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This is to certify that the
thesis entitled

Management of Soybean (Glycine max L.) White
Mold by Reducing Sclerotinia sclerotiorum
Population Using Beneficial Microorganisms

presented by

Wenting Zeng

has been accepted towards fulﬁllment
of the requirements for the

MASTER OF degree in PLANT PATHOLOGY
SCIENCE

 

 

 

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Date

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MANAGEMENT OF SOYBEAN (GL YCINE MAXL.) WHITE MOLD BY REDUCING
SCLEROTINIA SCLEROTIOR UM POPULATION USING BENEFICIAL
MICROORGANISMS
By

Wenting Zeng

A THESIS

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

MASTER OF SCIENCE
Plant Pathology

2010

ABSTRACT
MANAGEMENT OF SOYBEAN (GLYCINE MAX L.) WHITE MOLD BY REDUCING
SCLEROTINIA SCLEROTIOR UM POPULATION USING BENEFICIAL
MICROORGANISMS
By
Wenting Zeng
White mold of soybean [Glycine max (L.) Merr.], caused by Sclerotinia
sclerotiorum (Lib.) de Bary, is an important disease widely spread in the North Central
region of the US. Effectiveness of biocontrol agents (BCAs) was evaluated in the

laboratory, grth chamber and ﬁeld at two locations in Michigan. The result showed

that increased rates of BCAs decreased the sclerotial survival and carpogenic germination

of S. sclerotiorum. Coniothyrium minitans CON/M/9l-08 (Contans® WG) and

Streptomyces lydicus WYEC 108 (Actinovate® AG) survived throughout the season and

had the best efﬁcacy on disease suppression among the BCAs tested. C. minitans did not
fully eradicate infection by S. sclerotiorum, but over time it reduced the majority of
inoculum densities in soil. Coniothyrium minitans strain W09 was isolated in Michigan.
Morphological characteristics, effects of environmental factors on conidial production
and mycelia growth, and colonization of sclerotia of S. sclerotiorum were compared
between C. minitans W09 and CON/M/91-08. The optimal conditions were 20°C, pH 4.5
and 0 photoperiod h/d for mycelial growth, and 20°C, 24 photoperiod h/d for conidial

production. W09 outperformed CON/M/9l—08 in mycelia growth, conidial

production/plate, and colonization of sclerotia at 104 and 106 conidia/ml.

To my love Yuanteng Pei, my mom Changbin Sun and dad Jinxiang Zeng,

I dedicate this thesis

ﬁﬂﬂﬂ%ﬁ%ﬁk.ﬁmﬂ%ﬂx%

iii

ACKNOWLEDGEMENTS

I would like to sincerely thank my major advisor Dr. Jianjun Hao for bringing me
to Michigan State University and giving me an opportunity to pursue my masteris degree
in Plant Pathology. His instruction, patience, dedication, encouragement and friendship
have inspired me for any accomplishments. I graciously thank Drs. Raymond
Hammerschmidt, William K. Kirk, and Dechun Wang for serving on my advisory
committee. Their critiques and encouragement enable me to learn more about science and
move onto the right track of my professional career. I gratefully appreciate Drs. Martin
Chilvers, Dennis Fulbright and Noah Rosenzweig for reviewing my thesis. Gary Zehr and
Rob Schafer have given me vital support in the ﬁeld operation. I would not have survived
without their help. Justin Hodgins, Nicole Duchanne greatly assisted me both in the
laboratory and ﬁeld. Michigan Soybean Promotion Committee and GREEEN Program
have provided research funding for my project. Natural Industries, SipcamAdvan,
Bioworks, AgraQuest and PQ Corporation provided materials. Elizabeth Kelly has been
my favorite lab equipment consultant. Kimberley Lesniak has giving me precious advises
based on her research experience. Qingxiao Meng and Xiaohong Lu have provided me
kindhearted assistance and become my special friends. Wei Wang spent numerous hours
to help with my statistical analysis. Linglong Wei is supportive for being my best friend
in the past two years. Amy and Rick Oldejans, from MSU OISS friendship family
program, have moved me hundreds of times for their warm care. My love Yuanteng Pei
is always supportive. I am dedicated to my mom and dad, for their understanding,
encouragement, and their tolerance of the long period of missing me.

Wenting Zeng

iv

TABLE OF CONTENTS

Images in this thesis are presented in color.

LIST OF TABLES ........................................................................................................... viii
LIST OF FIGURES ........................................................................................................... ix
CHAPTER ONE: LITERATURE REVIEW ....................................................................... 1
INTRODUCTION ................................................................................................... 1
SOYBEAN ............................................................................................................... 2
SCLEROTINIA SCLEROTIOR UM .......................................................................... 4
SOYBEAN WHITE MOLD .................................................................................. 10
DISEASE MANAGEMENT ................................................................................. 15
FUTURE PERSPECTIVES ................................................................................... 25
OBJECTIVE OF THIS THESIS ............................................................................ 26
LITERATURE CITED .......................................................................................... 27

CHAPTER TWO: EFFECTS OF BIOLOGICAL CONTROL AGENTS ON
SCLEROTIAL SURVIVAL AND GERMINATION OF SCLEROTINIA

SCLEROTIOR UM .............................................................................................................. 37
INTRODUCTION ................................................................................................. 38
MATERIALS AND METHODS ........................................................................... 41

Isolate collection and inoculum production of Sclerotinia sclerotiorum...41
Soil inoculation with Sclerotinia sclerotiorum and treatment with

biocontrol agents ........................................................................................ 42
Apothecial observation and sclerotial retrieval .......................................... 43
RESULTS .............................................................................................................. 45
Effect of application rates of biological control agents on apothecial
production of Sclerotinia sclerotiorum ...................................................... 45
Effect of application rates of biological control agents on sclerotial
survival of Sclerotinia sclerotiorum .......................................................... 45
DISCUSSION ........................................................................................................ 53
LITERATURE CITED .......................................................................................... 55
CHAPTER THREE: MANAGEMENT OF SOYBEAN WHITE MOLD USING
BIOLOGICAL CONTROL STRATEGIES IN THE FIELD ............................................ 58
INTRODUCTION ................................................................................................. 59
MATERIALS AND METHODS ........................................................................... 61
Field plots ................................................................................................... 61

Soil infestation with Sclerotinia sclerotiorum ........................................... 61

Soil treatments ........................................................................................... 62
Population of sclerotia in soil .................................................................... 65
Disease evaluation ..................................................................................... 65
Soybean yield evaluation ........................................................................... 67
Population dynamics of Coniothyrium minitans, Trichoderma,
Streptomyces and Bacillus spp. in soil ....................................................... 67
RESULTS .............................................................................................................. 69
Disease and yield evaluation ...................................................................... 69
Population of sclerotia in soil .................................................................... 70
Population dynamics of Coniothyrium minitans, Trichoderma,
Streptomyces and Bacillus spp. in soil ....................................................... 71
DISCUSSION ........................................................................................................ 75
LITERATURE CITED .......................................................................................... 79

CHAPTER FOUR: GROWTH CHARACTERIZATION OF CONIOTHYRIUM
MINITANS ISOLATES UNDER VARIOUS ENVIORNMENTAL CONDITIONS AND

THEIR COLONIZATION ON SCLEROTINIA SCLEROTIOR UM .................................. 82
INTRODUCTION ................................................................................................. 83
MATERIALS AND METHODS ........................................................................... 86

Isolation of Coniothyrium minitans and the comparison of mycelial
growth ........................................................................................................ 86
Identiﬁcation of Coniothyrium minitans .................................................... 87
Effect of temperature on mycelial growth and conidial production of
Coniothyrium minitans ............................................................................... 88
Effect of pH on mycelial growth of Coniothyrium minitans ..................... 88
Effect of light on mycelial growth and conidial production of
Coniothyrium minitans ............................................................................... 89
Colonization of Sclerotinia sclerotiorum by Coniothyrium minitans on
agar plates .................................................................................................. 89
Colonization of Sclerotinia sclerotiorum sclerotia by Coniothyrium
minitans in the growth chamber ................................................................. 91
Effect of Coniothyrium minitans concentration on colonization of
Sclerotinia sclerotiorum in the growth chamber ....................................... 92
RESULTS .............................................................................................................. 93
Identiﬁcation of Coniothyrium minitans and the comparison of mycelial
growth ........................................................................................................ 93
Effect of temperature on mycelial growth and conidial production of
Coniothyrium minitans ............................................................................... 93
Effect of pH on mycelial growth of Coniothyrium minitans ..................... 94
Effect of light on mycelial growth and conidial production of
Coniothyrium minitans ............................................................................... 95
Colonization of Sclerotinia sclerotiorum by Coniothyrium minitans on
agar plates .................................................................................................. 95
Colonization of Sclerotinia sclerotiorum by Coniothyrium minitans in the
growth chamber ............................... ' .......................................................... 96

vi

Effect of Coniothyrium minitans concentration on colonization of

Sclerotinia sclerotiorum in the growth chamber ....................................... 97
DISCCUSSION ................................................................................................... 1 13
LITERATURE CITED ........................................................................................ l 16

vii

LIST OF TABLES

Table 1-1. Beneﬁcial microorganisms that affect Sclerotinia

sclerotiorum ........................................................................................... 20
Table 1-2. Commercialized biocontrol strains, product names and the producers. .. 22
Table 2-1. Biocontrol agents and their application rates in the study ............................... 43
Table 3-1. Treatment arrangement, application rate and date in each ﬁeld and year. ...... 64

Table 3-2. Effects of biological control agents and fungicide at planting to soybeans on
soil production of Sclerotiorum sclerotiorum, white mold severity and yield of soybean at
PLP .................................................................................................................................... 72

Table 3-3. Effects of biological control agents and fungicide on soil population of
Sclerotinia sclerotiorum and white mold severity and yield of soybean at CLK ............. 73

Table 4-1. Diversity of mycelial growth rates of Coniothyrium minitans isolated from
different ﬁelds in Michigan on potato dextrose agar ............................................... . ........ 99

Table 4-2. Origin, colony type and conidia size of Coniothyrium minitans isolates W09
and CON/M/9l-08 on potato dextrose agar .................................................................... 100

viii

LIST OF FIGURES

Figure 2-1. Effect of biological control agents on apothecial production of Sclerotinia
sclerotiorum. Soil was treated with one of the products at various rates: Coniothyrium
minitans (A), Trichoderma harzianum (B), Bacillus subtilis (C), and Streptomyces
lydicus (D). Repeated trials were combined for regression analysis. In the equation, x =
log (rate of application), and y = number of sclerotia/L soil. ........................................... 47

Figure 2-1 continued. ........................................................................................................ 48

Figure 2-2. Effect of biological control agents on survival of Sclerotinia sclerotiorum
sclerotia in group 1 trials (no carpogenic germination occurred). Soil was treated with one
of the BCA products at various rates: Coniothyrium minitans (A), Trichoderma
harzianum (B), and Bacillus subtilis (C). Data from all trials were averaged for regression
analysis. In the equation, x = log (rate of application), and y = number of sclerotia/L soil
........................................................................................................................................... 49

Figure 2-2 continued. ........................................................................................................ 50

Figure 2-3. Effect of biological control agents on sclerotial survival of Sclerotinia
sclerotiorum in group 2 (carpogenic germination occurred). Soil was treated with one of
the BCA products at various rates: Coniothyrium minitans (A), Trichoderma harzianum
(B), Bacillus subtilis (C), and Streptomyces lydicus (D). Data from all trials were
averaged for regression analysis. In the equation, x = log (rate of application), and y =
number of sclerotia/L soil ................................................................................................. 51

Figure 3-1. Population dynamics of Coniothyrium minitans in C. minitans CON/M/91-
08-treated soil (A), T richoderma spp. in T. harzianum T-22-treated soil (B), and
Streptomyces spp. in S. lydicus WYEC 108-treated soil (C) in 2009. Each microorganism
was detected and enumerated on semi-selective media. Soil samples were collected at 3,
28, 71, and 169 days after soil treatment. Error bars were determined by repeated
measurements by Tukey-Kramer adjustment using RROC GLIMMIX at P = 0.05. *
indicate signiﬁcant difference between treated and control plots on the same sampling
day ..................................................................................................................................... 74

Figure 4-1. Morphology of C. minitans W09 (400 X magniﬁcation), showing mycelia and
conidia. ............................................................................................................................ 101

ix

Figure 4-2. Colonization of Sclerotinia sclerotiorum sclerotia by C. minitans strain W09.
Pycnidia (black spherical bodies) and conidia of Coniothyrium minitans oozing out of
sclerotial surface (arrows) of Sclerotinia sclerotiorum under a dissecting microscope (25
O magniﬁcation) .............................................................................................................. 102

Figure 4-3. Effect of temperature on mycelial growth of C. minitans isolates, W09 and
CON/M/91-08. Mycelial growth was measured for eight consecutive days. Growth rates
were the slope of regression equations derived from eight-day mycelial growth. Bars on
each column are standard deviation. Uppercase and lowercase letters are used for mean
separation for C. minitans W09 and CON/M/91-08, respectively. Means with the same
letters are signiﬁcantly different. Growth at each temperature was compared between the
two isolates, and * indicates signiﬁcant difference of means between the two isolates at
the same temperature ...................................................................................................... 103

Figure 4-4. Effect of temperature on conidial production/plate (90 mm in diam.) of C.
minitans isolates W09 (upper panel) and CON/M/91-08 (lower panel). Conidial
production was determined at 3, 6, 9 and 12 d using a hemacytometer. Colored lines
indicate different temperatures. Comparisons were performed using F isheris least
signiﬁcant difference (LSD, P = 0.05) for each column. Bars on each column are
standard deviation, and * indicates signiﬁcant difference between the strains at the same
temperature (P = 0.05) .................................................................................................... 104

Figure 4-5. Effect of temperature on conidial production/mm2 mycelial area of C.

minitans isolates W09 and CON/M/91-08. Conidia were counted at the sixth day using a
hemacytometer. Comparisons were conducted using Fisheris least signiﬁcant difference
(LSD, P = 0.05) for each column. Uppercase and lowercase letters are used for means
separation of C. minitans W09 and CON/M/91-08, respectively. Means with same letters
are not signiﬁcantly different. Bars on each column are standard deviation, and *
indicates signiﬁcant difference between strains at the same temperature (P = 0.05) ..... 105

Figure 46 Effect of pH values on mycelial growth of C. minitans isolates W09 and
CON/M/91-08. Mycelial grth was measured for eight consecutive days. Growth rates
were determined by the slope of equations in linear regression based on the data of eight
days. Growth rates of the two isolates were compared at each pH value. Uppercase and
lowercase letters are used for means separation of C. minitans W09 and CON/M/91-08,
respectively. Means with same letters are not signiﬁcantly different. Bars on each column
are standard deviation, and * indicates the signiﬁcant difference between isolates at the
same pH (P = 0.05) ......................................................................................................... 106

Figure 4-7. Effect of light on mycelial growth of C. minitans isolates W09 and
CON/M/91-08. Mycelial growth was measured for eight consecutive days. Growth rate
under each photoperiod was determined by the slope of equations in linear regression
based on the growth data of eight days. Growth rates of the two isolates were compared
at each photoperiod. Uppercase and lowercase letters are used for means separation of C.
minitans W09 and CON/M/91-08, respectively. Means with same letters are not
signiﬁcantly different. Bars on each column are standard deviation, and * indicates the
signiﬁcant difference between isolates at the same photoperiod (P = 0.05) .................. 107

Figure 4-8. Effect of light on conidial production/plate (90 mm in diam.) of C. minitans
isolates W09 (upper panel) and CON/M/91-08 (lower panel). Conidial production was
determined at 3, 6, 9 and 12 d using the hemacytometer. Colored line indicated different
photoperiod (h/d). Multiple comparisons were performed for each photoperiod using least
signiﬁcant difference (LSD, P = 0.05), and * indicates signiﬁcant difference between the
strains under the same photoperiod. Bars on each column are standard deviation ......... 108

Figure 4-9. Effect of light on conidial production/mm2 mycelial area of C. minitans

isolates W09 and CON/M/91-08. Conidia were counted at the sixth day using a
hemacytometer. Uppercase and lowercase letters are used for means separation of C.
minitans W09 and CON/M/91-08, respectively. Means with same letters are not
signiﬁcantly different. Values of the two isolates were compared at each photoperiod
using least signiﬁcant difference (LSD, P = 0.05), and * indicates signiﬁcant difference
between the strains. Bars on each column are standard deviation .................................. 109

Figure 4-10. Colonization of S. sclerotiorum sclerotia by C. minitans isolates W09 and

CON/M/91-08 via mycelia (upper panel) and conidial concentration of 108 conidia/ml
(lower panel). The sclerotium was diced into slices with same thickness. Colonization
was calculated as the number of infected sclerotial slices divided by total sclerotia slices
examined. Linear regression estimated the correlation between time (day, x axis) of
incubation and percentage of colonization (y axis) ......................................................... 110

Figure 4-11. Effect of inoculation of C. minitans isolates W9 and CON/W91-08 on
colonization of sclerotia (upper panel) and sclerotial survival (lower panel) of S.
sclerotiorum in the growth chamber. A total of 25 sclerotia were placed in each pot

followed by 100 ml C. minitans conidial suspension (108 conidia/ml) was placed in each

pot with three replications. The percentage of colonization was determined as the infected
sclerotia divided by total sclerotia buried. The survival rate was calculated after four
weeks. Non-linear regression determined the correlation between time (day, x axis) from
inoculation and the percent of colonization or sclerotial number (y axis) ...................... 111

xi

Figure 4-12. Effect of concentration of C. minitans isolates W09 and CON/M/9l-08 on
colonization of sclerotia (upper panel) and sclerotial survival (lower panel) of Sclerotinia
sclerotiorum. A total of 25 sclerotia were placed in each pot. Conidial concentrations at 0

(NT), 102, 104, 106, 108 conidia/ml in a volume of 100 ml were inoculated in each plot
with four replications. Sclerotial survival was determined after four weeks. Comparisons
using Fisher’s least signiﬁcant difference (LSD) were performed for each week. Growth
at each temperature was compared between the two isolates. Bars on each column are
standard deviation, and * indicates signiﬁcant difference between the means of isolates at
the same temperature ...................................................................................................... 112

xii

CHAPTER ONE: LITERATURE REVIEW

INTRODUCTION

Sclerotinia sclerotiorum (Lib.) de Bary is a destructive fungal plant pathogen
which poses a threat to a great number of economically important crops worldwide
(Abawi and Grogan 1979; Steadman 1979). White mold caused by S. sclerotiorum is
ranked as the second most important disease of soybean in the United States next to
soybean cyst nematode (Heterodera glycines Ichinohe) (Wrather et a1. 2001). Annual
loss of soybean production due to S. sclerotiorum is estimated to be $70 million in the
United States (U. S. Canola Association 2005). Extensive economic losses have been a
driving force for substantial research on this disease. This review focuses on the biology,
physiology, pathogenicity of S. sclerotiorum, as well as disease development and

management of soybean white mold.

SOYBEAN

Soybean [Glycine max (L.) Merr.] is derived from Glycine ussuriensis (Regel and

Mack), a legume originating from Central China. Soybean has been cultivated as a food

crop in Eastern China since the 11th century BC (Hymowitz and Shurtleff 2005). The

crop was ﬁrst introduced into the US in 1765 by Samuel Bowen (Hymowitz and Shurtleff
2005). At present, the United States, Brazil, China, Argentina, India, Canada, Australia,
and South Africa produce over 90% of the worldis total soybean crop (Dill 2005). In
2010, 78.1 million acres of soybeans have been planted in the US, among which two
million acres have been planted in Michigan (National Agricultural Statistics Service
2010). Soybean varieties grown in the United States are divided into indeterminate and
determinant growth habit, and separated by 13 maturity groups (Tian et al. 2010).
Soybean genome sequence has been released in public data base recently (Schmutz er al.
2010). Analysis from sequence will assist the identiﬁcation of the genetic basis of traits
and facilitate the creation of disease resistant varieties (Schmutz et al. 2010).

Soybean is an important crop, providing protein and oil. It is a high-quality food
source because it contains high concentrations of ﬁber, vitamins, minerals,
polyunsaturated fats, low concentrations of saturated fats, and all essential amino acids
for humans (Sacks et al. 2006). Isoﬂavones from soybean help decrease low-density
lipoprotein cholesterol concentrations (Sacks et al. 2006). Because of these constituents,
soy products have been widely used in food supplies including cooking oil, meal, ﬂour,
infant formula, and dairy product substitutes. In addition, soybean is a major resource for

biodiesel production in the United States. Over 80% of biodiesel in the US. is derived

from domestic soybean Oil (Hill et al. 2006). However, only 6% of domestic diesel
demand could be met if the entire US. soybean crop was processed (Hill et al. 2006).
Therefore, the ability to produce sufﬁcient biodiesel without affecting food supplies is
urgently needed.

To increase yield and enhance the efﬁciency of soybean production, advanced
technologies are the key to achieving disease and pest control and improving yield. For
example, glyphosate-resistant soybeans released in 1996 have greatly impacted soybean

production and weed management. Monsanto Company produces a genetically modiﬁed

Roundup Ready® soybean with an inserted glyphosate-resistant gene CP4 EPSPS from

A grobacterium tumefaciens strain CP4. With these glyphosate-resistant soybeans,
growers can spray the non-selective herbicide glyphosate without damaging the crop
(Dill 2005). In 2005, more than 80% Of soybeans grown in the US. were glyphosate-
resistant (Dill 2005).

In spite of the success of weed control, disease control needs to be improved.
Soybean is still prone to be attacked by a number of phytopathogens, including six
bacteria, 38 fungi and oomycetes, 12 nematodes, and 15 viruses (Sinclair and Hartman
1999). Among them, Sclerotinia sclerotiorum (Lib.) de Bary is one of the most common

and important fungal pathogens. Therefore, it is a research focus for plant pathologists.

SCLEROTINIA SCLEROTIORUM

Taxonomy

Sclerotinia sclerotiorum (Lib.) de Bary is characterized by the production of
melanized tuber-like sclerotia, apothecia, ascospores and lack of conidia (Tourneau
1979). A sclerotium is a compact mycelial mass containing food reserves. Apothecia
initials arise from sclerotia and form on stipes. Apothecia are brown and cup-shaped
structures full of asci. An ascus contains eight ascospores which form after meiosis. At
maturity, the ascospores are forcibly discharged into the air (Tourneau 1979).

Since S. sclerotiorum was ﬁrst studied as a plant pathogen in 1837, its taxonomy
and name have been changed several times (Purdy 1979). Sclerotinia sclerotiorum was
ﬁrst named as Peziza sclerotiorum in 1837 by Libert. Later in 1924, Fuckel renamed it as
Sclerotinia libertiania Fuckel, which was in the same genus with S. candolleana, S.
fuckeliana, S. libertinia, S. tuberose, and S. baccata. The binomial name stood until
Wakeﬁeld demonstrated its conﬂict with the International Rules of Botanical
Nomenclature. Waterﬁeld incorrectly changed the name from S. libertiania F uckel to S.
sclerotiorum (Lib.) Massee. It wasnit until 1979 when Purdy recognized that de Bary
used the name ﬁrst in 1884 and gave the proper authority as Sclerotinia sclerotiorum
(Lib.) de Bary. The detailed history of taxonomy is described by Purdy (1979). The
classiﬁcation of the organism was further conﬁrmed by Kohn using restriction fragment
length polymorphism ﬁnger printing (Kohn et al. 1988). Sclerotinia sclerotiorum is now
classiﬁed in the kingdom Fungi, phylum Ascomycota, class Discomycetes, order

Helotiales, family Sclerotiniaceae, and genus Sclerotinia.

Host range

The host range of S. sclerotiorum expanded from 64 plant families, 225 genera,
361 species in a total of 383 species compiled by Purdy (1979) to at least 75 families, 278
genera 408 species by Boland and Hall (1994). The hosts include both economically
important dicotyledonous species, such as soybean, dry bean, peanut, canola, chickpea,
lentils and many vegetables, and monocotyledonous species such as tulip and onion
(Boland and Hall 1994). However, S. sclerotiorum does not infect corn, small grains or
grasses (Rousseau et al. 2007). More than 60 diseases caused by S. sclerotiorum have
been named for speciﬁc hosts, such as soybean white mold (Yang et al. 1999), lettuce
drop (Chitrampalam et al. 2008), Sclerotinia wilt Of sunﬂower (Eva 2003), head rot Of
sunﬂower (Huang et al. 2006), pod rot of pea (Huang et al. 2006), stem rot of common
bean and tomato (Huang and Erickson 2007), watery soft rot of cabbage (Bolton et al.

2006), and Sclerotinia blight of peanut and canola (Huang et al. 2006).

Geographic distribution and diversity

Sclerotinia sclerotiorum usually occurs in cool (bellow 20°C) and moist areas Of
the world (Abawi and Grogan 1979). The growth of S. sclerotiorum is less active at
temperatures below 0 or above 32°C than at temperatures in between (Abawi and Grogan
1979). These temperature ranges enable the fungus to have broad geographical niches
across all continents (Purdy 1979). Whetzel (1945) believed that S. sclerotiorum
originated from the Northern Hemisphere considering most of the sclerotiniaceous fungi
are populated in this cool environment. However, DNA ﬁngerprinting studies

demonstrate that clones of S. sclerotiorum are scattered, dispersed and disconnected from

their points of origin; they are geographically mixed within ﬁelds and widely separated
between ﬁelds (Anderson and Kohn 1995). Therefore, the precise geographical and
ecological origin of S. sclerotiorum is unclear. Although S. sclerotiorum is more active in
cool and moist areas, it does exist in hot and dry areas such as Florida, California, and
Arizona (Purdy 1979; Subbarao 1998; Chitrampalam et al. 2008).

Genetic diversity of S. sclerotiorum is associated with geographic distribution.
Sequence-related ampliﬁed polymorphism (SRAP) and random ampliﬁed polymorphic
DNA (RAPD) have been applied to investigate the diversity of S. sclerotiorum isolates in
China, Canada, and United Kingdom. The polymorphic loci of S. sclerotiorum isolates
vary from 31% to 98% using SRAP (Li et al. 2009). These genetic variations can be
considered as adaptation under environmental stress. Variations in oxalic acid production
potential and mycelial compatibility of S. sclerotiorum isolates have also been observed

from different geographic locations and hosts (Durman et al. 2005).

Sclerotia

Sclerotia are an overwintering structure of S. sclerotiorum. The development of
sclerotia is practically divided into three phases: mycelial initiation, sclerotial
development, and sclerotial maturation (Tourneau 1979). At the beginning, white round
mycelial masses appear with tiny liquid droplets on the surface (Tourneau 1979). As this
structure continues to expand, it forms a sclerotium initial. The medulla inside the
sclerotium consists of compacted hyphae. Rind cells start to assemble beneath the

sclerotial surface (Tourneau 1979). At maturity, a sclerotium is composed of black rind

cells, medulla of prosenchymatous tissues, ﬁbrillar matrix and a cortical layer, which
forms a black hardened compact mass of mycelia (Tourneau 1979).

The formation of sclerotia is inﬂuenced by the type of nutrition available and
environmental factors (Humphersonjones and Cooke 1977). Sclerotinia sclerotiorum
consumes organic compounds, such as carbohydrates and amino acids, to produce
proteins, crude fat, and inorganic compounds required for growth and formation of
sclerotia (Tourneau 1979). Different nutrition sources, for example, artiﬁcial media
versus ﬁeld soil, result in different biochemical composition of sclerotia (Tourneau
1979). Light, temperature, and pH value affect the sclerotial formation (Wong and
Willetts 1974). Sclerotia are properly formed at temperatures between 0 to 30°C and at
pH between 2.5 to 9.

Mature sclerotia are able to germinate either carpogenically or myceliogenically.
For carpogenic germination, sclerotia ﬁrst produce stipes from surface, followed by
apothecia production on tip of stipes. Abawi and Grogan (1979) suggest that average
three apothecia are produced from a single sclerotiorum, but the actual number of
apothecia is highly correlated with sclerotial sizes (Hao et al. 2003).

The survival of S. sclerotiorum sclerotia is affected by many factors, such as
moisture, temperature, depth of burial in soil, soil microﬂora, and soil proﬁle. Soil
moisture, temperature, and preconditioning are important factors that affect the
carpogenic germination of S. sclerotiorum. The optimal condition for carpogenic
germination in the laboratory is around 16°C, -0.03 MPa water potential for about 10
continuous days, and 24 hours of continuous light exposure/day (Hao et‘ al. 2003;

Harikrishnan and del Rio 2006). Temperature ﬂuctuations Of 8°C around 20°C lead to

maximum sclerotial germination (Mila and Yang 2008). On the contrary, ﬂuctuations of
water potential have negative effect on sclerotial germination and apothecial production
(Mila and Yang 2008). Carpogenic germination can be enhanced by chilling sclerotia at
4°C for 2 weeks or longer at soil moisture near saturation and rinsing of sclerotia under
running water prior to burial in soil (Dillard et al. 1995). In addition, soil texture and light
intensity also affect sclerotial germination (Hao et al. 2003; Wu el al. 2008).

Not only do many abiotic factors affect survival of sclerotia in soil,
microorganisms also play a critical role in sclerotia degradation (Lockwood 1977; Budge
et al. 1995; Abdullah et al. 2008). Microorganisms in soil affect the germination of
sclerotia. Lockwood (1977) explains this phenomenon as fungistasis. Mycoparasites of
sclerotia such as C. minitans and T. harzianum, T. koningii, T. gamsii, T. asperellum, and
T. virens, colonize sclerotia and therefore reduce the germination of S. sclerotiorum
(Budge and Whipps 1991; Budge et al. 1995; Escande et al. 2002; Abdullah et al. 2008;
Kim and Knudsen 2009). Bacteria such as Bacillus subtilis, have demonstrated the ability
to degrade sclerotia (Yang et al. 2009). Volatile antiftmgal compounds produced by
bacteria including aldehydes, alcohols, ketones, and sulﬁdes inhibit carpogenic
germination of S. sclerotiorum (Fernando et al. 2005). The soil bacterium Serratia
plymuthica is able to completely suppress apothecial formation by producing a
chlorinated macrolide (Thaning et al. 2001). In addition, the larvae of the fungus gnat
(Bradysia coprophila Lintner) directly damage sclerotia by feeding and indirectly
increase the colonization Of sclerotia by mycoparasites (Gracia-Garza et al. 1997). A

number of organisms that feed on or parasitize sclerotia include nematodes, earthworms,

centipedes, snails, mites may lead to the degradation of sclerotia as well (Coley-Smith

and Cooke 1971).

SOYBEAN WHITE MOLD

Symptoms

The name white mold comes from the visual sign of the disease: white ﬂuffy
mycelia on leaves, stems, and pods. The earliest symptoms are necrotic foliage of
soybean (Abawi and Grogan 1979). Water soaked lesions may be seen on leaves and
leaves will turn brown and senesce. Stem nodes become bleached, which is a distinctive
symptom of an infected host. At later stages of disease development, ﬂuffy white mycelia
may cover the bleached stem. Finally, the infection may result in death of the entire plant,

leaving sclerotia formed in/on the pods and the stems of the plant debris.

Disease epidemiology

Sclerotia of S. sclerotiorum serve as survival structures and primary inocula.
Sclerotia can survive for up to eight years in soil, but viability of sclerotia decrease over
time (Coley-Smith and Cooke 1971). Sclerotinia sclerotiorum can initiate disease of
soybean via two mechanisms. If sclerotia are in contact or in close proximity to soybean
plants, mycelial germination may result in infection of the plant. However, this form of
infection plays a minor role in disease development compared to carpogenic germination.
Ascospores released by carpogenic germination of sclerotia account for the most
common source of inoculum (Abawi and Grogan 1979). Apothecia are formed from
sclerotia at germination and ascospores are forcibly discharged from matured apothecia

into the air (Boland and Hall 1988). Under naturally infested ﬁeld conditions, the density

of apothecia in the ﬁeld normally ranges from zero to six apothecia/m2 (Gerlagh et al.

10

1999). However, up to 180 apothecial m2 has been reported in artiﬁcially inoculated soil

(Huang et al. 2006). Released ascospores are spread by precipitation (rain and irrigation)

and air movement (wind). A single apothecium can produce as many as 3 X 107

ascospores with the maximum ascospore production occurring at days four to nine during
ascospore release (Steadman 1979). Ascospores have been documented to travel at a
speed of 1600 m/h (Clarkson et al. 2003) and travel up to four kilometers under ﬁeld
conditions, but 90% of ascospores are deposited within 100 to 150 m (Abawi and Grogan
1979; Steadman 1979). Ascospores can be trapped from neighboring ﬁelds as well
(Hammond et al. 2008). Clearly, external sources of ascospores can lead to disease
development in distant crops. Adjacent ﬁelds with plant debris are prone to sclerotial
survival (Hammond et al. 2008). Ascospores exposed to ﬁeld environment desiccate and
lose their viability in three to four days (Olivier and Seguin-Swartz 2006). However,
under optimal conditions at temperatures of 30°C and relative humidity over 90%, 50%
of ascospores are able to last two to three weeks (Clarkson et al. 2003)

Ascospore infection includes ascospore germination, host penetration, and
colonization (Jamaux et al. 1995). Abawi and Grogan (1979) demonstrated that
ascospores require exogenous nutrients for germination and penetration of the host. For
soybeans, senescent ﬂower petals or necrotic tissues, serve as the nutrient resource for
ascospore germination (Abawi and Grogan 1979; Bolton et al. 2006). Consequently,
early reproductive stages are the most critical period for white mold development in
soybean and many other crops (Boland and Hall 1988; Clarkson et al. 2003; Hammond et
al. 2008). Ascospores can germinate with substantial moisture without nutrients in the

environment, but the ability to form appresoria and to penetrate the host is reduced

11

(Harikrishnan and del Rio 2006). A period Of approximately 48 to 72 hrs of free moisture
is required for infection (Abawi and Grogan 1979), which may come from dew, fog, and
rain (Young et al. 2004). In the ﬁeld, a period of continuous wetness for 10 days is the
key to initial infection and white mold development (Young et al. 2004).

Infection hyphae developed from ascospores grow through the ﬂower pedicel and
petals down to the stem and move all over the host tissue in the cortex (Lumsden and
Dow 1973). The infection hyphae go intercellularly and intracellularly, consuming host
nutrients from the senescent tissues (Lumsden and Dow 1973). The infected host tissues
undergo histological changes including alteration in cell wall structure, hypersensitive
response of cell, accumulation of ﬂuids and phytoalexins, and other enzymes related to

pathogenicity (Hancock 1972; Lumsden and Dow 1973).

Pathogenesis

Sclerotiorum sclerotiorum pathogenesis is associated with hydrolytic enzymes
and toxins (Bolton et al. 2006). Sclerotinia sclerotiorum has several weapons to infect
host plants, such as cell wall degrading enzymes (CWDEs), oxalic acid, and toxins
(Maxwell and Lumsden 1970; Fraissinet-Tachet er al. 1995; Cessna et al. 2000; Rollins
and Dickman 2001; Cotton et al. 2003; Guimaraes and Stotz 2004; Bolton et al. 2006).
Plant cell walls consist of primary cell wall (cellulose, and pectin), the middle lamella
(pectin), and secondary cell wall (cellulose). The enzyme complex produced by S.
sclerotiorum facilitates cell wall degradation and tissue maceration, which includes
pectinases, B-l, 3-glucanases, cutinases, and cellulases (Cotton et al. 2003). Pectinase

breaks down the primary cell wall and the middle lamella (Cotton et al. 2003).

12

Sclerotinia sclerotiorum produces polygalacturonase (PG), a pectinase serving as
a virulence factor (Bolton et al. 2006). PGs release oligo-galacturonides and degrade
unesteriﬁed pectin (Fraissinet-Tachet et al. 1995). Based on their function, PGs are
divided into endoPGs and exoPGs. Bolton (2006) has listed 18 genes encoding for
CWDEs. The multiple copies of polygalacturonase isozymes of endoPGs produced by S.
sclerotiorum give the pathogen ﬂexibility and adjustability in the infection process to a
wide range of hosts (Fraissinet-Tachet et al. 1995). I

Oxalic acid (0A) is considered as a fundamental pathogenic factor that
contributes to the early infection process of S. sclerotiorum (Maxwell and Lumsden
1970). 0A targets host tissue and functions pathogenicity with several modes of action.
(1) 0A accumulates in early infected tissues; increased concentration of 0A reduces the

pH value of host tissue gradually (Maxwell and Lumsden 1970). Low pH values around 4

to 5 favor various cell wall degrading enzymes (Maxwell and Lumsden 1970). (2) 0A

activity is closely associated with chelation of Ca2+ (Bateman and Beer 1965). During

penetration and infection, OA binds to the cell wall Ca2+, resulting in host cell wall

collapse and tissue maceration (Bateman and Beer 1965). (3) Oxidative burst is

characterized by the release of 02 and H202 at the site of pathogen infection and is

. 2+ . .
determmed by H202 and Ca concentratlons In most defense responses (Cessna et al.

2000). 0A inhibits oxidative burst in soybean and tobacco (Cessna et al. 2000). (4) 0A
also functions as an elicitor of plant programmed cell death (PCD) which is responsible
for induction of mammalian apoptotic-like features during white mold development (Kim

et al. 2008). (5) In addition, OA inhibits polyphenol oxidase production, regulates pH-

.13--

regulated gene expression in molecular signaling pathways (Rollins and Dickman 2001),

and operation of guard cells which control stomatal opening (Guimaraes and Stotz 2004).

14 —.

DISEASE MANAGEMENT

Management of soybean white mold is based upon the epidemiology of the
disease. Efﬁcient management requires implementation of an integrated disease control
approach. Strategies of disease management include a) pathogen refusal, such as using
plant resistance; b) pathogen elimination, such as using chemicals, soil ﬁrmigation,
biological control, cultural practice, and soil amendments; c) mistiming of pathogen
germination and soybean ﬂowering, such as rearranging planting dates and suppressing

or stimulating sclerotial germination.

Disease resistance

The most effective way to manage white mold is to plant resistant or less
susceptible soybean cultivars (Kim et al. 2000). To date, resistant germplasms to white
mold have not been found, but the disease severity of soybean cultivars vary responding
to S. sclerotiorum in controlled environments (Kim et al. 2000; Hoiﬁnan et al. 2002;
Cober et al. 2003). For example, cultivars Corsoy, COrsoy 79, Hodgson 78, $19-90, and
Asgrow A2506, demonstrate the greatest partial resistance to S. sclerotiorum (Kim et al.
1999; Yang et al. 1999; Kim et al. 2000).

The reason that immunity has not been developed for soybean is partially because
the resistance is multigenic (Arahana et al. 2001; Vuong et al. 2008). Inconsistent ﬁeld
performance and undiscovered resistance in commercial cultivars support this conclusion.
In recent years, quantitative molecular approaches have been developed. A number of

resistance quantitative trait loci (QTLs) on molecular linkage groups (LGs) of soybean

have been identiﬁed by a large range of genetic markers such as RAPD, restriction
fragment length polymorphism (RFLP), and simple sequence repeat (SSR) (Arahana et
al. 2001; Guo et al. 2008; Vuong et al. 2008). These QTLs have been mapped and can
provide valuable information for marker-assisted soybean breeding programs (V uong et
al. 2008). QTLs or speciﬁc resistant genes evaluation in controlled environment will
make disease resistance evaluation more consistent.

Although genes for resistant to white mold have not been found, it is possible to
develop resistance via genetic modiﬁcation of soybean. For example, soybean is
modiﬁed by gene transfer to obtain the oxalic acid degrading enzymes (Donaldson et al.
2001). Genes in soybean do not encode oxalic-acid-degrading enzymes. However, a
number of microorganisms are capable of degrading oxalic acid, and therefore contain
the genes encoding for oxalic acid degrading enzymes. Soybean introduced with an
oxalate oxidase gene f_rom wheat germin reduces disease incidence after cotyledon and

stem inoculation of S. sclerotiorum (Donaldson et al. 2001). Oxygen oxidoreductase

oxidase (0x0) is encoded to degrade oxalic acid and generate H202, which plays a role

in oxidative burst and hypersensitive response (Donaldson et a1. 2001). Transgenic
tobacco plants over expressed an oxalate decarboxylase gene (oxdc) contain less 0A,
reduce colonization, and show resistance to S. sclerotiorum (Dias et al. 2006; Walz et al.
2008). Decarboxylase gene has been introduced in lettuce by Agrobacterium-mediated
transformation and the transgenic lettuce was symptomless after inoculation of S.
sclerotiorum (Dias et al. 2006).

Soybean produces polygalacturonase-inhibiting protein (PGIP) to target fungal

PGs (Ferrari et al. 2003). PGIPs induce a number of defense responses. Pgip gene family

16

in Arabidopsis thaliana (L.) Heynh have been characterized by expressed sequence tag
(EST) and genomic libraries (Ferrari et al. 2003). D'Ovidio (2006) compared four
members of the legume Pgip gene family and determined the distinct regulation
properties of each encoded protein product. These researches provide useful information

for developing disease resistance.

Chemical control

Chemical fungicides play an important role in current management strategies for
Sclerotinia diseases, although they are not ﬁrlly effective. The efﬁcacy of ﬁmgicides on
white mold has been evaluated on snap bean (Hunter et al. 1978), soybean (Mueller et al.
2002), dry bean (Mueller et al. 1999), sunﬂower (Mueller et al. 1999), white bean
(Morton and Hall 1989), canola (Bradley et al. 2006), and lettuce (Chitrampalam et al.
2008). Fungicides such as azoxystrobin (Quadris), benomyl (Benlate), boscalid (Endura),
dicloran (Botran), iprodione (Rovral), prothioconazole (JAU6476),
pentachloronitrobenzene (PCNB), tebuconazole (Folicur), triﬂoxystrobin (Gem),
thiophanate-methyl (Topsin M), and vinclozolin (Ronilan) have provided moderate
disease control with inconsistency (Hunter et al. 1978; Mueller et al. 2002; Bradley er al.
2006; Chitrampalam et al. 2008). Vinclozolin was the most effective fungicide in
inhibiting mycelial growth of S. sclerotiorum in vitro (Mueller et al. 2002). At low
disease incidence, foliar sprays of thiophanate methyl reduced the incidence of white
mold by 50% (Mueller et al. 2002). However, high disease incidences result in non-

consistent control (Mueller et al. 2002).

Thorough coverage of the canopy and timing of application are essential to
improve the efﬁcacy of fungicides (Mueller et al. 2002). Fungicides are recommended to
be applied during the early reproductive stage of host plants, when plants are more
susceptible to S. sclerotiorum (Mueller et al. 2002). However, the long period of soybean
ﬂowering (one to ﬁve weeks) limits the efﬁcacy of foliar fungicides, and even two
applications provide a maximum 28-day protection (according to manufactures’
recommended application protocol) (Mueller et al. 2002). Because timing is critical, a
forecasting system would be useful to maximize application efﬁcacy, especially when
disease risk is high.

Many other chemical compounds have been tested for managing white mold.
Herbicides chlorsulfuron, cyanazine, metribuzin, triallate, and triﬂuraline signiﬁcantly
reduced carpogenic germination of sclerotia (Teo et al. 1992). A diphenyl ether herbicide
such as lactofen can reduce white mold incidence ﬁ'om 40 to 60% (Dann et al. 1999). In
addition, the essential oil Orihanum syriacum var. bevanii and F oeniculum vulgare were
reported to have effect on apothecial germination of S. sclerotiorum sclerotia (Soylu et al.
2007).

Fungicide resistance in S. sclerotiorum remains a concern. Although no fungicide
(benomyl) resistance was found based on 100 S. sclerotiorum isolates collected from a
snap bean ﬁeld in New York (Hunter et al. 1978), Michigan (Detweiler 1983), and
Virginia (Smith et a1. 1991), it may be possible that S. sclerotiorum also demonstrate

resistance to these fungicides. However, further tests are needed.

Biological control

Biological control (biocontrol) has been used in the past decades for managing
soybean white mold (Budge and Whipps 2001; Jones 2002; Abdullah et al. 2008).
Beneﬁcial microorganisms such as Coniothyrium minitans Campbell (syn.
Paraconiothyrium minitans) can eliminate sclerotia of S. sclerotiorum via
mycoparasitism (Campbell 1947; Budge et a1. 1995; Gerlagh et al. 1999; Budge and
Whipps 2001; Gerlagh et al. 2003; Li et al. 2005; Chitrampalam et al. 2008). Beside C.
minitans, many other microorganisms have been demonstrated inhibitory effects on S.
sclerotiorum (Table 1 -1).

To date, a total of eight biocontrol products have been commercially available to
control Sclerotinia diseases (Table 1-2). Among these formulations, products containing
C. minitans have been well studied due to highly specialized mycoparasitism of C.
minitans to sclerotia-forming fungi, including S. sclerotiorum, S. minor, S. trifoliorum,
and Sclerotium cepivorum (Campbell 1947; Budge et al. 1995; Huang et al. 2000; Jones
2002; Jones et al. 2004a; Yang et al. 2007). Since its discovery from S. sclerotiorum
(Campbell 1947), C. minitans has been evaluated as soil treatment on crops, such as dry
bean, potato, oilseed rape, carrot, lettuce, celery, sunﬂower, bean, chicory and cowpea,

and applied as a foliar spray on onion, kiwi fruit, rapeseed, and bean (Whipps et al.

2008)

19

Table 1-1. Beneﬁcial microorganisms that affect Sclerotinia sclerotiorum.

 

 

Speﬁes Mode of action Reference

Conio'thyrium minitans Mycoparasitism Campbell 1947; Budge 1995
Bacillus subtilis Antibiosis Schmiedeknecht et al. 2001
Bacillus amyloliquefaciens Antibiosis Abdullah 2008
Sporidesmium sclerotivorum Mycoparasitism del Rio et al. 2002

T richoderma harzianum

T richoderma koningii

T richoderma aureoviride

T richoderma gamsii

T richoderma asperell um

T richoderma Iongibrachiatum
T richoderma hamatum

T richoderma virens
Ulocladium aﬁ‘um
UlocIadium atrum

Pseudomonasﬂuorescens

Pseudomonas chlororaphis
Ophiostoma mitovirus

Cryptococcus albidus

Mycoparasitism, antibiosis, SAR
Nutrient competition, antibiosis
Nutrient competition, antibiosis
Nutrient competition, antibiosis
Nutrient competition, antibiosis
Nutrient competition, antibiosis
Mycoparasitism
Mycoparasitism

Unknown

Competitive colonization
Antibiosis

Toxins

Hypovirulence

Unknown

Menendez and Godeas 1998
Escande et al. 2002
Escande et al. 2002

Van Beneden et al. 2010
Van Beneden et al. 2010
Escande et al. 2002
Gracia-Garza et al. 1997
Tu 1980; Budge 1995
Huang and Erickson 2007
Li et al. 2003

Ashofteh ei al. 2009
Selin et al. 2010

Boland 2004

Reeleder 2004

 

2O

uh

I.”

The mechanisms of C. minitans attack on S. sclerotiorum are mycoparasitism and
nutrient competition (Whipps et al. 2008). The latter is less important because C.
minitans does not grow on/in plant tissue even with wound (Gerlagh et al. 1996). The
suppressive effect on S. sclerotiorum has peen studied using detailed co-culture methods
to characterize the interaction (Smith et al. 2008). Coniothyrium minitans produces a
wide range of cell wall degrading enzymes such as glucanases and chitinases that are
associated with the mycoparasitic process (Whipps et al. 2008). C oniothyrium minitans is
able to degrade oxalic acid produced by S. sclerotiorum, which interrupts the infection by
S. sclerotiorum (Ren et al. 2007). Coniothyrium minitans can not only degrade oxalic
acid but can also survive in the soil (Ren et al. 2007). The detection of antimicrobial
metabolites in culture indicates that chemical compounds are associated with
mycoparasitic processes (Tomprefa et al. 2009).

There is little information of molecular studies on mycoparasite-host interaction.
Speciﬁc enzyme and metabolite production in the progress of mycoparasitism remains
unclear. Several genes have been sequenced and 11 mutants of C. minitans were
identiﬁed by Agrobacterium tumefaciens-mediated transformation (Whipps et al. 2008).
A cosmid library of C. minitans has been established and pathogenic genes, PKAC and
PMKI genes have been sequenced (Whipps et al. 2008). Muthumeenakshi (2007)
conducted suppression subtractive hybridization (SSH) to determine genes regulating the
mycoparasitism process using SSH, a cDNA library was established and 251 putative
genes were identiﬁed (Muthumeenakshi et al. 2007). More than 20% of the genes were

considered to have novel function during sclerotial mycoparasitism (Whipps et al. 2008).

21

Table 1-2. Commercialized biocontrol strains, product names and the producers.

 

 

Product name Active ingredient Company

Contans® WG C. minitans CON/M/9l-08 SipCamAdvan, Roswell, GA

lntercept® WG C. minitans CON/W9l-08 Encore Technologies, Minnetonka, MN

Serenade® MAX B. subtilis QST 713 AgraQuest, Davis, CA

Companion® B. subtilis Growth Products Ltd., White Plains,
NY

PlantShield® HC T. harzianum T-22 Bioworks, Victor, NY

Supresevit® T. harzianum Binab USA, Bridgeport, CT

Tenet®WP T. asperellum and T. gamsii SipCamAdvan, Roswell, GA

Soilgard® T. virens Certis U.S.A., Columbia, MD

 

In addition to C. minitans, a number Of other species have been proved to be
inhibitive to S. sclerotiorum. For example, T richoderma harzianum is effective to control
S. sclerotiorum (Kim and Knudsen 2009) with multiple modes of action including
mycoparasitism, antibiosis, nutrient competition, and promotion of plant growth (Ousley
et al. 1994); Bacillus subtilis is also effective in suppressing mycelia of S. sclerotiorum
and reducing white mold incidence and severity in the ﬁeld (Schmiedeknecht et al. 2001;
Yang et al. 2009); Streptomyces lydicus demonstrates a strong antagonistic effect on
Pythium ultimum and R. solani by producing extracellular antifungal metabolites (Yuan
and Crawford 1995) and cell wall degrading enzymes (Mahadevan and Crawford 1997).

Application of biocontrol products is challenging as their efﬁcacy can vary
depending on environmental conditions. The microorganisms are affected by a number of

abiotic and biotic factors. For example, soil moisture (Tu 1999), temperature (McQuilken

22

v."

V.
m-

>3

1‘:

et al. 1997; Tu 1999), soil type, light (McQuilken et al. 1997), water tension (Ciotola et
al. 2007), and pH (McQuilken et al. 1997) all affect the activity of biocontrol agents. The
timing of application of biocontrol agents is important. Earlier application can beneﬁt the
strains that survive and establish better in soil (Jones et al. 2004b). Soil temperature is
also an important factor to be considered prior to application. For example, C. minitans
and T. virens were applied as integrated control for white mold. But C. minitans infected
sclerotia at a lower temperature range (4 to 25°C) than T. virens (10 to 30°C) and

therefore C. minitans had better efﬁcacy (Budge et al. 1995).

Cultural practices and soil amendments

Cultural practices and cultivar selection can improve the management of S.
sclerotiorum. Planting date, plant density, crop rotation, tillage, and weed management
have signiﬁcantly affected soybean white mold (Gracia-Garza et al. 2002; Rousseau et al.
2006). For example, early planting may result in lower disease incidence because of the
mismatch in timing between the period covering soybean ﬂowering and apothecial
germination (Hammond et al. 2008). Increasing within-row distance of soybean results in
decreased incidence of white mold due to improved air circulation and alteration of the
microclimate (Vieira et al. 2010). Two or four years of soybean rotation with corn lowers
the risk of white mold (Gracia-Garza et al. 2002; Rousseau et al. 2007). However, the
longevity of sclerotia survival in soil limits the effect of rotation (Coley-Smith and Cooke
1971) and the effectiveness of cultural practices is inﬂuenced by inoculum density and

environmental factors.

23

Soil amendments have been used in the ﬁeld to control soybean white mold
(Huang et al. 2006; Rousseau et al. 2006; Huang and Erickson 2007). Crop residues of
mustard, wheat, broccoli, canola, barley, cat, and lentil have been amended in soil and
reduced carpogenic germination of S. sclerotiorum (Huang and Sun 1991; Huang et al.
2007). Urban compost, mineral compost and fermented industrial wastes are also
effective (Rousseau et al. 2006; Rousseau et al. 2007). Most of the soil amendments
change the soil microbial community that favored disease suppression. However, all
these soil amendments lack consistency, as changing the microbial community can result
in either positive or negative effects (Rousseau et al. 2006). The results also vary
depending on soil types, environmental conditions, and biochemical characteristics of
pathogens (Rousseau et al. 2006). In addition, the large amount of soil amendment
materials required for successful trails is often not practical for large-scale commercial

application.

24

FUTURE PERSPECTIVES

Screening highly resistant varieties of soybean is the ultimate goal of disease
management, although ﬁnding genes regulating resistance is challenging. In the future,
more efforts need to be made on breeding programs for white mold resistance. Among
the other research directions, utilization of oxalic acid degrading enzymes is attractive.
Since the gene that encodes oxalic acid degrading enzymes has been transferred into
soybeans, we anticipate that the efﬁciency and consistency of gene expression can be
further improved. Alternatively, microorganisms that produce oxalic acid degrading
enzymes can be directly used as biological control agents. Biocontrol research needs to
focus on reproducibility and improved efﬁcacy of disease control. Finding compounds or
microorganisms that can stimulate or suppress the germination of S. sclerotiorum
sclerotia is another possible way to manage the disease. In addition, a disease forecasting
system is urgently needed to assess disease risk and suggest timings for chemical
application, cutting down the cost of application and preventing severe diseases. The

theme of the above researches is to help the establishment of sustainable agriculture.

25

OBJECTIVE OF THIS THESIS

White mold (caused by Sclerotinia sclerotiorum) signiﬁcantly affects soybean
production in Michigan. Since current strategies provide unsatisfactory control, better
approaches are required for disease management. Studying the interactions between
sclerotia of S. sclerotiorum and antagonistic biocontrol strains allows researchers to
develop better strategies for disease control, Optimize the efﬁcacy of control, and reduce
overall disease development.

Even though the successful biocontrol of white mold has been documented on
various crops such as lettuce and dry bean, biocontrol of white mold on soybean have
received mixed and inconsistent results. In addition, little information is available about
the survival of biocontrol strains under environmental conditions in Michigan. Evaluating
the effect of commercialized biocontrol products to control white mold on soybean,
optimizing the application rates of biocontrol products, and studying the characteristics
and survival of biocontrol strains in comparison to local strains will directly beneﬁt

Michigan soybean production in sustainable agricultural systems.

26

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Wu, B. A., Subbarao, K. V., and Liu, Y. B. (2008). Comparative survival of sclerotia of
Sclerotinia minor and S. sclerotiorum. Phytopathology 98: 659-665.

Yang, D., Wang, B., Wang, J ., Chen, Y., and Zhou, M. (2009). Activity and efﬁcacy of
Bacillus subtilis strain NJ -1 8 against rice sheath blight and Sclerotinia stem rot of
rape. Bio]. Control 51: 61-65.

Yang, R., Han, Y. C., Li, G. Q., Jiang, D. H., and Huang, H. C. (2007). Suppression of
Sclerotinia sclerotiorum by antifungal substances produced by the mycoparasite
Coniothyrium minitans. Eur. J. Plant Pathol. 119: 411-420.

Yang, X. B., Lundeen, P., and Uphoff, M. D. (1999). Soybean varietal response and yield
loss caused by Sclerotinia sclerotiorum. Plant Dis. 83: 456-461.

Young, C. S., Clarkson, J. P., Smith, J. A., Watling, M., Phelps, K., and Whipps, J. M.
(2004). Environmental conditions inﬂuencing Sclerotinia sclerotiorum infection
and disease development in lettuce. Plant Pathol. 53: 387-397.

Yuan, W. M., and Crawford, D. L. (1995). Characterization of Streptomyces lydicus

WYEC108 as a potential biocontrol agent against fungal root and seed rots. App].
Environ. Microbial. 61: 3119-3128.

36

CHIP
SILE
Sflf

Q5

5111

of

CHAPTER TWO: EFFECTS OF BIOLOGICAL CONTROL AGENTS ON
SCLEROTIAL SURVIVAL AND GERMINATION OF SCLEROTINIA
SCLEROTIORUM

ABSTRACT
Biological control agents (BCAs) were evaluated on affecting sclerotial survival

and germination of Sclerotinia sclerotiorum in the grth chamber. The BCAs included

Coniothyrium minitans CON/M/91-08 [Contans® WG, 5.3% active ingredient (a.i.)] at

rates of 0, 0. 001, 0.005, 0.025, 0.125, 0.625, 3.125 g/L soil, Streptomyces lydicus WYEC

108 (Actinovate® AG, 0.04% a.i.) and Trichoderma harzianum T-22 (PlantShield® HC,

1.15% a.i.) at 0, 0.025, 0.050, 0.100, 0.250, 0.500, 1.000 g/L soil, and Bacillus subtilis

QST 713 (Serenade® MAX, 14.6% a.i.) at 0, 0.002, 0.010, 0.050, 0.250, 1.250, 6.250 g/L

soil. Twenty-ﬁve sclerotia were buried in soil, followed by soil treatment with the BCAs.
Five soybean seeds were sown in each pot. Presence and number of S. sclerotiorum
apothecia were recorded daily. Sclerotia of S. sclerotiorum were retrieved after six weeks
from soil treatment and viability was assessed on water agar plates. Increasing the rates
of C. minitans and T. harzianum resulted in decreased S. sclerotiorum sclerotia survived.
Increasing the rates of C. minitans and S. lydicus led to reduced apothecial production of
S. sclerotiorum. The most effective rate for C. minitans was around 0.2 g/L soil with
90.1% apothecial reduction and 50.0% of sclerotial reduction. The most effective rate for
T. harzianum was 0.2 g/L soil with 80.5% apothecial reduction and 31.7% of sclerotial
reduction. The most effective rate for S. lydicus was 0.2 g/L soil with 100% apothecial
reduction and 29.5% of sclerotial reduction. The effective rate for B. subtilis was 0.9 g/L

soil with 81.24% reduction of apothecia and 29.6% of sclerotial reduction.

37

:Il 4
KIM

In

INTRODUCTION

Soybean white mold, caused by Sclerotinia sclerotiorum, can result in signiﬁcant
soybean yield losses (Abawi and Grogan 1979; Purdy 1979; Boland and Hall 1994;
Bolton et a]. 2006). Sclerotia of S. sclerotiorum serve as a survival structure that is
resistant to drought, heat and remain viable in the soil for up to eight years (Coley-Smith

and Cooke 1971). With continuous moisture, sclerotia germinate carpogenically and

produce 107 ascospores per apothecium (Abawi and Grogan 1979; Clarkson et al. 2003),

which may infect soybean. Currently, strategies for controlling white mold include use of
partial resistant cultivars, foliar fungicides applications and cultural practices (Vuong et
a]. 2008, Mueller eta]. 2004, and Gracia-Garza et al. 2002). However, no soybean
cultivars are completely resistant to S. sclerotiorum (Vuong et al. 2008). Foliar fungicide
sprays, even with multiple applications, cannot prevent the disease (Mueller et al. 2004).
Cultural practices are inconsistent because of the variation of environmental conditions
and the longevity of sclerotia in soil (Gracia-Garza et al. 2002). The desire for higher
level of disease management and the concern for using chemicals drive the search for
biological approaches as alternatives.

A range of commercially available biocontrol reagents (BCAs) were selected for
screening their potential for control of white mold. The active ingredients of biocontrol
agents are derived from fungal and bacterial strains. Speciﬁc antagonistic fungi have
been reported to effectively suppress S. sclerotiorum. The mycoparasites Sporidesmium
sclerotivarum and Coniothyrium minitans are able to speciﬁcally parasitize sclerotia and

mycelia of Sclerotinia spp. (Budge et al. 1995; del Rio et al. 2002). Coniothyrium

38

minitans produces a broad range of cell wall degrading enzymes such as chitinases and
glucanases as well as antifungal metabolites that enhance colonization and degradation of
sclerotia of S. sclerotiorum (Hu et a]. 2009). Sporidesmium sclerotivarum has been
applied in the ﬁeld to control white mold of soybean reducing 50% to 100% white mold
incidence (del Rio et a]. 2002). Coniothyrium minitans has been applied in soil to manage
S. sclerotiorum sclerotia in lettuce reducing 50% disease incidence (Chitrampalam eta].
2008). It signiﬁcantly reduced 90% of sclerotia in a snap bean ﬁeld (Gerlagh et al. 2003).
F oliar application of C. minitans on detached common bean leaves reduced lesion
development from ascospore infection (Bremer 2000).

Besides speciﬁc mycoparasites of S. sclerotiorum, a number of non-speciﬁc
bacterial and fungal antagonists have also shown to inhibit sclerotia or mycelia of S.
sclerotiorum, such as Bacillus amyloliquefaciens, B. subtilis, T richoderma asperellum, T.
aureaviride, T. gamsii, T. harzianum, T. koningii, T. langibrachiatum, T. virens,
Ulacladium afrum, and Pseudomonasﬂuarescens (Tu 1980; Budge eta]. 1995;
Menendez and Godeas 1998; Schmiedeknecht et al. 2001; del Rio et a]. 2002; Escande et
a]. 2002; Huang and Erickson 2007; Abdullah et al. 2008; Ashofteh 2009; Van Beneden
et a]. 2010). Trichoderma harzianum is widely used to suppress various pathogens
including S. sclerotiorum (Kim and Knudsen 2009), which has multiple modes of action
including mycoparasitism, nutrient competition, antibiosis, and plant growth promotion
(Ousley eta]. 1994). Bacillus subtilis has broad inhibitory activities against pathogens
such as Rhizoctania solani, F usarium axysparum, Gaeumannamyces graminis var. tritici,

and S. sclerotiorum (Schmiedeknecht eta]. 2001; Yang eta]. 2009).

39

Coniothyrium minitans, T. harzianum, B. subtilis, and Streptomyces lydicus have
been registered as commercial products for biological control (Budge and Whipps 2001;
Chitrampalam et al. 2008). High speciﬁcity of C. minitans to S. sclerotiorum has the
advantages of avoiding undesirable side effects to human and the environment, although
limits the market size and market expansion (Gerlagh et a]. 2003). Although T.
harzianum and B. subtilis have been used to control a range of diseases, little information
is available for their efﬁcacies on management of white mold on soybean. Streptomyces
lydicus strongly inhibits Pythium ultimum and R. salani (Yuan and Crawford 1995) by
producing cell wall degrading enzymes (Mahadevan and Crawford 1997) and
extracellular antifungal metabolites (Yuan and Crawford 1995). With this strong
antimicrobial activity, we were interested in testing it against Sclerotinia sclerotiorum on
soybean

The objectives of this study were to select effective BCAs on sclerotial and
apothecial reduction of S. sclerotiorum, compare the efﬁcacies of selected BCAs, and
determine application rates for each product for suppressing S. sclerotiorum in soil. The
efﬁcacy of C. minitans, T. harzianum, B. bacillus, and S. lydicus was evaluated by

measuring the sclerotial survival, and carpogenic germination of S. sclerotiorum.

40

 

151

{:44

MATERIALS AND METHODS

Isolate collection and inoculum production of Sclerotinia sclerotiorum

Sclerotinia sclerotiorum isolates, 1001, 1004 and 1005 were collected from
infected soybean plants in Michigan and isolated in the laboratory. Sclerotia were surface
sterilized for 3 min by submerging in 0.6% sodium hypochlorite, rinsed three times in
sterile distilled water, and plated on water agar. A mycelial plug was excised from the
edge of a 2-d-old culture using a 3 mm diameter cork borer, transferred to a new 100 mm
diameter Petri plate containing potato dextrose agar (PDA), plates were then sealed with
Paraﬁlm.

Sclerotia of S. sclerotiorum were produced in quantity in the laboratory on peeled

and cubed potato tubers. Tubers were cut into 1 cm3 cubes, and placed in a flask (1 L) to

a volume of approximately 25% ﬂask capacity. The ﬂask was covered with aluminum
foil and twice autoclaved for 20 min with a 24 h rest period between. Three 3 mm
diameter mycelial plugs from 2-3 day old S. sclerotiorum cultures on PDA were
transferred onto the potato cubes. The ﬂasks were incubated at 22°C for at least 15 d until
sclerotia matured. Sclerotia were harvested by wet sieving (Hao et a]. 2003). Brieﬂy, soil
was washed through constant water in series of three-layer sieves (U. S. standard sieves
series IOO-mesh, ZOO-mesh and 325-mesh, Fisher Scientiﬁc Inc., Pittsburgh, PA) to
separate sclerotia and other soil particles. Harvested sclerotia were dried in a transfer

hood on autoclaved paper towel and stored in plastic bags at 4°C until use.

41

Soil inoculation with Sclerotinia sclerotiorum and treatment with biocontrol agents

The products Actinovate® AG (Streptomyces lydicus strain WYEC 108, Natural
Industries, Houston, TX), Contans® WG (Coniothyrium minitans strain CON/M/91-08,
SipCamAdvan LLC, Roswell, GA), PlantShield® HC (Trichoderma harzianum strain T-

22, Bioworks, Victor, NY), and Serenade® MAX (Bacillus subtilis strain QST 713,

AgraQuest, Davis, CA) were used in this study. Products were applied based on

manufactures’ recommended rates (Table 2-1).

Plastic pots (1 L with 10 X 10 cm2 Opening), with bases lined with a single coffee

ﬁlter paper (24.5 in diam), were ﬁlled with potting mix (BACCTO Professional Planting

Mix, Michigan Peat Company, Houston, TX; 70% - 80% Sphagnum peat) and sandy soil
(v:v = 1:2), and placed in two 1.3 m2 growth chambers (Model: PGR14, Conviron,

Winnipeg, Manitoba, Canada) using a completely randomized design. Twenty-ﬁve
laboratorial produced S. sclerotiorum sclerotia (isolate 1005) were buried one centimeter
below the surface, spread evenly in the pots. BCAs were suspended and applied in 100
ml distilled water per pot. Four weeks after the soil treatment, soybean seeds (cv.
Olympus) were soaked for two hours in distilled water before sowing. Five seeds were
individually placed in soil in the pot, two centimeters in depth from top, with even

distance between.

42

Table 2-1. Biocontrol agents and their application rates in the study.

 

 

Biological Application rates (g/L soil) Active No. Rec.
control ingredient of rates
agents (%) trials (g/L)‘

C. minitans 0 0.250 0.500 1.000 2.500 5.000 10.000 5.3 1" 0.008
C. minitans 0 0.001 0.005 0.025 0.125 0.625 3.125 5.3 3 0.008
B. subtilis 0 0.250 0.500 1.000 2.500 5.000 10.000 14.6 1 0.005
B. subtilis 0 0.002 0.010 0.050 0.250 1.250 6.250 14.6 3 0.005
T. harzianum 0 0.025 0.050 0.100 0.250 0.500 1.000 1.15 4 0.003
S. lydicus 0 0.025 0.050 0.100 0.250 0.500 1.000 14.6 4 0.001

 

a
Recommended rates were converted from the rates on product labels for ﬁeld
application.

b Application rates of C. minitans and B. subtilis modiﬁed based on the results of ﬁrst
trial.

Apothecial observation and sclerotial retrieval

Numbers of apothecia of S. sclerotiorum were recorded every other day after the
ﬁrst apothecium was found. After six weeks from sclerotial placement in soil, soybean
plants were removed, and total fresh weight of plants from each pot was recorded. To test
viability, sclerotia of S. sclerotiorum were retrieved from each pot by wet sieving, surface
sterilized (as described above) and placed on water agar medium in Petri plates, with ten
sclerotia/plate. The plates were incubated at 20°C in the dark. Mycelial grth was

Observed after three to seven days of incubation (Budge and Whipps 1991).

43

Data

5111.

Data Analysis

The trials were divided into two groups for analyses: 1) no carpogenic
germination occurred; 2) carpogenic germination was observed. If there was no
interaction between trials and treatments, data were combined. Statistical analysis was
performed using SAS package (v.9.2, SAS Institute, Cary, NC). PROC NLIN (non-linear

model) was used to correlate between rates of BCA application and sclerotial survival or

apothecial production. The curves were ﬁt by the exponential model: Y = a + b X eH/c),

where x is rate of BCAs, Y is number of viable sclerotia or produced apothecia, and a, b,
and c are parameters. Rates of BCAs were transformed to logarithmic form prior to

analysis.

44

RESULTS

Effect of application rates of biological control agents on apothecial production of
Sclerotinia sclerotiorum

Coniothyrium minitans, T. harzianum, B. subtilis and S. lydicus were all effective,
at different levels of efficacy, to reduce the apothecial production of S. sclerotiorum (Fig.
2-1). Increasing the rates of BCA application resulted in an exponential decrease in
apothecial production of S. sclerotiorum (Fig. 2-1). The rate at 0.22 g/L soil of C.
minitans reached an efﬁcacy plateau by 84.3% apothecial reduction of S. sclerotiorum
(Fig. 2-1 A). T richoderma harzianum reached the maximum apothecial reduction at 0.20
g/L soil (Fig. 2-1 B). Bacillus subtilis had the maximum reduction of apothecia at 0.15
g/L soil, and its highest efﬁcacy was under 90% of apothecial inhibition (Fig. 2-1 C).
Streptomyces lydicus reached the plateau of apothecial reduction around 0.25 g/L soil,
where apothecial production was completely suppressed (Fig. 2-1 D). No further
reduction in production of apothecia was Observed at asymptotic application rates greater
than 0.25 g/L of all the BCAs, indicating that rates above 0.25 g/L cannot further enhance

the efficacy and therefore they are not recommended.

Effect of application rates of biological control agents on sclerotial survival of
S cleratinia sclerotiorum

Coniothyrium minitans, T. harzianum, and B. subtilis reduced the number of
Survived sclerotia in soil (Fig. 2-2, 2-3). The number of sclerotia was negatively

cofrelated with application rates of BCAs. In group 1, where no carpogenic germination

45

occurred, the concentrations for the maximal efﬁcacy were 0.15 g/L soil for C. minitans
(Fig. 2-2A), 0.22 g/L soil for T. harzianum (Fig. 2-2B), and 0.89 g/L soil for B. subtilis
(Fig. 2-2C). However, no 100% reduction of sclerotia was observed for all BCA
treatments. The highest level of sclerotial reduction for each individual BCA was 60.9%
for C. minitans (Fig 2-2 A), 30.9% for T. harzianum (Fig 2-2 B), and 38.7% for B.
subtilis (Fig 2-2 C).

In group 2, where carpogenic germination occurred, the number of sclerotia
continued to decrease with increased application rates of BCAs (Fig. 2-3). The maximum
reduction of sclerotia for C. minitans was 84.5% (Fig. 2-3A); T. harzianum was 23.5%
(Fig. 2-3 B); B. subtilis wasl2.3% (Fig. 2-3 C); and S. lydicus was 27.0% (Fig. 2-3 D) in

this group of trials.

46

 

20 A

 

 

 

 

= 15 (q
o .
:3 10 y =0.89+1.15 x10-6.e 0.06)
E R2 =0.81

‘6 5

2. O O

0 I I H H——‘-—O—

-1.00 -0.50 0.00 0.50 1.00

C. minitans (Log glL soil)

 

 

 

 

 

 

20 - B
= 15 f‘ (1)
5 y =1.55+0.004°e “-13
10 -
f; A ‘ R2 =0.66
% 5 -
2' A
o . ‘ i .
-1.00 -0.50 0.00 0.50

 

 

1'. harzianum Log glL soil)

 

Figure 2-1. Effect of biological control agents on apothecial production of Sclerotinia
sclerotiorum. Soil was treated with one of the products at various rates: Coniothyrium
minitans (A), T richaderma harzianum (B), Bacillus subtilis (C), and Streptomyces
lydicus (D). Repeated trials were combined for regression analysis. In the equation, x =
log (rate of application), and y = number of apothecia/L soil.

47

 

20] c

.5?

y = 2.13+ 3.31 x 10"8 - em]

 

 

E
U,
_|
~ 2
-§10‘ R =0.61
0
:5
8 5
< I I
J—
I
o T- . '

 

 

 

-1.00 -O.5O 0.00 0.50 1.00
B. subtilis (Log gIL soil)

 

 

 

 

 

 

 

 

D
_ 15 - i
'5 y = —0.06 + 0.0024012)
2 1° R2 = 0.98
3%
-1.00 -0.50 0.00 0:50

8. lydicus (Log glL soil)

 

 

 

Figure 2-lcontinued.

48

 

  
 

 

 

 

25 (1) A
20 y=11.38+1.11x10‘6°e°'0°
'5‘: R2 =0.77
315 - o o
‘2‘
§1o - ° v
5 I I I
4.00 cm 0.00 0.50
C. minitans (Log gIL soil)

 

 

 

 

 
 

 

 

 

25 (1) B
y = 14.78 + 0.01 ° e ”"5
20 -
73 A R2 =0.82
U)
a A A
E A
810 -
5 I I I
4.00 .050 0.00 0.50

 

1'. harzianum (Log glL soil)

 

 

Figure 2-2. Effect of biological control agents on survival of Sclerotinia sclerotiorum
sclerotia in group 1 trials (no carpogenic germination occurred). Soil was treated with one
of the BCA products at various rates: Coniothyrium minitans (A), Trichoderma
harzianum (B), and Bacillus subtilis (C). Data from all trials were averaged for regression
analysis. In the equation, x = log (rate of application), and y = number of sclerotia/L soil.

49

 

 
   

 

 

 

 

25 (.12) C
y = 13.55 + 0.47 . e 038
20
g 1'?2 = 0.66
in u
.r
g 15 - D n
2
2 El
0‘; 10 -
5 I l T r
-1.00 -0.50 0.00 0.50 1.00
B. subtilis (Log glL soil)

 

 

Figure 2-2 continued.

50

 

  

 

   

 

 

 

 

 

 

 

 

 

25 ("“l A
20 y=—17.56+28.10°e 5-‘3
s R2 = 0.68
d 15
g
o
E 10
0
a:
5 .
o
O l I I I
-1.00 -0.50 0.00 0.50 1.00
C. minitans (Log glL soil)
25 ( -x )B
y = 3471769 — 3471756- c ”15790
_ 20
'g R2=009
.r A ‘
.31 - A
i 5 .
2 A A
810 - A j ‘
5 I I I
-1.00 -0.50 0.00 0.50
1'. harzianum (Log gIL soil)

 

 

Figure 2-3. Effect of biological control agents on sclerotial survival of Sclerotinia
sclerotiorum in group 2 (carpogenic germination occurred). Soil was treated with one of
the BCA products at various rates: Coniothyrium minitans (A), Trichoderma harzianum
(B), Bacillus subtilis (C), and Streptomyces lydicus (D). Data from all trials were
averaged for regression analysis. In the equation, x = log (rate of application), and y =
number of sclerotia/L soil.

5].

 

 

 

 

 

 

 

 

 

 

25 C
= 20 I I I
3 I
7“! 15 -
'5 _x I
g 10 - y=l4.l9+1.22°e(67_°) '
0
m 2
5 R = 0.34
-1.00 -0.50 0.00 0.50 1.00
B. subtilis (Log gIL soil)
20 1 D
_ 15 4 y = -1067829 + 10678331116291“)
.3 .
3 R2 = 0.11
g C . .
one: 10 i~r\:\'o\
8 o
. o
5 . r .
-1.00 -O.50 0.00 0.50
S. lydicus (Log glL soil)

 

 

 

Figure 2-3 continued.

52

DISCUSSION

The results indicate that B. subtilis, C. minitans, S. lydicus, and T. harzianum can
reduce soil population and suppress apothecial production by S. sclerotiorum, with varied
efﬁcacy depending on the biological control agent. The efﬁcacy of BCAs is positively
correlated with the rate of application. The results of C. minitans are consistent with
previous study (Gerlagh et al. 2003). The manufacture recommended rates are less than
the rates with best efﬁcacy. This implies that increasing the rates for ﬁeld application
may reduce more primary inocula of white mold. However, once the rate passes certain
level, increasing the amount of products will not give any beneﬁt.

Mycoparasites will not degrade the pathogen unless they have direct contact
(Adams and F rave] 1990). This can be done by active (extension of growing mycelia) or
passive (carried and moved by other organisms or environment) modes (Williams et al.
1998). Lacking aggressive growth of C. minitans in the soil limits its efﬁcacy (Adams
and Fravel 1990). At this point, large amount of inocula and thorough mixing of BCAs in
soil may help to enhance direct contact between sclerotia and BCAs. Fortunately, soil
mesofauna can transport and relocate conidia of C. minitans in soil (Williams et al.
1998). Because of the transportation of conidia by soil mesofauna, the amount of
inoculum may be reduced. This partially explains the efﬁcacy of BCAs in this study
reaches a limit of rates with no further reduction of apothecia or sclerotia.

Fungal (C. minitans and T. harzianum) and bacterial (B. subtilis and S. lydicus)
BCAs have different mechanisms for affecting S. sclerotiorum sclerotia. Our data

demonstrate that C. minitans and T. harzianum are more effective at reducing sclerotial

53

survival than B. subtilis and S. lydicus at comparable rates. Apparently, mycoparasitism is
the key for the ﬁmgal BCA to colonize S. sclerotiorum sclerotia and eventually cause the
sclerotia degraded. Although S. lydicus has no effect on sclerotial reduction, it greatly
suppresses the carpogenic germination of S. sclerotiorum and its effectiveness is even
superior to C. minitans. It is interesting to further study the mechanism how S. lydicus
inhibits the carpogenic germination without the degradation of sclerotia.

For some unknown reasons, two trials had no carpogenic germination. This is not
expected, but the results generate interesting information. In the group (1) with no
carpogenic germination, the efﬁcacy of BCAs reaches the plateau at certain application
rates or above. The highest sclerotial reduction is less than 50%. While in the group (2)
with carpogenic germination, the number of sclerotia continues to decrease as application
rates of BCAs increases. In group 2 (sclerotia carpogenic germination occurs), sclerotial
reduction is caused by both carpogenic germination and sclerotial degradation. We
hypothesize that sclerotia in group 1 are in dormancy, which could be an indication why
they are not germinated carpogenically. This status could prevent them from degradation
by other parasitic microorganisms.

In summary, C. minitans and T harzianum are effective in reducing sclerotial
population of S. sclerotiorum. Coniothyrium minitans and S. lydicus are effective in
reducing apothecia production. Coniothyrium minitans are more effective than other
products on reduction of both sclerotia and apothecia. Its potential to reduce S.
sclerotiorum population in soil over time can be an alternative strategy to chemical

fungicides and play an important component in sustainable agriculture.

54

 

 

ll

LITERATURE CITED

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species. Phytopathology 69: 899-904.

Abdullah, M. T., Ali, N. Y., and Suleman, P. (2008). Biological control of Sclerotinia
sclerotiorum (Lib.) de Bary with Trichoderma harzianum and Bacillus
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Adams, P. B., and Fravel, D. R. (1990). Economical biological control of Sclerotinia
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Ashofteh, F., Ahmadzadeh, M., and Fallahzadeh-Mamaghani, V. (2009). Effect of
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Boland, G. J ., and Hall, R. (1994). Index of plant hosts of Sclerotinia sclerotiorum. Can.
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Bolton, M. D., Thomma, B., and Nelson, B. D. (2006). Sclerotinia sclerotiorum (Lib.) de

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Budge, S. P., and Whipps, J. M. (2001). Potential for integrated control of Sclerotinia
sclerotiorum in glasshouse lettuce using Coniothyrium minitans and reduced
fungicide application. Phytopathology 91 : 221-227.

Chitrampalam, P., F iguili, P. J ., Matheron, M. E., Subbarao, K. V., and Pryor, B. M.

(2008). Biocontrol of lettuce drop caused by Sclerotinia sclerotiorum and S.
minor in desert agroecosystems. Plant Dis. 92: 1625-1634.

55

Clarkson, J. P., Staveley, J ., Phelps, K., Young, C. S., and Whipps, J. M. (2003).
Ascospore release and survival in Sclerotinia sclerotiorum. Mycol. Res. 107: 213-
222.

Coley-Smith, J ., and Cooke, R. C. (1971). Survival and germination of fungal sclerotia.
Annu. Rev. Phytopathol. 9: 65-92.

del Rio, L. E., Martinson, C. A., and Yang, X. B. (2002). Biological control of Sclerotinia
stem rot of soybean with Sporidesmium sclerotivorum. Plant Dis. 86: 999-1004.

Escande, A. R., Laich, F. S., and Pedraza, M. V. (2002). Field testing of honeybee-
dispersed T richoderma spp. to manage sunﬂower head rot (Sclerotinia
sclerotiorum). Plant Pathol. 51: 346-351.

Gerlagh, M., Goossen-van de Geijn, H. M., Hoogland, A. E., and Vereijken, P. F. G.
(2003). Quantitative aspects of infection of Sclerotinia sclerotiorum sclerotia by
Coniothyrium minitans - timing of application, concentration and quality of
conidial suspension of the mycoparasite. Eur. J. Plant Pathol. 109: 489-502.

Gracia-Garza, J. A., Neumann, S., Vyn, T. J ., and Boland, G. J. (2002). Inﬂuence of crop
rotation and tillage on production of apothecia by Sclerotinia sclerotiorum. Can.
J. Plant Pathol 24: 137-143.

Hao, J. J ., Subbarao, K. V., and Duniway, J. M. (2003). Germination of Sclerotinia minor
and S. sclerotiorum sclerotia under various soil moisture and temperature
combinations. Phytopathology 93: 443-450.

Hu, Y., Li, G., and Yang, L. (2009). Characterization of factors affecting activity of

chitinase produced by mycoparasite Coniothyrium minitans. Chin. J. Appl.
Environ. Biol. 15: 226-229.

Huang, H. C., and Erickson, R. S. (2007). Ulocladium aﬁ'um as a biological control agent
for white mold of bean caused by Sclerotinia sclerotiorum. Phytoparasitica 35:
15-22.

Kim, T. G., and Knudsen, G. R. (2009). Colonization of Sclerotinia sclerotiorum
sclerotia by a biocontrol isolate of Trichoderma harzianum, and effects on
myceliogenic germination. Biocontrol Sci. Technol. 19: 1081-1085.

Mahadevan, B., and Crawford, D. L. (1997). Properties of the chitinase of the antifungal

biocontrol agent Streptomyces lydicus WYEC108. Enzyme Microb. Technol. 20:
489-493.

Menendez, A. B., and Godeas, A. (1998). Biological control of Sclerotinia sclerotiorum
attacking soybean plants. Degradation of the cell walls of this pathogen by

56

Trichoderma harzianum (BAFC 742) - Biological control of Sclerotinia
sclerotiorum by Trichoderma harzianum. Mycopathologia 142: 153-160.

Mueller, D. S., Bradley, C. A., Grau, C. R., Gaska, J. M., Kurle, J. E., and Pedersen, W.
L. (2004). Application of thiophanate-methyl at different host grth stages for
management of sclerotinia stem rot in soybean. Crop Prot. 23: 983-988.

Ousley, M. A., Lynch, J. M., and Whipps, J. M. (1994). Potential of Trichoderma spp as
consistent plant-growth stimulators. Biol. Fertil. Soils 17: 85-90.

Purdy, L. H. (1979). Sclerotinia sclerotiorum: History, diseases and symptomatology,
host range, geographic distribution and impact. Phytopathology 69: 875-880.

Schmiedeknecht, G., Issoufou, I., Junge, H., and Bochow, H. (2001). Use of Bacillus
subtilis as biocontrol agent. V. Biological control of diseases on maize and
sunﬂowers. J. Plant Dis. Prot. 108: 500-512.

Tu, J. C. (1980). Gliocladium virens, a destructive mycoparasite of Sclerotinia
sclerotiorum. Phytopathology 70: 670-674.

Van Beneden, S., Leenknegt, I., Franca, S. C., and Hofte, M. (2010). Improved control of
lettuce drop caused by Sclerotinia sclerotiorum using Contans combined with
lignin or a reduced fungicide application. Crop Prot. 29: 168-174.

Vuong, T. D., Diers, B. W., and Hartman, G. L. (2008). Identiﬁcation of QTL for
resistance to Sclerotinia stem rot in soybean plant introduction 194639. Crop Sci.
48: 2209-2214.

Williams, R. H., Whipps, J. M., and Cooke, R. C. (1998). Role of soil mesofauna in
dispersal of Coniothyrium minitans: Transmission to sclerotia of Sclerotinia
sclerotiorum. Soil Biol. Biochem. 30: 1929-1935.

Yang, D., Wang, B., Wang, J ., Chen, Y., and Zhou, M. (2009). Activity and efﬁcacy of
Bacillus subtilis strain NJ -18 against rice sheath blight and Sclerotinia stem rot of
rape. Biol. Control 51: 61-65.

Yuan, W. M., and Crawford, D. L. (1995). Characterization of Streptomyces lydicus

WYEC108 as a potential biocontrol agent against fungal root and seed rots. Appl.
Environ. Microbiol. 61: 3119-3128.

57

[H
811

501

U3
C 1

51:1

 

CHAPTER THREE: MANAGEMENT OF SOYBEAN WHITE MOLD USING
BIOLOGICAL CONTROL STRATEGIES IN THE FIELD

ABSTRACT
Biological control agents (BCAs) were evaluated for their efﬁcacy on reducing

soybean white mold and Sclerotinia sclerotiorum population in soil in Michigan. BCAs

included Coniothyrium minitans CON/M/9l-O8 (Contans® WG), Streptomyces lydicus
WYEC 108 (Actinovate® AG), Trichoderma harzianum T-22 (PlantShield® HC), and

Bacillus subtilis QST 713 (Serenade® MAX). At two ﬁeld locations, artiﬁcially infested

soil with S. sclerotiorum sclerotia was treated by incorporating the above BCAs and
boscalid (control) in the topsoil before planting. Populations of Bacillus, Streptomyces,
Trichoderma spp., and C. minitans were monitored at 3, 28, 71, and 169 days after BCA
application. Coniothyrium minitans had the best efficacy, reducing disease severity index
(DSI) by 68.5% and sclerotia of S. sclerotiorum in soil by 95.3%. Streptomyces lydicus
reduced DSI by 43.1% and sclerotia in soil by 90.6%. Trichoderma harzianum reduced
DSI by 38.5% and sclerotia by 70.8%. Bacillus subtilis had marginal effect on S.
sclerotiorum. The population of Streptomyces, T richoderma spp., and C. minitans did not
change signiﬁcantly throughout the season. Bacillus spp. population was not signiﬁcantly
higher in B. subtilis-treated plots. In our trials, C. minitans was more effectively managed

S. sclerotiorum in the ﬁeld than other BCAs tested.

58

INTRODUCTION

White mold [Sclerotinia sclerotiorum (Lib.) de Bary] of soybean [Glycine max
(L.) Merrill] is a devastating disease. It signiﬁcantly affects the soybean industry, and is
responsible for annual losses of about $70 million in the US (U. S. Canola Association
2005). Sclerotinia sclerotiorum has a wide host range; causing disease on a reported 408
plant species in 75 plant families and 278 genera of economically important crops
worldwide (Bolton et al. 2006). Severe outbreaks of white mold are often associated with
consistent cool and humid conditions at the time of soybean blossom (Abawi and Grogan
1979). In these conditions, sclerotia germinate and produce apothecia. Mature apothecia
generate copious numbers of ascospores from in and outside soybean ﬁelds, causing
initial infection (Abawi and Grogan 1979). If the peak time of ascospore release
corresponds with soybean blossom, an outbreak of white mold may occur (Subbarao
1998; Bolton et al. 2006). Once epidemics initiate, white mold is difﬁcult to manage.
Sclerotia develop in infected pods and stems, leaving plant debris full of sclerotia in the
ﬁeld after harvest. The disease cycle results in accumulation of sclerotia in soil from
season to season. The production of sclerotia is a key ecological stage in the life cycle of
the pathogen for disease development. Therefore, reducing the sclerotial population may
be the most efﬁcient way to manage the disease, interrupting the disease cycle.

Crop rotation breaks down the disease cycle so as to reduce the rate of
accumulation of sclerotia of S. sclerotiorum in the ﬁeld, but one- or two-year rotations is
unreliable and unpractical because S. sclerotiorum can survive in soil up to eight years

and has a broad host range and (Coley-Smith and Cooke 1971). Fungicides such as

59

azoxystrobin, boscalid, thiophanate-methyl, iprodione and vinclozolin have provided
moderate disease suppression (Bradley et al. 2006), but foliar spray on a 14-day interval
is costly (Mueller et al. 2002; Bolton et al. 2006). Although soil fumigation can eliminate
sclerotia in soil, it is expensive and may not meet expectations of environmental
stewardship (Budge and Whipps 1991). Partially resistant soybean varieties have
provided inconsistent protection under ﬁeld conditions (Chen and Wang 2005). The
concerns over environmental effects of chemical applications, the unsatisﬁed results from
cultural practice or resistant cultivars to S. sclerotiorum, as well as the drive for higher
efﬁciency and lower cost of disease control support the development of effective,
consistent and durable biocontrol strategies.

Substantial studies have been conducted in evaluating biological control agents
for controlling white mold in many cropping system such as lettuce (Budge and Whipps
1991; Chitrampalam et al. 2008), soybean (del Rio et al. 2002), sunﬂower (Eva 2003),
dry bean (Huang et al. 2000), and celery (Budge and Whipps 1991). Biocontrol agents
derived from antagonistic fungi and bacteria showed suppression of S. sclerotiorum. It
was our interest to evaluate the performances of BCAs in Michigan and to determine the
best candidates to manage soybean white mold. The objectives of this study were to
evaluate the efﬁcacy of the above BCAs to reduce the number of S. sclerotiorum sclerotia
of and soybean white mold disease, and track the population density of BCAs in the soil
after application. This may provide information for soybean white mold management in

areas of North America with similar climates to Michigan.

60

MATERIALS AND METHODS

Field plots

Field trials were conducted at the Plant Pathology Research Farm (PLP) at
Michigan State University, East Lansing, MI (N 42°41.477’; W 84°29.153’) from 2007
to 2009, and at Clarksville (CLK) Horticulture Experiment Station, Clarksville, MI (N
42°42.626’; W 85°33.958’) in 2008 and 2009. The soil texture of at PLP was a sandy
loam, with 54.2% sand, 35% silt, and 10.8% clay, and a pH of 7.4. The trial at PLP was

designed as a randomized complete block design (RCBD) with four replications (blocks).

Plot size at PLP was 3.05 x 7.62 m2 in 2007 and 6.10 x 9.14 m2 in 2008 and 2009. The

texture of soil at CLK was a sandy loam, with 70.2% sand, 25% silt, and 2.8% clay, and a
pH of 6.8. Plots were established at CLK in a split-plot design with three replications.
Each plot was split into sub-plots and treatments consisting of an application of C.
minitans CON/M/91-08 applied on 1 Nov 2007 or no treatment were randomized over

the sub-plots. No further treatments of C. minitans were applied in subsequent trial years

2008 and 2009. Individual sub-plot size was and 3.05 x 15.2 m2 at CLK.

Soil infestation with Sclerotinia sclerotiorum

Sclerotia produced in the ﬁeld and laboratory were used to inoculate the soil.
Sclerotinia sclerotiorum isolates 1001, 1004, and 1005 from infected soybean plants in
Michigan were used to produce sclerotia on autoclaved potato as described previously in

chapter 2. Sclerotia produced in commercial soybeans were collected from a Michigan

61

grain elevator. Approximately 2.3 kg of ﬁeld sclerotia and 3.3 kg of laboratory sclerotia
were evenly distributed each year by hand or seed spreader onto the soil surface on 16
Apr 2007, 18 May 2008, and 21 Apr 2009 at PLP. Sclerotinia sclerotiorum density in soil
was 2.25 sclerotia/L soil in 2007, and 2008, 2.0 sclerotia/L soil in 2009, respectively.
Sclerotia were spread on 30 May 2008, and 24 Apr 2009 at CLK, and the ﬁnal population
was 1.25 sclerotia/L soil in 2008 and 2009, respectively. The sclerotial inoculum was
incorporated into the top 10 cm of soil with a cultivator (2210 Field Cultivator, Deere &

Company, Moline, IL).

Soil treatments

Three (in 2007) and four (2008 and 2009) BCAs were applied at PLP (Table 3-1).
On 7 May 2007, C. minitans CON/M/9l-O8 (120 g a.i.lha, SipcamAdvan, Research
Triangle Park, NC), T. harzianum T-22 (4 g a.i/ha, BioWorks, Inc., Victor, NY), B.
subtilis QST 713 (60 g a.i.Iha, AgraQuest, Inc., Davis, CA), and boscalid (180 g a.i./ha,
Endura, BASF Corporation, Research Triangle Park, NC) were applied. On 7 May 2008
and 12 May 2009, C. minitans (240 g a.i./ha), T. harzianum (50 g a.i.lha), B. subtilis (60
g a.i.lha), S. lydicus (2 g a.i./ha) and boscalid (380 g a.i./ha) were applied in water
suspension. The products were sprayed with a tractor-driven boom sprayer with TEEJ ET
nozzles (model XR8008VS) set at xx cm apart. Applications were made at speed of 1.77
km/hr with a nozzle pressure of 103.4 kPa and ﬁnal application volume rate of 280.6
tha. The treatment, application rate and date were summarized in Table 3-1.

Four BCAs were applied at CLK from 2008 to 2009 (Table 3-1). At CLK on 1

Nov 2007, half of the split-blocks were treated with C. minitans (240 g a.i./ha) as the fall

62

treatment. On 1 May 2008 C. minitans (240 g a.i./ha), T. harzianum (50 g a.i./ha), B.
subtilis (60 g a.i./ha), and S. lydicus (2 g a.i.lha) were applied. On 8 Jul 2008, boscalid
(380 g a.i./ha) was sprayed onto the foliage at 5% soybean bloom (stage R1 of soybean
growth). On 15 May 2009, C. minitans (240 g a.i./ha), T. harzianum T-22 (50 g a.i.lha),
B. subtilis (60 g a.i./ha), S. lydicus (2 g a.i./ha), and boscalid (380 g a.i./ha) were applied

as described above (Table 3-1).

63

Table 3-1. Treatment arrangement, application rate and date in each ﬁeld and year.

 

 

 

Field Year 17W? Treatment Rate (g a.i.lha) Date DPPb
PLP 2007 None B. subtilis QST 713 60 7-May 7
C. minitans CON/M/9l-08 120 7-May 7
T. harzianum T-22 4 7-May 7
Boscalid 180 7-May 7
2008 None B. subtilis QST 713 60 7-May 2
C. minitans C0N/M/91-08' 240 7-May 2
T. harzianum T-22 50 7-May 2
S. lydicus WYEC 108 2 7-May 2
Boscalid 380 7-May 2
Control 0 7-May 2
2009 None B. subtilis QST 713 60 12-May 14
C. minitans CON/M/9l-08 240 12—May 14
T. harzianum T-22 50 lZ-May 14
S. lydicus WYEC 108 2 12-May 14
Boscalid 380 12-May 14
Control 0 12-May 14
CLK 2008 C. minitans B. subtilis QST 713 60 l-May 9
CON/M/9l-O8 C. minitans CON/M/91-08 240 l-May 9
T. harzianum T-22 50 l-May 9
S. lydicus WYEC 108 2 l-May 9
Boscalid 380 8-Jul -60
Control 0 l-May 9
None B. subtilis QST 713 60 l-May 9
C. minitans CON/M/9l-O8 240 l-May 9
T. harzianum T-22 50 l-May 9
S. lydicus WYEC 108 2 l-May 9
Boscalid 380 8-Jul -60
Control 0 l-May 9
2009 C. minitans B. subtilis QST 713 60 lS-May 7
CON/M/9l-O8 C. minitans CON/M/9l-08 240 lS-May 7
T. harzianum T-22 50 lS-May 7
S. lydicus WYEC 108 2 lS-May 7
Boscalid 380 lS-May 7
Control 0 15-May 7
None B. subtilis QST 713 60 15-May 7
C. minitans CON/M/9l-08 240 lS-May 7
T. harzianum T-22 50 lS-May 7
S. lydicus WYEC 108 2 lS-May 7
Boscalid 380 15-May 7
Control 0 lS-May 7

 

a Treatments were applied on 1 Nov, 2007 at 240 g a.i./ha

64

b
. DPP = days prior to planting.

A; A

 

 

Varieties M99927 (susceptible) and CL968413 (partially resistant) were planted
at PLP on 20 April 2007. Olympus (susceptible) was used in 2008 and North Ripking
(susceptible) in 2009 at PLP and CLK in early May. Soybean seeds were planted in 18-
cm row space with average seeding rate around 444,790 seed/ha. A sprinkler irrigation
system was set up to maintain canopy moisture in the canopy above about 80% RH and
were run twice each day for 20 minutes at 11:00 am and 16:00 pm at a volume of 1000
L/ha/h. Irrigation was also applied when soil moisture was fell below 80% of ﬁeld
capacity as measured with soil moisture sensors placed at 6 and 12 cm below the soil

surface (CRIOX Measurement and Control System, Campbell Scientiﬁc, Logan, Utah).

Population of sclerotia in soil

Prior to soil inoculation, soil samples were obtained to determine the density of
sclerotia in soil. Two liters of soil per sample from ﬁve random points to a depth of 10
cm in each plot were collected. Sampling was replicated three times for each plot. Soil
samples were also collected at harvest. The samples were transported to the laboratory in
an ice chest and stored at 4°C until processing. Sclerotia of S. sclerotiorum were retrieved
by wet sieving described earlier in chapter two and viability was tested on water agar

plates.

Disease evaluation

Counts of S. sclerotiorum apothecia were made when apothecia were visible.

Counts were performed by randomly placing a l-m2 circles in each plot, counts were

65

replicated three times. The number of apothecia/m2 of S. sclerotiorum was counted on 21

July and 28 July 2009 at PLP and 21 July 2009 at CLK (at bloom, stage R1 of soybean
growth). Disease severity was measured using disease severity index (D81), and the
number of sclerotia/kg of harvested beans, depending on disease incidence each year.
When disease incidence was low (<5%) in 2007 at PLP, sclerotia/kg bean was used and
in 2008 at PLP and CLK, D81, and sclerotia/kg bean were used. When disease incidence
was moderate (5%-50%) in 2009 at PLP and CLK, sclerotia/kg harvested bean were
used.

Disease severity index (DSI) (Kim et al. (2000) was determined at R7 stage of
soybean growth as deﬁned by the maturity of soybean pods on the main stem. Fifty plants
from the center two rows were rated for DSI 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, and 3 = symptoms on main stem leading to plant death and poor or no pod ﬁll
(Kim et al. 2000). The DSI was calculated by the following formula:

the sum of disease scale rating of each plant
051 = X 100
3 x number of plants rated

 

Number of sclerotia per kg of harvested beans was recorded as a proxy for disease

severity. Soybean samples were distributed in the Rubbermaid® white polypropylene

pans (29.2 X 34.3 cm, Newell Rubbermaid, Atlanta, GA, 30328). Sclerotia were

separated from beans by hand and the numbers of sclerotia were recorded.

66

Soybean yield evaluation

Soybeans were harvested with a small-plot combine harvester (Massey Harris 35
plot combine, with a 1.8 m cutting bar). Only 66% of plants in the center of each plot
were harvested (the outside rows were not harvested and served as guard rows) and
soybean yield was evaluated. Two to three liters of beans were sub-sampled from the
harvested beans and sclerotia were retrieved. Soybean moisture content and 100 bean
weight were determined from each plot with a Steinlite Moisture Tester SL95 (Seedburo
Equipment Co. Chicago, IL) and Count-A-Pak Seed Totalizer (Seedburo Equipment Co.

Chicago, IL), respectively.

Population dynamics of Coniothyrium minitans, T richoderma, Streptomyces and
Bacillus spp. in soil

Soil samples were collected from PLP and CLK in 2009 four times: 3, 28, 71
(soybean bloom), and 169 (harvest) days after application of BCAs, respectively. From
each plot, three samples with 3 liters of soil for each were collected. Each sample
contained soil cores from ﬁve arbitrarily selected locations in the plot. In the laboratory,
soil samples were homogenized by breaking soil clods and shaking the sample bag for l
min. To determine soil moisture content, 50 g of soil from each sample was weighed,
placed in a glass Petri plate, and dried in an oven at 50°C for 48 h.

Soil dilution plating was conducted to enumerate the population of BCAs,
measured as colony forming units (CFU)/g soil. Brieﬂy, 10 g of soil was agitated in 90
ml of phosphate buffered saline (1.2 g NazHPO4, 0.18 g NaHzPO4, 8.5 g NaCl, and

adjusted pH to 7.4) for 10 min on a rotary shaker at 200 rpm. A series of dilutions were

67

prepared from 1:10 to 1:102 for C. minitans, 1:102 to 1:103 dilutions for Trichoderma

and Streptomyces spp., and 1:104 to 1:103 for Bacillus spp. Streptomyces spp. were

detected on Streptomyces selective media (STR) (Conn et al. 1998). T richoa’erma spp.
were measured on T. harzianum selective media (THSM) (Williams et al. 2003).
Bacillus spp. were detected on Tryptic Soy Agar (TSA): 3 g tryptic soy broth (EMD
Chemicals, Gibbstown, NJ), and 17 g agar (EMD Chemicals, Gibbstown, NJ) amended
with 100 mg cycloheximide (MP Biomedical, Solon, OH) and 100 mg Benlate (DuPont

Co., Wilmington, DE). Aliquots of 100 pl were spread onto the three semi-selective

media using Spiral Autoplate® 4000 (Advanced Instruments, Norwood, MA), using Even

Deposition mode feature. Each plate was incubated at 20°C in the dark for 2 to 4 d for
Trichoderma and Streptomyces, and 5 to 10 d for C. minitans, respectively. Individual
colonies on each plate were enumerated twice within 7 d and CFU/ g soil for each sample

was calculated.

Data Analysis

Statistical analysis was performed using SAS package (v.9.2, SAS Institute, Cary,
NC) with PROC GLIMMIX to analyze the effects of soil treatments. Treatments were
compared using Tukey-Kramer multiple comparison at P = 0.05. Repeated measurements
in Tukey-Kramer grouping were conducted to estimate the BCA population variation
over time. Colony forming units (CFU/ g soil) were log transformed to satisfy the

assumption of homogeneity of variance.

68

RESULTS

Disease and yield evaluation

Soybean varieties M99927 (susceptible) and CL968413 (tolerant) demonstrated
no signiﬁcant difference in disease suppression. Variety M99927 had 9.2 sclerotia/kg
harvested bean in control plots and CL968413 had 9.0 sclerotia/kg bean in the control. In

2009 at PLP, production of apothecia from boscalid treated plots signiﬁcantly reduced

0.17 apothecia/m2 in comparison to 4.58 apothecia/m2 from control plots to lower

number than the control (Table 3-2). The apothecia/m2 in BCA treated plots were not

signiﬁcant from the control (Table 3-2). In 2009 at CLK, apothecia production is under

detectable level, and only 0.33 apothecia/m2 was observed in the non-treated plot (Table

3-3).

In 2007 at PLP, disease severity, measured as number of sclerotia/kg harvested
bean was not signiﬁcantly different between treated and non-treated plots (Table 3-2).
However, BCAs and boscalid signiﬁcantly reduced the D81 and sclerotia/kg harvested
bean in 2008 and 2009 (Table 3-2). In 2008, C. minitans, S. lydicus, T. harzianum, B.
subtilis, boscalid reduced DSI by 53.9%, 30.8%, 38.5%, 15.4%, 30.8% respectively
compared to the control, and reduced sclerotia/kg harvested bean by 94.0%, 93.8%, 79.0
%, 77.5%, and 52.5% respectively, compared to the control. In 2009, with moderate
disease pressure, the D81 was reduced by 68.5% for C. minitans, 43.2 % for S. lydicus,
4.7% for T. harzianum, 44.0% for B. subtilis, and 53.4% for boscalid. Sclerotia/kg

harvested bean reduction was 80.5% for C. minitans, 85.8% for S. lydicus, 56.4% for T.

69

harzianum, 54.7% for B. subtilis, and 60.0% respectively (Table 3-2). Boscalid treated
plots had signiﬁcant lower number of apothecia than control plots. Soybean moisture
content (15.3% in average) and 100 bean weight (69.8 kg/hL in average) were not
signiﬁcantly different between treatments. The yield of soybean (kg/ha) was not
signiﬁcant from different treatments in 2007 to 2009 (Table 3-2).

In 2008 at CLK, sclerotia/kg harvested bean and D81 were not different between
treated and non-treated plots. However, in 2009, all treatments having fall application had
a signiﬁcant lower number of sclerotia/kg bean in comparison to the untreated control
(Table 3-3). However, only sclerotia from C. minitans plots had signiﬁcantly lower
sclerotia/kg bean than the untreated control without fall treatment (Table 3-3). The yield
of soybean (kg/ha) was not signiﬁcant from different treatments in 2008 to 2009 (Table

3-3).

Population of sclerotia in soil

At PLP, the population of sclerotia in the soil, examined at harvest, was below 2
sclerotia/L soil for all the treatments for the three years, and not signiﬁcantly different
among the treatments in 2007 and 2009 (Table 3-2). However, in 2008, the number of
sclerotia/L soil was signiﬁcantly different, and reduced by 953.8%, 90.6%, 76.0%,
70.8%, and 59.1%, in C. minitans, S. lydicus, B. subtilis, T. harzianum and boscalid-
treated plots respectively in comparison to the non-treated control (Table 3-2). In all
years at CLK the number of sclerotia/L soil was also bellow 2 sclerotia/L soil, and not

signiﬁcantly different for BCA and boscalid-treated plots compared to the control (Table

70

3-3). Plots treated with C. minitans in the fall (1 Nov 2007) had no signiﬁcantly lower

number of sclerotia than plots without fall treatment.

Population dynamics of Coniothyrium minitans, T richoderma, Streptomyces and
Bacillus spp. in soil
Population of C. minitans, T richoderma, and Streptomyces spp. was signiﬁcantly

higher (P = 0.05) in soil treated with C. minitans CON/M/9l-08, T. harzianum T-22, and

S. lydicus WYEC 108, respectively, compared with control plots without soil treatment

(Fig. 3-lA, B, C). The population of the BCAs maintained the level above 103 CFU/g

soil throughout the season (Fig. 3-1). The population of C. minitans changed from an
average of 3.2 log CF U/ g soil at application to 2.96 log CFU/g soil as measured at 169
days. Occasionally, one or two colonies were observed from non-treated soils, indicating
that the majority of C. minitans colonies counted were from applied inocula (Fig. 3-1 A).
Trichoderma spp. remained at 3.8 log CFU/ g soil throughout the season in T. harzianum
T-22-treated plots while the background population increased from 1.6 log CFU/g soil to
2.5 log CFU/ g soil in untreated plots (Fig. 3-1 B). Streptomyces spp. decreased slightly
during the season in S. lydicus WYEC 108-treated plots, but it was signiﬁcantly higher
than in non-treated plots, although the latter increased later in the season (Fig. 3-1 C).
The population of Bacillus spp. remained at the same level in treated and non-treated soil

(data not shown).

71

Table 3-2. Effects of biological control agents and ﬁmgicide at planting to soybeans on
soil production of Sclerotiorum sclerotiorum, white mold severity and yield of soybean at
PLP.

 

 

 

Year Sclerotial D51“ Apothecia/mix Sclerotial Yield
Treatment L soilv km“). (kg/ha)
2007
B. subtilis 1.99 32 16.5 a 2538 a
C. minitans 0.62 a 12.9 a 2514 a
T. harzianum 0.50 a 10.1 a 2392 a
Boscalid l .42 a 12.8 a 2294 a
Control 0.80 a 7.5 a 2538 a
HSDons 1.519 31.32 679.1
2008
B. subtilis 0.41 b 5.5 ab 15.2 be 3214 a
C. minitans 0.08 b 3.0 b 4.1 c 3356 a
T. harzianum 0.50 ab 4.0 ab 14.2 be 3681 a
S. lydicus 0.16 b 4.5 ab 4.2 c 3417 a
Boscalid 0.70 ab 4.5 ab 32.1 b 3417 a
Control 1.71 a 6.5 a 67.6 a 3315 a
P130005 1.388 2.78 27.51 1613.4
2009
B. subtilis 0.64 a 19.2 b 1.50 ab 74.3 b 2400 a
C. minitans 0.57 a 10.8 b 2.50 ab 31.9 b 2684 a
T. harzianum 0.70 a 32.7 a 1.75 ab 71.5 b 2725 a
S. lydicus 0.56 a 19.5 b 1.25 ab 23.3 b 2502 a
Boscalid 0.57 a 16.0 b 0.17 b 65.5 b 2359 a
Control 0.64 a 34.3 a 4.58 a 163.9 a 2481 a
HSDn as 0.742 20.0 3.378 315.7

 

v Sclerotia were retrieved at harvest on 10 Oct 2007, 22 Oct 2008, and 26 Oct 2009.

w DSI = disease severity index, evaluated from 50 plants in the center row of plots 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, 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 Xtotal plants rated)>< 100. x Apothecia were observed
during blossom stage of soybean. Number of apothecial m2 was recorded in three sub-

samples in each plot. y the number of sclerotia/kg harvested beans, evaluated at harvest.

2 Tukey-Kramer multiple comparisons were conducted for each column each year in the
table. Values followed by the same letter are not signiﬁcantly different at P = 0.05 .

72

Table 3-3. Effects of biological control agents and fungicide on soil population of
Sclerotinia sclerotiorum and white mold severity and yield of soybean at CLK.

 

 

 

Year Spring Sclerotia Sclerotia/ DSIy Apothecia Yield
Treatment treatment IL soilw beanx lm2 (kg/ha)
2008 T . T '
C. minitans B. subtilis 0.0 a 0.50 a 8.0 a 2702 a
C. minitans 0.0 a 0.16 a 6.7 a 3743 a
T. harzianum 0.0 a 1.13 a 6.0 a 3255 a
S. lydicus 0.2 a 0.73 a 8.0 a 2832 a
Boscalid 0.0 a 0.69 a 6.7 a 3157 a
Control 0.0 a 0.16 a 6.7 a 2929 a
P1313005 0.27 2.146 6.77 2086.
None B. subtilis 0.28 a 3.14 a 6.7 a 4134 a
C. minitans 0.00 a 0.45 a 4.7 a 2962 a
T. harzianum 0.16 a 1.08 a 6.0 a 3808 a
S. lydicus 0.13 a 1.91 a 8.0 a 3450 a
Boscalid 0.49 a 0.88 a 6.0 a 3548 a
Control 1.36 a 9.77 a 4.7 a 3548 a
“SD”05 1.634 13.397 6.32 2670.
2009
C. minitans B. subtilis 0.20 a 7.22 b 0.0 2995 a
C. minitans 0.14 a 3.02 b 0.0 4036 a
T. harzianum 0.33 a 7.17 b 0.0 4166 a
S. lydicus 0.39 a 6.00 b 0.0 4492 a
Boscalid 0.26 a 6.86 b 0.0 3437 a
Control 0.42 a 30.50 a 0.0 3124 a
HSD005 0.423 1 1.937 2016.
None B. subtilis 0.10 a 9.51 ab 0.0 2376 a
C. minitans 0.12 a 3.14 b 0.0 3776 a
T. harzianum 0.19 a 10.95 ab 0.0 4459 a
S. lydicus 0.32 a 10.93 ab 0.0 4166 a
Boscalid 0.16 a 13.26 ab 0.0 4160 a
Control 0.04 a 20.52 a 0.3 4069 a
HSDn as 0.367 16.034 2345.

 

V Treatment was applied on 1 Nov, 2007 at 0.24 kg a.i./ha. w Sclerotia were retrieved at

harvest on 15 Oct 2007, 24 Oct 2008, and 28 Oct 2009. x the number of sclerotia/kg

harvested beans. y DSI = disease severity index, evaluated 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, 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 Xtotal plants rated)>< 100. Z Tukey-Kramer multiple comparisons were conducted
for each column each year in the table. Values followed by the same letter are not
signiﬁcantly different at P = 0.05.

73

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

4- A
E 3 H: *1— + _.*
5?
D 2 ~
u.
0
a: 1 —~ . .
3 +C. minitans -A—Control
0 4* + at a
3 28 71 169
E
m
2’
:1
IL
0
8’
-| -A—Control +I harzianum
1 I 1 1 1
3 28 71 169
4.5 , C
* 4*
= 3.5 -
a L a:
2 /
a 2.5 .
0 +8. lydicus -&-Control
8’1 5
_I . I 1 l
3 28 71 169
Day

 

 

 

Figure 3-1. Population dynamics of Coniothyrium minitans in C. minitans CON/M/91-08-treated
soil (A), T richoderma spp. in T. harzianum T-22-treated soil (B), and Streptomyces spp. in S.
lydicus WYEC 108-treated soil (C) in 2009. Each microorganism was detected and enumerated
on semi-selective media. Soil samples were collected at 3, 28, 71, and 169 days after soil
treatment. Error bars were determined by repeated measurements by Tukey-Kramer adjustment
using RROC GLIMMIX at P = 0.05. * indicate signiﬁcant difference between treated and control
plots on the same sampling day.

74

DISCUSSION

The biological control agents (BCA) tested suppressed white mold caused by
Sclerotinia sclerotiorum but the results were inconsistent over multiple years and
locations. In our study, C. minitans signiﬁcantly reduced DSI and the number of
sclerotia/kg bean in 2008 and 2009 at PLP and reduced sclerotial density of S.
sclerotiorum in soil in 2008 at PLP, this result is in agreement with previous studies on
bean (Bremer 2000; Gerlagh et al. 2003), lettuce (Chitrampalam et al. 2008) and celery
(Budge and Whipps 1991). However, disease severity and sclerotial density in soil in
2007 at PLP and in 2008 at CLK were not signiﬁcantly different from the control.
Hammond et al. (2008) had the same mixed results in potato ﬁeld in the Columbia Basin
and concluded that the ascospores originating external to ﬁelds were an abundant source
of inoculum for white mold development. Trichoderma harzianum also reduced
sclerotia/kg harvested bean in 2008 at PLP, as demonstrated by others on soybean
(Menendez and Godeas 1998), celery (Budge and Whipps 1991), and lettuce. However,
the efﬁcacy of T. harzianum varied compared to C. minitans. Bacterial agents Bacillus
subtilis and Streptomyces lydicus have different modes of action against S. sclerotiorum.
Bacillus subtilis had only a marginal effect on reducing the sclerotia in soil and disease
severity, and the suppression of carpogenic germination was sporadic. Streptomyces
lydicus had a signiﬁcant reduction of sclerotial density in 2008 at PLP, but the result was
inconsistent across years and locations in our trials.

Efﬁcacy of BCAs is affected by disease pressure, the timing of application and

soil conditions. At PLP, the impact of BCAs was more signiﬁcant in 2008 and 2009

75

when the disease pressure was higher than in 2007. In 2007, disease incidence was not
signiﬁcantly different due to a low disease pressure. Factors such as soil temperature,
moisture, and microbial diversity can also affect the efﬁcacy of BCAs (Hao et al. 2003;
Reeleder 2003). Early application beneﬁts the establishment and growth of biocontrol
strains in soil. Application BCAs prior to tillage are preferred because tillage can enhance
the even distribution of BCAs in soil and help direct contact between BCAs and
pathogen. Pathogen population and non-target soil microbial diversity can be changed by
the introduction of biocontrol strains (Cordier and Alabouvette 2009). The microbial
diversity of total bacterial and fungal CFU/ g dry soil was modiﬁed for a nine month
period (Cordier and Alabouvette 2009). In addition, biocontrol agents are sensitive to

some fungicides or bactericides that are applied in the ﬁeld. For example, C. minitans are

highly sensitive to iprodione (EC50 7 to 18 ug a.i./m1) and moderately sensitive to thiram

(EC50 52 to 106 ug a.i./ml) (Budge and Whipps 2001). All these factors should be

considered in integrated pest management programs.

Soybean yields in treated plots were not signiﬁcantly different from untreated
controls. This was partially due to the low incidence of white mold. According to del Rio
et al., no signiﬁcant canola yield differences were detected between fungicide-treated and
control plots when the average white mold incidence or severity was low (del Rio et al.
2007). Thus, evaluation of yield losses under low disease pressure could result in either
overestimation underestimation of the damage.

Despite signiﬁcant differences in DSI and the number of sclerotia per kg of
harvested beans in 2009 at PLP, the number of sclerotia recovered from the soil was not

signiﬁcantly different between control and treated plots. Although the sample size may

76

need to be increased to detect signiﬁcant differences in sclerotial density, it is possible
that two different mechanisms of sclerotial degradation resulted in non-signiﬁcant
differences. Perhaps sclerotia in the control plot degraded predominantly via the
production of apothecia, while those in treated plots were predominantly degraded by the
BCAs. Although the number of apothecia recorded at PLP was not signiﬁcantly different,
the absolute number of apothecia recorded in the control was higher than those in the
treated plots. Signiﬁcant differences in DSI and the number of sclerotia per kg of
harvested beans between control and treatments lend further support to this hypothesis,
however further studies are needed to test this theory.

The populations of Coniothyrium minitans, Trichoderma and Streptomyces spp.
maintained throughout the season. Maintenance of BCA populations is the key for
effective control of sclerotia. In our greenhouse trials, a linear relationship between rates
of BCA application and sclerotia/apothecia reduction was demonstrated (Chapter 2). The
population of C. minitans decreased gradually over the season indicating that a single
application may not be extended for more than one year. Since sclerotia are produced at
the end of the crop season, either fall or spring application of C. minitans or other
biocontrol agents may work. Therefore, multiple applications may not be necessary if a
substantial population is established. The population of Bacillus spp. was not different
between B. subtilis-treated and control plots. This is because the media is not highly
selective and cannot separate the applied and the native Bacillus species, and high
population of naturally occurred Bacillus spp. in the soil. This can be improved by

ﬁnding a better selective media for B. subtilis.

77

In summary, the efﬁcacy of BCAs varies. Coniothyrium minitans was more
effective on reduction of disease severity and sclerotial density among the products
tested. The key of this activity is to maintain a consistent high population of the
biological control agents throughout the season. Multiple applications have a potential to

improve the efficacy of C. minitans by increasing the total amount of applications.

78

LITERATURE CITED

Abawi, G. S., and Grogan, R. G. (1979). Epidemiology of diseases caused by Sclerotinia
species. Phytopathology 69: 899-904.

Bolton, M. D., Thomma, B., and Nelson, B. D. (2006). Sclerotinia sclerotiorum (Lib.) de
Bary: biology and molecular traits of a cosmopolitan pathogen. Mol. Plant Pathol.
7: 1-16.

Bradley, C. A., Lamey, H. A., Endres, G. J ., Henson, R. A., Hanson, B. K., McKay, K.
R., Halvorson, M., LeGare, D. G., and Porter, P. M. (2006). Efﬁcacy of
fungicides for control of Sclerotinia stern rot of canola. Plant Dis. 90: 1129-1134.

Bremer, E., Huang, H. C., Selinger, L. J ., and Davis, J. S. (2000). Competence of
Coniothyrium minitans in preventing infection of bean leaves by Sclerotinia
sclerotiorum. Plant Pathol. Bull. 9: 69-74.

Budge, S. P., and Whipps, J. M. (1991). Glasshouse trials of Coniothyrium minitans and
Trichoderma species for the biological control of Sclerotinia sclerotiorum in
celery and lettuce. Plant Pathol. 40: 59-66.

Budge, S. P., and Whipps, J. M. (2001). Potential for integrated control of Sclerotinia
sclerotiorum in glasshouse lettuce using Coniothyrium minitans and reduced
fungicide application. Phytopatholog» 91: 221-227.

Chen, Y., and Wang, D. (2005). Two convenient methods to evaluate soybean for
resistance to Sclerotinia sclerotiorum. Plant Dis. 89: 1268-1272.

Chitrampalam, P., Figuili, P. J ., Matheron, M. E., Subbarao, K. V., and Pryor, B. M.
(2008). Biocontrol of lettuce drop caused by Sclerotinia sclerotiorum and S.
minor in desert agroecosystems. Plant Dis. 92: 1625-1634.

Coley-Smith, J ., and Cooke, R. C. (1971). Survival and germination of fungal sclerotia.
Annu. Rev. Phytopathol. 9: 65-92.

Conn, K. L., Leci, E., Kritzman, G., and Lazarovits, G. (1998). A quantitative method for
determining soil populations of Streptomyces and differentiating potential potato
scab-inducing strains. Plant Dis. 82: 631-63 8.

Cordier, C., and Alabouvette, C. (2009). Effects of the introduction of a biocontrol strain

of T richoderma atroviride on non target soil micro-organisms. Eur. J. Soil Biol.
45: 267-274.

79

del Rio, L. E., Bradley, C. A., Henson, R. A., Endres, G. J ., Hanson, B. K., McKay, K.,
Halvorson, M., Porter, P. M., Le Gare, D. G., and Lamey, H. A. (2007). Impact of
Sclerotinia stem rot on yield of canola. Plant Dis. 91: 191-194.

del Rio, L. E., Martinson, C. A., and Yang, X. B. (2002). Biological control of Sclerotinia
stem rot of soybean with Sporidesmium sclerotivorum. Plant Dis. 86: 999-1004.

Eva, B. (2003). Biological control of pathogens Sclerotinia sclerotiorum (Lib.) de Bary
and Botrytis cinerea (Pers.) from sunﬂower (Helianthus annuus L.) crops. Agron.
Res. Moldova 36: 73-80.

Gerlagh, M., Goossen-van de Geijn, H. M., Hoogland, A. E., and Vereijken, P. F. G.
(2003). Quantitative aspects of infection of Sclerotinia sclerotiorum sclerotia by
Coniothyrium minitans - timing of application, concentration and quality of
conidial suspension of the mycoparasite. Eur. J. Plant Pathol. 109: 489-5 02.

Hammond, C. N., Cummings, T. F ., and Johnson, D. A. (2008). Deposition of ascospores
of Sclerotinia sclerotiorum in and near potato ﬁelds and the potential to impact
efﬁcacy of a biocontrol agent in the Columbia Basin. Am. J. Potato Res. 85: 353-
360.

Hao, J. J ., Subbarao, K. V., and Duniway, J. M. (2003). Germination of Sclerotinia minor
and S. sclerotiorum sclerotia under various soil moisture and temperature
combinations. Phytopathology 93: 443-450.

Huang, H. C., Bremer, E., Hynes, R. K., and Erickson, R. S. (2000). Foliar application of
fungal biocontrol agents for the control of white mold of dry bean caused by
Sclerotinia sclerotiorum. Biol. Control 18: 270-276.

Kim, H. S., Hartman, G. L., Manandhar, J. B., Graef, G. L., Steadman, J. R., and Diers,
B. W. (2000). Reaction of soybean cultivars to Sclerotinia stem rot in ﬁeld,
greenhouse, and laboratory evaluations. Crop Sci. 40: 665-669.

Menendez, A. B., and Godeas, A. (1998). Biological control of Sclerotinia sclerotiorum
attacking soybean plants. Degradation of the cell walls of this pathogen by
T richoderma harzianum (BAFC 742) - Biological control of Sclerotinia
sclerotiorum by Trichoderma harzianum. Mycopathologia 142: 153-160.

Mueller, D. S., Dorrance, A. E., Derksen, R. C., Ozkan, E., Kurle, J. E., Grau, C. R.,
Gaska, J. M., Hartman, G. L., Bradley, C. A., and Pedersen, W. L. (2002).
Efﬁcacy of fungicides on Sclerotinia sclerotiorum and their potential for control
of Sclerotinia stem rot on soybean. Plant Dis. 86: 26-31.

U. S. Canola Association. (2005). 2005 Sclerotinia White Paper. U. S. Canola
Association. Online publication.

80

Reeleder, R. D. (2003). Fungal plant pathogens and soil biodiversity. Can. J. Soil Sci. 83:
331-336.

Subbarao, K. V. (1998). Progress toward integrated management of lettuce drop. Plant
Dis. 82: 1068-1078.

Williams, J ., Clarkson, J. M., Mills, P. R., and Cooper, R. M. (2003). A selective medium

for quantitative reisolation of Trichoderma harzianum from Agaricus bisporus
compost. Appl. Environ. Microbiol. 69: 4190-4191.

81

CHAPTER FOUR: GROWTH CHARACTERIZATION OF CONIOT H YRI UM
MINITANS ISOLATES UNDER VARIOUS ENVIORN MENTAL CONDITIONS
AND THEIR COLONIZATION ON SCLEROTINIA SCLEROTIORUM

ABSTRACT

Coniothyrium minitans has been shown to be an effective biological control agent
against Sclerotinia sclerotiorum. To improve the efﬁcacy of biological control agents in
the area of application, biological characteristics of commercial and local strains of C.
minitans were compared. Local strains of C. minitans were isolated from sclerotia of S.
sclerotiorum found in Michigan. Eighty-eight percent of the Michigan isolates grew
faster than the commercial strain. The isolate W09, with the fastest growth rate, was
compared with commercial strain CONfM/91-08 for morphological and biological
characteristics. Colonization rate of C. minitans isolates on S. sclerotiorum sclerotia was
evaluated in the laboratory and growth chamber. Daily mycelial growth of C. minitans
isolates was recorded at temperatures of 5, 10, 15, 20, 25, and 30°C; at pH values of 4.0,
4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0; and at photoperiods of 0, 6, 12, 18 and 24 h in a
day for eight consecutive days. Production of conidia of C. minitans was counted 3, 6, 9,
and 12 days after incubation using a hemacytometer. The optimal conditions for mycelial
growth of C. minitans W09 and CON/M/91-08 was 20°C, pH 4.5 in darkness. Moreover,
the optimal condition for conidial production was at 20°C, pH 4.5 and a photoperiod of
12 or 24 h in a day. Strain W09 grew faster and produced more conidia than CON/M/9l-
08. W09 had relative similar colonization capability compared to CON/M/91-08.
Therefore, commercial strain CON/M/9l-08 has shown the ability of adaptation, although

Michigan strain W09 has better overall growth characteristics.

82

INTRODUCTION

White mold [caused by Sclerotinia sclerotiorum (Lib.) de Bary] is a major disease
of soybean in northern regions of the United States, including Michigan (Boland and Hall
1994; McQuilken et al. 1997; Workneh and Yang 2000; Mila et al. 2003). Overwintering
sclerotia of S. sclerotiorum in the soil serve as the primary inocula for infection. Once
sclerotia germinate and produce apothecia, millions of ascospores are released and under
favorable environmental conditions for disease can infect plants (Abawi and Grogan
1979). The best strategy to manage the disease is to suppress sclerotia from germination
or to eliminate their survival in soil (Coley-Smith and Cooke 1971; Adams and Ayers

1979; Subbarao 1998; Bardin and Huang 2001). Fungicides such as azoxystrobin,

boscalid, iprodione, vinclozolin and herbicides including lactofen (Cobra®) have

provided a moderate level of disease suppression (Dann et al. 1999; Matheron and
Porchas 2004; Bradley et al. 2006). However, these chemicals are used for canopy
sprays, which may require multiple applications. Crop rotation with non-host crops such
as corn or cotton has a minor effect on suppressing white mold since sclerotia survive in
soil for up to 8 years (Coley-Smith and Cooke 1971). The concerns over regular use of
fungicides, the unsatisfactory cultural control practices, and an overall for higher level of
disease control lead to the development of biological control alternatives.

Beneﬁcial microorganisms have been used for decades to control plant diseases
(Yuan and Crawford 1995; Subbarao 1998; Compant et al. 2005; Bolton et al. 2006;
Whipps et al. 2008; Yang et al. 2009). Successful cases of biological control have been

reported in different crop systems under laboratory conditions, controlled environments,

83

and in the ﬁeld (Bolton et al. 2006; Whipps et al. 2008). For example, Coniothyrium
minitans Campbell (syn: Paraconiothyrium minitans) has been used to control multiple
diseases caused by Sclerotinia sclerotiorum (Campbell 1947; Jones et al. 2004;
Chitrampalam et al. 2008), because C. minitans has been shown to effectively degrade
sclerotia of S. sclerotiorum in the soil (Gerlagh et al. 1999; Jones 2002; Chitrampalam et
al. 2008). Control of Sclerotinia diseases using C. minitans has been achieved on lettuce,
celery, sunﬂower, bean, cowpea carrot, onion, kiwi fruit, rapeseed and alfalfa both in the
greenhouse and ﬁeld (Jones and Stewart 2000; Bardin and Huang 2001; Jones et al.
2004; Li et al. 2005; Whipps et al. 2008).

Since C. minitans is a strong mycoparasite of S. sclerotiorum (Campbell 1947;
Budge and Whipps 1991; Jones et al. 2004; Chitrampalam et al. 2008), it has the

advantage of removing S. sclerotiorum sclerotia from soil. For example, C. minitans at

concentrations of 104 and 106 conidia/ml effectively controlled lettuce drop in the

greenhouse, which can be enhanced by incorporating solid-substrate into soil (Jones

2002; Jones et al. 2004). Coniothyrium minitans strain CON/M/9l-08 (active ingredient

of biocontrol product Contans® WG, SipCamAdvan LLC, Roswell, GA) has been used

in the US. The commercial strain was originally isolated from a natural sclerotium stored
in the Deutsche Sammlumng von Microorganismen culture collection in Germany
(Hutton 2007). As C. minitans has been developed to be a commercial product, it is
important to know whether it can be adapted to different regions, and how environmental
factors impact the efficacy of mycoparasitic activity of C. minitans. Microorganisms used
as biological control strains may have the potential to establish better in the same areas

where they are isolated. Therefore, local C. minitans isolates may serve as a good

84

comparison to the commercial strain CON/M/91-08 and may even provide additional
isolates for future commercialization.

We have collected a number of C. minitans isolates from Michigan and selected
one isolate (W09) with the fastest growth rate for this comparative study. The objectives
of this study were to compare C. minitans commercial strain CON/M/9l-08 and a
Michigan isolate (W09) on their growth characteristics under various temperatures, pH,

light regimes, and the colonization of S. sclerotiorum.

85

MATERIALS AND METHODS

Isolation of Coniothyrium minitans and the comparison of mycelial growth

Sclerotia of Sclerotinia sclerotiorum were collected from various locations in
Michigan, including soybean ﬁelds in experimental plots at the Plant Pathology Farm
(PLP) in East Lansing, MSU Horticulture Research Station at Clarksville (CLK), Lubeski
Farm in Huron County, farms in Sanilac County, as well as a black bean ﬁeld near Kinde
from Huron County. From each ﬁeld, 100 sclerotia were collected, surface sterilized for 3
min by submerging in 10% chlorine bleach solution (0.6% sodium hypochlorite) and

rinsed three times in sterile distilled water. Sclerotia were dissected by a scalpel on sterile

paper (Envision® Embossed, Georgia-Paciﬁc Resins, Atlanta, GA), and placed on 25%

strength potato dextrose agar (PDA, EMD Chemicals, Gibbstown, NJ ): 10 g potato
dextrose broth, and 15 g agar, amended with 100 mg tetracycline (MP Biomedical, Solon,
OH) in 1 L volume. The sclerotia were incubated at 20°C in the dark for six to ten days.
Fungi were isolated from the cultures and transferred to 25% strength PDA plates.
Cultures were further puriﬁed by single-spore methods (Ho and Ko 1997). Coniothyrium

minitans strain CON/M/9l -08 was also revived and puriﬁed from commercial product

Contans® WG by single-spore methods for this study.

Agar plugs (3 mm diam.) of C. minitans isolates were transferred to full strength
PDA plates and the cultures were incubated at 20°C in the dark for four days. The

diameters of C. minitans mycelia were recorded with three replicates.

86

Identification and conﬁrmation of Coniothyrium minitans

Morphological characteristics of C. minitans strain CON/M/9l-08 and isolate
W09 were observed. The morphology of mycelia and pycnidia, and the size of conidia
were observed with a dissecting microscope (Leica M2 12.5 stereomicroscope, Leica
Microsystems, Bannockbum, IL, 25 X magniﬁcation) and recorded with a microscope
(Leica DM 2500, Leica Microsystems, Bannockbum, IL, 400>< magniﬁcation). The sub-
grouping of C. minitans was based on the classiﬁcation scheme of Sandys-Winsch et al.
(1993). Colony type based on the characteristics of top/reverse colors growing on media
and distribution of pycnidia (Sandys-Winsch 1993) was recorded and conidia size of C.
minitans was measured.

To further identify and conﬁrm the taxonomy of W09, polymerase chain reaction
(PCR) protocols were used. A mycelial plug of C. minitans culture was transferred into
potato dextrose broth (PDB, same as PDA, but without agar) and incubated at 20°C for
six days. The mycelia were collected on ﬁlter paper (12.5 cm in diam., Fisher Scientiﬁc
Inc., Pittsburgh, PA) in a ceramic Bﬁchner funnel (top diam. 83 mm and perf. area diam
60 mm, The Lab Depot, Dawsonville, GA). Mycelia were rinsed by sterilized distilled
water using vacuum ﬁltration. The mycelial mat was blotted dry on sterile paper, and

ground into powder in liquid nitrogen with a mortar and pestle. Genomic DNA of the
isolate was extracted using a DNeasy® Plant Mini Kit (GIAGEN, Valencia, CA)
according to the manufacturer’s protocol. The internal transcribed spacer (ITS) region of
C. minitans was ampliﬁed using PCR with primers ITSl and ITS4 (White et al. 1990).

PCR products were puriﬁed using an UltraClean PCR Clean-up DNA Puriﬁcation Kit

(MO BIO, Carlsbad, CA), and sequenced at the Michigan State University Research

87

Technology Support Facility. The sequence was analyzed using the BLAST algorithm

against the NCBI GenBank (http://blast.ncbi.nlm.nih.gov).

Effect of temperature on mycelial growth and conidial production of Coniothyrium
minitans

Agar plugs (4 mm in diam.) of four-day culture of C. minitans isolates
CON/M/9l-08 and W09 were placed on PDA plates (pH 6.5), and incubated at 5, 10, 15,
20, 25, and 30°C in a temperature-controlled portable cabinet (PTC-l, Sable Systems
International, Las Vegas, NV). Three replicated cultures were used for each treatment.
Mycelial growth was determined by measuring the mycelial radius daily for eight
consecutive days. Conidial production was determined using two methods: 1) calculating
the total number of conidia from each Petri plate (90 mm in diam.) at 3, 6, 9, and 12 days;
2) calculating conidia per unit of mycelial area at the sixth day. To measure the number
of conidia, the mycelial culture growing on PDA was cut into pieces and blended at
21,000 rpm in a Waring Laboratory Blender (The Lab Depot, Dawsonville, GA) for one
minute with 40 ml sterile distilled water (Ooijkaas et al. 1999). Conidial concentration
was counted using a hemacytometer. The experiment was repeated a second time as

described.

Effect of pH on mycelial growth of Coniothyrium minitans
The pH values of PDA media were adjusted to 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5,
and 8.0. The pH values were determined with a standard laboratory benchtop pH unit

(UltraBasic, model UB-10, Denver Instrument, Bohemia, NY) before and after

88

autoclaving. Each treatment was replicated three times. Agar plugs (4 mm diam.) of four-
day culture of C. minitans CON/M/91-08 and W09 were placed on the pH-adjusted PDA
plates, and incubated at 20°C in the dark. Mycelial growth was determined by measuring
colony diameter for eight consecutive days. The experiment was repeated twice as

described.

Effect of light on mycelial growth and conidial production of Coniothyrium minitans
Cultures of C. minitans isolates CON/M/91-08 and W09 were grown on PDA (pH

6.5) as described above, incubated in the grth chamber at 20°C for 12 d. The

photoperiods were: 0, 6, 12, 18 or 24 h/d with a light intensity of 153.96 umol s-1 m-2 per

uA. Each treatment was replicated three times. Mycelial grth was determined by
measuring the culture colony diameter daily for eight consecutive days. The conidial
production per plate was measured at days 3, 6, 9 and 12. Conidia per area of culture
were estimated at the sixth day. Conidial concentration was determined using a

hemacytometer. The experiment was repeated a second time as described.

Colonization of Sclerotinia sclerotiorum by Coniothyrium minitans on agar plates

Conidial suspensions of C. minitans isolates CON/M/9l-08 and W09 were
prepared by ﬂooding lO-d-old cultures on PDA with 10 ml of sterile distilled water and
gently scrapping the surface of the colony with a sterile bent glass rod. Conidia

concentration was determined with a hemacytometer (Hausser Scientiﬁc, Horsham, PA)

and adjusted to a ﬁnal value of 108 conidia/ml prior to use.

89

Sclerotinia sclerotiorum isolates 1004, 1005 and 1006 collected from infected
soybean plants in Michigan were used in the study. Nine sclerotia of each isolate were

placed on water agar plates. This was repeated three times. A 5 ul aliquot of conidial

. 8 . . . . .
suspensron (10 comdra/ml) of the C. minitans lsolates were placed on the top and center

of each sclerotium. The plates were incubated at 20°C in the dark. Sclerotial colonization
of S. sclerotiorum by C. minitans was observed for seven consecutive days after
inoculation. Subsamples of sclerotia were collected on each day, surface sterilized, and
cut into ten 0.5 mm slices with razor blade. To avoid possible cross contamination,
sclerotia were washed individually (Jones et al. 2003). The sclerotial slices were
incubated on 25% strength PDA at 20°C for six days. Coniothyrium minitans recovery
was observed on each slice. The frequency of colonization was calculated by the number
of infected slices divided by total number of slices. The experiment was repeated a
second time as described.

In the second experiment, sclerotial colonization of S. sclerotiorum initiated by
mycelia of C. minitans was observed. Nine sclerotia of each S. sclerotiorum isolate were
placed on water agar with three plates as replications. A mycelial plug (4 mm diam.) of
each C. minitans isolate was placed on water agar two mm from the sclerotium. The
plates were incubated at 20°C in the dark and sclerotial colonization was observed as
described above. The frequency of colonization was calculated by the number of infected
slices divided by the number of total slices. The experiment was repeated a second time

I as described.

90

Colonization of Sclerotinia sclerotiorum sclerotia by Coniothyrium minitans in the
growth chamber

A single coffee ﬁlter paper (62913 Junior Basket, 24.5 in diam., Melitta USA,
Clearwater, Florida) was placed at the bottom of each pot (one liter with 10 X 10 cm2
opening) prior to soil ﬁlling to avoid soil leakage. Each pot was ﬁlled with one liter of
potting soil (BACCTO Professional Planting Mix, Michigan Peat Company, Houston,
TX; ingredient: 70% - 80% horticultural Sphagnum peat). Twenty-ﬁve laboratory-

produced sclerotia from isolate 1006 (isolated from soybean in Michigan) were buried
one centimeter below soil surface. The pots were placed in the 1.3 in2 growth chamber
(Model: PGR14, Conviron, Winnipeg, Manitoba, Canada). An 100 ml aliquot of conidial
suspension of C. minitans CON/M/9l-08 or W09 (108 conidia/ml) was evenly sprayed on
the top soil, with three replications, at 20°C and a photoperiod of a 14:10 h light-dark
circle. Light intensity was 73.56 umol s-lm-2 per uA. Control pots were applied with 100

m1 of distilled water. Sclerotia were retrieved 1, 2, 3 and 4 wk after inoculation by wet
sieving (Hao et al. 2003). The retrieved sclerotia were surface sterilized and placed on
water agar at 20°C for three to seven days. Sclerotia that were viable were determined by
mycelial growth on water agar cultures. Sclerotia infected by C. minitans were
determined by growth of black pycnidia and conidia. The experiment was repeated a

second time as described.

91

Effect of Coniothyrium minitans concentration on colonization of Sclerotinia
sclerotiorum in the growth chamber

Sclerotia of S. Sclerotiorum (isolate 1006) were buried in pot soil as described

. . . . . . 2 4 6 8
above. Comdlal concentratlons of C. minitans Isolates were 0, 10 , 10 , 10 and 10

conidia/ml. There were four replications for each treatment. The pots with sclerotia were

placed in the grth chamber at 20°C and a photoperiod of 14/10 h. Light intensity was

-1 -2 . . . .
73.56 umol s m per uA. Inoculatron, incubation, and sclerotial recovery were referred

to the procedure as described above. The experiment was repeated a second time as

described.

Statistical analysis

Statistical analysis was performed using SAS package (v.9.2, SAS Institute, Cary,
NC). PROC GLM was used to compare mycelial grth rates and conidial sizes of C.
minitans isolates, and analyze the effect of environmental factors (temperature, pH value,
and light regime). Means were compared using least signiﬁcant difference (LSD) at P =
0.05. Since there was no interaction between treatments and trials (P>0.05), data from
two trials were combined. Because there was no difference among S. sclerotiorum
isolates 1004, 1005 and 1006 (P = 0.05), data from the three isolates were pooled
together for analysis. PROC NLIN was used to ﬁt the curve for the colonization rates of
Sclerotinia sclerotiorum sclerotia by Coniothyrium minitans. Conidial concentrations
were transformed to a logarithmic scale prior to analysis. PROC REG was used to
analyze the relationship between mycelial growth rates of C. minitans and colonization

on sclerotia of S. sclerotiorum through time.

92

RESULTS

Identification of Coniothyrium minitans and the comparison of mycelial growth

The frequency of C. minitans colonization on sclerotia varied in different
sampling locations (Table 4-1). A total of 108 C. minitans isolates were collected from
500 sclerotia, in which 20 isolates were from PLP, 9 from CLK, 22 from Lubeski, 28
from Kinde and 28 from Sanilac County. The mycelial growth of isolates collected from
CLK, Lubeski, PLP and Sanilac County were signiﬁcantly faster than the commercial
strain CON/M/9l-08 and isolates from Kinde. A total of 88% of Michigan isolates grew
faster than CON/M/91-08. Coniothyrium minitans isolates from Michigan had similar
morphology in culture (Fig. 4-2). Among all the isolates mycoparasitic to S. sclerotiorum,
isolate W09 from Sanilac County grew the fastest (diam. 33.6 mm) was used for further
study. W09 had similar morphology to C. minitans strain CON/M/91-08, with black
pycnidia and conidia oozing out on the sclerotial surface in black, watery slime (Fig. 4-
2). There was no signiﬁcant difference in conidial length and width between W09 and
CON/M/9l-O8 at P = 0.05 (Fisher’s LSD, Table 4-2). However, W09 had different color
on PDA agar in comparison to CON/M/9l-08. The sequence of ITS gene of W09 was

100% identical to C. minitans on NCBI database (access number AJ293811.1).

Effect of temperature on mycelial growth and conidial production of Coniothyrium
minitans
For both C. minitans isolates CON/M/91-08 and W09, temperature signiﬁcantly

affected daily mycelial growth (Fig. 4-3). At 20°C, both strains had the highest mycelial

93

growth rates: 4.10 and 8.43 mm/d for CON/M/9l-08 and W09, respectively. At 15°C, the
growth rate was the second highest: 2.69 and 6.08 mm/d for CON/M/91-08 and W09,
respectively. At 5°C, limited mycelia growth (less than 1 mm) was observed. No growth
occurred at 30°C. W09 had signiﬁcantly (P = 0.05) faster growth than CON/M/91-08 at
temperatures of 5, 10, 15, 20, and 25°C (Fig. 4-3).

Temperature signiﬁcantly affected conidial production (Fig. 4-4, 4-5). C. minitans
initiated conidia within the ﬁrst three days at 10, 15, 20, and 25°C, but the conidial
production was not observed until the ninth day at 5°C. In general, the overall conidial
production /plate increased as incubation progressed, and the maximal conidial

production occurred at 20°C. Both strains stopped producing conidia at 30°C as pycnidia

were not produced at this temperature (Fig. 4-4, 4-5). At the sixth day, conidia/mm2

mycelial area of CON/M/91-08 was greater (P = 0.05) at 10, 15, 20, and 25°C than W09,
but there was no difference at 10°C (Fig. 4-5). However, conidia/plate of W09 was

greater than CON/M/91 aﬁer six days (Fig. 4-4).

Effect of pH on mycelial growth of Coniothyrium minitans

Mycelial growth of C. minitans CON/M/91-08 and W09 occurred at pH values
from 4 to 8, with the maximal grth rate at pH 4.5 (Fig. 4-6). The mycelial growth was
gradually decreased when pH values increased from 5.0 to 8.0. W09 had higher rates of

mycelial growth at pH values of 4.0 to 8.0 than CON/M/91-08 (Fig. 4-6).

94

Effect of light on mycelial growth and conidial production of Coniothyrium minitans

For both CON/M/91-08 and W09, mycelial growth was negatively correlated with
the length of photoperiods (Fig. 4-7). The maximal mycelial growth of C. minitans
occurred without light and the minimum grth rate with a 24 h photoperiod. At each
point of all photoperiods, W09 had a signiﬁcantly (P = 0.05) greater grth rate than
CON/M/9l-08 (Fig. 4-7).

Light also affected conidial production of C. minitans isolates. The highest

conidia/mm2 of mycelial area was observed with a 24 h photoperiod (Fig. 4-9), but the

maximum conidia/plate was at 12 h/d photoperiod (Fig. 4-8). W09 produced more

conidia/plate (Fig. 4-8), but fewer conidia/mm2 mycelial area (Fig. 4-9) than CON/M/91-

08. At day three, both CON/W9l-08 and W09 produced more conidia under photoperiod
of 12, 18 and 24 h/d compared to 1 and 6 h/d. However, at the 6th, 9th and 12th day, the
maximal conidial production was 6, 12 and 18 h/d. Under a photoperiod of 12 h/d, W09
had a signiﬁcant greater number of conidia/plate than CON/M/9l-08 at 3, 6, 9 and 12 d

(Fig. 4-8, 4-9).

Colonization of Sclerotinia sclerotiorum by Coniothyrium minitans on agar plates
Coniothyrium minitans isolates W09 and CON/M/9l-08 had a linear relationship
between inoculation time and the rates of colonization, using either mycelia or conidia as

inocula (Fig. 4-10). When mycelia were used as inocula, the coefﬁcient of determination

(R2) in the regression equation was 0.9 for W09 and 0.85 for CON/M/9l-08 at P <

0.0001 (Fig. 4-10). Earliest colonization started within the ﬁrst day by W09 and the

95

second day by CON/M/91-08. After ﬁve days, 100% of the sclerotia tested were infected
by both W09 and CON/M/91-08. There was no signiﬁcant difference between the rate of
colonization between W09 and CON/M/91-08 except on the ﬁfth day, when W09 had a

higher colonization frequency (30.8%) than CON/M/9l-08 (28.75%) (F isher's LSD: P =

. . . 2 . . .
0.05, data not shown). When comdra were used as inocula, R in the regressron equation

was 0.9 and 0.85 for W09 and CON/M/91-08, respectively, at P < 0.0001. The earliest
colonization started within the ﬁrst day by W09 and the second day by CON/M/91-08
while100% sclerotia were colonized by CON/M/9l-08 after the ﬁfth day and by W09
after the sixth day and. There was no signiﬁcant difference of colonization rates between
strains W09 and CON/M/9l-08 except at the third day (Fisher’s LSD: P = 0.05, data not

shown).

Colonization of Sclerotinia sclerotiorum by Coniothyrium minitans in the growth
chamber

Coniothyrium minitans isolates W09 and CON/M/9l-08 were re-isolated from
inoculated sclerotia. Colonization frequency was increased as time of incubation was
extended (Fig. 4-11). In contrast, C. minitans was not recovered from sclerotia of control
pots (Fig. 4-11). Colonization frequency and time of incubation had a curve linear
relationship. The regression equation was y = a.ln(x) + b, where y is colonization rate, x is
incubation time, a is slope, and b is the intercept of the curve. During the ﬁrst week post
inoculation, 47.0% of sclerotia were colonized by CON/M/91-08 and 49.0% by W09. In
week two, 72.0% and 84.2% of sclerotia were colonized by CON/M/9l-08 and W09,

respectively. The portion of sclerotia colonized by W09 was higher than by CON/M/91-

96

08 in the second and third week (Fisher’s LSD: P = 0.05, data not shown). In week four,
all the sclerotia retrieved from soil were colonized. The frequency of colonized sclerotia
was 63.6% from CON/M/91-08-treated pots and 65.2% from W09-treated pots (Fig. 4-
11). Thus, W09 and CON/M/9l-08 demonstrate similar efﬁcacy for sclerotial
colonization.

The survival of sclerotia from soil treated with W09 and CON/M/91-08 was

signiﬁcantly lower compared to control. When calculated in the equation: y = a + b 'ex, y

is the number of retrieved sclerotia, x is the time post inoculation, a and b are intercept
and slope, respectively. In week one, 15.0% and 12.9% of sclerotia were reduced by
CON/M/91-08 and W09, respectively. In week 2, 11.6% and 21.9% of sclerotia were
reduced by CON/M/91-08 and W09, respectively. The number of retrieved sclerotia from
the W09-treated pot was signiﬁcantly lower than the number of sclerotia treated with
CON/M/91-08 (Fisher’s LSD: P = 0.05). The third week showed a similar trend as in
week two: W09 had a signiﬁcantly higher efﬁcacy on sclerotial reduction than
CON/M/9l-08 (Fisher’s LSD: P = 0.05). In week four, 63.1% and 70.8% of sclerotia
were degraded by CON/M/9l-08 and W09, respectively (Fig. 4-11). Overall, W09 and
CON/M/9l-08 demonstrated a similar trend for sclerotial reduction, but W09 was more

aggressive in week two and three.

Effect of Coniothyrium minitans concentration on colonization of Sclerotinia
sclerotiorum in the growth chamber
There was a linear relationship between conidial concentration and the rate of

colonization for both C. minitans isolates CON/M/9l-08 and W09. Increasing conidial

97

concentration of C. minitans enhanced the efﬁcacy of colonization in S. sclerotiorum

(Fig. 4-12). For CON/M/91-08, the colonization frequency was from 77.64% at

concentration of 102 conidia/ml to 91.6% at 108 conidia/m1 after four weeks. For W09,

the colonization frequency ranged from 75.3% at 102 conidia/ml to 92.8% at 108
conidia/ml. There was no difference of colonization efﬁciency between the two C.
minitans isolates, except W09 had a signiﬁcant higher colonization frequency at 104

conidia/ml (Fig. 4-12). No C. minitans colonization of sclerotia was observed from
control pots (Fig. 4-12).

There was a negative relationship between C. minitans concentration and survival
of S. sclerotiorum sclerotia (Fig. 4-12). For both C. minitans isolates, lower numbers of
sclerotia were retrieved from the soil treated with higher conidial concentration of C.
minitans. The number of sclerotia from all treatments was signiﬁcantly lower than the

control (P = 0.05). For CON/M/9l-08, S. sclerotiorum sclerotia were reduced by 27.7,

33.3, 47.6, and 65.7% at concentrations of 102,104, and 108 conidia/ml, respectively. For

W09, sclerotia were reduced by 36.1, 47.6, 63.6, and 73.5% at concentration of C.

minitans 102, 104, 106, and 108 conidia/ml, respectively. The CON/M/91-08 had a
signiﬁcant effect on the number of sclerotia retrieved from soil at concentrations of 104
and 106 conidia/ml. There was no difference between the two C. minitans isolates on

sclerotial colonization at the highest concentrations (106 and 108 conidia/ml) (Fig. 4-12).

98

Table 4-1. Mycelial growth of Coniothyrium minitans isolated from different ﬁelds in
Michigan on potato dextrose agar.

 

Mycelial Colony diam. (mm) range (%)
colony (mm

in diameter)x 0-10.0 10.1-20.0 20.1-30.0 30.1-40.0

Location Isolate

 

 

CLK 9 26.4 ay 0.0 0.0 88.9 11.1
Kinde 28 21.6 b 3.6 25.0 71.4 0.0
Lubeski 22 25.3 a 0.0 13.6 68.2 18.2
PLP 20 24.1 a 0.0 5.0 95.0 0.0
Sanilac 28 27.0 a 0.0 3.6 89.3 7.1
CON/M/91-O8Z 1 13.0 b 0.0 100.0 0.0 0.0

 

Z Mycelial growth diameter were determined after four days of incubation at 20°C in the
dark.

y Fisher’s least signiﬁcant difference (LSD) at P = 0.05was used to compare the average
mycelial diameter of C. minitans isolates.

2 Commercial strain of C. minitans CON/M/91-08 from product Contans® WG was listed
to compare the mycelial growth rate with Michigan isolates.

99

Table 4-2. Origin, colony type and conidia size of Coniothyrium minitans isolates W09
and CON/M/91-08 on potato dextrose agar.

 

Source Conidia size (um)y’ z
of isolate

 

Isolate Origin Colony Length Width

typex
Mean Max Min Mean Max Min

 

CON/M/9l-08 Germany Sclerotia Typel 5.9a 7.7a 4.1a 4.4a 6.2a 3.0a

W09 Michigan Sclerotia Type 11 5.9a 7.6a 3.9a 4.2a 5.3a 3.5a

 

x Colony type is described with top/reverse colors and distribution of pycnidia (Sandys-
Winsch 1993). Type I: Isabelline (moderate yellowish brown)/Isabelline. Many black,
mature pycnidia visible from top and reverse; type II: Hazel (light-moderate yellowish
brown)/Isabelline. Many black, mature pycnidia were visible from both top and reverse
sides of the media plate.

y Conidia size was measured under the microscope (Leica DM 2500, Leica
Microsystems, Bannockbum, IL, 400 X magniﬁcation).

Z Fisher’s least signiﬁcant difference (LSD) at P = 0.05was used to compare conidial
sizes of C. minitans isolates

100

 

Sum

Figure 4-1. Morphology of C. minitans W09 (400 x magniﬁcation), showing mycelia
and conidia.

101

 

Figure 4-2. Colonization of Sclerotinia sclerotiorum sclerotia by C. minitans strain W09.
Pycnidia (black spherical bodies) and conidia of Coniothyrium minitans oozing out of
sclerotial surface (arrows) of Sclerotinia sclerotiorum under a dissecting microscope (25
x magniﬁcation).

102

 

 

 

 

 

   

 

9 ‘ DCON/M/91-08
8 ‘ IW09
A 7 -
'O
E 3*
E 6 ‘
‘13 4~
9 b
u: 3 -
E:
8 2~
1 .1
f 1:
0 "' l 1
5 15 20 25 30
Temperature (°C)

 

 

Figure 4-3. Effect of temperature on mycelial growth of C. minitans isolates, W09 and
CON/M/91-08. Mycelial growth was measured for eight consecutive days. Growth rates
were the slope of regression equations derived from eight-day mycelial growth. Bars on
each column are standard deviation. Uppercase and lowercase letters are used for mean
separation for C. minitans W09 and CON/M/91-08, respectively. Means with the same
letters are signiﬁcantly different. Growth at each temperature was compared between the
two isolates, and * indicates signiﬁcant difference of means between the two isolates at
the same temperature.

103

 

 

 

 
 
 

 

 

 

*
‘*
Temperature
6 .(°C)
_ +5
E
3 +10
'0
'g 4 +15
° -x—2o
8’ .
.1 2 -H--25
”ﬁe-30
O 1,. J
3 6 9 12
8 _
emperature
6 - (°C)
_ -O-5
E 4 .4
g +15
0
8, —)-(—20
‘l 2 a +‘ 25
wan-430
0 C .3 Q
3 6 9 12
Incubation time (day)

 

 

Figure 4-4. Effect of temperature on conidial production/plate (90 mm in diam.) of C.
minitans isolates W09 (upper panel) and CON/M/91-08 (lower panel). Conidial
production was determined at 3, 6, 9 and 12 d using a hemacytometer. Colored lines
indicate different temperatures. Comparisons were performed using Fisher’s least
signiﬁcant difference (LSD, P = 0.05) for each column. Bars on each column are
standard deviation, and * indicates signiﬁcant difference between the strains at the same
temperature (P = 0.05).

104

 

 

 

 

 

 

 

 

 

 

5 - DCON/M/91-08
IW09
as b*
4 — A d c*
A
5 3 - B
N
1":
'E a
o
u —
.5." 2 ‘
1 4
e D e D
O 7 1
S 10 15 20 25 30
Temperature (°C)

 

 

 

Figure 4-5. Effect of temperature on conidia production/mm2 mycelial area of C.
minitans isolates W09 and CON/M/91-08. Conidia were counted at the sixth day using a
hemacytometer. Comparisons were conducted using Fisher’s least signiﬁcant difference
(LSD, P = 0.05) for each column. Uppercase and lowercase letters are used for means
separation of C. minitans W09 and CON/M/9l-08, respectively. Means with same letters
are not signiﬁcantly different. Bars on each column are standard deviation, and *
indicates signiﬁcant difference between strains at the same temperature (P = 0.05).

105

DCON/M/91-08
C* .W09
6 J 0* 05* Ea:

* .
a b C G H II:

Daily growth rate (mm/d)
.5
o.
o.
m

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 4-6. Effect of pH values on mycelial grth of C. minitans isolates W09 and
CON/M/9l-08. Mycelial grth was measured for eight consecutive days. Growth rates
were determined by the slope of equations in linear regression based on the data of eight
days. Growth rates of the two isolates were compared at each pH value. Uppercase and
lowercase letters are used for means separation of C. minitans W09 and CON/M/9l -08,
respectively. Means with same letters are not signiﬁcantly different. Bars on each column
are standard deviation, and * indicates the signiﬁcant difference between isolates at the
same pH (P = 0.05).

106

8 ‘ A* DCON/M/91-08
7 « . -w09

C*

Daily growth rate (mm/d)
h
m
w
:-

 

 

 

0 6 12 18 24
Photoperiod (hid)

 

 

 

Figure 4-7. Effect of light on mycelial grth of C. minitans isolates W09 and
CON/M/91-08. Mycelial growth was measured for eight consecutive days. Growth rate
under each photoperiod was determined by the slope of equations in linear regression
based on the grth data of eight days. Growth rates of the two isolates were compared
at each photoperiod. Uppercase and lowercase letters are used for means separation of C.
minitans W09 and CON/M/91-08, respectively. Means with same letters are not
signiﬁcantly different. Bars on each column are standard deviation, and * indicates the
signiﬁcant difference between isolates at the same photoperiod (P = 0.05).

107

   

 

 

 

 

 

 

 

 

7 _
. Photoperiod
E (h/d)
U
'E +6
8 +12
8’
_, +18
—-24
3 6 9 12
7 s
6.5 1 Photoperiod
E (h/d)
3 6 *0
u q
5 +6
0 +12
°’ 5.5 -
3 -)-(-18
+24
5
4.5 . . .
3 6 _ 9 12
Incubation time (day)

 

Figure 4-8. Effect of light on conidial production/plate (90 mm in diam.) of C. minitans
isolates W09 (upper panel) and CON/M/9l-08 (lower panel). Conidial production was
determined at 3, 6, 9 and 12 d using the hemacytometer. Colored line indicated different
photoperiod (h/d). Multiple comparisons were performed for each photoperiod using least
signiﬁcant difference (LSD, P = 0.05), and * indicates signiﬁcant difference between the
strains under the same photoperiod. Bars on each column are standard deviation.

108

 

 

     

 

 

 

 

 

 

 

 

6 _
DCON/M191-08 IW09
5 - a a*
b* a A
‘ B
_ 4 e c*
E C
E
u
.g 3 _ D
0
a:
o
-' 2 1
1 —1
0
0 6 12 18 24
Photoperiod (hld)

 

 

 

Figure 4-9. Effect of light on conidial production/mm2 mycelial area of C. minitans

isolates W09 and CON/M/91-08. Conidia were counted at the sixth day using a
hemacytometer. Uppercase and lowercase letters are used for means separation of C.
minitans W09 and CON/M/91-08, respectively. Means with same letters are not
signiﬁcantly different. Values of the two isolates were compared at each photoperiod
using least signiﬁcant difference (LSD, P = 0.05), and * indicates signiﬁcant difference
between the strains. Bars on each column are standard deviation.

109

 

60 -
y = 7.34x - 3.49 3
R2: 0.90 P<0.0001 (W09) °

00

    
 

h
C
1

Colonization (%)

N
O
1

y =87.46x - 7.30 ° CONN/9198
3 R2: 0.85 P<0.0001 . w09
(CON/M/91-08)

 

o

4

" o o
o

 

 

  
  

1 2 3 4 5 6 7
a 7 7 A I
O
y= 7.34x- 3.49 . g
R2 = 0.90 P<0.0001 (W09) ‘
SE40 -
E
320 - .

  
 

8
y = 7.46X - 7.30 o CON/M/91-08
R2: 0.85 P<0.0001 . wog
(CON/M/91-08)

I I j

 

 

o
{
4) ..
w 4)

4 5 6
ncubation time (day)

 

 

 

Figure 4-10. Colonization of S. sclerotiorum sclerotia by C. minitans isolates W09 and

CON/M/91-08 via mycelia (upper panel) and conidial concentration of 108 conidia/ml
(lower panel). The sclerotium was diced into slices with same thickness. Colonization
was calculated as the number of infected sclerotial slices divided by total sclerotia slices
examined. Linear regression estimated the correlation between time (day, x axis) of
incubation and percentage of colonization (y axis).

110

     
 
  

y = 36.32|n(x) + 52.17
R2 = 0.93.(W09)

   

Colonization (96)
Ch
0

4:.
O

0
y = 37.00|n(x) + 46.30 0 CON/M/91-08
R2 = 0.93 (CON/M/91-08) a Control

 

 

 

 

 

 

0 W09
20
0 a as
1 2 3 4
3° y =24.76-0.06e"
25 Rz =

 
 
  

 

 

 

9 (Control) U
y - 23.02- .26e
R2 = 0.96

y = 21.84+0.28ex COMM/081

Sclerotial L soil
H
U1

 

10 R2 = 0.99 (W09)
o CON/M/91-08
5 a Control
0 . 0 W09 1 .
1 2 3 4

 

 

Incubation time (week)

 

Figure 4-11. Effect of inoculation of C. minitans isolates W9 and CON/M/91-08 on
colonization of sclerotia (upper panel) and sclerotial survival (lower panel) of S.
sclerotiorum in the grth chamber. A total of 25 sclerotia were placed in each pot

followed by 100 ml C. minitans conidial suspension (108 conidia/ml) was placed in each
pot with three replications. The frequency of colonization was determined as the infected
sclerotia divided by total sclerotia buried. The survival of sclerotia was recorded after
four weeks. Non-linear regression determined the correlation between time (day, x axis)
from inoculation and the percent of colonization or sclerotial number (y axis).

111

H120 -
-e—CON/M/91-08
100 ~ +W09

80 -

60*

Infection (%)

40-

20-

 

 

 

 

 

 

25 +CONIM/91-08
+W09

SclerotialL soil

 

 

 

NT 2 4 6 8
Log conidia/ml

 

 

 

Figure 4-12. Effect of concentration of C. minitans isolates W09 and CON/M/91-08 on
colonization of sclerotia (upper panel) and sclerotial survival (lower panel) of Sclerotinia
sclerotiorum. A total of 25 sclerotia were placed in each pot. Conidial concentrations at 0

(NT), 102, 104, 106, 108 conidia/ml in a volume of 100 ml were inoculated in each plot

with four replications. Sclerotial survival was determined after four weeks. Comparisons
using Fisher’s least signiﬁcant difference (LSD) were performed for each week. Growth
at each temperature was compared between the two isolates. Bars on each column are
standard deviation, and * indicates signiﬁcant difference between the means of isolates at
the same temperature.

112

DISCUSSION

Coniothyrium minitans has a worldwide distribution (Sandys-Winsch 1993), and
has been found in many regions, such as the United States, Australia, Belgium, Canada,
Denmark, France, Germany, Israel, Japan, Korea, Netherlands, New Zealand, Portugal,
South Africa, Sri Lanka, Sudan, Switzerland and the UK (Campbell 1947; Turner and
Tribe 1976; Sandys-Winsch 1993; Jones and Stewart 2000; Budge and Whipps 2001;
Ren et al. 2010). This report adds Michigan to this list.

Coniothyrium minitans isolates grew at temperatures between 5 and 30°C, with
the optimum of 20°C for mycelial growth and conidial production. Similar results were
found with European strains in previous studies (Campbell 1947; McQuilken et al. 1997).
However, the optimal environmental temperature for mycoparasitic activity is higher than
that for mycelial growth and conidial production. For example, the temperature for most
efﬁcient colonization on sclerotia of Sclerotinia sclerotiorum is 30°C (Tu 1999).

This study found that the optimal pH for mycelial growth is 4 to 5, which is also
suggested by McQuilken (1997). Light was found to have a negative effect on the
mycelial growth of C. minitans, which is different to a previous study that found that
light has no effect (McQuilken et al. 1997). Regardless of this, light is necessary to
promote the conidial production. Besides photoperiod, UV radiation and its effects on the
survival of the fungus will be important in the future to determine viability in when
employed under ﬁeld conditions.

The colonization frequency of C. minitans W09 and CON/M/91-08 on S.

sclerotiorum in the growth chamber was consistent with results from agar plate tests. Half

113

of the sclerotia were infected during the ﬁrst week, indicating a strong mycoparasitic
ability of C. minitans on sclerotia of S. sclerotiorum. After four weeks, sclerotia treated
with both W09 and CON/M/9l-08 were 100 percent colonized. This is in agreement with
Turner and Tribe (1976), indicating that both W09 and CON/M/91-08 are more
aggressive than other European isolates tested on sclerotial colonization.

C oniothyrium minitans W09 was similar in growth pattern with CON/M/9l-08,
but outperformed CON/M/91-08 on mycelial growth and conidial production/plate under

most of the experimental conditions, including temperatures, pH and light. W09 had a

lower conidia/mm2 mycelial area than CON/M/91-08. However, since W09 grows faster,

its overall conidial production/plate is higher than CON/M/91-08. From this, W09 is
more efﬁcient than CON/M/9l-08 for large amounts of conidial production. The mycelial
growth rate at 20°C for CON/M/9l-08 was similar to many European isolates
(McQuilken et al. 1997). However, W09 had a considerably higher daily growth rate,
which doubled the growth rate of CON/M/9l-08. At pH 4.5, the mycelial growth rate of
W09 is 55.2% higher than CON/M/91-08. In dark conditions, W09 also doubled the
average daily growth rate of CON/M/91-08. Therefore, under these artiﬁcial conditions
W09 has a faster growth than CON/M/9l-08.

Coniothyrium minitans W09 has a faster colonization at an early stage than
CON/M /91-08. W09 can penetrate into S. sclerotiorum sclerotia and establish
colonization within 24 h but CON/W91-08 needs at least 48 h. W09 had signiﬁcantly

higher mycoparasitic activity in the growth chamber at moderate conidial concentrations

(104 and 106 conidia/ml) than CON/M/91-O8. This indicates that w09 may be more

114

efficient until its conidial concentrations reach the plateau. This may reduce the conidial
concentration without scarifying the efﬁcacy if it is used as a biocontrol product.

Sclerotinia sclerotiorum produces oxalic acid that decreases the pH in infected
tissue and favors cell wall degrading enzymes to macerate tissue, and low pH (around. 4.0
to 5.0) was the most favorable condition for colonization (Cessna et al. 2000).
Interestingly, this range of pH values (4.0 to 5.0) is also favorable for mycelial growth of
C. minitans. Oxalic acid produced by S. sclerotiorum can promote the growth of and be
colonized by C. minitans (Ren et al. 2007). Its outstanding ability to grow in low pH
could be one of the reasons that C. minitans colonizes sclerotia so successfully.

Care needs to be taken when implementing results from laboratory or grth
chamber to the ﬁeld. Several data showed the subtle differences under tested conditions
although statistical signiﬁcances appear. For example, mycelial growth rate of C.
minitans W09 had signiﬁcant difference ranging from pH 7.5 to 8.0. However, the
absolute mycelial growth rate was 4.5 min/day and 4.1 mm/day, respectively. This
growth difference is hardly noticeable under ﬁeld conditions. It is interesting to further
evaluate the optimal grth and mycoparasitism of C. minitans under ﬁeld conditions.

In conclusion, commercial strain CON/M/9l-08 grows well in responding to
various pH values and temperatures, and has a similar colonization ability to C. minitans
W09, although the latter was more effective in mycelial growth, conidial production, and
colonization frequency at certain conidial concentrations overall. This shows the
advantage of using endemic isolates for effective disease control if they were developed

into a biocontrol product.

115

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119

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