spiral gradient dilution (SGD) method................................................ 32
Table 3-1. Biocontrol agents and chemical treatments used in this study with rates of application
and dates used to test the efficacy against Sclerotinia stem rot of soybean at the Plant
Pathology Farm (PLP) and Clarksville Horticultural Experimental Station (CKL) from
2010, 2011 and 2012. ........................................................................................................ 49
Table 3-2. Effect of biological control agents Coniothyrium minitans, Trichoderma asperellum,
T, gamsii, Streptomyces lydicus; a plant extract from Reynoutria sachalinensis or a
fungicide boscalid on Sclerotinia sclerotiorum viable sclerotia in soil and harvested
beans, severity of Sclerotinia stem rot as expressed as a disease severity index (DSI) and
yield of soybean in 2010 at two field locations: the Plant Pathology Farm (PLP) and the
Clarksville Horticulture Experimental Station (CLK). ..................................................... 54
Table 3-3. Effect of biological control agents and the herbicide lactofen applied individually or
in combination on severity of Sclerotinia stems rot, number of sclerotia in harvested
beans and yield of soybean. Different times of application were included for the C.
minitans treatments: Fall, Spring and Fall-Spring. The experiment was conducted at the
Plant Pathology Farm in 2011........................................................................................... 55
Table 3-4. Effect of biological control agents applied at varying times in absence/combination
with the herbicide lactofen on soil levels of Sclerotinia sclerotiorum viable sclerotia,
severity of Sclerotinia stems rot, number of sclerotia in harvested beans and yield of
soybean at Clarksville Horticulture Experiment Station in 2011. .................................... 57
Table 3-5. Effect of biocontrol agents with foliar or ground application in absence/presence of
the herbicide lactofen or with thiophanate-methyl on soil levels of Sclerotinia
sclerotiorum viable sclerotia, severity of Sclerotinia stem rot, number of sclerotia in
harvested beans and yield of soybean at the Plant Pathology Farm in 2012. ................... 59

vii

Table 3-6. Effect of biocontrol agents with foliar or ground application in absence/presence of
the herbicide lactofen or with thiophanate-methyl on soil levels of Sclerotinia
sclerotiorum viable sclerotia, severity of Sclerotinia stem rot, number of sclerotia in
harvested beans and yield of soybean at the Clarksville Horticulture Experiment Station
in 2012. ............................................................................................................................. 60
Table 3-7. Summary effects of the biological control agents evaluated at different time, mode of
application or in absence/combination with the herbicide lactofen on Sclerotinia stem rot
severity and yield of soybean at Plant Pathology Farm in 2010, 2011 and 2012. ............ 61
Table 3-8. Summary effects of the biological control agents evaluated at different time, mode of
application or in absence/combination with the herbicide lactofen on Sclerotinia stem rot
severity and yield of soybean at Clarksville Horticultural Experimental Station in 2010,
2011 and 2012. .................................................................................................................. 62

viii

LIST OF FIGURES

Figure 2-1. Effect of 5 herbicides (below) and a bio-fertilizer (Accomplish LM®) on
Coniothyrium minitans population under tank mix conditions. Active ingredient,
commercial name, percentage of active ingredient (a.i.) and field rate are listed below:
metolachlor (Stalwart® 86.4% a.i., 4,677 g/ha), sulfentrazone and cloransulam -methyl
(Sonic®, 62.1% a.i., 7.9% a.i., 210.1g/ha), sulfentrazone and chlorimuron-ethyl
(Authority XL® 33.33% a.i, 6.67% a.i.; field rate 451.83 g/ha), simazine (Simtrol®
42.8% a.i., 4,677 g/h), glyphosate (Roundup Power Max®, 48.7% a.i., 607.8 g/ha),
Bacillus spp., Acidovorax facilis and Rhodococcus rhodochorus (Accomplish LM®,
Loveland Products Inc. Loveland, CO; 4,677 g/ha). Sterile water was used as a control.
Each column is the average of 32 observations (mean of three replicates per isolate) and
the experiment was conducted twice. Bars on each column are standard deviation. ....... 33
Figure 2-2. Time effect on the survival of Coniothyrium minitans. Samples were collected at 1,
2, and 7 days after treatment. Coniothyrium minitans population was detected and
enumerated by plating on semi-selective media. Columns represent the average of 32
observations of combined experiments. Error bars indicate standard deviation. Mean of
C. minitans growth followed by same letter are not significantly different at α = 0.05
(Tukey-Kramer HSD). ...................................................................................................... 35
Figure 2-3. Biological control activity of Coniothyrium minitans on viability of sclerotia of
Sclerotinia sclerotiorum in a mixture of potting mix (BACCTO Professional Planting
Mix, Michigan Peat Company, Houston TX 70-80% sphagnum peat) and a natural field
soil with sand and bailed peat (v:v=1:1). Ten sclerotia were used per pot. The sclerotia
were buried in potting mix: soil mixture and retrieved 10 days after C. minitans
application. Their viability was determined by observing mycelial germination on water
agar. Since no significant difference between C. minitans alone or in combination with
pesticide treatments were found (P = 0.601 or P = 0.522 respectively), the treatments
were combined over C. minitans treated and non-treated pots. Columns represent the
average of 48 observations of combined experiments. Error bars indicate standard
deviation. Means of number of viable sclerotia in each treated and non-treated control
were compared by Tukey-Kramer HSD at α = 0.05. “*” Significant difference at P <
0.001.................................................................................................................................. 36
Figure 4-1. Cotton pad inoculation method and Sclerotinia stem rot symptoms. A. The
inoculation was done on the petiole of the lowest node bearing flowers of the soybean cv.
GL2449R2 at the R1 growth stage with a cotton pad saturated with a mycelial suspension
of S. sclerotiorum (10 4 cfu/ml). B. Typical symptoms of Sclerotinia stem rot: stem
discoloration and leaf wilting 5 d after inoculation. ......................................................... 74
Figure 4-2. Panel A and Panel B. Effect of the herbicide lactofen application at the V2 (A) and
the V6 (B) soybean growth stages on Sclerotinia stem rot severity in soybean (cv.
ix

GL2449R2). The plants were all inoculated with Sclerotinia sclerotiorum at the R1
growth stage and were rated 20 to 25 days after inoculation. Disease severity was
evaluated on a 0 to 5 scale (0 = no symptoms and 5 = general wilting/plant dead). A
disease severity index (DSI) was calculated using the formula: DSI (%) = Σ (scores of all
plants)/[5 × (total number of plants)] × 100. A total of 30 plants were rated per lactofen
concentration and the experiments were repeated. Soybean plants treated with destilled
water were used as control for both trials. Bars indicate standard deviation. Means
followed by same letter are not significantly different at α = 0.05 (Ryan-Gabriel-WelshMultiple Range Test). ....................................................................................................... 75

x

CHAPTER ONE: LITERATURE REVIEW
SOYBEAN
The soybean (Glycine max L. Merr.) is an important crop globally that is cultivated
widely in countries such as Brazil, Argentina, China, India, and the United States (Singh and
Hymowitz 1999). Soybeans were first domesticated in Northeast China around 1500 B.C.
(Hymowitz 2004, Stacey 2008) and introduced in the United States (U.S.) in 1765 but were not
formally recognized as a crop until the early 19th century (Singh and Shivakumar 2010). The
U.S. now leads the world in soybean production followed by Brazil (Singh and Hymowitz 1999).
From 2000 to 2008, soybeans were grown on an average of more than 29 million hectares
in 31 states occupying approximately 22% of the roughly 138 million hectares of harvested
cropland in the U.S. This makes soybean the second largest crop in acreage after corn (USDANASS 2008). In 2012, Michigan reported 809,371.3 hectares planted to soybean, with
approximately 2.3 billion of kg harvested. This is worth about $1.2 billion (Heatherly et al. 2009,
USDA-NASS 2012).
Soybeans have been called the “miracle golden bean” because of high mineral (P, K, Ca,
and Zn) and protein (42 to 45%) content (Rahman et al. 2011). In the U.S., soybeans also are a
major resource for biodiesel production, as soybean has the advantage of lower nitrogen input as
compared to crops such as sunflower (Pimentel and Patzek 2005).
An important agronomic trait associated with soybean physiology is the maturity groups
that determine its geographical adaptation (Zhang et al. 2007). Soybeans have been classified
into 13 maturity groups (MG) of which I and II are typically grown in the northern region of the
U.S., whereas higher maturity groups (III- VI) are grown farther south (Heatherly et al. 2009) .
This pattern of adaptation from north to south is due primarily to differences in sensitivity to
1

photoperiod (Scott and Aldrich 1970). To maximize yield, one must tailor the physiological
events of plant development with favorable environmental conditions (Singh and Shivakumar
2010).
The modern soybean germplasm collection generated through U.S.breeding programs
contains cultivars and breeding lines with a relatively low genetic diversity (Zhu et al. 2003).
This lack of diversity increases the potential vulnerability of soybean to plant diseases (Sleper
and Poehlman 2006). The most important soybean diseases in the U.S. are soybean cyst caused
by Heterodera glycines Ichinoche (Heatherly et al. 2009), seedling diseases, Phytophthora root
and stem rot caused by Phytophthora sojae Kaufman & Gerdemann (Wrather and Koenning
2006), and sudden death syndrome caused by Fusarium virguliforme Akoi, O’Donnell, Homma
& Lattanzi (Koenning and Wrather 2010). Sclerotinia stem rot (also known as white mold),
caused by Sclerotinia sclerotiorum (Lib.) de Bary has caused significant yield and economic
losses of soybean, particularly in northern states (Wrather et al. 2001). In the U.S. the Sclerotinia
stem rot epidemic in 2009 was ranked as the second most damaging disease following by
soybean cyst (reviewed in Peltier et al. 2012). Yield losses have been estimated at 60 million
bushels (Sleper and Poehlman 2006, Wrather et al. 2001). Thus, many efforts have aimed to
reduce the impact of the disease through appropriate disease management. Disease management
practices include cultural control (Boland and Hall 1988a, Gracia-Garza et al. 1998, Mueller et
al. 2002b) and chemical control (Dann et al. 1998, Dann et al. 1999, Mueller et al. 2002a,
Mueller et al. 2004). Biological control practices have been also evaluated (Del Rio et al. 2002,
Huang et al. 2000a, Zeng et al. 2012a, Zhang et al. 2011).

2

SCLEROTINIA SCLEROTIORUM
Biology and life cycle
Sclerotinia sclerotiorum is a fungus belonging to the family Sclerotiniaceae, the order
Helotiales, and the phylum Ascomycota (Whetzel 1945). Like all other species in the Helotiales,
S. sclerotiorum forms cup- or disk-shaped apothecia from sclerotia; the apothecia produce
inoperculate asci which contain ascospores (reviewed in Bolton et al. 2006, Whetzel 1945). A
sclerotium is a hyphal aggregate with an outer black rind composed of thick cells containing
melanin, a compound that is believed to play an important role in protection from adverse
conditions and microbial degradation (Butler et al. 2001, reviewed in Henson et al. 1999).
Sclerotia of S. sclerotiorum serve as survival structures that can remain viable for 3-5 years in
soil under natural conditions (Ayers and Adams 1979, Cook et al. 1975). Changes in
environmental conditions such as pH can prevent sclerotial formation. This was demonstrated
with a culture media assay showing that at pH 5 complete development and maturation of
sclerotia were observed in 4 d. However, under controlled conditions, when neutral pH was
evaluated, complete inhibition of sclerotia development was obtained (Rollins and Dickman
2001). Therefore the manipulation of external factors may provide alternatives to the control of
the Sclerotinia stem rot through disease cycle disruption.
Sclerotinia sclerotiorum overwinters as sclerotia in soil (Cook et al. 1975, Merriman
1976), as mycelium in infected seeds (Cook et al. 1975, Nicholson et al. 1972) or as mycelia in
dead or living hosts other than soybean (Boland and Hall 1994). When environmental conditions
are favorable, sclerotia germinate carpogenically and produce apothecia. Over a period of 2-3
weeks, large numbers of ascospores are discharged from mature apothecia into the air (Clarkson
et al. 2003). These ascospores can be spread by wind (Williams and Stelfox 1979) to nearby
3

hosts and serve as primary inoculum for infection (Abawi and Grogan 1975, Boland and Hall
1988b). It has been documented that ascospores can travel up to four kilometers under field
conditions; however, the majority are deposited within 100 to 150 m (Abawi and Grogan 1979,
Steadman 1979). In addition to production of ascospores, myceliogenic germination of sclerotia
results in the production of mycelial strands which can directly infect soybean seedlings (Grau
and Hartman 1999). The greatest potential for long distance dissemination of Sclerotinia spp. is
by transportation of seeds infested with mycelia or by sclerotia mixed with seeds (Mueller et al.
1999).
Host range
Sclerotinia sclerotiorum is a necrotrophic pathogen that has a wide host range. Host
include cultivated plants such as canola (oilseed rape, Brassica napus L.) (Morrall and Dueck
1982), soybean (Glycine max L. Merr.) (Boland and Hall 1988a), bean (Phaseolus vulgaris L.)
(Abawi and Grogan 1975), and sunflower (Helianthus annuus L.) (Hoes and Huang 1976). Wild
plants species are also reported as S. sclerotiorum host and this is considered to be an important
factor in Sclerotinia stem rot epidemiology (Boland and Hall 1994). A survey in 1994 reported
408 plant species from 278 genera and 75 families are susceptible to S. sclerotiorum. Most of
hosts belonging to the Dicotyledoneae subclass of Angiospermae (Boland and Hall 1994). The
wide host range of S. sclerotiorum often limits the opportunities for disease management through
crop rotation (Krupinsky et al. 2002, Tu 1989) but when combines with zero-tillage an effective
strategy is obtained, reducing the primarily inoculum of S. sclerotiorum in infested soybean
fields (Garza et al. 2002).

4

Molecular mechanisms of pathogenicity
Disease development by S. sclerotiorum requires secretion of virulence factors like oxalic
acid (Godoy et al. 1990, Magro et al. 1984), extracellular hydrolytic enzymes such as cellulases,
hemicellulases, pectinases, aspartyl protease, and/or endopolygalacturonases, and acidic protease
(Li et al. 2004, Marciano et al. 1982, Martel et al. 2006, Riou et al. 1992). Pectinolytic enzymes
and pectin- methyesterases (Lumsden 1976) are highly active under acidic conditions such as
those provided by oxalic acid (Marciano et al. 1983). The synergistic relation between the oxalic
acid and endopolygalacturonases (pectinolytic enzymes) promote the disruption of the calciumpectate complex in the middle lamella and primary cell wall of the host. (Bateman and Beer
1965). Other polymers such as cellulose and hemicelluloses in the cell wall are more exposed to
other enzymes (i.e. cellulases, hemicellulases, and xylanases) that contribute to tissue maceration
and pathogen invasion (reviewed in Lagaert et al. 2009). At early stages of disease development
polygalacturonases (PGs) are associated with pathogenicity in S. sclerotiorum (Lumsden 1976).
Oxalic acid has three modes of action with respect to pathogenesis (Cessna et al. 2000,
Marciano et al. 1983). First, oxalic acid directly blocks host-defense by suppressing the oxidative
burst and callose deposition (Cessna et al. 2000, Kim et al. 2008, Williams et al. 2011) at early
stages of infection, thus gaining important time for S. sclerotiorum establishment prior to plant
recognition. Second, the secretion of oxalic acid may be toxic to host plants, leading to the loss
of plant tissue integrity (Noyes and Hancock 1981). It was shown that oxalic acid moves
systemically to the leaves where it apparently accumulates to levels that produce wilt in infected
plants (Noyes and Hancock 1981). Third, the oxalic acid can chelate the cell wall Ca2+, thereby
destabilizing the cell wall host integrity and favoring the action of polygalacturonases (Bateman
and Beer 1965, Marciano et al. 1983).
5

Once S. sclerotiorum is established in the host tissue, the induction of an apoptotic-like
programmed cell death of host tissue occurred through a manipulation of plant defense response.
The nutrients that are derived from the dying plant cells benefit the pathogen (Kabbage et al.
2013, Williams et al. 2011). Overall, the infection process by this necrotrophic fungus is
complex.
SCLEROTINIA STEM ROT
Symptoms
In soybeans symptoms appear after crop flowering and canopy closure and are observed
on the main stem 15 to 40 cm above the soil surface and also appear on lateral branches (Boland
and Hall 1988a). The initial symptoms of Sclerotinia stem rot in soybean consist of brown and
water-soaked spots located at the junction of the petiole and the stem , similar to that described in
dry beans (Tu 1989) and the lower branches of the plant where senescent leaves or flower petals
have fallen and adhered (Natti 1971). Leaves of infected branches turn yellow and wilt. As the
infection progresses, the affected tissue show discoloration and a soft rot results in dieback of the
branches (Tu 1989). The lesions generally turn into necrotic tissues that show patches of fluffy
white mycelium, often with sclerotia, which is a typical sign of plants infected with S.
sclerotiorum (reviewed in Bolton et al. 2006).
Disease cycle
The major infection propagules for S. sclerotiorum on soybean are the ascospores derived
from carpogenic germination of sclerotia (Boland and Hall 1988a, Le Tourneau 1979). The
ascospores can germinate on the surface of healthy tissues but cannot infect the plant without an
exogenous nutrient source (Abawi and Grogan 1975, Sutton and Deverall 1983). Initial

6

colonization of dead or senescing tissue is required for successful infection of the plant organs
such as blossoms, cotyledons, leaves, seeds or injured plant tissue (Abawi and Grogan 1979,
Purdy 1979).
Details of early events of infection by the scanning electron microscopy have shown that
the formation of appressoria is a prerequisite to penetration of healthy plant tissues by S.
sclerotiorum (Tariq and Jeffries 1984). The development and complexity of the appresoria at
early infection stage depends on the nutritional status of the inoculum and the physical resistance
of the surface under attack (Tariq and Jeffries 1984). The formed appresorium is usually
complex, multicelled, dome-shaped structure (reviewed in Lumsden 1979, Lumsden and Dow
1973). The complex appressoria were observed in bean hypocotyls 6 hours after inoculation.
They are formed by three different types of hyphae which are involved in penetration and
infection of bean tissue formed via dichotomous branching from a single hyphae, with terminal
swelling of the apex followed by repeated branching and secretion of adhesive mucilage
(Lumsden and Dow 1973, Tariq and Jeffries 1984). Pore-like infection pegs formed at the tips of
appresoria allow the ingress of S. sclerotiorum into the host tissue and their development
contributes to the cellular disorganization of the leave (Abawi and Grogan 1975). Penetration of
the host surface may be mediated by the secretion of enzymes and toxins in addition to the
mechanical force exerted by the specialized hyphae when a tight interface is formed with the
host surface (Abawi and Grogan 1975, Tariq and Jeffries 1987). Once the stems and petioles are
infected, vascular tissues are disrupted, and stems, pods, or leaves beyond the site of infection
die. After consumption of plant nutrient, fungal mycelia aggregate into sclerotia, which form
both inside and outside the stem. These sclerotia then fall to the ground with the potential to
remain viable for an extended period of time (Coley-Smith and Cooke 1971).

7

Epidemiology
The release of ascospores from apothecia of Sclerotinia sclerotiorum is considered the
initial step for Sclerotinia stem rot epidemics (Abawi and Grogan 1975, Cook et al. 1975,
Schwartz and Steadman 1978) and environmental conditions constituted an important factors for
its occurrence (Boland and Hall 1988a, Pennypacker and Risius 1999).
Studies on the conditions for carpogenic germination indicated that different ranges of
temperatures and soil moistures impact the development of apothecia (Abawi and Grogan 1975,
Hao et al. 2003 , Mila and Yang 2008). The range of temperature for sclerotial germination
indicates an adaptation pattern of the pathogen to different geographic regions (Mila and Yang
2008, Phillips 1987). Size of sclerotia (Hao et al. 2003) and sclerotial burial (Mitchell and
Wheeler 1990, Wu and Subbarao 2008) appear to contribute to apothecia development. The
information from such studies may lead to a better understanding of the epidemiology of the
Sclerotinia stem rot and thus provide better strategies for its management.
Sclerotinia stem rot development on soybean and beans is also associated with canopy
closure (Boland and Hall 1988a, Natti 1971, Schwartz and Steadman 1978) that provides the
moisture conditions for carpogenic germination of sclerotia (Weiss et al. 1980). However, the
high relative humidity provided by canopy closure is not the only factor required for an effective
disease development (Boland and Hall 1988a). The amount and source of inoculum of S.
sclerotiorum are also critical factors. Boland et al. (1988) indicated that Sclerotinia stem rot
incidence was determined mainly by inoculum produced within the field. It is evident that more
than one factor is required for the onset of infection under field conditions, making the prediction
of this disease difficult.

8

DISEASE MANAGEMENT
The management of Sclerotinia stem rot involved the integration of several measures
(Abawi and Grogan 1979 , Boland and Hall 1988a, Kurle et al. 2001, Whipps et al.
2007).Strategies of disease management can include a) eradication through the reduction of
inoculum density of sclerotia in the soil, such as cultural practices, and soil amendments. b)
selection of tolerant varieties; and c) protection through chemical application, impairing the
pathogen ability to infect the susceptible soybean stage.
Tillage and crop rotation. Integration of zero-tillage with crop rotation can reduce Sclerotinia
stem rot (Garza et al. 2002). Under zero-tillage, S. sclerotiorum sclerotia are generally located
close to the soil surface (2 to 3 cm in depth) favoring the rapid degradation of sclerotia mainly by
soil microflora (Ayers and Adams 1979, Merriman 1976, Tu 1997). In contrast, tilled soils
promote the burial of sclerotia providing conditions for the sclerotia to survive for several years
(Duncan et al. 2006, Subbarao et al. 1996). Thereby, reduction of the inoculum potential can be
achieved through zero-tillage and subsequent infection of host plants by the pathogen may be
minimized (Tu 1986).
Cultivation of non-host crops is recommended to reduce sclerotia populations in soil
(Kurle et al. 2001, Steadman 1979); however, the dissemination of ascospores from field to field
by wind (Williams and Stelfox 1979) or long distance dissemination through contaminated seed
with sclerotia (Ayers and Adams 1979) might limit the efficacy of this strategy.
Resistant cultivars. Plant breeding for resistance to Sclerotinia stem rot has had limited success
(Cober et al. 2003). Genetic resistance to S. sclerotiorum is difficult to identify and evaluate
because both physiological resistance (i.e. incomplete resistance conditioned by multiple genes
of partial effect) and escape mechanisms (i.e. agronomic traits or modification in physiological

9

plant events that are involved in halt pathogen infection) contribute to differences in plant
disease responses under field conditions (Boland and Hall 1987, Grau et al. 1982, Kim and Diers
2000). Advances in genetic engineering, however, have allowed the development of Sclerotinia
stem rot-resistant soybean through the introduction of defense genes, such as oxalate oxidase, an
oxalic-acid degrading enzyme (Cober et al. 2003). Although the biotechnology approach may
provide potentially useful management tools, to date there is no transgenic commercial soybean
variety resistant to Sclerotinia stem rot or any fungal diseases (Hudson et al. 2013). In general,
some factors to be considered for commercialization of new improved varieties include: the
acceptance of farmers, society and regulatory agencies as well as the cost required during the
experimental and regulatory stages. Altogether, these factors may prevent the practical
application of these new technologies in a short term (Sleper and Shannon 2003).
Chemical control. According to Peltier et al. (2012), three different classes of fungicides are
registered for Sclerotinia stem rot control on soybean: methyl benzimidazole carbamate,
succinate dehydrogenase inhibitors, and demethylation inhibitors. These fungicides have limited
movement, and none of them move down in the plant (Yuan and Crawford 1995). In this regard,
Natti (1971) showed the importance of the systemic activity of a fungicide to achieve an
effective method of Sclerotinia stem rot control, as well as, the permanence of fungicidal activity
in senescent and dead bean blossoms.
For Sclerotinia stem rot control on bean and canola, foliar applications of fungicides have
been used but have not proven economically feasible on soybeans (Mila et al. 2003, Steadman
1979). In soybean good canopy penetration and foliar coverage with fungicides along with
appropriate application timing are critical for effective Sclerotinia stem rot control (Mueller et al.
2002a). This is particularly true for indeterminate soybean growth pattern, in which there are

10

many infection courts because of extended flowering (1-5 wk). This increases the opportunity for
pathogen infection over the time and more than two fungicide applications may be required
(Mueller et al. 2002a).
Chemicals are used for seed treatment against Sclerotinia stem rot in different crops
(Herd and Phillips 1988, Mueller et al. 1999, Tu 1989). In beans (Tu 1988) and soybeans
(Mueller et al. 1999) the sclerotia produced from the mycelium-infected seed may increase the
amount of inoculum for the next season. Even though the sclerotia from contaminated seeds may
occur in low proportion for both crops, under conducive environmental conditions the potential
for high disease incidence can increase (Mueller et al. 1999, Tu 1988). This indicates that seed
treatment may be an appropriate measure to prevent the introduction of S. sclerotiorum into
uncontaminated fields.
Herbicides constitute a class of chemicals that is routinely used in soybean production
for weed control. As well as their impact on weeds control, some herbicides affect S.
sclerotiorum directly (Altman and Campbell 1977, Radke and Grau 1986). The soil-applied
herbicides pendimethalin and trifluralin increased the number of apothecia produced per sclerotia
of S. sclerotiorum, whereas chloramben, alachlor and DNBP suppressed apothecia production
(Radke and Grau 1986). Casale et al. (1986) reported that soil amended with atrazine and
simazine induced abortion of apothecial disk with subsequent production of stipes or apothecia
abnormally shaped without ascospore production. Huang et al. (1995) confirmed that the triazine
family of herbicides enhanced branching of stipes and induced malformation of the apothecia of
S. sclerotiorum. These results provide evidence that it is possible that the routine application of
some herbicides can potentially alter the carpogenic germination of S. sclerotiorum sclerotia,
contributing to the reduction of a primary source of inoculum under field conditions.

11

Resistance inducing chemicals. The induction of plant resistance to pathogens is classified as
systemic acquired resistance (SAR) (reviewed in Hammerschmidt 1999a) or induced systemic
resistance (ISR) (reviewed in Van Loon et al. 1998). Both types of responses can be induced by
chemical or synthetic elicitors and may result in promising strategies for managing crop pest.
Systemic acquired resistance can be induced by biotic factors (Caruso and Kuć 1979,
Cohen and Kuc 1981) or chemical inducers of SAR such as 2,6-dichloro-isonicotinic acid (INA)
(Dann et al. 1998) or S-methyl benzo [1,2,3] thiadiazole-7-carbothioate (ASM) (Dann et al.
1998, Friedrich et al. 1996). Both INA and ASM are considered as functional analogs of salicylic
acid (reviewed in Vallad and Goodman 2004). In soybeans, multiple applications of both
chemicals (INA and ASM) results in a significant reduction of Sclerotinia stem rot severity
especially in susceptible cultivars compared to partially resistant varieties (Dann et al. 1998).
However reductions of Sclerotinia stem rot varied among field and years which might influence
its adoption in commercial soybean production.
Some herbicides modify the plant defense response either by inducing antimicrobial
compounds (e.g. phytoalexins) to stop the pathogen growth or conversely, reducing the plant
defense response making it vulnerable to diseases and pests (reviewed in Duke et al. 2012, Johal
and Huber 2009). In soybean, the diphenyl-ether, lactofen, has been shown to reduce Sclerotinia
stem rot severity under field conditions (Dann et al. 1999). High phytoalexin content (glyceollin)
in lactofen-treated leaves was associated with reduction in lesion size caused by S. sclerotiorum
(Dann et al. 1999). The increase in phytoalexin induction by lactofen treatment is restricted to
treated leaves with a systemic resistance response that protects the untreated leaves after
challenge with the pathogen (Nelson et al. 2002b). The mechanism associated with the increase
in glyceollin is the inhibition of protoporphyrinogen IX oxidase (protox), an enzyme involved in

12

the biosynthesis of heme (Ahrens and Edwards 1994). Although lactofen has been shown to
reduce disease severity in some cases, it needs further study as soybean cultivars vary in their
response to the chemical (Dann et al. 1999, Nelson et al. 2002a).
Biological control. Difficulties in disease control related with long-term survival structure,
sporadic occurrence and widespread distribution (Zhou and Boland 1998) of sclerotia-forming
pathogens in different crops have been promote studies on biological control (Adams and Ayers
1982, Budge and Whipps 2001, Campbell 1947, Huang 1980).
Several mechanisms have been associated with biological control including but not
limited to competition for space and nutrients (Baker 1968, Handelsman and Stabb 1996),
parasitism (Papavizas and Lumsden 1980, Whipps 2001), siderophore production (Duffy and
Défago 1999, Kloepper et al. 1980), synthesis of antibiotics (reviewed in Baker 1968, Fravel
1988), and induced resistance (reviewed in Handelsman and Stabb 1996, reviewed in Whipps
2001). The mechanisms of biocontrol associated with S. sclerotiorum that have been reported
include: 1) mycoparasitism (Budge et al. 1995, Campbell 1947, Del Rio et al. 2002, GraciaGarza et al. 1997, Trutmann and Keane 1990), 2) antibiosis (Abdullah et al. 2008, Fernando et al.
2007, McLoughlin et al. 1992) and 3) nutrient competition (Huang et al. 1993, Savchuk and
Fernando 2004, Yuan and Crawford 1995).
Even though some microorganisms have proven to be effective biocontrol agents against
S. sclerotiorum under controlled conditions, their application under field conditions were not
always effective (reviewed in Alabouvette et al. 2006, Spadaro and Gullino 2005, reviewed in
Van Veen et al. 1997). Since the soil is a dynamic and heterogeneous system in which diverse
forms of organisms interact under variable conditions (reviewed in Van Veen et al. 1997), a

13

complete understanding of the mode(s) of action(s) of biocontrol agents in an ecological context
is required to improve the reproducibility of disease control.
According to Whipps et al (2007), few commercial biological control agent products are
available in the market for Sclerotinia-induced diseases. Those available include Contans®,
Intercept® and KONI® (Coniothyrium minitans Campbell) and BioTrek ®, Harzian-10®,
Rootshield®, and T-22G® (Trichoderma species). Among these biological control agents, C.
minitans with mycoparasitic activity on Sclerotinia spp. has been the focus of extensive research
(Campbell 1947, Sandys-Winsch et al. 1993, Turner and Tribe 1976). This biological control
agent has been used successfully under controlled and field conditions, suppressing S.
sclerotiorum in numerous crops (Budge et al. 1995, Budge and Whipps 1991, Gerlagh et al.
1999, Huang 1977, Huang et al. 2000a, McQuilken et al. 1995). Despite the good performance in
preliminary trials, different factors may constrain the adoption of commercial biological control
in commercial crop systems: the levels of suppression may not be below to the economical
threshold for growers (Gnanamanickam 2002), the inconsistencies in disease control associated
with environmental factors and high pathogen inoculum levels (Whipps et al. 2007), and the lack
of effective technology transfer to growers (Glare et al. 2012).
The mechanism of sclerotia parasitism by C. minitans starts with the formation of
appressorium-like hyphae on S. sclerotiorum hyphae or sclerotia, follow by inter-and
intracellular growth within subcortical layers of the target pathogen (Grau and Radke 1984).
Since no depression was observed at every point of penetration, Tu (1984) suggested the action
of cell wall degrading enzymes were involved in the initial stage of S. sclerotiorum infection by
C. minitans. This observation was later confirmed by the detection and identification of a gene
encoding an exo-β-glucanases during sclerotia parasitism by C. minitans (Giczey et al. 2001).

14

Chitinases and exo-β-1,3-glucanases have been directly involved in the mycoparasitism
interaction between Trichoderma species and its hosts (Harman et al. 2004, Howell 2003).
Evidence of the wide range of enzymes produced by C. minitans can be supported by expression
analysis, which showed that the wide range of hydrolytic enzyme genes identified are likely to be
important in fungal-fungal interaction (Muthumeenakshi et al. 2007). The same study revealed
that nearly 20% of the identified unisequences may have potential novel function during
sclerotial colonization. The possibility to explore novel functions of promising biocontrol agents
through comparative genomics or other molecular tools, may allow a better exploitation of these
microorganism for more successful plant disease management.
Similarly, Trichoderma spp. have shown biological control properties associated with the
successful reduction of disease incidence caused by Sclerotinia spp. (Abd-El-Motty and Shatla
1981, Clarkson et al. 2006). The process of mycoparasitism exerted by Trichoderma spp. is
complex and involves a sequence of events. It has been shown that Trichoderma secretes
exochitinases constitutively at low levels while growing towards the target fungus (Chet et al.
1981, Haran et al. 1996, Inbar and Chet 1995). When chitinases degrade fungal cell walls, the
target fungus releases oligomers that induce the expression of these enzymes by Trichoderma
spp. and the attack of the host begins. (reviewed in Benitez et al. 2004). Once in contact with the
target fungus, the Trichoderma spp. produce several hydrolytic enzymes (e.g. β-1,3-glucanase,
chitinase, protease and cellulase) which initiate the fungal cell wall degradation (Elad et al. 1982,
Rey et al. 2001). Examination of sclerotia colonization by fluorescent microscopy revealed
intracellular growth of the antagonist in the medulla S. sclerotiorum (Sarrocco et al. 2006). The
uniform distribution of T. virens Miller, Giddens & Foster strain I10 just beneath the sclerotia
ring suggests that the sclerotia became infected randomly without any preferential point of entry

15

(Sarrocco et al. 2006). In the same study, after 9 and 14 d of colonization, it was demonstrated
that T. virens strain I10 was growing in the medulla and that the colonization was characterized
by intercellular growth. At 20 days complete colonization and formation of chlamydospores was
observed (Sarrocco et al. 2006). The time required for partial or complete degradation of
sclerotia by Trichoderma spp. and the sequential events that might occur during the colonization
and infection process by the antagonist constitute important information in terms of the practical
application and improvement of the antagonist activity for an effective disease control.
Some bacteria have been tested experimentally for managing Sclerotinia stem rot (Boland
1997, Huang et al. 1993, Thaning et al. 2001, Tu 1997). In soybean, the effect of different
Bacillus subtilis (Ehrenberg) Cohn strains on control has been variable. Zeng et al. (2012) found
that treatment with B. subtilis strain QST 713 (Serenade MAX®) resulted in a modest disease
severity index (DSI) reduction of Sclerotinia stem rot compared to other biological control
agents and reduced the disease severity 15.4% compared with the control treatment. Cell
suspension of Bacillus subtilis strains SB01 or SB24 successfully reduced Sclerotinia stem rot
severity and their effectiveness was maintaining during 15 days of evaluation (Zhang et al.
2011).
Fluorescent Pseudomonas spp. have been the subject of detailed studies on antibiotic
production (Duffy and Défago 1999, Keel et al. 1996, Raaijmakers et al. 1997) for disease
control (Raaijmakers et al. 2002, reviewed in Whipps 1997). Preliminary studies on detached
canola petals identified Pseudomonas cholororaphis Guignard & Sauvageau strains PA-23 and
DF41 as potential effective inhibitors of S. sclerotiorum mycelial growth and germination of
sclerotia and ascospores (Savchuk and Fernando 2004). The results showed significant disease
severity reduction when the bacterial strains were co-applied with the pathogen and subsequent

16

inhibition of ascospore germination compared to control treatments (Savchuk and Fernando
2004). Overall, these results clearly show that biological control by bacterial strains may be a
viable measure in addition to S. sclerotiorum control. However more efforts are needed to
evaluate these candidates on soybean and under field conditions, identifying the different factors
that might prevent an effective disease control to improve their application.
Integrated disease management. Integrated pest management (IPM) is a systems approach that
combines different crop protection practices with careful monitoring of pests (Chandler et al.
2011, Flint and Bosch 1981) looking to manage disease below levels that cause economic
damage (Chandler et al. 2011).
Synthetic pesticide in combination with biological control agents have been reported to
successfully manage Sclerotinia spp. in diverse cropping systems such as lettuce (Budge and
Whipps 2001), witloof chicory (Benigni and Bompeix 2010), peanut (Partridge et al. 2006),
garlic (Clarkson et al. 2006) and bean (Li et al. 2002). This demonstrated that integrated control
of S. sclerotiorum may be an additional tool to other more conventional disease management
measures.
The combined use of biocontrol agents and chemical pesticides has gained much attention
(Abd-El Moity et al. 1982, Spadaro and Gullino 2005) aimed at obtaining synergistic or additive
effects in the control of soil-borne pathogens (Sarkar et al. 2010 ). For controlling sclerotiaproducing fungi, combination of pesticides with biological control agents may have benefits such
as reduction in the use of synthetic pesticides (Budge and Whipps 2001). It is also possible that
using more than one biocontrol agent may prove effective (Jain et al. 2012). Combining
biocontrol agents with cultural practices such as adding organic matter, compost or the use of
soil steaming may provide other management approach (Whipps et al. 2007). For example, the

17

combination of T. viride with either tebuconazole or onion waste compost in greenhouse tests
enhanced disease control in comparison with the individual treatments with complete
suppression of Allium white rot caused by S. cepivorum Berk. (Clarkson et al. 2006). The
integration of partial soil sterilization (pasteurization) with a subsequent C. minitans application
for a sustainable control of S. sclerotiorum, contribute to a rapid colonization of sclerotia
compared to non-pasteurized soil (Bennett et al. 2005). Nevertheless, consideration of the
economic benefit for growers needs to be evaluated before the adoption of any of the disease
control measures mentioned above.

18

OBJECTIVES OF THIS STUDY
The integration of different management practices has the potential to provide an
effective strategy for Sclerotinia stem rot control. Even though some biological control agents
have shown as a promising additional tool to disease management, their use in practice is limited
due to inconsistent results under field conditions that prevent their practical application in
commercial soybean production. The successful integration of biological control agents for
disease management would be achieved, if under a routine regimen of agrochemicals, the
introduced soil microorganisms maintain their biological control properties as well as adequate
population levels in the soil. Thereby, a significant reduction of the inoculum levels of S.
sclerotiorum might be expected. Since any escaped sclerotia from biological control agent action
can potentially become a threat for the plant, an additional treatment during the soybean growing
season may strengthen the disease control.
The combination of biological control agents with chemicals, can be an additional
measure to enhance the levels of Sclerotinia stem rot control. To achieve this goal, the following
objectives were proposed:
1. Assess the sensitivity of Coniothyrium minitans (CON/M/91-08), Trichoderma asperellum
Samuels, Lieckf. & Nirenberg (ICC 012); and T. gamsii Samuels & Druzhin (ICC 080) to
select agrochemicals in vitro.
2. Determine the efficacy of biological control agents combined with chemical applications for
managing Sclerotinia stem rot of soybean under field conditions.

19

CHAPTER TWO: SENSITIVITY OF BIOCONTROL AGENTS TO PESTICIDES
ABSTRACT
Trichoderma asperellum (strain ICC 012), T. gamsii (ICC 080), and Coniothyrium
minitans (CON/M/91-08), which have been studied by others for managing Sclerotinia stem rot
(Sclerotinia sclerotiorum) were examined for their compatibility with pesticides used in soybean
production. This was accomplished by determined in vitro sensitivities to 15 conventional
pesticides used in soybean production. The herbicide lactofen, and the fungicides propamocarb
HCl, mefenoxam and mandipropamid had no inhibitory effects on mycelial growth of
Trichoderma spp. and C. minitans. At the tested levels, Trichoderma spp. were additionally
insensitive to boscalid, cyazofamid, and thiram. Based on responses in agar plate tests,
propamocarb HCl, tetraconazole, and lactofen were applied at 1,166.2; 58.5 and 105.2 g a.i./ha
respectively in a soil-mixture containing S. sclerotiorum sclerotia and C. minitans under growth
chamber conditions to assess their effects on survival and biocontrol activity of C. minitans.
Coniothyrium minitans growth was evaluated at1, 2 and 7 days after being inoculated into the
soil. The tested chemicals did not significantly reduce the survival as determined by C. minitans
growth on agar plates (P = 0.541) or biocontrol activity as determined by viability of infected
sclerotia (P = 0.601). The C. minitans population decreased significantly as the time of
incubation extended in the soil-mixture whether or not they were treated with the chemicals (P <
0.001). To test for the potential to tank mix biological control agents, a suspension of five
selected herbicides and a biofertilizer were incubated with C. minitans for up to 4 hours. No
significant change in C. minitans growth was observed (P = 0.944). These results indicated that
some chemicals used in soybean production could be co-applied with C. minitans.

20

INTRODUCTION
The management of Sclerotinia stem rot of soybean relies on multiple strategies including
biological control agent application (Del Rio et al. 2002). However, its practical implementation
against soilborne pathogens has not been consistently effective, and alternatives aimed to
improve the efficacy of biocontrol have been explored (reviewed in Spadaro and Gullino 2005).
These alternatives include combinations with other biological control agents, with chemicals, or
different agronomic o cultural practices (reviewed in Fravel 2005).
Currently in the U.S, the soybean production include the application of different
fungicides for managing different fungal diseases (NCRA 2013) as well as herbicides for weed
control (USDA-NASS 2012). The registered fungicide for Sclerotinia stem rot management
included thiophanate methyl, boscalid, tetraconazole and prothioconazole (reviewed in Peltier et
al. 2012). Accordingly with a recent survey, the used of herbicides are widely extensive
compared to compared to other agrochemicals (USDA-NASS 2012). Some of the most
commonly applied in soybean crop are: glyphosate potassium salt, glyphosate isopropylamine
salt, chlorimuron-ethyl, and flumioxazin (USDA-NASS 2012).This list of pesticides provide
possibilities to explore alternatives for the combined application of biological control agents with
pesticides or their integration with different agronomical practices aimed to promote plant health,
towards to a practical application of the biological control agents.
Evidence of the combination of pesticides and biological control agents are documented
for management of diseases caused by Sclerotinia spp. These are aimed at reducing fungicide
application and promoting an eco-friendly environment (Abd-El Moity et al. 1982, Budge and
Whipps 2001, Van Beneden et al. 2010). In a greenhouse experiment, Trichoderma viride Pers.
strains L14 and S17A combined with a tebuconazole-based seed treatment reduced Allium white

21

rot caused by S. cepivorum Berk. compared with using either T. viride or tebuconazole alone
(Clarkson et al. 2006). Lettuce drop suppression caused by S. minor Jagger. was achieved with a
single foliar application of iprodione combined with C. minitans similar to that obtained with
prophylactic sprays with iprodione every 2 weeks. This indicates that reduction foliar iprodione
applications could be a feasible option for lettuce drop control (Budge and Whipps 2001).
Nevertheless, when studies are extended to field conditions (for example, Clarkson et al., 2006),
the level of Allium white rot control was less consistent than in the glasshouse assays. These
inconsistencies might constitute one of the principal limitations for adoption of biological control
agents in commercial crop production.
An additional approach to improve biocontrol efficacy for Sclerotinia stem rot
management is the integration of C. minitans with other cultural control measures. For instance,
in oilseed rape, the combination of C. minitans with a N–P–K compound fertilizer application
reduced carpogenic germination of S. sclerotiorum in vitro (Yang et al. 2011). However, in field
the experiments, the application of C. minitans either alone or in combination with the compound
fertilizer had limited effect on disease suppression, despite the fact that such applications
significantly reduced carpogenic germination of S. sclerotiorum sclerotia (Yang et al. 2011).
Agronomical practice techniques such as tank mixes (“the associations among two or
more chemical products (pesticides) or among chemical products and fertilizers in a unique tank
for application in crops”) (Tornisielo et al. 2013), could be used as a means to combine
biological control agents with other management tools. This may help to the adoption of
biological control agents by farmers.
It is essential to recognize the limitations of biological control agents as living organisms
and explore different approaches to Sclerotinia stem rot management. Thus, attempts towards the

22

integration of biological, chemical and cultural methods for successful disease control (Warrior
2000) need to be considered. The objectives of this study were 1) to test the sensitivity of the
commercial biocontrol agents T. asperellum, T. gamsii, and C. minitans to selected pesticides in
vitro; 2) to assess the effect of selected pesticides on the survival and mycoparasitism activity of
C. minitans in a potting mix-natural field soil trial in controlled conditions; and 3) to test the
survival of C. minitans with herbicides or a biofertilizer under tank mix conditions.

23

MATERIALS AND METHODS
Sensitivities of biological control agents to pesticides in vitro
Isolation of biological control agents from commercial products. Isolates of Trichoderma
asperellum (ICC 012), T. gamsii (ICC 080) and Coniothyrium minitans (CON/M/91-08), were
obtained by plating out the commercial products Tenet® WP (Isagro USA, Inc., Morrisville,
NC) and Contans® (Prophyta Biologischer Pflanzenshutz GmbH, malchow/Poel, Germany) on
potato dextrose agar (PDA; EMB Chemical Inc., Gibbstown, NJ, USA) at room temperature for
3 d under constant fluorescent light for Trichoderma spp. and for 5-10 d in the dark for C.
minitans. The colonies selected by morphological features were isolated from single conidia and
grown in potato dextrose broth (PDB, EMB Chemical Inc., Gibbstown, NJ, USA) at 20◦C on a
rotatory shaker at 120 rpm for 3 d for Trichoderma spp. and 7 d for C. minitans. Mycelia were
harvested by vacuum filtration through sterile filter paper (Whatman # 1, Clifton, NJ, USA) and
used for genomic DNA extraction using the DNeasy Plant Mini kit (QIAGEN, Inc. Valencia,
CA. USA) according to the manufacturer’s instructions. Fungal identity was confirmed by
amplification of the internal transcribed spacer region (ITS) using universal primers ITS 1 and
ITS 4 (White et al. 1990). Visualization of PCR products was confirmed by electrophoresis on a
1.5% (w/v) agarose gel containing Tris-Acetate-EDTA [(TAE) 40mM Tris acetate, pH 8.0, 1mM
ethylenediaminetetractic acid (EDTA)] as the running buffer, and stained with GelGreen
(Biotium Inc., Hayward, CA, USA). Purification of PCR products was done using a PCR
purification kit (Denville Scientific Inc., Metuchen, NJ, USA) and products were sequenced at
the Michigan State University Genomic Technology Support Facility (East Lansing, MI, USA).
The sequences were analyzed using the BLAST algorithm against the National Center of
Biotechnology Information (NCBI) GenBank database.
24

Preparation of inoculum of biological control agents and pesticides. Trichoderma asperellum
and T. gamsii strains were cultured on malt extract agar (MEA; Difco Laboratories, Detroit MI,
USA) under constant fluorescent light exposure for 5 d to enhance sporulation (Schrüfer and
Lysek 1990). Coniothyrium minitans was grown on PDA for 10 d in the dark at 20◦C. The plates
were flooded with 5 ml of sterile water and the surface was gently scraped using a plastic rod to
dislodge the conidia. The resulting suspensions were collected in a 1.8 ml sterile tube for each
fungal isolate.
Fourteen pesticides, selected based on their routine use in soybean production in the U.S.
(NCRA 2013), and one herbicide reported to reduced Sclerotinia stem rot severity (Dann et al.
1999) were included and evaluated with each fungal isolate (Table 2-1).
Table 2-1. Pesticides used in the soybean cropping system in the U.S. included for the evaluation
of commercial biological control agents sensitivity test. All chemicals are fungicides with the
exception of lactofen, a post emergence herbicide.
Compounds

Trade name

Manufacturer

Fludioxonil
Mefenoxam
Metconazole
Trifloxystrobin
Cyazofamid
Tetraconazole
Boscalid
Tebuconazole
Azoxystrobin
Mandipropamid
Propamocarb HCl
Pyraclostrobin
Propiconazole
Thiram
Lactofen

Apron maxx
Apron XL
Caramba
Compass
Ranman
Domark
Endura
Folicur
Heritage 50WG
Revus
Previcur flex
Pristine
Stratego
Thiram
Cobra

Syngenta crop protection Greensboro, NC
Syngenta
BASF Corp., Research Triangle Park, NC
Bayer Corporation, Kansas City, MO
Ishihara Sangyo Kaisha, Ltd
Valent USA Corp., Walnut Creek, CA
BASF
Bayer
Syngenta
Syngenta
Bayer
BASF
Bayer
Bayer
Valent

25

Active
ingredient
(%)
2.30
33.3
8.6
50
34.5
20.5
70
38.7
50
23.3
66.5
12.8
11.4
42
24

Sensitivity of biological control agents to pesticides using a spiral gradient dilution method
The effective concentration leading to a 50% reduction in mycelial growth (EC50) was
determined for all fungal isolates. The spiral gradient endpoint (SGD) method was used as
described by Förster et al. (2004). Fifty ml of PDA was poured into 15 cm diameter petri dishes
at least 24 h before fungicide solution were applied. Stock concentrations of each of the
pesticides (Table 2-1) were prepared at 1,000 mg/L of active ingredient (a.i) in sterile distilled
water. A total of 50 μl of pesticides suspension was applied individually to each plate with a
Spiral Autoplate ® 4000 (Advanced Instruments, Norwood, MA, USA) using the exponential
deposition mode. No salicylhydroxamic acid (SHAM) as inhibitor of alternative respiratory
pathway (Olaya et al. 1998) was added to the agar plates for QoI fungicide (azoxystrobin,
pyraclostrobin and trifloxystrobin) evaluation.
The plates were incubated at room temperature in a horizontal-flow transfer hood, for 3 h
to allow the diffusion of the chemical into the medium, forming a gradient of concentrations
along the radius of the plate. The untreated center portion of the plate was removed with a sterile
cork borer (25 mm diameter) to avoid growth of the fungus across the plate’s center. A droplet of
conidial suspension of each isolate was spread along the radial lines using a template at
predetermined positions with a sterile glass rod. Duplicate samples for each Trichoderma spp.
were placed at opposite sides of the plate. Coniothyrium minitans was evaluated on different
plates due to its slow growth rate (Whipps and Gerlagh 1992) compared to the Trichoderma spp.
The plates were incubated at 20◦C for 3 d and 5-7 d for Trichoderma spp. and C. minitans,
respectively.
The growth of the fungus along the pesticide gradient concentration was recorded based
on two parameters: ending radius (ER) (point at which mycelia starts to grow) and the end of

26

mycelial growth, defined as the tail-ending radius (TER). The mid-distance point between the
ER and the TER was calculated for each fungal strain on each treatment. The incubation option
in the SGD software was used for calculation of the local concentrations at which 50% growth
inhibition was observed. Isolates were rated qualitatively and classified based on SGD software
calculations as highly sensitive (if the obtained EC50 is below the calculated range), highly
insensitive (if the obtained EC50 is above the calculated range) or moderate (if the obtained EC50
within the calculated range) to the tested pesticide at the concentration evaluated (Wharton
2010). The experiment was repeated with four replicates per isolate.
Survival of Coniothyrium minitans with herbicides and fertilizers under tank mix
conditions
Inoculum preparation. Conidial suspensions of C. minitans (CON/M/91-08) were prepared
from cultures grown on PDA for 10 d in the dark at 20°C as described above. The conidial
concentration was estimated with a hemocytometer and adjusted approximately to 1 × 10 7
conidia/ml with sterile water. The conidial suspension (5 ml) was placed in 15 ml centrifuge
tubes (Denville, Scientific Inc., Metuchen, NJ).
Herbicides and fertilizer tested. Commercial formulations of herbicides commonly used in the
U.S. soybean production (Heatherly et al. 2009). These included: metolachlor (Stalwart® 86.4%
a.i., Sipcam Agro USA, Inc., Roswell, GA; field rate 2.3 Kg a.i/ha); sulfentrazone and
chlorimuron ethyl (Authority XL® 62.22% a.i, 7.78% a.i., FMC Corporation Agrochemical
Products Group, Philadelphia, PA; field rate 0.4 Kg a.i./ha); sulfentrazone and cloransulammethyl (Sonic®, 62.1% a.i.,7.9% a.i., Dow AgroSicence LLC, Indianapolis, IN; field rate 0.2 Kg
a.i./ha), simazine (Simtrol® 42.8% a.i., Sipcam Agro USA, Inc., Roswell, GA; field rate 4.6 Kg
a.i./ha ), glyphosate (Roundup Power Max®, 48.7% a.i., Monsanto Co., St Louis, Mo; field rate
27

1.6 Kg a.i./ha) and a biofertilizer Accomplish LM® (Loveland Products Inc. Loveland, CO; field
rate 4.6 Kg a.i./ha). The chemicals were prepared according to the recommended field rate and
adjusted to a final volume of 5 ml.
Evaluation of Coniothyrium minitans survival. A volume of 2.5 ml of the conidial suspension
prepared as described was mixed thoroughly with 500 µl of one of the prepared chemical
solutions with each chemical tested separately. Suspensions were transferred to 15 ml sterile
plastic centrifuge tubes. The prepared suspensions were incubated at 23˚C for 0, 1, 2, and 4
hours, followed by centrifugation at 7,600 × g for 10 min. The conidial pellet was washed three
times by centrifugation with sterile water to remove chemicals and resuspended in 1 ml of sterile
water. An aliquot of 100 μl of the spore suspension was added to 900 μl of sterile water in a 1.5
ml tube. Serial dilutions with three replications were made on PDA and colony forming units
(CFU) ml -1of C. minitans were estimated after incubation of 10 d at 20°C. Sterile water was
used as a control.
Effect of pesticides on Coniothyrium minitans survival and its biocontrol activity under
growth chamber conditions
Pesticides and commercial biological control preparation. The commercial formulation of
propamocarb HCl (Previcur flex® 66.5% a.i. Bayer CropSience, Research Triangle Park, NC) at
1,166.2 g/ha; tetraconazole (Domark® 20.5% a.i. Valent Bioscience Corporation) at 58.5 g/ha
and lactofen (Cobra® 24% a.i. Valent U.S.A Corporation, Walnut Creek, CA) at 105.2 g/ha,
were selected based on the C. minitans sensitivity test. The chemicals were prepared in a water
solution and adjusted to the appropriate volume based on the surface area of the treated pots. A
commercial formulation of C. minitans (CON/M/91-08) ContansWG® was used at the

28

recommended field rate (2,241.2 g/ha) and applied as an aqueous suspension in sterile distilled
water based on the surface area as above.
Soil infestation with pesticides and Coniothyrium minitans. Plastic pots (1L with 15 x 15 cm2
opening) were filled with a mix of: potting mix (BACCTO Professional Planting Mix, Michigan
Peat Company, Houston TX 70-80% sphagnum peat) and a mixture of natural field soil with
sand and bailed peat (v:v=1:1) obtained from Michigan State University greenhouse facilities.
Sclerotia of S. sclerotiorum were produced artificially using the method of Jones et al. (2003).
Briefly, mushroom spawn bags containing 150 g of wheat and 400 ml of distilled water were
autoclaved, allowed to cool for 6 h and inoculated with 5 discs (3 mm diameter) cut from the
colony margin of a 5 day-old S. sclerotiorum culture grown on PDA. The spawn bags were
incubated at room temperature for 4 weeks and exposed to ambient light. Ten sclerotia between
approximately 2 to 5 mm long visually were selected and buried at 1 cm depth from the surface
of each pot and placed approximately 1 cm apart. The treatments are described in Table 2-2. The
pots were placed in a growth chamber (Model: PGR14, Conviron, Winipeg, Manitoba, Canada)
adjusted to 20°C with a 14:10 h light-dark circle. Light intensity was 73.56 µmol -1 m -2 per µA.
Soil sample collection and processing. Samples were collected three times: 1, 2 and 7 d after
the C. minitans application. Dilution plating was conducted to enumerate C. minitans colony
forming units (CFU/g of soil). Briefly, 1 g of soil randomly selected from the pots was agitated
in 9 ml of phosphate buffered saline (1.2 g Na2HPO4, 0.18 g NaH2PO4, 8.5 g NaCl, adjusted to
pH 7.4 with 1M HCl) for 10 min on a rotary shaker at 150 rpm. A dilution series was prepared
from 1:10 to 1:102 and plated onto PDA amended with chlortetracycline (25µg/ml) (AMRESCO,
Solon, OH) and Triton X-100 (2 ml/l) (Research Product International Corp, Mt. Prospect, IL).
Aliquots of 50 µl were spread onto a semi selective media for C. minitans isolation (Bennett et

29

al. 2003) using the Spiral Autoplate ® 4000 with the even deposition mode feature. Each plate
was incubated at 20◦C in the dark for 5-10 d. Each dilution was done in duplicate and the
experiment was repeated twice.
Table 2-2. Description of treatments combinations included to evaluate the effect of two selected
fungicides and one herbicide on a commercial formulation of Coniothyrium minitans
(CON/M/91-08) survival and its biological control activity against Sclerotinia sclerotiorum
under 20°C, 14h light:10 h dark.
Treatment 1
Treatment 2
Treatment 3
5
Sclerotia
Sclerotia
Sclerotia
Sclerotia + C. minitans 1
Sclerotia + C. minitans
Sclerotia + C. minitans
2
3
Sclerotia + Propamocarb HCl
Sclerotia + Tetraconazole
Sclerotia + Lactofen 4
Sclerotia + C. minitans +
Sclerotia + C. minitans +
Sclerotia + C. minitans +
Propamocarb HCl
Tetraconazole
Lactofen
1
Coniothyrium minitans was applied at the recommended field rate based on the surface area
treated. A total volume of 50 ml was applied to the corresponding treated pots.2, 3, 4 Pesticide
treatments were applied at the equivalent field rate of the treated surface area at a total volume of
10 ml per pot. 5 Pots with only sclerotia of S. sclerotiorum were treated with sterile water (10 ml)
as a control.
Sclerotia viability. Three days after the last sample collection sclerotia were removed from all
the evaluated treatments, surface disinfested for 3 min by submerging in 0.6% sodium
hypochlorite, rinsed three times in sterile distilled water and placed onto 1.5 % water agar plates
(15-mm). The viability of the sclerotia was assessed visually by production of typical mycelia
after 3 d of incubation at room temperature in the dark.
Data Analysis
Statistical analysis was performed with SAS Version 9.2 (SAS Institute, Inc., 100 SAS
Campus Drive, Cary NC). The tank mix experiment was analyzed with PROC MIXED to test for
the significance of the treatments for the main effects and their interaction. Significant difference
between treatment means were compared using Tukey-Kramer multiple comparison at α = 0.05.
30

Repeated measurements in Tukey-Kramer grouping were conducted to estimate the C. minitans
population variation over time. Colony forming units (CFU per gram of soil-mix) were log
transformed to satisfy the assumption of homogeneity of variance. The effect of pesticides on C.
minitans survival experiment was analyzed with PROC MIXED. Significant differences between
treatment means were compared using Tukey-Kramer multiple comparison at α = 0.05. Analysis
of variance for C. minitans biocontrol activity and sclerotia viability were evaluated with PROC
GLIMMIX. Significant differences between treatment means were compared using TukeyKramer multiple comparison at α = 0.05. For all the experiments the residual analysis performed
by PROC UNIVARIATE confirmed the hypothesis that the experimental errors were normally
distributed with a common variance.
RESULTS
Sensitivity of biological control agents to pesticides in vitro
Of the fifteen pesticides evaluated, three fungicides mefenoxam, propamocarb HCl,
mandipropamid, and the herbicide lactofen did not inhibit mycelial growth of any of the
biological control agents evaluated. Although no inhibition of mycelial grown with lactofen
treatment was detected, a delay (3-5 d) in conidia production was observed for C. minitans
compared with the untreated control. Trichoderma spp. were highly insensitive to cyazofamid or
thiram (Table 2-3). Coniothyrium minitans and Trichoderma spp. showed moderate sensitivity to
fludioxonil, metconazole, trifloxystrobin, tetraconazole, tebuconazole, azoxystrobin,
pyraclostrobin and propiconazole (Table 2-3). Sensitivities of the biological control agents to the
fungicides mentioned above at the level tested varied depending on the fungus but overall the
range was between 0.05 to 1 µg/ml.

31

Survival of Coniothyrium minitans with herbicides and fertilizers under tank mix condition
No significant differences were found between the pesticides (P= 0.944), time of
incubation (P= 0.993) or agrochemical × time effect interaction (P= 0.425) on the survival of C.
minitans in the simulate tank mix conditions (Figure 2-1). Similar levels of C. minitans were
recovered with simazine, sulfentrazone and cloransulam- methyl, sulfentrazone and chlorimuronethyl and the bio-fertilizer booster product (Accomplish LM) compared with the control
treatment (Figure 2-1).
Table 2-3. Effective concentration for 50% reduction of mycelial growth (EC50) values for
fifteen fungicides and one herbicide. Data are for three biological control fungi as determined by
the spiral gradient dilution (SGD) method.
EC50 (µg/ml)
Compound

Dilution range
T. asperellum
T. gamsii
C. minitans
b
(SGD)
Fludioxonil
0.03 – 4.34
0.35 a
0.06
0.10
c
Mefenoxam
0.03 – 4.33
≥ 4.33
≥ 4.33
≥ 4.33
Metconazole
0.02 – 4.32
0.06
0.07
0.09
Lactofen
0.02 – 4.20
≥ 4.20
≥ 4.20
≥ 4.20
Trifloxystrobin
0.02 – 4.20
0.08
0.12
0.07
Cyazofamid
0.02 – 4.31
≥ 4.31
≥ 4.31
0.09
Tetraconazole
0.02 – 4.27
0.27
0.19
0.05
Boscalid
0.02 – 4.30
≥ 4.30
≥ 4.30
0.10
Tebuconazole
0.02 – 4.32
0.14
0.10
0.08
Azoxystrobin
0.02 – 4.25
0.19
0.21
0.12
Mandipropamid
0.02 – 4.24
≥ 4.24
≥ 4.24
≥ 4.24
Propamocarb HCl
0.03 – 4.28
≥ 4.28
≥ 4.28
≥ 4.28
Pyraclostrobin
0.02 – 4.26
0.43
0.10
1.00
Propiconazole
0.02 – 4.30
0.2
0.71
0.05
Thiram
0.03 – 4.34
≥ 4.34
≥ 4.34
0.24
a
Each number is the average of four replicates per each fungal isolate per pesticide. The
experiment was repeated twice and combined. b Range of pesticide concentrations (µg/ml)
calculated by the spiral plate dilution (SGD) method. c The (≥) indicates that the EC50 value is
greater than the concentration evaluated and this was classified as highly insensitive.

32

C. minitans (Log CFU/ml)

7
6
5
4
3
2
1

Figure 2-1. Effect of 5 herbicides (below) and a bio-fertilizer (Accomplish LM®) on
Coniothyrium minitans population under tank mix conditions. Active ingredient, commercial
name, percentage of active ingredient (a.i.) and field rate are listed below: metolachlor
(Stalwart® 86.4% a.i., 4,677 g/ha), sulfentrazone and cloransulam -methyl (Sonic®, 62.1% a.i.,
7.9% a.i., 210.1g/ha), sulfentrazone and chlorimuron-ethyl (Authority XL® 33.33% a.i, 6.67%
a.i.; field rate 451.83 g/ha), simazine (Simtrol® 42.8% a.i., 4,677 g/h), glyphosate (Roundup
Power Max®, 48.7% a.i., 607.8 g/ha), Bacillus spp., Acidovorax facilis and Rhodococcus
rhodochorus (Accomplish LM®, Loveland Products Inc. Loveland, CO; 4,677 g/ha). Sterile
water was used as a control. Each column is the average of 32 observations (mean of three
replicates per isolate) and the experiment was conducted twice. Bars on each column are
standard deviation.

33

Effect of pesticides on Coniothyrium minitans survival and its biocontrol activity under
growth chamber conditions
Three pesticides with differing effects on C. minitans growth in the plate test were
evaluated. These included the herbicide lactofen and the fungicides propamocarb HCl (no
inhibitory effect on mycelial growth) and the fungicide tetraconazole (inhibitory effect on
mycelial growth). No significant differences in the survival of C. minitans were found for the
three treatments (P = 0.5409) compared to the levels for the biological control agent applied
alone.
The interaction of the pesticide treatments with the time of exposure did not significantly
affect C. minitans population (P = 0.5762). However, the main effect of time on the BCA
population was statistically significant (P < 0.001).
The initial level of C. minitans before any pesticide application was 4.5 log CFU/g soil.
By the second day after its incorporation into the soil and immediately after pesticide application
a significant reduction in the level of C. minitans (4.12 Log CFU/g soil) was observed compared
to the initial population (P < 0.001). At the 7 d sample, C. minitans was lower than the initial
levels (4.32 Log CFU/g soil) (P =0.007). At the second day of sampling, lower levels of the
biological control agent was found compared with the 7 d (P = 0.0024) (Figure 2-2).
Biocontrol activity of C. minitans was evaluated after pesticide application and compared
with the untreated control. There was a significant reduction in sclerotial viability of S.
sclerotiorum with C. minitans application alone (without pesticide application) or in combination
with pesticides (P < 0.001) (Figure 2-3). No significant difference between biological control
agent alone (P = 0.601) or BCA in combination with pesticide treatments (P = 0.522) was found.

34

C. minitans (Log CFU/gr soil)

4.70
4.60

a

4.50
4.40

c

4.30
4.20

b

4.10
4.00
3.90
3.80
1

2
Time (days)

7

Figure 2-2. Time effect on the survival of Coniothyrium minitans. Samples were collected at 1,
2, and 7 days after treatment. Coniothyrium minitans population was detected and enumerated by
plating on semi-selective media. Columns represent the average of 32 observations of combined
experiments. Error bars indicate standard deviation. Mean of C. minitans growth followed by
same letter are not significantly different at α = 0.05 (Tukey-Kramer HSD).

35

10

Number of viable sclerotia

9
8
7
6
5

*

4
3
2
1
0
Treatments
C. minitans

Non treated

Figure 2-3. Biological control activity of Coniothyrium minitans on viability of sclerotia of
Sclerotinia sclerotiorum in a mixture of potting mix (BACCTO Professional Planting Mix,
Michigan Peat Company, Houston TX 70-80% sphagnum peat) and a natural field soil with sand
and bailed peat (v:v=1:1). Ten sclerotia were used per pot. The sclerotia were buried in potting
mix: soil mixture and retrieved 10 days after C. minitans application. Their viability was
determined by observing mycelial germination on water agar. Since no significant difference
between C. minitans alone or in combination with pesticide treatments were found (P = 0.601 or
P = 0.522 respectively), the treatments were combined over C. minitans treated and non-treated
pots. Columns represent the average of 48 observations of combined experiments. Error bars
indicate standard deviation. Means of number of viable sclerotia in each treated and non-treated
control were compared by Tukey-Kramer HSD at α = 0.05. “*” Significant difference at P <
0.001.

36

DISCUSSION
In this study, Coniothyrium minitans (CON/M/91-08), Trichoderma asperellum (ICC
012) and T. gamsii (ICC 080) showed different ranges of sensitivities to the selected pesticides at
the level tested (Table 2-3). The commercial biological control agents were classified as
insensitive (range above the threshold calculated by SGD) to lactofen, mefenoxam, propamocarb
HCl, and mandipropamid. This result was expected since these chemicals are registered either
for weed control (lactofen) (Vidrine et al. 1993) or for control of oomycetes (Cohen et al. 2007,
Papavizas et al. 1978, Taylor et al. 2002). In addition, other researchers have demonstrated that
propamocarb HCl does not have an adverse effect on beneficial microorganisms such as
mycorrhizae and Trichoderma spp., so it is considered a good component of IPM programs that
use biological control (Hu et al. 2007, May and Kimati 2000).
A moderate sensitivity of mycelial growth of Trichoderma spp. and C. minitans was
observed to the quinone outside inhibitors (QoI) (i.e. trifloxystrobin, azoxystrobin,
pyraclostrobin), and sterol demethylation inhibitors (DMI) (i.e. metconazole, tetraconazole,
tebuconazole and propiconazole) (Table 2-3). The field rates of these fungicide are higher when
compared to the EC50 concentrations for mycelial growth for both of the biological control agent
evaluated. The fungicides are applied in the field in a range of 258 to 1520 µg a.i./ml. This
suggests that Trichoderma spp. and C. minitans might not be combined with the tested
fungicides. Although, since this biocontrol agents are applied before planting at the soil level, the
detrimental effect of late season foliar applications of these fungicides might be lower than early
sprays. Factors such as rainfall or irrigation can contribute to reduction pesticide concentration at
the soil surface (Moorman 1989).

37

Fungicides used as a soybean seed treatment, fludioxonil and thiram, (Mueller et al.
1999) showed different effects on mycelial growth of C. minitans whereas Trichoderma spp.
were inhibit only by fludioxonil under level tested (Table 2-3). A complete inhibition of
fludioxonil on C minitans mycelial growth was reported previously (Benigni and Bompeix
2010). Similarly, fludioxonil was proved to have an inhibitory effect on mycelial growth for T.
virens strain G-6 (Howell 2007). Inhibitory effect of thiram on mycelial growth of C. minitans
was reported as moderate by Budge et al. (2001). An in vitro study showed that the combination
of thiram with T. harzianum and T. viride was feasible, since no significant inhibition was
detected (Bagwan 2010). These observations might open the possibility to use Trichoderma spp.
as a soybean seed treatment in combination with thiram to control S. sclerotiorum in
contaminated seeds. In addition, since a pesticide is concentrated near the treated seed and not in
the bulk soil around (Moorman 1989), it might be also possible the soil application of C minitans
after sowing the treated seeds.
The mycelial growth of C. minitans was not inhibited by the herbicide lactofen but a
delay in the conidiation of the fungus was observed. Partridge et al. (2006) found that the
herbicide flumioxazin reduced the spore germination, growth and biocontrol activity of C.
minitans. Since flumioxazin and lactofen belong to the same group of diphenyl-ether herbicide, it
might be expected to have the same effect on fungal growth; however, unlike the lactofen, the
flumioxazin did reduce mycelial growth and conidia germination of C. minitans while lactofen
only retarded the conidiation without any restriction in vegetative growth. It may be useful to
determine the effects of the different components in commercial formulations of pesticides as
some of these may interfere with different biological properties of beneficial microorganisms.

38

This is the first report that the mycelial growth of at least two Trichoderma spp. is not
inhibited by lactofen. A similar result has been reported for oxyflorofen (diphenyl-ether
herbicide, belonging to the same group of herbicides as lactofen), for which no inhibition of T.
viride occurred (Madhavi et al. 2011). The compatibility of the Trichoderma spp. evaluated with
lactofen, indicate the potential combination of these treatments for Sclerotinia stem rot of
soybean management as new additional strategy.
Propamocarb HCl, tetraconazole, and lactofen did not affect mycelial growth or
biocontrol activity of C. minitans population in a soil potting mixture. The persistence of lactofen
in the soil is considered low (Wauchope et al. 1992) and herbicides of the diphenyl ether family
can be degraded by soil microorganism (Chakraborty et al. 2002, Keum et al. 2008). This may
explain why it did not have any effect on the biological properties on C. minitans in pot trials.
Studies with fungicides in the triazole (Strickland et al. 2004, White et al. 2010) and carbamate
families (Knowles and Benezet 1981, Myresiotis et al. 2012) have shown no effect on microbial
communities in the soil. Some of the other factors involved in the impact of pesticides on soil
microbial communities include physical, chemical (Beulke and Malkomes 2001) and
biochemical properties of the soil (Monkiedje and Spiteller 2002), and the nature and
concentration of the chemicals applied (Kah et al. 2007). All of these factors may play an
important role in the potential effect of pesticides on biological control agent’s survival when
applied to soil environments.
In this study, the levels of C. minitans decreased in soil over time (Figure 2-2). Since the
sclerotia of S. sclerotiorum serve as a reservoir of C. minitans and the biocontrol agent has been
shown to survive up to 10 months (Bennett et al. 2006), it might expect a progressive increase in
the C. minitans levels as the sclerotia were infected by the mycoparasite (Gerlagh and Vos 1991,

39

Jones et al. 2003). However, Jones and Stewart (2011) reported a significant decrease in C.
minitans recovery from soil immediately after application and during the subsequent evaluation
from 1 up to 24 weeks. In other words, there was no return to the initial population levels. In
contrast, in other studies the C. minitans population did not change during the testing period
(Jones et al. 2004, McQuilken et al. 1995). For example, the population remained at the same
level (1 × 106 CFU/g) for 30 d, whether the soil was sterile or not (Bennett et al. 2003). In
contrast, at the lowest rate (1 × 103 CFU/g) the recovery of C minitans in sterile soil versus nonsterile soil was significantly different. This suggests that the successful establishment and
survival of C. minitans in the soil might be affected either by the interaction with other
indigenous soil microorganisms or by the rate of application.
The main purpose of applying herbicides is to eliminate weeds that compete with crop
growth and, in particular, for the Sclerotinia stem rot epidemiology because some weeds may
serve as potential hosts of S. sclerotiorum (Boland and Hall 1994). A secondary advantage may
be that some herbicides, such as lactofen, can reduce the severity of Sclerotinia stem rot under
field conditions (Dann et al. 1999, Nelson et al. 2002a). This study showed that C. minitans is
compatible with lactofen, allowing them to be applied together, which might be an advantage in
terms of reduced cost of applications. It means that with a single application it may be possible to
obtain a dual-purpose treatment, hence reducing the operational cost. A specific cost analysis is
required to determine the actual impact to farmers in the adoption of this particular practice.
No inhibitory effect on C. minitans growth was observed with the biofertilizer tested
under tank mix conditions. It has been documented that the antimicrobial compounds produced
by Bacillus spp. may inhibit the growth of some fungal pathogens (Fernando et al. 2007, He et
al. 1994). However, this was not the case in this study. A possible explanation is that the

40

production of some secondary metabolites may be modified by external factors such as carbon
source, mineral concentration, and environmental conditions (Duffy and Défago 1999, Haas and
Keel 2003). It is also possible that the simulated tank mix conditions were not conducive to the
production of antimicrobial compounds. However, it would require additional evidence to
understand the microbial metabolism under this particular environment. Production of an
antimicrobial compound (macrosphelide A) by C. minitans is also dependent on other factors:
temperature, pH and fungal strain (Tomprefa et al. 2011). This antimicrobial compound has an
inhibitory effect to some Bacillus spp. (Tomprefa et al. 2009). This suggests that a contrary
effect might occur and it may be interesting to establish if is there any negative effect caused by
C. minitans against Bacillus spp. under tank mix conditions.
In conclusion, the in vitro study provide preliminary evidence towards the incorporation
of the Trichoderma spp. and C. minitans into an integrated Sclerotinia stem rot management
program with some pesticide evaluated. Simulated soil and tank mix conditions indicate that C.
minitans can be applied together with selected chemicals without limiting its biocontrol ability
and growth respectively. Future studies may include the in vitro test on Trichoderma spp. and C.
minitans conidial germination with the selected pesticides as well as their effect on the
antagonistic activity of Trichoderma spp. under soil environment context.

41

CHAPTER THREE: MANAGEMENT OF SCLEROTINIA STEM ROT BY
INTEGRATION OF BIOCONTROL AND CHEMICAL STRATEGIES UNDER FIELD
CONDITION
ABSTRACT
Bacillus amyloliquefaciens (BAC03) and the commercial biological control products
Contans WP® (Coniothyrium minitans CON/M/91-08), Tenet® (mixture of Trichoderma
asperellum ICC 012 and T. gamsii ICC 080), Actinogrow ® (Streptomyces lydicus WYEC 108),
and Regalia® (Reynoutria sachalinensis) were evaluated for their efficacy in reducing
Sclerotinia sclerotiorum sclerotial populations and Sclerotinia stem rot severity in field trials at
two locations in Michigan from 2010 to 2012. The biocontrol materials were either applied to the
soil alone or co-applied with the herbicide lactofen, which has been shown to reduce Sclerotinia
stem rot severity in some soybean cultivars. Two fungicides, boscalid and thiophanate methyl,
were included as foliar treatments. The biological control agents were applied to soil at planting
in field plots artificially infested with S. sclerotiorum sclerotia. Reductions in the severity of
Sclerotinia stem rot by 78 %, 71 % and 70 % were obtained with the co-application of C.
minitans, Trichoderma spp., and S. lydicus with lactofen respectively at Clarksville Horticulture
Experimental Station (CLK) under high disease pressure in 2011 and 2012. Coniothyrium
minitans and Trichoderma spp. reduced the sclerotia viability in the soil by 90 % and 86 %
respectively at CKL in 2011 and 2012. The co-application of lactofen with Trichoderma spp. and
C. minitans significantly increased the yield compared to the non-treated control in some cases
but results depended on the year and location. In 2010, no significant difference in Sclerotinia
stem rot reduction was observed with boscalid compared with the non-treated control.
Thiophanate methyl reduced the severity of Sclerotinia stem rot at CKL in 2012.

42

INTRODUCTION
Sclerotinia stem rot of soybean is a threat to soybean production and is a major concern in
the north central region of the United States (Grau and Radke 1984, Mueller et al. 2002b,
Workneh and Yang 2000).Both the severity of Sclerotinia stem rot and the frequency of
outbreaks have increased after the 1990s (Kurle et al. 2001). The adoption of management
practices intended to increase soybean yield have been associated with the increase of Sclerotinia
stem rot prevalence in north-central region of the U.S. (Mila et al. 2003).
Cultural and management practices associated with reduction of incidence and severity of
Sclerotinia stem rot include planting tolerant cultivars (Boland and Hall 1987), zero-tillage
(Kurle et al. 2001), wider row spacing and reduction of irrigation regimens (Grau and Radke
1984), and fungicide application in situations where susceptible cultivars must be grown, or
where modification of cultural practices is not an option (Mueller et al. 2004). Fungicides such
as azoxystrobin, boscalid, thiophanate-methyl, and iprodione have been used against S.
sclerotiorum with moderate disease suppression (Bradley et al. 2006).
The diphenyl ether herbicides, such as lactofen, may be used as an additional strategy to
fungicides. The reduction in Sclerotinia stem rot severity after lactofen application has been
associated with the induction of phytoalexins, modification of canopy structure and delay in
reproductive development (Dann et al. 1999, Nelson et al. 2002a). In field trials, lactofen
treatment resulted in a significant reduction in Sclerotinia stem rot severity of 40-60% (Dann et
al. 1999) and higher levels of glyceollin accumulation compared with other herbicides of the
same class. However, the reduction in disease severity does not necessarily imply an increase in
yield (Nelson et al. 2002a, Nelson et al. 2002b). According to da Rocha and Hammerschmidt

43

(2005), the poor effect on yield would be the result of the plant's energy resources invested in
defense rather than development and reproduction.
Studies have been conducted in evaluating biological control agents to reduce the
inoculum population of S. sclerotiorum in soil (McQuilken et al. 1995, Santos and Dhingra 1982,
Trutmann et al. 1980). Different biocontrol agents derived from antagonistic fungi with diverse
modes of action have been shown to suppress S. sclerotiorum. Fungal antagonists such as
Coniothyrium minitans Campbell, have been tested on different crops as a means of reduction of
sclerotia levels in soil (Budge and Whipps 1991, Huang 1977, McQuilken et al. 1995, Zeng et al.
2012a). Similarly, Trichoderma spp. also have the ability to directly parasitize and degrade the
sclerotia of S. sclerotiorum (Menendez and Godeas 1998, Santos and Dhingra 1982, Trutmann
and Keane 1990). Although successful reduction of S. sclerotiorum inoculum has been
observed, a better understanding of the ecological context of the fungal antagonist and the
pathogen, might contribute to enhance the performance of biological control.
Filamentous bacteria in the genus Streptomyces, such as S. lydicus have been attractive as
biocontrol agents because of the ability to produce secondary antifungal compounds (Yuan and
Crawford 1995). Field evaluation showed that Streptomyces lydicus WYEC-108 reduced
Sclerotinia stem rot severity of soybean by 43.1% and sclerotia levels in soil by 90.6% (Zeng et
al. 2012a). This indicated its potential as an effective biological control under natural conditions.
Similarly, Bacillus spp. also have been recognized for the production of a vast array of
biologically active molecules potentially inhibitory to phytopathogen growth (Emmert and
Handelsman 1999) such as a novel class of antibiotics (He et al. 1994, Silo-Suh et al. 1994).
Biological control agents including C. minitans, Trichoderma harzianum Rifai, and
Streptomyces lydicus WYEC-108 have been previously studied to manage Sclerotinia stem rot in

44

Michigan (Zeng et al. 2012). The results showed a potential use of some biological control
agents as additional strategy to Sclerotinia stem rot management. Although, it may be
advantageous to combine different strategies for disease management. For instance, the use of
biological control agents that reduce pathogen inoculum in soil (Gerlagh et al. 1999, Santos and
Dhingra 1982) and the chemical (lactofen) that is correlated with the induction of defense
response in soybean (Dann et al. 1999). Therefore, to enhance the disease management, the
objective of this study was to examine the efficacies of these products individually or in
combination to affect Sclerotinia stem rot of soybean under field conditions.

45

MATERIALS AND METHODS
Field plots
Field trials were conducted at the Michigan State University Plant Pathology Research
Farm (PLP) (N 42°41.477’; W 84°29.153’) in East Lansing, and at the Clarksville Horticulture
Experiment Station (CLK) (N 42°42.626’; W 85°33.958’), Clarksville, MI in 2010, 2011 and
2012. The soil at PLP is a sandy loam, with 54.2% sand, 35% silt, and 10.8% clay, and a pH of
7.4. At CLK the soil is also a sandy loam, with 70.2% sand, 25% silt, and 2.8% clay, and a pH of
6.8. In 2010 the plots were arranged as a randomized complete block design (RCBD) with a plot
size of 6.1 × 9.1 m2 at PLP and 9.1× 15.2 m2 at CKL. In 2011 and 2012 the trials were arranged
in a split-plot design. At PLP the plot sizes were 13.7 × 9.14 m2 in 2011 and 9.1 × 9.1 m2 in
2012; at CKL the plot sizes were 10.6 × 12.2 m2 and 9.14 x 9.14 m2 in 2011 and 2012
respectively. There were four replications at PLP and three replications at CLK.
Soil infestation with Sclerotinia sclerotiorum
Sclerotia from commercial soybean production fields were collected from a Michigan
grain elevator for the 2010 and 2011 trials, and from D.F Seed Inc., Dansville, MI in 2012. The
sclerotia were separated from the seed by a gravity table and collected in burlap seed sacks and
stored until used for soil infestation. Approximately 6 kg of sclerotia were evenly distributed
each year by hand, onto the soil surface of plots. This was done on the 23rd of April 2010, the
5th of May 2011, and the 11th of May 2012 at PLP. Sclerotia were spread on the 20th of April
2010, the 7th of May 2011, and the 2nd of May 2012 at CLK. The sclerotia were incorporated
into the top 10 cm of soil with a cultivator (2210 Field Cultivator, Deere & Company, Moline,
IL).

46

Soil and foliar treatments
The treatments for PLP and CLK trials were applied at the manufacturers recommended
field rates. Coniothyrium minitans strain CON/M/91-08 (Contans WG, SipcamAdvan, Research
Triangle Park, NC), Trichoderma asperellum ICC 012 -T. gamsii ICC 080 (Tenet WP,
SipcamAdvan), Streptomyces lydicus WYEC 108 (Actinogrow, SipcamAdvan), Reynoutria
sachalinensis (Regalia, Marrone Bio Innovations Inc., Davis, CA). Bacillus amyloliquefaciens
(BCA03) were applied either to the soil or as foliar treatment at soybean R1growth stage as
water suspension (Table 3-1).
The chemical fungicides boscalid (Endura, BASF Corp., Research Triangle Park, NC)
and thiophanate-methyl ( Topsin M 70 WP, Nisso TM LLC, New York, NY) and the herbicide
lactofen (Cobra, Valent U.S.A. Corp., Walnut Creek, CA) (Table 3-1) were applied foliarly to
the plants and arranged in split-plot designs. All the treatments were applied with a tractor-driven
boom sprayer with TEEJET nozzles (model XR11003VS) set 18 cm apart and 45 cm above the
soil. Applications of biocontrol agents and chemicals (at rates shown in Table 3-1) were made at
speed of 1.77 km/hr with a nozzle pressure of 103.4 kPa.
In 2010, soybean cultivar Northup King S-20 p3 was planted on the 24th of May at PLP
and on the 19th of May at CLK; In 2011 and 2012, Great Lakes hybrids GL2449R2 was planted
the 12th of May at PLP and the 4th of June at CLK; In 2012, the GL2449R2 was planted on the
29th of May at PLP and the 6th of June at CLK. Soybean seeds were planted in 20 cm row
spacing with average seeding rate around 444,790 seed/ha. A sprinkler irrigation system was
used to maintain canopy moisture by watering twice each day for 20 minutes at 11:00 and 16:00
h at a volume of 1000 L/ha/h. Irrigation was also applied when soil moisture was below 80% of

47

field capacity, as measured with soil moisture sensors (CR10X Measurement and Control
System, Campbell Scientific, Logan, Utah) placed at 6 cm and 12 cm below the soil surface.
Viability of sclerotial population in soil
Sclerotia were counted at both pre-planting and at harvest. Prior to soil treatment, soil
samples were obtained to determine the base line density of S. sclerotiorum sclerotia in soil. Four
liters of soil were collected from each plot using a trowel. Each bag contained soil samples
randomly collected from each plot to a depth of 10 cm. Soil samples were also collected at
harvest using the same method. The samples were transported to the laboratory in an ice chest
and stored at 4C until processed. Sclerotia of S. sclerotiorum were harvested by wet sieving
(Hoes and Huang 1975). The soil was rinsed through a series sieves (U. S. standard sieves series
100-mesh, 200-mesh and 325-mesh, Fisher Scientific Inc., Pittsburgh, PA) using cool running
tap water to separate sclerotia. A subsample of ten sclerotia were taken and surface disinfested
with 0.6 % sodium hypochlorite for 3 min, and rinsed three times with sterile water for 1min
each. The disinfested sclerotia were air-dried on sterile filter paper. Once dried, sclerotia were
placed on water agar (1.5%) plates with a sterile forceps and incubated at room temperature in
the dark for 5 d. Sclerotia that germinated were considered viable.
Disease evaluation
Disease severity was estimated using a disease severity index (DSI). Plots were rated for
disease severity based on the rating system of Grau et al. (1982) at approximately the R7 stage of
soybean growth (Fehr et al. 1971) as defined by the maturity of soybean pods on the main stem.
Fifty plants from the center two rows were rated on a scale of 0 to 3, with 0 = no symptom, 1 =
symptoms on lateral branches, 2 = symptoms on the main stem with little or no damage on pods,

48

Table 3-1. Biocontrol agents and chemical treatments used in this study with rates of application
and dates used to test the efficacy against Sclerotinia stem rot of soybean at the Plant Pathology
Farm (PLP) and Clarksville Horticultural Experimental Station (CKL) from 2010, 2011 and
2012.
Year

Treatments

Rate a

Date
(day-month) b
28-April
28-April
28-April

Date
(day-month) c
4-May
4-May
4-May

Coniothyrium minitans
2.2 × 1012 cfu/ha
Streptomyces lydicus
1.1 × 1010 cfu/ha
Trichoderma asperellum 2.8 ×1010 cfu/ha
+ T. gamsii
C. minitans + T.
4-May
1.1 × 1012 cfu/ha + 28-April
10
asperellum + T. gamsii
1.4 × 10 cfu/ha
Reynoutria sachalinensis 9,354 ml/ha
19-July
16-July
extract
Boscalid
1,525 g/ha
19-July
16-July
Non-treated control
15-Nov
16-Nov
2011 C. minitans
2.2 × 1012 cfu/ha
12
C. minitans
15-Nov -9-May 16-Nov 11-May
1.1 × 10 cfu/ha
12
C. minitans
9-May
11-May
2.2 × 10 cfu/ha
10
S. lydicus
9-May
11-May
1.1 × 10 cfu/ha
10
T. asperellum + T.gamsii 2.8 ×10 cfu/ha
9-May
11-May
Lactofen
105 g/ha
20-July
3-August
Non-treated control
17-May
26-May
2012 C. minitans
2.2 × 1012 cfu/ha
12
C. minitans
17-May
26-May
1.1 × 10 cfu/ha
11
B. amyloliquefaciens
19-July
24-July
8 × 10 cfu/ha
BAC03
S. lydicus
17-May-19-July 26-May-24-July
1.1 × 1010 cfu/ha
10
T. asperellum + T.gamsii 2.8 × 10 cfu/ha
17-May-19-July 26-May-24-July
Lactofen
105 g/ha
12-July
18-July
Thiophanate-methyl
1,000 g/ha
27-July
1-August
Non-treated control
a
Biological control agents and chemicals were applied as the rate given in 280 L/ha of water .
b
Dates for treatments application at Plant Pathology Farm (PLP). c Dates for treatments
application at Clarksville Horticultural Experimental Station (CLK).
2010

49

and 3 = symptoms on main stem leading to plant death and poor or no pod fill. The DSI was
calculated by the formula:
DSI =

Ʃ disease rating of each plant
× 100
3 × number of plants rated

Soybean yield evaluation
Soybeans were harvested with a small-plot combine harvester (Massey Harris 35 plot
combine, with a 1.8 m cutting bar in 2010 or ALMACO Small Plot Combine (SPC20) with a 1.5
m cutting bar in 2011 and 2012). To avoid plot interactions, only the center of each plot was
harvested (one run of the harvester, covering about 66% of the width of the plot). The collected
soybean samples were weighed and yield. The yield was calculated as amount of soybeans
expressed in kg per harvested area (hectare). Two to three liters of beans were collected from the
pile of harvested beans from each plot and transported to the laboratory for examination.
Soybean moisture content and 100 bean weight were determined with a Steinlite
Moisture Tester SL95 (Seedburo Equipment Co. Chicago, IL) and seed counter (Count-A-Pak
Seed Totalizer Seedburo Equipment Co).
Number of sclerotia in harvested beans
Soybean samples were distributed in white polypropylene pans (29 × 34 cm, Newell
Rubbermaid, Atlanta, GA, 30328). Sclerotia were separated from beans by hand and the numbers
of sclerotia per kg of harvested beans was recorded.
Data analysis
Statistical analysis was performed using the SAS statistical package Version 9.2 (SAS
Institute, Inc., Cary, NC). Analysis of variance was performed using PROC GLM to test the
significance of the treatments for main effects and their interaction. Significant difference
50

between treatment means were compared using the Ryan Einot -Gabriel-Welsh Multiple Range
Test with α = 0.05. The residue analysis was performed by PROC UNIVARIATE and confirmed
the hypothesis that the experimental errors are normally distributed with a common variance.
RESULTS
Disease severity index (DSI) and density of sclerotia in beans
The treatment effects on the DSI were significant at both CLK and PLP (P < 0.001) in
2010. At PLP, the DSI was reduced by 75, 77, and 81 % with the S. lydicus, the Trichoderma
spp. /C. minitans combined treatment, and the extract of R. sachalinensis, respectively, as
compared to the non-treated control (Table 3-2). At CLK, the extract of R. sachalinensis and the
Trichoderma spp. /C. minitans combined treatment were the most effective, reducing the DSI by
55 % and 53 % respectively compared to the control (Table 3-2). In contrast, in the Trichoderma
spp. treatment an increase of the disease severity was observed when compared with the nontreated control (Table 3-2).
Significant effects of the treatments on the number of sclerotia in harvested beans were
observed at CLK (P = 0.006) and PLP (P = 0.027), in 2010 (Table 3-2). At PLP, the C. minitans
applied as single treatment had the highest number of sclerotia/kg of bean and differed
significantly from the non-treated control as well as S. lydicus and Trichoderma spp. /C. minitans
(Table 3-2). No significant differences were observed between the non-treated control with
boscalid, extract of R. sachalinensis, Trichoderma spp. (Table 3-2). At CLK, the C. minitans, the
Trichoderma spp. individually applied, Trichoderma spp. /C. minitans combined application, S.
lydicus and extract of R. sachalinensis had lower sclerotia numbers compared to the non-treated
control (Table 3-2). No difference was observed in number of sclerotia/kg of bean with boscalid
compared to the non-treated control (Table 3-2).
51

The combination of the biological control agents and lactofen significantly impacted the
DSI at PLP (P < 0.0004) and CLK (P < 0.0001) in 2011. At PLP, none of the treatments were
significantly different from the non-treated control in the reduction of Sclerotinia stem rot
severity except the C. minitans/Fall treatment in the absence of lactofen. In this treatment, the
DSI was higher than the non-treated control (Table 3-3). At CLK, significant DSI reductions
were obtained with all the treatments when compared with the non-treated control with the
exception of the Trichoderma spp. individually applied (Table 3-4).
The effect of the interaction between biological control agents and pesticide treatments on
the numbers of sclerotia in the beans was significant (P < 0.001) at CLK and PLP (P = 0.003) in
2011. At PLP, a reduction in the number of sclerotia in the beans was significant for
lactofen/Trichoderma spp., lactofen/C. minitans/Spring combined application, and the S. lydicus
individually applied compared to the non-treated control (Table 3-3). The highest numbers of
sclerotia in the harvested beans were observed in the C. minitans/Fall-Spring and Trichoderma
spp. alone application compared to the non-treated control (Table 3-3). At CLK, significant
reduction in the number of sclerotia in harvested beans were found in the lactofen/C.
minitans/Fall, lactofen/S. lydicus and lactofen/Trichoderma spp. treatments compared to the
same treatment applications without lactofen. No significant difference in sclerotia density were
observed when compared the lactofen treatment and the non-treated control (Table 3-4).
A significant chemical and biological control agent interaction for the severity of
Sclerotinia stem rot was observed at PLP and CLK (P < 0.001) in 2012. At PLP, the most
effective treatment was Trichoderma spp. alone reducing the severity of Sclerotinia stem rot by
84% followed by 42% for the combined treatment of lactofen/C. minitans at full rate (2.2 × 1012
cfu/ha) compared to the non-treated control (Table 3-5). The highest levels of DSI were observed

52

for BCA03 and lactofen/Trichoderma spp. when compared to the non-treated control (Table 35). Significant reductions in the DSI at CLK were observed for the C. minitans full rate (2.2 ×
1012 cfu/ha) and BAC03 in individual or combined application with lactofen when compared to
non-treated control. Similarly, S. lydicus applied individually, C. minitans applied alone at 1.1 ×
1012 cfu/ha or combined with thiophanate methyl and lactofen/Trichoderma spp. significantly
reduced the DSI compared with the non-treated control (Table 3-6).
In 2012 at PLP, the interaction of the biological control agent treatments with the
chemicals was significant (P = 0.037) for the number of sclerotia in the harvested beans.
Significant differences were observed in thiophanate methyl application; foliar application of S.
lydicus combined with lactofen; and one test with C. minitans alone applied at 1.1 × 1012 cfu/ha
compared to the non-treated control. No differences were detected among the other evaluated
treatments compared with the non-treated control (Table 3-5). At CLK, the interaction of
biological control agents with chemical treatments was significant (P < 0.001). Application of
thiophanate methyl significantly reduced the sclerotia in harvested beans compared to the nontreated control (Table 3-6). Individual application of S. lydicus, BCA03 and C. minitans at 1.1 ×
1012 cfu/ha significantly increased the number of sclerotia in harvested beans compared with the
non-treated control. No significant differences were found among the other treatments (Table 36).

53

Table 3-2. Effect of biological control agents Coniothyrium minitans, Trichoderma asperellum,
T, gamsii, Streptomyces lydicus; a plant extract from Reynoutria sachalinensis or a fungicide
boscalid on Sclerotinia sclerotiorum viable sclerotia in soil and harvested beans, severity of
Sclerotinia stem rot as expressed as a disease severity index (DSI) and yield of soybean in 2010
at two field locations: the Plant Pathology Farm (PLP) and the Clarksville Horticulture
Experimental Station (CLK).

Location
Treatment

Viable
Sclerotia/L
soil v

DSI w

Sclerotia/Kg
bean x

Yield
(Kg/ha)

PLP
Coniothyrium minitans
0.4 a y
18.2 a
3.0 a
4521.0 ab
T. asperellum and T.gamsii
0.4 a
14.2 a
1.8 ab
3888.0 b
Trichoderma asperellum +T. gamsii 0.4 a
4.2 b
1.1 b
5018.3 a
(50%) + C. minitans (50%)
S. lydicus
0.4 a
4.7 b
1.4 b
4656.3 ab
Reynoutria sachalinensis extract
0b
3.6 b
2.0 ab
5154.0 a
Boscalid
0.5 a
13.9 a
1.9 ab
3797.7 b
Control
0.5 a
15.9 a
1.5 b
4385.3 ab
CLK
C. minitans
0.2 b
21.1 b
3.8 c
2414.3 a
T. asperellum and T.gamsii
0.2 b
35.1 a
3.8 c
2821.0 a
Trichoderma spp. + C. minitans
0.2 b
16.4 c
4.4 bc
2889.0 a
S. lydicus
0.4 a
23.6 b
4.1 bc
2522.3 a
Reynoutria sachalinensis extract
0c
15.8 c
4.3 bc
2495.3 a
Boscalid
0c
23.8 b
5.2 ab
2604.0 a
Control
0c
23.8 b
5.6 a
2522.7 a
v
Sclerotia were retrieved by washing thorough sieves at harvest on 6 Oct and 7 Oct at PLP and
CLK respectively. w DSI = disease severity index of Sclerotinia stem rot, evaluated from 90
plants in the plot center on a scale of 0 to 3, with 0 = no symptoms, 1 = symptoms on lateral
branches, 2 = symptoms on the main stem with little or no damage on pods, and 3 = symptoms
on main stem leading to plant death; poor or no pod fill. The DSI was calculated by [(the sum of
disease scale rating of each plant)/ (3 × total plants rated)] × 100. x Sclerotia were counted at
harvest. y The Ryan Einot -Gabriel-Welsh Multiple Range test was conducted for each category
of data (in columns). The statistical analysis was done separately for each location.Values
followed by the same letter are not significantly different at α= 0.05.

54

Table 3-3. Effect of biological control agents and the herbicide lactofen applied individually or
in combination on severity of Sclerotinia stems rot, number of sclerotia in harvested beans and
yield of soybean. Different times of application were included for the C. minitans treatments:
Fall, Spring and Fall-Spring. The experiment was conducted at the Plant Pathology Farm in
2011.
Treatment

Timing of
applicationv

C. minitans
Lactofenu
no-lactofen
C. minitans
Lactofen
no-lactofen
C. minitans
Lactofen
no-lactofen
Trichoderma spp.
Lactofen
no-lactofen
S. lydicus
Lactofen
no-lactofen
No BCA
Lactofen
non-treated control

Fall

DSIw

Sclerotia/
Kg beanx

Yield
(Kg/ha)

3.2 cdy

2.9 cd

2936.0 a

15.0 a

2.0 cd

2634.5 abc

4.3 bcd
3.8 bcd

0d
3.2 cd

2652.7 abc
2762.3ab

4 bcd
0d

17.5 bcd
37.2 a

2483.5 bc
2401.3 c

2.7 cd
0d

0.9 d
30.7 ab

2524.7 bc
2506.3 bc

10.3 ab
8.7 abc

4.2 cd
1.3 d

2812.8 ab
2469.7 bc

2.0 cd
3.5 bcd

11.3 cd
19.2 bc

2671.0 abc
2579.5 bc

Spring

Fall + Spring

Spring

Spring

Spring

u

The herbicide lactofen was applied as a split treatment at the R1 growth stage of soybean. v The
BCAs were applied as a complete plot treatment before planting as a spray suspension on the soil
surface. w DSI = disease severity index, evaluated from 150 plants in the plot center on a scale of
0 to 3, with 0 = no symptoms, 1 = symptoms on lateral branches, 2 = symptoms on the main stem
with little or no damage on pods, and 3 = symptoms on main stem leading to plant death and
poor or no pod fill. The DSI was calculated by (the sum of disease scale rating of each plant)/ (3
× total plants rated) × 100. x Sclerotia were counted and evaluated at harvest. y The Ryan Einot Gabriel-Welsh Multiple Range test was conducted for each category of data (in columns). Values
followed by the same letter are not significantly different at α=0.05.

55

Sclerotia viability in the soil
In 2010 at PLP, a significant reduction in sclerotia viability was observed with the extract
of R. sachalinensis treatment compared with the non-treated control and the other treatments
evaluated (Table 3-2). In 2010 at CLK, the number of viable sclerotia in the soil with S. lydicus
application was significantly higher compared to the extract of R. sachalinensis, boscalid and the
non-treated control (Table 3-2). Similarly, viability of sclerotia in soil was higher for the C.
minitans, Trichoderma spp. and C. minitans/Trichoderma spp. treatments compared with the
non-treated control (Table 3-2). In 2011 at PLP, no sclerotia were found from any plots
evaluated. At CLK, the interaction of chemical with biological control agents was significant (P
< 0.001). A significant reduction of 98 % was observed for the Fall application of C. minitans in
the absence of lactofen, compared to lactofen applied alone and the non-treated control (Table 33). Coniothyrium minitans applied at Fall or Spring combined with lactofen or in individual
application reduced the sclerotia viability in the soil by 98, 91, and 90%, respectively (Table 34).
In 2012, the biocontrol and chemical treatments interaction for the reduction of sclerotia
viability in soil was significant at PLP (P < 0.002). Coniothyrium minitans at the full rate (2.2 ×
1012 cfu/ha) combined with lactofen reduced sclerotial viability in soil by 82 % when compared
to the same treatments applied individually. The highest number of viable sclerotia in soil was
found in the lactofen/Trichoderma spp. combined treatment. No significant differences were
found between the other treatments evaluated compared to the non-treated control (Table 3-5).
The biocontrol agent treatments effect was significant for viability of sclerotia in the soil at CLK
(P<0.001). Significant reductions by 79 % and 86 % for foliar applied S. lydicus and
Trichoderma spp. respectively were observed compared to C. minitans applied at 2.2 × 1012

56

cfu/ha and 1.1 × 1012 cfu/ha of the recommended field rate; BCA03; S. lydicus and non-treated
control. Trichoderma spp. ground application combined with lactofen had the highest number of
viable sclerotia in soil (Table 3-6).
Table 3-4. Effect of biological control agents applied at varying times in absence/combination
with the herbicide lactofen on soil levels of Sclerotinia sclerotiorum viable sclerotia, severity of
Sclerotinia stems rot, number of sclerotia in harvested beans and yield of soybean at Clarksville
Horticulture Experiment Station in 2011.
Treatment t

Timing of
applicationv

Viable
Sclerotia/L soilw

DSIx

Sclerotia/
Kg beany

Yield
(Kg/ha)

Fall
828.3 a
C. minitans
Lactofenu
0.7 dez
14.1 ef
2.9 bc
no-lactofen
0.1 f
23.7 d
55.9 a
Spring
723.8 ab
C. minitans
Lactofen
0.4 ef
16.1 ef
2.0 bc
no-lactofen
0.5 ef
16.0 ef
19.1 bc
Fall + Spring
585.6 b
C. minitans
Lactofen
0.9 cde
13.0 f
2.7 bc
no-lactofen
2.1b
37.7 c
22.4 b
Spring
688.6 ab
Trichoderma spp.
Lactofen
2.3 b
18.8 de
7.2 bc
no-lactofen
1.1cd
65.1 a
63.8 a
Spring
803.5 a
S. lydicus
Lactofen
1.1 cd
19.5 de
0.6 c
no-lactofen
1.9 b
19.9 de
64.2 a
Spring
743.2 ab
No BCA
Lactofen
5.3 a
10.7 f
8.2 bc
non-treated control
1.2 c
52.7 b
4.4 bc
u
The herbicide lactofen was applied as a split treatment at the R1 growth stage of soybean. v The
biological control agents were applied as a complete plot treatment before planting as spray
suspension on the top soil surface. w Sclerotia were retrieved by washing through sieves at
harvest on 15 Nov. x DSI = disease severity index, evaluated from 150 plants in the plot center
on a scale of 0 to 3, with 0 = no symptoms, 1 = symptoms on lateral branches, 2 = symptoms on
the main stem with little or no damage on pods, and 3 = symptoms on main stem leading to plant
death and poor or no pod fill. The DSI was calculated by (the sum of disease scale rating of each
plant)/ (3 × total plants rated) × 100. y Sclerotia were counted and evaluated at harvest. z The
Ryan Einot -Gabriel-Welsh Multiple Range test was conducted for each category of data (in
columns). Values followed by the same letter are not significantly different at α=0.05.

57

Yield evaluation
A significant effect of the interaction between biological control agent and chemical
application on yield was observed at PLP in 2010 (P = 0.006). Yield was significantly higher
with the extract of R. sachalinensis and the combination of C. minitans and Trichoderma spp.
compared with Trichoderma spp. alone and boscalid. No significant differences were found
between S. lydicus, and C. minitans compared with the non-treated control (Table 3-2). At CLK,
no significant effect of biological control agent or fungicide was observed (P = 0.802) on the
yield (Table 3-2). At PLP the effect of biological control agent and chemical interaction on yield
was significant (P = 0.02) in 2011. The Fall application of C. minitans had a higher yield than
the non-treated control. No significant differences were observed on yield among the other
evaluated treatments (Table 3-3). At CLK the effect of biological control agent was significant
(P = 0.02). The Fall application of C. minitans and S. lydicus was significantly different from
Fall/Spring application of C. minitans. None of the evaluated treatments were significantly
different from the control (Table 3-4). In 2012 at PLP, no significant differences were found
among the treatments compared with the non-treated control except the thiophanate-methyl
application compared with Trichoderma spp. alone application (Table 3-5). At CLK, there was
an interaction between biological control agent and chemical treatments for soybean yield
response (P = 0.024). The yield was increased with Trichoderma spp. in combination with
lactofen when compared to the non-treated control. No significant differences were observed
among the other treatments (Table 3-6).

58

Table 3-5. Effect of biocontrol agents with foliar or ground application in absence/presence of
the herbicide lactofen or with thiophanate-methyl on soil levels of Sclerotinia sclerotiorum
viable sclerotia, severity of Sclerotinia stem rot, number of sclerotia in harvested beans and yield
of soybean at the Plant Pathology Farm in 2012.
Treatmentt

Type of
applicationu

C. minitans 2.2 ×1012cfu/ha
Lactofen

Ground

Viable
DSI w
Sclerotia
/L soil v

Sclerotia/
Kg bean x

Yield
(Kg/ha)

0.7 cy

13.3 abc

4478.7 ab

39.3 e

3.2 ab
73.8 b
11.7 bcd
4326.0 ab
no-lactofen
12
C. minitans 1.1 ×10 cfu/ha Ground
1.6 abc
55.1 cd 13.5 abc
4610.3 ab
Thiophanate-methyl
1.4 abc
52.1 d
3.0 e
4563.0 ab
no-thiophanate-methyl
BCA03
Ground
1.7 abc
86.8 a
8.3 cde
4501.7 ab
Lactofen
2.0 abc
94.7 a
13.0 abc
4336.7 ab
no-lactofen
Trichoderma spp.
Foliar
2.0 abc
63.3 bcd 12.8 abc
4599.5 ab
Lactofen
1.6
abc
66.8
bc
20.1
a
4282.0 ab
no-lactofen
Trichoderma spp.
Ground
3.9 a
86.7 a
9.3 cde
4167.5 ab
Lactofen
1.5 abc
14.8 f
5.4 cde
4065.0 b
no-lactofen
S. lydicus/lactofen
Foliar
1.0 bc
64.0 bcd 3.6 e
4565.0 ab
S. lydicus/no-lactofen
Ground
1.0 bc
64.3 bcd 18.4 ab
4682.3 ab
Thiophanate methyl
Foliar
2.0 abc
53.7 cd 4.7 de
4896.3 a
Non-treated control
1.2 bc
66.5 bc 13.3 abc
4467.0 ab
t
Chemicals were applied as subplot treatments at the V6 and R1 stages of soybean growth for
lactofen and thiophanate methyl respectively. u Foliar application of biological control agents was
done at the R1 stages of soybean growth and ground application was done before planting as a
spray suspension on the soil surface. v Sclerotia were retrieved by washing through sieves at
harvest on 2 Nov. w DSI = disease severity index, evaluated from 150 plants in the plot center on
scale of 0 to 3, with 0 = no symptoms, 1 = symptoms on lateral branches, 2 = symptoms on the
main stem with little or no damage on pods, and 3 = symptoms on main stem leading to plant
death and poor or no pod fill. The DSI was calculated by [(the sum of disease scale rating of each
plant)/ (3 × total plants rated)] × 100. x Sclerotia were counted and evaluated at harvest. y The
Ryan Einot -Gabriel-Welsh Multiple Range test was conducted for each category of data (in
columns). Values followed by the same letter are not significantly different at α = 0.05.

59

Table 3-6. Effect of biocontrol agents with foliar or ground application in absence/presence of
the herbicide lactofen or with thiophanate-methyl on soil levels of Sclerotinia sclerotiorum
viable sclerotia, severity of Sclerotinia stem rot, number of sclerotia in harvested beans and yield
of soybean at the Clarksville Horticulture Experiment Station in 2012.
Treatmentt

Viable
DSI w
Sclerotia/ Yield
Sclerotia/
Kg bean x (Kg/ha)
v
L soil
12
9.2 bcdy
C. minitans 2.2 ×10 cfu/ha Ground
36.5 f
69.0 bcd
1147.5 b
Lactofen
38.7 ef
60.0 cde
2252.5 ab
no-lactofen
12
8.4 cd
C. minitans 1.1 ×10 cfu/ha Ground
86.1 bc 66.2 cd
1718.5 ab
Thiophanate-methyl
71.3 d
164.1 a
845.0 b
no-thiophanate-methyl
BCA03
Ground
8.1 d
47.0 ef
31.8 ef
1733.0 ab
Lactofen
51.0 e
94.6 b
1515.0 ab
no-lactofen
Trichoderma spp.
Foliar
2.2 e
99.0 a
42.4 de
1725.0 ab
Lactofen
89.8 abc 78.7 bc
2939.0 ab
no-lactofen
Trichoderma spp.
Ground
14.6 a
40.8 ef
51.8 cde
3536.0 a
Lactofen
97.0 ab 56.5 cde
1208.0 b
no-lactofen
S. lydicus/lactofen
Foliar
3.0 e
91.2 abc 42.0 de
1781.5 ab
S. lydicus/no-lactofen
Ground
11.3 b
45.0 ef
95.7 b
2196.5 ab
Thiophanate methyl
Foliar
11.0 bc
82.5 c
14.0 f
1006.5 b
Non-treated control
8.2 cd
100.0 a 51.0 cde
1421.0 b
t
Chemicals were applied as subplot treatments at the V6 and R1stages of soybean growth for
lactofen and thiophanate methyl respectively. u Foliar application of biological control agents
was done at the R1soybean growth stage and ground application of was done before planting as a
spray suspension on the soil surface. v Sclerotia were retrieved by washing through sieves at
harvest on 2 Nov. w DSI = disease severity index, evaluated from 150 plants in the plot center on
scale of 0 to 3, with 0 = no symptoms, 1 = symptoms on lateral branches, 2 = symptoms on the
main stem with little or no damage on pods, and 3 = symptoms on main stem leading to plant
death and poor or no pod fill. The DSI was calculated by [(the sum of disease scale rating of each
plant)/ (3 × total plants rated)] × 100. x Sclerotia were counted at harvest. y The Ryan Einot Gabriel-Welsh Multiple Range test was conducted for each category of data (in columns). Values
followed by the same letter are not significantly different at α = 0.05.
Type of
applicationu

60

Table 3-7. Summary effects of the biological control agents evaluated at different time, mode of
application or in absence/combination with the herbicide lactofen on Sclerotinia stem rot severity
and yield of soybean at Plant Pathology Farm in 2010, 2011 and 2012.
Treatment

2010
2011
2012
DSI Yield DSI Yield DSI
Yield
+
0
+
0
+
0

Streptomyces lydicus
C. minitans + T. asperellum + T. gamsii
Reynoutria sachalinensis extract
C. minitans Fall
Lactofen
0
+
no-lactofen
0
C. minitans 2.2 ×1012cfu/ha
Lactofen
+
0
C. minitans 1.1 ×1012cfu/ha
no-thiophanate-methyl
+
0
BCA03
Lactofen
0
no-lactofen
0
Trichoderma spp.
Lactofen
0
no-lactofen
+
0
(+) Reduction of Sclerotinia stem rot severity or yield increase by the biological control agents,
chemical treatment or their combination compared to the non-treated control. (0) No significant
effect of the biocontrol, chemical treatment or their combination in reduction Sclerotinia stem rot
severity or yield increase compared to the non-treated control. (-) Increase in Sclerotinia stem rot
severity of soybean by the biological control agents, chemical treatment or their combination
compared to the non-treated control.

61

Table 3-8. Summary effects of the biological control agents evaluated at different time, mode of
application or in absence/combination with the herbicide lactofen on Sclerotinia stem rot severity
and yield of soybean at Clarksville Horticultural Experimental Station in 2010, 2011 and 2012.
Treatment

2010
2011
2012
DSI Yield DSI Yield DSI
Yield
0
+
0
+
0
0
+
+
0
+
+
0
+
+
0
+
0
+
+
+
0

Trichoderma asperellum + T. gamsii
C. minitans + T. asperellum + T. gamsii
Reynoutria sachalinensis extract
C. minitans Fall
Lactofen
no-lactofen
C. minitans Spring
Lactofen
no-lactofen
C. minitans Fall + Spring
Lactofen
no-lactofen
Trichoderma spp.
Lactofen
no-lactofen
S. lydicus
Lactofen
no-lactofen
Lactofen
C. minitans 2.2 ×1012cfu/ha
Lactofen
+
0
no-lactofen
+
0
C. minitans 1.1 ×1012cfu/ha
Thiophanate-methyl
+
0
no-thiophanate-methyl
+
0
BCA03
Lactofen
+
0
no-lactofen
+
0
Trichoderma spp.
Lactofen
+
+
S. lydicus/no-lactofen
+
0
Thiophanate methyl
+
0
(+) Reduction of Sclerotinia stem rot severity or yield increase by the biological control agents,
chemical treatment or their combination compared to the non-treated control. (0) No significant
effect of the biocontrol, chemical treatment or their combination in reduction Sclerotinia stem rot
severity or yield increase compared to the non-treated control. (-) Increase in Sclerotinia stem rot
severity of soybean by the biological control agents, chemical treatment or their combination
compared to the non-treated control.
62

DISCUSSION
Coniothyrium minitans, Trichoderma spp. and S. lydicus can be effective in reducing the
severity of Sclerotinia stem rot, viability of sclerotia populations in the soil and sclerotia density
in harvested beans, but their performance varied among the years and locations. The lack of
consistency of microbial antagonist for disease suppression has been previously reported
(reviewed in Adams 1990, Boland 1997, Inglis and Boland 1992). It is not always clear which
factors may account for inconsistencies or which factors cause an antagonist to be effective in
biological control (Stolk et al. 1998). This situation may limit the adoption of these products in
commercial soybean production.
Effective reductions in the DSI and density of viable sclerotia in soil by Coniothyrium
minitans was observed in some instances, depending on the location and on the years with
particularly high disease levels (Table 3-4, 3-5 and 3-6). Other reports have shown that the
effective disease suppression by C. minitans was observed under low disease pressure (Budge et
al. 1995, Budge and Whipps 1991, Huang et al. 2000b). The extra ‘nutrient source’ provided by
the artificial soil infestation with sclerotia before planting during the current study might
stimulate the spore production of C. minitans fostering further propagation in the treated plots.
Similar observations was made by del Rio et al. (2002) with the mycoparasite Sporidesmium
sclerotivorum Uecker, Adams et Ayers in a plots with high disease pressure.
A significant reduction in sclerotia viability of S. sclerotiorum was observed at Fall and
Spring application of C. minitans compared with the non-treated control (Table 3-4). Under
controlled conditions, C. minitans strain CON/M/91-08 has a maximum growth rate at 18 to
20ºC (Zeng et al. 2012b). Temperatures either at fall or spring might provide the optimum
growth conditions and establishment for of C. minitans in the soil. The similarity between C.

63

minitans treated plots and the non-treated in regard of sclerotia viability in the soil, may be
related with the reported dispersal mechanisms of C. minitans such as irrigation water (Williams
et al. 1998a), and soil mesofauna (Williams et al. 1998b). The combination of any of these
dissemination factors with C. minitans application may improve the conidial dissemination
efficiency in the soil. An experiment with oilseed rape has reported a promissory result using
water-assisted application of C. minitans (Yang et al. 2009). Evaluation of this alternative mode
of C minitans application in soybean production may be useful to achieve consistent levels of
Sclerotinia stem rot control.
Trichoderma spp. reduced the DSI in 2011 and 2012 at CKL and 2012 only at PLP
(summary Table 3-8). Their role in reduction of viable sclerotia density in soil was minimal.
Infection of sclerotia under controlled conditions (Clarkson et al. 2004, Inbar et al. 1996,
Knudsen et al. 1991) and suppression of Sclerotinia diseases have been documented in other
crop-systems by Trichoderma spp. (Huang et al. 2000a, Zeng et al. 2012a), though under field
conditions the efficacy may be reduced (Budge and Whipps 1991, Ojaghian 2011). Different
abiotic factors (Duffy et al. 1997, Kredics et al. 2003) and biotic factors (Bae and Knudsen 2005,
Simon and Sivasithamparam 1988) may reduce growth and establishment of Trichoderma spp. in
soil ecosystems. The difference in clay content at PLP and CLK (10.8% and 2.8% respectively),
might impacted the biocontrol activity of the tested Trichoderma spp. Although, a report with T.
koningii indicated a positive correlation of clay contend with its biocontrol activity (Duffy et al.
1997). In fact, different mechanisms act synergistically to achieve disease control in some
Trichoderma spp. (reviewed in Howell 2003). According to Duffy et al. (1997), the relative
importance of individual mechanisms depending on the strain, environmental conditions, and
pathosystems; factors that might account for the results observed in the current study.

64

A relatively limited impact in reduction of sclerotia viability in the soil was observed with
Trichoderma spp. when compared with non-treated control for 2011 and 2012 at PLP. The
spatial pattern distribution of S. sclerotiorum sclerotia in the soil seems to play an important role
for a successful colonization of T. harzianum. Sclerotia in highly aggregated spatial pattern were
significantly colonized by the biocontrol in the soil compared to sclerotia in random distributions
(Bae and Knudsen 2007). It is possible that the even artificially infested sclerotia in the soil may
impact the biocontrol efficacy of Trichoderma spp. as a result of the modification of the natural
spatial pattern arrangement of sclerotia. This could be particularly important in terms of the
cultural practices that involved soil disturbance and the biocontrol application.
The bacterial strains included in this study had limited effect in Sclerotinia stem rot
suppression, although S. lydicus reduced the DSI in 2010 at PLP and 2012 at CLK and reduced
the sclerotial viability at CLK in 2012. Under growth chamber condition a complete reduction of
apothecia formed was observed with S. lydicus WYEC108 (Zeng et al. 2012b), which may
account for its effect in disease suppression, albeit the efficacy of S. lydicus was not consistent
under field conditions.
The co-application of biological control agents and the herbicide lactofen might be
advantageous strategy for disease control since they focus on different targets: the biocontrol
agents infect the primary inoculum of S. sclerotiorum (Abdullah et al. 2008, Froes et al. 2012,
McQuilken et al. 1995) and lactofen is associated with a plant defense response (Dann et al.
1999). The results were variable and dependent on the location and the year. Lactofen did
strengthen the disease suppression when was combined with Trichoderma spp., and C. minitans
particularly in years with high disease pressure (2011 and 2012) (summary Table 3-7 and 3-8). In
some cases the individual application of the biocontrol agents also reduced the Sclerotinia stem

65

rot severity compared the non-treated control. Based on these results it is difficult to provide any
conclusive recommendation due to the fluctuating results over the years evaluated. Many factors,
including biotic and abiotic environment (Bostock 2005), host genotype (reviewed in Da Rocha
and Hammerschmidt 2005, Herman et al. 2007) and the extent to which plants in the field are
already induced (reviewed in \Walters and Fountaine 2009) might modify or influence the
response of a resistance activators such as the herbicide lactofen.
Interestingly, disease reduction and yield increase was observed if Trichoderma spp. were
combined with lactofen at CKL in 2012 (summary Table 3-8). According with Nelson et al.
(2002a), the treatment of different soybean cultivars with postemergence herbicide such lactofen
reduced the number of flower per plant and influenced the peak flowering for all cultivars. This
could have direct implication in disease severity, as was indicated by Nelson and co-workers,
since it reduces the number of infection court (flowering stage) and convenient time (delay peak
of flowering) for S. sclerotiorum infection. The treatment of lactofen in combination with C.
minitans in 2011 at PLP (summary Table 3-7) did not reduced the Sclerotinia disease severity
but did increase the yield. The variation in the relationship between yield loss and disease
incidence in soybean may be the result of plant spatial compensation capacity. This correlation
between yield and disease is also affected by the environmental conditions after the infection
process has stared (Yang et al. 1999). Thus, recommendation of Sclerotinia stem rot management
with combined application of lactofen and biological treatment needs to be cautious given that
disease control and ultimately yield depend on multiple factors.
The fungicide application in the years evaluated also were inconsistent in the Sclerotinia
stem rot reduction and yield. Application of boscalid at PLP and CLK in 2010 (Table 3-2) did
not reduce the disease severity compared with the non-treated control. According with Mueller et

66

al. (2002a), poor coverage of the fungicide on the existing infection sites as well as additional
blossoms developing after that the fungicide has been applied might impact the efficacy of
fungicide. The application of thiophanate methyl at CLK in 2012, a year with a high disease
level, was effective in the diseases reduction but did not showed any benefits in yield when
compared with the non-treated control plot (summary Table 3-8). It is possible that although the
thiophanate methyl application reduced disease other factor such as environmental conditions, or
the presence of other soybean pathogen under field conditions might potentially limit the grain
yield.
In summary, the results indicated that reductions in the inoculum potential in the soil and
in Sclerotinia stem rot severity could be achieved by C. minitans and Trichoderma spp.
However, the effect varied over the years and locations. Similarly, combination of biocontrol
agents with lactofen provided effective reduction of Sclerotinia stem rot in some cases but not
others. The yield did not increase in all cases where disease severity was reduced. This variable
result emphasizes the need of evaluating different approaches that help to increase the stability of
disease suppression. For instance the selection of biocontrol strains natural adapted to soybeansoil ecosysistems. Further studies might evaluate the co-application of lactofen with selected
fungicides that may offer less variation in Sclerotinia stem rot management.

67

APPENDIX

68

THE EFFECT OF LACTOFEN ON REDUCTION OF SCLEROTINIA STEM ROT
UNDER CONTROLLED CONDITIONS

INTRODUCTION
In conventional agriculture synthetic chemical activators of resistance have been tested in
different pathosystems (Dann and Deverall 1996, Faoro et al. 2008, Katz et al. 1998), providing a
different approach to conventional disease management. Chemical activators such as 2,6dichloro-isonicotinic acid (INA) or S-methyl benzo [1,2,3] thiadiazole-7-carbothioate (ASM)
induced the defense response by mimicking the signal molecule salicylic acid (SA) and inducing
pathogenesis-related proteins (PR-proteins) gene expression (Friedrich et al. 1996, Uknes et al.
1993). In soybean multiple applications of these compounds under field and greenhouse
conditions resulted in a reduction on Sclerotinia stem rot severity by 70 % and 60% with INA
and ASM in susceptible varieties respectively (Dann et al. 1998). The adoption of these materials
in commercial soybean production may be hindered by their variable disease suppression over
the period of time and fields evaluated.
Variations in disease incidence and severity by the induction of defense response in
plants have been also documented with synthetic herbicides (reviewed in Altman and Campbell
1977, reviewed in Johal and Huber 2009). In soybean, lactofen, a postemergence herbicide of the
diphenyl ether class, has shown a protective effect against Sclerotinia stem rot under field
conditions (Dann et al. 1999). According to this study 40% to 60% disease reduction was
observed when lactofen was applied at an early soybean growth stage (V3-third trifoliolate- three
unfolded trifoliolate leaves) or at a later growth stage close to flowering. High levels of the
soybean phytoalexins were detected in herbicide injured leaves and a significant reduction in
lesion size of S. sclerotiorum were observed on plants treated at the V4 growth stage compared

69

with the untreated plant’s leaves (Dann et al. 1999). Subsequent studies of the effect of lactofen
on Sclerotinia stem rot revealed that the impact of the herbicide treatment is cultivar dependent
and the increase of phytoalexin production was detected up to 26 days after treatment (Nelson et
al. 2002a). Another study indicated that levels of glyceollin were observed in the leaves but were
not detected in the stem of the treated plants (Nelson et al. 2002b). This may suggest that an
additional mechanism(s) could be involved in the plant defense response of soybean treated with
lactofen.
The expression of plant induced resistance can be influenced by host genotype, biotic and
abiotic and environmental conditions (reviewed in Vallad and Goodman 2004). Hence, the
objective of this experiment are: 1. Evaluate the effect of Cobra® (a.i lactofen) on Sclerotinia
stem rot in a commercial soybean variety Great Lakes Hybrids GL2449R2 which belong to
maturity group 2.5 very suitable for Michigan condition, 2. Test two different growth stages at
the V2 and the V6 under greenhouse conditions to define the best time for an effective disease
reduction. 3. Evaluate three different concentrations of lactofen to determine the extent of
effective disease response.

70

MATERIALS AND METHODS
Plant material and growth conditions.
Glyphosate-resistant soybean (variety GL2449R2, Great Lakes® Hybrids) seeds with
partial resistance to Sclerotinia stem rot were planted in plastic pots 15.5 cm (height) x 20 cm
(diameter of top opening) filled with potting mix (BACTO Professional Planting Mix, Michigan
Peat Company, Houston, TX; 70%-80% sphagnum peat) sown at a 2.5 cm depth. Plants were
grown in a 1.3 m2 growth chamber (PGR14, Conviron, Winipeg, Manitoba, Canada) adjusted to
20°C and a 16:10 h light: dark cycle with light intensity of 73.56 µmol/m2/µA. At emergence of
cotyledons, seedlings were transferred to the greenhouse where the temperature ranged from 15°
C to 20° C. A 10 h photoperiod of natural sun light and supplemental lighting for 6 hours was
provided. Pots were watered on a regular basis as needed. Starting four weeks after planting,
soybeans were fertilized bi-weekly with a blood meal organic nitrogen supplement 12-0-0
(Scotts Miracle-Gro Products Inc., Marysville, OH).
Lactofen treatment
The application of lactofen was made at the V2 (second trifoliolate - two sets of unfolded
trifoliolate leaves) and the V6 (six trifoliolate – six unfolded trifoliolate leaves) growth stages of
soybean development. Treatments included water-treated control or lactofen (Valent, USA
Corporation, Walnut Creek, CA) treatments at 0.052 kg/ha, 0.11 kg/ha (field rate), and 0.22 kg/
ha. Ten ml of herbicide solution was applied per pot with a plastic household sprayer delivering
a fine mist to the leaf surface, covering the entire plant. A total of thirty plants were treated for
each herbicide concentration.

71

Sclerotinia sclerotiorum inoculum preparation
An isolate of Sclerotinia sclerotiorum originating from a single sclerotioum was collected
from an experiment plot with a history of Sclerotinia stem rot at the Pathology Farm (East
Lansing, MI) in 2011. A modification of the method of Bastien et al. (2011) for inoculum
production was used. Sclerotinia sclerotiorum was first growth on potato dextrose agar (EMB
Chemical Inc., Gibbstown, NJ, USA) at room temperature for 5 d. A pre-culture was initiated by
transferring three mycelial plugs (3 mm) to 200 ml of potato dextrose broth. After 5 days on a
rotary shaker at 110 rpm and 20°C the pre-culture was transferred to a 2 L flask containing 1 L
of potato dextrose broth and agitated on a rotary shaker at 150 rpm for 3 d at 20°C. The mycelial
suspension was homogenized for 30 s in a blender (Waring® Commercial New Hartford, CT).
Pieces (3 x 8 cm) of cotton pad (Top Care® Skokie, IL) were soaked in the fresh suspension and
immediately used for plant inoculation. Five ml of mycelial suspension was collected to
determine the fungal inoculum concentration by serial dilution plating. Approximately 5 ml of
fungal suspension was absorbed in the cotton pad as determined by estimating the remaining
volume in a 50 ml plastic centrifuge tube.
The inoculation was made when more than 80% of the plants reached the beginning of
the flowering stage (R1). The inoculum suspension was applied on the petiole of the lowest node
bearing flowers. The cotton pad covered the flower bud at the node but not the main stem (Figure
3-1). The inoculated plants were covered with plastic bags held to the pot with a rubber band.
After 5 d the plastics bags were removed along with the cotton pad. The disease was evaluated
approximately 20 d to 25 d after inoculation when the plants were at the R2-R3 (full floweringbeginning pod) growth stage. A disease severity index (DSI) was calculated, using a subjective
disease scale from 0 to 5, where 0 = no disease, 1 = inoculated nodes with bleached lesion, 2 =

72

extension of bleached lesion to lateral branches, 3 = bleached lesion on the main stem, 4 =
progression of bleached lesion along the stem, 5 = general wilting/plant dead. The DSI was
calculated by the following formula:
𝐷𝑆𝐼 =

Ʃ disease rating of each plant
× 100
5 × number of plants rated

Each experiment for the two soybean growth stage (V2 and V6) were done separately and
arranged as a two-factorial design with ten replications per treatment with pots as experimental
units for each the stage tested. Each experiment was repeated.
Data analysis
Statistical analysis was done separately for each soybean growth stage (the V2 and the V6
using the SAS statistical package Version 9.2 (SAS Institute, Inc., Cary, NC). The analysis of
variance was performed to test for significance of the treatments main effects. Means were
compared by the Ryan Einot -Gabriel-Welsh Multiple Range Test with α = 0.05. The residue
analysis was tested by PROC UNIVARIATE and confirmed the hypothesis that the experimental
errors were normally distributed with a common variance.
RESULTS
Sclerotinia stem rot evaluation
A statistically significant effect of lactofen on the Sclerotinia stem rot severity
was observed for the V2 and the V6Agrowth stages (P < 0.001). Lactofen application reduced
Sclerotinia stem rot severity at the V2 and the V6 soybean developmental stages, an average of
58.7 and 12.8 % respectively.
The different lactofen concentrations evaluated did not differ
B
statistically in their effect on disease severity at the V2 soybean growth stage (Figure 3-2A).
Significant differences were found among lactofen concentrations applied at the V6 growth stage
73

(Figure 3-2B). All the treatments were significantly different from the non-treated control. No
symptoms of Sclerotinia stem rot were observed in plants treated with the highest lactofen
concentration at the V6 (Figure3-2B).

Figure 4-1. Cotton pad inoculation method and Sclerotinia stem rot symptoms. A. The
inoculation was done on the petiole of the lowest node bearing flowers of the soybean cv.
GL2449R2 at the R1 growth stage with a cotton pad saturated with a mycelial suspension of S.
sclerotiorum (104 cfu/ml). B. Typical symptoms of Sclerotinia stem rot: stem discoloration and
leaf wilting 5 d after inoculation.

74

Disease severity index (%)

100

A

a

80
b

b

60

b

40
20
0
0

0.052

0.11

0.22

Lactofen a.i. kg.ha-1

Disease severity index (%)

100
B
80
60
40

a
b

20
c
0
0

0.052
0.11
Lactofen a.i. kg.ha-1

c
0.22

Figure 4-2. Panel A and Panel B. Effect of the herbicide lactofen application at the V2 (A) and
the V6 (B) soybean growth stages on Sclerotinia stem rot severity in soybean (cv. GL2449R2).
The plants were all inoculated with Sclerotinia sclerotiorum at the R1 growth stage and were
rated 20 to 25 days after inoculation. Disease severity was evaluated on a 0 to 5 scale (0 = no
symptoms and 5 = general wilting/plant dead). A disease severity index (DSI) was calculated
using the formula: DSI (%) = Σ (scores of all plants)/[5 × (total number of plants)] × 100. A total
of 30 plants were rated per lactofen concentration and the experiments were repeated. Soybean
plants treated with destilled water were used as control for both trials. Bars indicate standard
deviation. Means followed by same letter are not significantly different at α = 0.05 (RyanGabriel-Welsh-Multiple Range Test).
75

DISCUSSION
The severity of Sclerotinia stem rot was significantly reduced by lactofen application at
the V2 and V6 growth stages at the three concentrations evaluated for cv. GL2449R2. Similar
results were observed in soybean plants treated with lactofen under field conditions at early
growth stages (V3) or at flowering (R1) with a reduction of 40 to 60% in Sclerotinia stem rot
severity (Dann et al. 1999). In a previous report, lactofen treatment on soybean before flowering
resulted in a reduction of Sclerotinia stem rot but the extent of control varied among the cultivars
evaluated (Nelson et al. 2002a).This is particularly important in terms of recommendation for
growers since the impact of herbicide treatments on the incidence of disease may depend, among
other factors, on the cultivar (Nelson et al. 2002a, reviewed in Vallad and Goodman 2004). In
this study a reduction of Sclerotinia stem rot severity in the soybean variety Great Lakes Hybrids
GL2449R2 after lactofen treatments, indicate the potential applicability of the herbicide
treatment under field condition for the disease management.
Phytoalexin production is a good indicator of defense expression induced by a herbicide
treatment (Dann et al. 1999, Hammerschmidt 1999b). The levels of glyceollin induction depend
on seedling age, the chemical elicitor, and its concentration (Liu et al. 1992, Stössel 1982).
Preliminary results, using thin layer chromatography (TLC), suggest that the treatment with the
different lactofen rates (0.052; 0.11 and 0.22 kg/ha) at V6 induced glyceollin up to 20 days after
lactofen treatments compared with the glyceollin induction only with the highest rate when
lactofen was applied at the V2 growth stage (data not shown).
In summary, the application of lactofen to soybean cv. GL2449R2 at an early growth
stage or before flowering at the recommended field rate or even at half of the rate provided
significant reductions in Sclerotinia stem rot severity under controlled conditions when

76

compared with the non-treated plants. These results may contribute to appropriate
recommendations for Sclerotinia stem rot management with lactofen application under field
conditions for this variety.

77

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