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FUNGICIDE SENSITIVITY OF
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ERIN MARIE LIZO'I'I'E

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FUNGICIDE SENSITIVITY OF CHERRY PATHOGENS
By

Erin Marie Lizotte

A THESIS
Submitted to
Michigan State University
In partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE
Department of Plant Pathology

2007

ABSTRACT
FUN GICIDE SENSITIVITY OF CHERRY PATHOGENS
By
Erin Marie Lizotte
Michigan populations of Blumeriellajaapii (Rehm) Arx, the causal agent

of cherry leaf spot (CLS), have documented resistance to sterol demethylation inhibitor
fungicides (DMIs), which were frequently used to control the pathogen. A bioassay was
completed to determine the current sensitivity status of B. jaapii isolates to dodine.
Dodine has been used sporadically to control CLS for over 50 years, but the sensitivity of
B. jaapii to dodine was unknown. When B. jaapii isolates from Michigan were screened
in vitro; a wide range of minimum inhibitory concentrations (MIC) were found. MIC
values ranged from 0.05 ppm to greater than 400 ppm dodine, and demonstrated a
reduction in the sensitivity of B. jaapii isolates collected fi'om dodine-exposed sites.

American brown rot (ABR) is caused by Moniliniafi'ucticola (Wint.) Honey, and
significantly impacts cherry production. DMIs are currently the most effective and
fi'equently used fungicides for ABR control, but resistance to DMI fimgicides is well
documented in various fungal pathogens, including M. fiucticola from Georgia and New
York. Due to the single-site mode of action, and the history of resistance development in
DMIs, the risk of resistance developing in Michigan M. fi-ucticola populations is
substantial. An in vitro assay was performed to determine the status of M. fructicola
sensitivity to fenbuconazole (a DMI) in Michigan. Effective dose values, by orchard,
ranged from 0.0038-0.0379 ppm fenbuconazole, a significant amount of variability that

may be an indicator of the fungal populations ability to shift toward reduced sensitivity.

To my parents John and Joyce Taylor,
who never had a doubt.

iii

ACKNOWLEDGEMENTS

I would like to thank Dr. George Sundin for giving me the opportunity to work in
his lab over the past two years, and for literally forcing me to overcome my fear of public
speaking. Thank you Dr. Tyre Proffer, who I am convinced knows more about fungi than
anyone in the world, and graciously shared his space and knowledge with me. Thank you
to both of you for the patience, encouragement, cartoons, and support. Special thanks to
Dr. Nikki Rothwell for her belief in me right fiom the beginning, and continued support
through the arduous process of graduate school. My sincere gratitude to Dr. William
Kirk for enlightening me about some valuable resources (FRAC!) and saving me
hundreds of hours of time by teaching me how to run a regression analysis in under 30
minutes (that took years off of my project). Special thanks to Gail Ehret and William
Klein, whom deftly oversaw spray trials, answered endless questions, and helped with
statistical analysis of the results.

I would be remiss if I didn’t acknowledge my fellow lab members who affected
my research daily through technical assistance, general support and sometimes all-out
sabotage. It would have been a long and excruciating journey without you all. Thanks to
you, I have managed to get by with limited molecular knowledge.

I am also grateful to the Michigan Cherry Committee, which provided much of

the fimding for my research.

iv

TABLE OF CONTENTS

LIST OF TABLES ................................................................................... vi
LIST OF FIGURES ................................................................................ viii
LITERATURE REVIEW ........................................................................... 1
Introduction ................................................................................... 1
Important Diseases of Cherry .............................................................. 2
The History of Fungicides .................................................................. 8
Fungicide Resistance in Fruit Pathogens ................................................ 10
Fungicide Resistance Mechanisms ...................................................... 15
Managing to Delay F ungicide Resistance ............................................... 16
Summary ..................................................................................... 18
Literature Cited ............................................................................. 19
CHAPTER 1: SENSITIVITY OF BL UMERIELLA JAAPII TO DODINE ................. 26
Introduction ................................................................................. 26
Materials and Methods ..................................................................... 28
Results ....................................................................................... 37
Discussion ................................................................................... 43
Literature Cited ............................................................................. 46

CHAPTER 2: SENSITIVITY OF MONILINA FRUCTICOLA TO

FENBUCONAZOLE ............................................................................... 49
Introduction ................................................................................. 49
Materials and Methods ..................................................................... 51
Results ....................................................................................... 59
Discussion ................................................................................... 72
Literature Cited ............................................................................. 76

APPENDD( A ....................................................................................... 79

APPENDIX B ....................................................................................... 82

LIST OF TABLES

LITERATURE REVIEW
Table 1. List of fungicide or chemistry introductions relevant to tree fruit production, the
associated specificity, and resistance issues ........................................................................ 9
CHAPTER 1

Table 1.1. The 2005 spray dates (in bold), respective growth stages, and cherry leaf spot
infection periods as recorded at the Northwest Horticultural Research Station ................ 32

Table 1.2. The 2006 spray dates (in bold), respective growth stages, and cherry leaf spot
infection periods as recorded at the Northwest Horticultural Research Station ................ 33

Table 1.3. Incidence of cherry leaf spot infection during the 2005 grong season based
on use of copper and dodine in a resistance management spray regime ........................... 38

Table 1.4. Cherry leaf spot incidence during the 2006 growing season based on season
long applications of fungicides commonly used for control .............................................. 39

CHAPTER 2

Table 2.1. List of suspected Moniliniafiucticola isolates screened to determine species,
including the source, locus and associated PCR code ....................................................... 53

Table 2.2. In vitro, mean fenbuconazole ECso value and standard error, based on location
and inoculate source of Moniliniafructicola isolate ............................................ 64

Table 2.3. Percent tart cherries infected, fifteen days after inoculation with a lOS/ml
Moniliniafiucticola propagules. Cherries were dipped in four concentration of
fenbuconazole. . ................................................................................................................. 66

Table 2.4. Percent sweet cherries infected, fifteen days after inoculation with a 105/m1
Moniliniafiuctz’cola propagules. Cherries were dipped in four concentration of
fenbuconazole .................................................................................................................... 67

Table 2.5. Sensitivity of Michigan Moniliniafmcticola isolates to fenbuconazole and
propiconazole ..................................................................................................................... 70

vi

APPENDIX A

Table A. 1. Weather conditions during the 2005 growmg season in Suttons Bay,

Michigan, including; temperature, wetting period duration and rainfall ........................... 80

Table A2. Weather conditions during the 2006 growing season in Suttons Bay,

Michigan, including; temperature, wetting period duration and rainfall ........................... 81
APPENDIX B

Table B. 1. Agar recipes, based on 500ml, used to induce conidial development in
Moniliniafiucticola ........................................................................................................... 83

vii

LIST OF FIGURES

CHAPTER 1

Figure 1.A. Custom built research plot sprayer with a JBS model 705 hand gun, used to
apply fungicides for efficacy trials at the Northwest Horticultural Research Station ....... 30

Figure 1.B. Blumeriellajaapii isolates (16) growing on dodine amended modified malt
extract agar to determine the minimum inhibitory concentration ...................................... 36

Figure 1.C. Distribution of dodine minimum inhibitory concentration values for all
Blumeriellajaapiz' isolates ................................................................................................. 41

Figure 1D. Relationship of dodine exposure of the isolate source to in vitro sensitivity of
Blumeriellajaapii isolates, as measured by the minimum inhibitory concentration.
Unexposed isolate sources included dooryard trees, wild cherry, and black cherry.
Exposed isolates were collected fi'om commercial cherry orchards. The abandoned
orchard isolates source has not been exposed to dodine since the 19805. A Chi-square
test was performed and indicated a significant difference between the exposed and
unexposed sources (P = 0.1) .............................................................................................. 42

CHAPTER 2

Figure 2.A. Monliniafiucticola isolates growing on PDA plates amended with six
concentrations of fenbuconazole (0, 0.001, 0.005, 0.01, 0.05, 0.1 ug/ml)......................56

Figure 2.B. PCR amplification of the ITS regions using (A) Monilinia laxa ITS primers
1 and 4, or (B) Moniliniaflucticola ITS primers 1 and 4 for suspected Monilinia
fiucticola isolates (1-6) ..................................................................................................... 60

Figure 2.C. PCR amplifications of the ITS] and ITS4 regions using Moniliniafi-ucticola
ITS primers 1 and 4, for suspected Moniliniafiucticola isolates 7-24. ....................... 61

Figure 2.D. PCR amplifications of the ITSl and ITS4 regions using Moniliniafiucticola
ITS primers 1 and 4, for suspected Moniliniafiucticola isolates 13, 19 and 27-44.. ... ...62

Figure 2.B. Average incidence of tart cherries infected with Moniliniafiuctz'cola, based
on isolate sensitivity, and fenbuconazole rate (full rate is 146 m1/2,810 liter water/
hectare)68

Figure 2.F. Average incidence of sweet cherries infected with Moniliniafi-ucticola,

based on isolate sensitivity, and fenbuconazole rate (full rate is 146 ml/2,810 liter water/
hectare)69

viii

Figure 2.G. Comparison of fenbuconazole and propiconazole sensitivity of Michigan
Moniliniafiucticola isolates (LAB-3, LAR-3, RFS-lO, WIL-3, KHG-l, VNL-18, MRC-
20, BRC-l6) with baseline propiconazole levels from Georgia (DL), and an isolate
resistant to propiconazole (AP) .......................................................................................... 71

Images in this thesis are presented in color.

ix

LITERATURE REVIEW

FUNGICIDE RESISTANCE IN PATHOGENS OF STONE FRUIT

INTRODUCTION

Michigan’s cherry industry began in the late 1800’s with the planting of the first
tart cherry orchard on Old Mission Peninsula in northwest Michigan. The cultivation of
tart cherry trees (Prunus cerasus L.), and sweet cherry trees (Prunus avium L.) has
prospered in the region thanks to sloping terrain, well-drained soils and mild weather
mediated by the proximity of the area to Lake Michigan. According to the USDA
National Agriculture Statistics Service, the 2006 tart cherry crop was worth an estimated
$53.4 million nationally (78% of which was produced in Michigan), and the sweet cherry
crop was estimated at $487.4 million nationally (7% of which was produced in
Michigan); (74). Fungal diseases, including cherry leaf spot, American brown rot,
Armillaria root rot, European brown rot and powdery mildew cause significant losses
annually. P. cerasus (Montrnorency) is most desirable and prevalent tart cherry cultivar
in the US. Montrnorency cherry trees possess no innate resistance to the more damaging
fungal diseases, cherry leaf spot and American brown rot, the two diseases that are the
focus of this review. This lack of inherent disease resistance drives the cherry industry’s
heavy reliance on fungicides. Genetic mutations are the most prevalent cause of
fungicide resistance and exist at very low frequencies within the fungal population (8).
In conventionally managed cherry orchards, sterol demethylation inhibitor (DMI)
fimgicides are often applied more than five times in a single season, this is heavy

fungicide pressure and selects for resistant isolates. Under heavy fungicide pressure

resistant isolates may increase disproportionately to normal population structure.
Resistant mutants typically exist at a frequency of roughly 1:100 million, but the
proportion of resistant mutants in the population increases under fungicide pressure
because they tolerate the fungicide treatment and survive to propagate (8). The increase
in this less sensitive population may ultimately lead to a lack of disease control in the
field, or practical field resistance (40). An estimated 1:10 to 1:100 ratio of resistant
isolates is required in the population before the effect of resistance is readily detectable in
the field (8). Resistance has developed to most major chemical classes of fungicides in
various plant pathogens. The fungicides affected include; dodine (35), dicarboxirnides
(17, 63), benzimidazoles (35, 49) organic fimgicides (24), strobilurins (29), and DMI’s
(20, 30). Resistance is a major concern for Michigan cherry growers because of heavy
fungicide pressure and the history of fimgicide resistance development in many fungicide

classes.

IMPORTANT DISEASES OF CHERRY
Cherry Leaf Spot

Cherry leaf spot (CLS), caused by the ascomycete Blumeriellajaapii (Rehm) Arx,
is arguably the most damaging fungal pathogen of tart cherry, and has the potential to
significantly reduce profits for growers in the Great Lakes region of the US. (54). There
has been relatively little information published on the biology and resistance issues
associated with B. jaapiz'. CLS primarily affects foliage and as a result reduces the
photosynthetic ability of the tree. Infected tart cherry leaves will turn yellow and

defoliate prematurely if disease is not effectively controlled. Sweet cherry tree leaves

turn yellow but are retained. Less than 50% defoliation by early September is considered
acceptable CLS control in Michigan. When significant defoliation occurs before harvest,
fruit is soft and immature, has low soluble solids and ripens unevenly (37). Significant
defoliation can be quantified based on the standard that at least two leaves are needed to
effectively ripen each cherry on tart cherry trees (57). Following two or more years with
significant defoliation early in the season, trees are susceptible to winter injury. This is
due to the loss of photosynthates and therefore stored carbohydrates in roots. Blossom
production is also reduced for at least two subsequent years (3 7).

B. jaapii overwinters on fallen leaves on the orchard floor and produces apothecia
(sexual spore-bearing structures) in the spring (3 7). The primary infection period may
last 2-6 weeks depending on conditions. Optimal apothecia] development occurs
between 6-16°C, with ascospore discharge increasing with temperature between 8-30°C
(23). Ascospore release occurs as tissue dries following the thorough wetting of mature
asci (39). Germination occurs on the surface of the leaf and infection occurs through the
leaf stomata. Leaves remain susceptible throughout the grong season (3 7), contrary to
the in vitro evidence of ontogenic, or age-related resistance in leaves (18). Following
infection, acervuli develop on the underside of the leaf and produce a visible mass of
white conidia. Conidia are dispersed from leaf to leaf by wind or rain and the infection
cycle can be repeated several times during a single season, depending on conditions. The
conidial stage was the basis for the development of an effective infection model by
Eisensrnith, an adaptation of which is still in use today (18).

All commercially cultivated cherry cultivars are susceptible to cherry leaf spot

(37), although less susceptible cultivars have been found (77). The primary method of

CLS management is through fungicide application, with five to seven applications
recommended per season, depending on disease pressure (62). The most common
fimgicides used to control CLS include: chlorothalonil, captan, strobilurins, and several
sterol demethylation inhibitors (fenbuconazole, tebuconazole, myclobutanil, and
fenarimol); (54). Salts containing the Cu2+ ion (copper hydroxide or copper sulfate), and
dodine are also used. B. jaapii has a history of cross resistance to DMI fungicides in
Michigan (62).
American Brown Rot

American brown rot (ABR) is caused by the ascomycete Monilinia fructicola
(Wint.) Honey, and is an important pathogen on apricot, peach, nectarine, plum and
cherry (particularly sweet cherry varieties); (3 7). The fiingus attacks fruit, blossoms,
spurs, and shoots with ideal infection conditions initiating epidemic inoculum levels in as
little as 24 hours. ABR causes fruit rot before and after harvest, greatly reducing the
quality and quantity of the yield (3 7). Fungicide applications help control the disease
until harvest, but the development of ABR in harvested fruit remains a problem in fresh
fi'uit markets. Processors suffer losses from ABR during the storage of fruit and ripening
process (31, 34). The most distinctive sign of infection is gray/tan conidia that appear on
the surface of discolored and decaying fiuit. Infected fi'uit may rot and abscise, or persist
through the winter in a mummified form. In cherry, mummified fruit provide the vast
majority of the primary inoculum for infection the following spring (37) in the form of
asexual conidia. Conidia] production is greatest between 15 and 23°C (76). The optimal
temperature for conidial germination is between 20 and 25°C (1 l, 59). Apothecia (sexual

fruiting structures) form on fruit on the orchard floor producing ascospores in the spring,

but only in areas where the soil is moist (31); generally apothecia formation on cherry is
rare in Michigan. Although injury to the fi'uit may lead to increased infection, the fungus
readily infects when no wound or fi'uit-to-fruit contact is present (55). Direct infection
occurs through the fruit cuticle (37), or the trichomes (31) and conidial production can
occur within as little as three days (37).

ABR infection is dependent on inoculum levels and occurs more readily on
mature mm (31, 48, 56); therefore the secondary infections that occur immediately prior
to harvest should be emphasized in disease management strategies (47, 48, 56). Losses
during shipment and at market can be great, as the ftmgus spreads rapidly when fruit is
removed from cold storage (1). Additionally, avoiding damage to the fiuit is an
important factor in minimizing disease (3 7), and there is evidence that hot water
treatments can decrease postharvest fruit infection (34). Removal of inoculum sources
such as mummies is recommended but not practical for cherry orchards. Fungicides
(commonly sterol demethylation inhibitors) are heavily relied upon for control (78).
Resistance of M. fiucticola to DMI fungicides has been reported in stone fruit production
outside of Michigan (12, 20, 67, 81).

Armillaria Root Rot

Armillaria root rot (ARR) is caused by fungi in the genus Armillaria, including;
A. gallica (Marxmfiller and Romagnesi), A. mellea (V ahl: Fr.) Kummer, A. ostoyae
(Romagnesi) Herink, and A. bulbosa (Barla, Kile and Watling) (37). These fungi are also
pathogenic to many forest and ornamental species (79). When land is cleared for cherry
production, Armillaria spp. may already be present in the soil, colonizing debris left over

from previously infected forest trees. The situation is exacerbated by the fact that the

Armillaria species appear able to persist saprophytically on woody debris for many years
and populations are not eliminated by fumigation. Trees on sandy, well drained soils are
more susceptible to ARR, often showing reduced growth of the terminal shoots. Leaves
on infected trees may change color earlier in the season and fail to abscise normally in the
fall (3 7). Tree mortality typically spreads in a circular pattern, emanating fi'om the initial
infection site. Armillaria root rot is diagnosed by removing soil from the base of a
suspected tree and looking for a thick, white, mycelial fan between under the bark and
woody xylem. Black rhizomorphs (root-like structures) and honey-colored mushrooms,
which form in autumn around the base of recently killed trees, are also signs of
Armillaria spp. Infrequently, the disease may also be introduced through contaminated
soil or equipment (37).

On Mazzard rootstock, sweet cherry cultivars are more resistant than tart cherry.
Currently there is no known control for Armillaria spp., except planting on sites where
infection has not occurred (3 7). The acreage of useable land for cherry production will
continue to diminish until an effective treatment for Armillaria spp. is found. Apple
rootstocks are considered moderately susceptible to armillaria root rot.

European Brown Rot

European brown rot is caused by the ascomycete Monilz'm'a laxa (Aderhold and
Ruhland), and infects tart cherry, specifically the cultivars Meteor, English Morello and
Balaton. The most common sweet cherry cultivars and Montrnorency tart cherry are
reasonably resistant to M. laxa, rarely requiring fungicide use, except during years with
cool, wet spring weather. M. laxa damages blossoms and spurs, with a wetting period of

only one day required for severe blossom infection. Newly infected blossoms turn brown

and the frmgus sporulates on withered tissue. Leaves at the base of infected blossoms
may also be killed; systemic infection of the spurs quickly follows. The systemic
infection causes the formation of cankers (2.5-7.6 cm long) at the base of the spur. The
cankers, as well as blossom debris and dead spurs, produce conidia during subsequent
seasons. Fruit infections are rare and require direct contact with infected blossoms.
Decreasing leaf drying time, and the application of DMI fungicides can help reduce
disease development in susceptible varieties (3 7).
Powdery Mildew

Powdery mildew of cherry is caused by the ascomycete Podosphaera clandestina
(Wall. Fr) Lev, an obligate parasitic. The infection first appears as circular, felt-like
patches of mycelium on the leaf surface and can spread rapidly. Severely infected leaves
become brittle and bunch up. Fruit infections manifest as red shiny blotches, often with
mycelium and spores in the center. Conidia are spread via wind and reinfect new leaves
during the repeating, secondary infection cycle. As the season progresses, cleistothecia
(dark, spherical fruiting bodies) are produced. The fungus overwinters as cleistothecia on
fallen leaves which then initiate the primary infection the following spring when
ascospores are released. The disease commonly reduces the growth of tart cherry trees in
nurseries and young orchards. Fungicide application is the primary method used to
control powdery mildew. Additional cultural practices such as pruning and the removal

of hedges may aid in creating a less favorable environment for disease development (3 7).

THE HISTORY OF FUN GICIDES

Since the first European settlers reached North America, tree fruit have been an
important commodity. Losses due to disease were an issue from the beginning and only
increased in severity with the development of high density agriculture (10). By the early
1900’s, relief arrived from Europe after grape growers in France began using Bordeaux
mixture, lime, and sulfur to control fimgal diseases. These chemistries were phytotoxic
and marginally effective at controlling tree fruit diseases (70); however, these mixtures
were the only known control for fungal pathogens. In Michigan, these fungicides became
important in controlling fimgal pathogens in newly established tart cherry orchards. A
1937 University of Wisconsin research bulletin recommended using Bordeaux mixture
and lime sulfur (a mixture of calcium polysulfides) to control CLS (39).

When discussing the history of fungicide development, it is important to note the
evolution of fungicide modes of action, from multi-site to single-site; as well as the
associated resistance issues. An overview of the history of important fruit fungicides,
their modes of action and resistance histories is shown in Table 1. Copper was the first
effective fungicide and is still considered valuable today (70). Broad spectrum
protectants, such as dithiocarbamates (mancozeb), were patented as early as 1934 (64).
During the 1950’s, captan and dodine were introduced and had multi-site modes of action
that affected the critical processes of the fungi.

In the late 19603, new site-specific fungicides were introduced and widely
adopted by growers. The mechanism of action in single-site fimgicides involves the

disruption of a single metabolic pathway or structural site of an enzyme. These

Table 1. List of fungicide, or chemistry introductions relevant to tree fruit production, the
associated specificity, and resistant pathogens.

 

 

Resistant
Chemistry Pathogens
Date Group or Target Risk of (date
introduced Fungicide Site Specificity Resistance" published)
1900 Copper Multi-site Low N/A"
. . Botrytis
1952 Captan Multr-snte Low ci n 9 re a
. . . V. inaequalis
1959 Dodine MultI-srte Low (1968)
1966 Chlorothalonil Multi-site Low N/A"
V. inaequalis,
. . . . . M. fructicola
1972 Benzrmldazole Single-Site High and B.jaapii
(1975-80)
. . . . . M. fructicola
1974 charbOXImIde Multr-srte Low (1979)
Sterol V. inaequalis
1986 demethylation Single-site High (1989) and 8.
inhibitors jaapii (2006)
. . . . . P. viticola
1996 Strobilunns Single-sue ngh (2002)

 

* Risk of resistance as determined by the Fungicide Resistance Action Committee. Table
modified from Sutton, 1996 and Russell, 1995. *N/A means no known resistant fungal

pathogens.

fungicides also often possessed post-infection activity, meaning that the fungicide could
be absorbed by plant tissue and kill the fungi that had already invaded. The expanded use
of single-site fungicides in the 1970’s resulted in the increased appearance of resistant
pathogen populations (62).

Contemporary public opinion is forcing many regulatory agencies to eliminate
effective fimgicides from the market and pushing the agrochernical industry toward
discovering new and safer modes of action. The demand for new fungicides with low
toxicity to non-target organisms is increasing as heightened concern over agricultural
inputs increase and more stringent regulatory rules are enforced. As of the mid 1990’s,
an estimated $570 million was spent annually on research and development by leading
agrochernical companies (52). Novel modes of action are also important in combating
pathogen resistance and reduced sensitivity in existing chemistries. Specialty crops such
as cherry are often overlooked when new ftmgicides are introduced to the market. Many
specialty crops are only added to the list of approved crops long after introduction of the

firngicide.

FUNGICIDE RESISTANCE IN FRUIT PATHOGENS

Due to the lack of innate resistance to pathogenic fungi in tart cherry cultivars and
sweet cherry cultivars; cultural practices and sanitation are frequently helpful in
controlling fungal diseases. Fungicides are ultimately are required to maximize yield and
quality. The intensive use of site-specific fungicides is particularly risky in terms of
resistance development. Intensive use provides a potent selective pressure that increases

the frequency of fungicide-resistant isolates in a pathogen population (62). As the

10

frequency of resistant isolates increases, a resistant subpopulation may develop. This
subpopulation can increase over time and lead to a reduced level of disease control in that
population (40). This is commonly referred to as practical field resistance, and is defined
as the point at which the fi'equency and levels of resistance are great enough to limit the
effectiveness of disease control in the field (62). In the late 1960’s, the first reports of
sporadic fungicide resistance were documented in Europe (24). In 1969, reduced
sensitivity of Venturia inaequalz’s (Cke.) Wint., the causal agent of apple scab, to dodine
was reported in New York (71). This report was the first documented case of field
resistance to any fungicide used in fruit production in the United States. By the late
1980’s, over 60 resistant fungal genera in hundreds of crop systems had been documented
(69). Currently, all of the systemic fungicide groups (sterol inhibitors, benzimidazoles,
strobilurins, phenylamides, and dicarboximides) have been affected by resistance (69).
Dodine

Dodine was introduced to the US. market in 1959. Dodine, a broad spectrum
fungicide, was used intensively to control important tree fruit diseases such CLS and
apple scab, caused by Venturia inaequalis. Dodine is a guanidine-based, protectant
ftmgicide currently classified as having multi-site activity (21). Ten years after the
introduction of dodine, resistance was reported in V. inaequalis fiom New York (71),
Michigan (36) and EurOpe (53, 80). Resistance appears to be stable (there is no fitness
penalty for resistance) and is present in orchards where Dodine has not been used for 20
years (41). The development of resistance in V. inaequalis populations to dodine suggest
resistance problems may develop in other pathogens. Dodine use for the control of apple

scab and CLS was greatly reduced when DMI fungicides were introduced in 1986. With

11

the recent confirmation of DMI cross resistance in Michigan B. jaapii populations (62)
dodine is needs to be reevaluated to determine the feasibility of widespread use to help
control CLS in Michigan.
Benzimidazole

Benzirnidazole fungicides (e. g. benomyl and thiophanate) are locally systemic,
single-site fungicides with a high potential for resistance development (73). After the
introduction of benzimidazole in 1972, the scientific community thought that fiingicide
resistance would not affect the fi'uit industry, and were optimistic about the use of
benomyl to control M. fi‘ucticola (46). In the early 1970’s, many pathogens, on a diverse
range of crops, rapidly developed resistance to benzimidazoles. The first case of
benzimidazole resistance developed in the peanut leaf spot fungus, Cercospora
arachidicola (Mulder and Holliday), in the southeastern US. (13). Within three years of
the 1972 registration for use on stone fi'uit, and 1973 registration for use on pome fruit,
benomyl-tolerant isolates of M. fructicola and V. inaequalis were found in Michigan (35,
36). Benzimidazole-resistant phenotypes of V. inaequalis are fit and have continued to
persist in the field; consequently benzimidazoles are rarely used in Michigan (35). New
technological advances have been made in testing for fungicide resistance, and PCR
based assays are now available for detecting benzimidazole resistance in M. fructicola
from California stone fruit (49).
Dicarboximides

Dicarboximides (iprodione or vinclozolin) were introduced to the market in 1974
(43) and were used globally. Dicarboximides are locally systemic and have a multi-site

mode of action (73). Dicarboximide resistance first appeared in Botrytis cinerea (Pers:

12

Fr) on the European grape crop (33). Reduced sensitivity also developed in M. fi-ucticola
isolates (58, 63, 72), and M. [am (38). Lab and field studies were conducted to assess the
general risk of resistance to dicarboximides. The results indicated that resistant isolates
were easily selected in vitro, but development was slower in the field (17, 45). In the
absence of dicarboximide, resistant strains were generally less fit than their wild-type
counterparts and returned at a very low level in the in vitro population (51).
Sterol Demethylation-Inhibiting Fungicides

Sterol demethylation inhibiting fungicides (e. g fenbuconazole, tebuconazole, or
propiconazole) are single-site, locally systemic, and are considered at high risk for
resistance development (73). DMI fungicides inhibit the sterol C-14 alpha-demethylation
of 24-methylenehydrolanosterol, a precursor of ergosterol, a critical fungal cell
membrane component (7). When first introduced to the market, DMI fungicides were
effective and contributed greatly to the relatively new management strategies of
integrated pest management programs (46). DMI fungicides have become essential in the
management of fimgal pathogens in fruit production (42) and have been used by
commercial fruit growers since the mid to late 1980’s. DMI fungicides can work as a
protectant, but are also effective after infection occurs (41), allowmg for some flexibility
in spray timing if an application is missed. In vitro testing proved early on that resistance
and cross resistance could be readily induced in Aspergillus nidulans (Eidarn) G. Wint,
(14). Resistance of V. inequalis to DMI fimgicides has been reported in experimental
apple orchards (6, 30, 68). As of 2004, statewide cross-resistance of B. jaapii isolates to
four DMI fungicides was reported in all the commercial cherry producing regions in

Michigan (62). Cherry pathogens such as M. fi-ucticola should be evaluated for DMI

13

sensitivity to aid in preventing fungicide resistance development. Monitoring pathogen
sensitivity now may help develop spray recommendations in subsequent seasons and
detect a significant change in DMI sensitivity that would lead to practical field resistance.

Resistance to DMI fungicides appears to be due to the combined effects of many
incremental genetic changes (32, 65, 75). These small genetic differences cumulatively
cause a reduction in fimgicide sensitivity, and decrease the efficacy of DMI fungicides in
the field (8). The slow development of DMI resistance makes it difficult to monitor for
practical field resistance. The loss of efficacy is generally not complete, and can
sometimes be overcome by shortening the spray interval or increasing the spray rate.
(69); not always feasible options due to application guidelines and cost.
Strobilurins

Strobilurin fungicides were introduced on 1996 and are important to the
management of many pant pathogens, and account for $620 million in frmgicide sales
during 1999, or over 10% of the global fungicide market (60). Strobilurin frmgicides act
as both a contact and translaminar fungicide, with a single-site mode of action (73).
Strobilurin fungicides are based on a substance produced by wood-rotting fungi fiom the
subphylum Basidiomycotina. Strobilurins inhibit mitochondrial respiration by binding to
the Q0 site, blocking electron transfer and subsequently disrupting the production of ATP
(5).

Azoxystrobin (synthetic strobilurin) is a broad—spectrum fungicide and oomycete
pesticide that controls organisms from four major taxonomic groups of plany pathogens;
Ascomycota, Basidiomycota, Zygomycota and Oomycota (25, 28). Strobilurin

fungicides strongly inhibit spore germination and have both preventative and curative

l4

properties. Strobilurins are particularly effective against P. clandestina and V. inaequalis
in tree fruit (2, 9).

Possible modes of resistance were recognized before the release of the product,
but the speed at which resistance developed was not expected. In 2002 Heaney reported
that Plasmopara viticola (Berk. and MA. Curtis) Berl & De Toni in Sacc., on grape was
resistant to strobilurins (29). The group is now considered high risk, and when used strict

resistance management strategies are recommended (8).

FUNICIDE RESISTANCE MECHANISMS

There are numerous known fungicide resistance mechanisms including: (i)
alteration of the biochemical target site so that it is no longer sensitive; (ii) increased
production of the target protein; (iii) development of an alternative metabolic pathway
that bypasses the target site; (iv) metabolic breakdown of the fungicide; and (v) exclusion
or expulsion of the fungicide through ATP-dependent transporter proteins (8).

The most common resistance mechanism appears to be an alteration of the
biochemical target site of the fungicide (8). This could explain why many of the earlier,
multi-site products have not encountered resistance problems. Once they have penetrated
the fimgal cell, the earlier frmgicides act as general enzyme inhibitors, affecting many
target sites. All of these target sites in the fungus would have to change concurrently in
order to stop the fungicide from working. The chances of the numerous, needed genetic
changes happening at the same time are slight, and would likely cause the organism to

become avirulent if not completely nonviable (8).

15

In contrast to the earlier, multi-site fungicides, modern firngicides act on single
target sites and are referred to as “single—site” fungicides. DMI fimgicides have a single-
site mode of action. A single gene mutation can cause an alteration of the target site,
resulting in decreased sensitivity to the fungicide, making modern fungicides much more
susceptible to resistance development. The rapid advances over the past ten years of
PCR-based diagnostic methods for detection of point mutations has aided in the
identification of resistance mechanisms, especially those with changes in the target site
(8).

In the case of DMI fungicides, three major mechanisms for resistance have been
described, including: mutations in the target enzyme, 14 alpha-demethylase (C YP5 1),
leading to a decreased affinity of DMI fungicides to the target protein (3, 15, 16);
overexpression of the CYP51 gene leading to increased production of the target enzyme
(26, 66); and overexpression of the ATP-binding cassette (27, 82). Recently published
data has shown that the overexpression of the CYP51 gene in B. jaapii mediates DMI
resistance (50). A PCR detection test that screens for the presence of a promoter

associated with the overexpression of the CYP51 gene was also developed (50).

MANAGMENT TO DELAY FUNGICIDE RESISTANCE

To increase the time a fungicide can effectively control a given pathogen
management strategies should minimize the risk of resistance development. New
cherrristries need to be carefully analyzed before widespread use on a particular pathogen
in a given agricultural system (69). Thoroughly scrutinizing the interaction of a pathogen

and fungicide before widespread use can make withdrawal from the market less likely by

16

anticipating possible issues (69). Predicting the risk of fungicide is difficult (69) and
predictions are often unreliable. In the case of dicarboxirnides resistance developed more
slowly than expected (44) and DMI fungicides developed resistance despite predictions
that it was at low risk for pathogen resistance (22). Monitoring the response of a
pathogen population to repeated fungicide exposures is the only way aspects of pathogen
fitness, such as the disproportionate survival of resistant isolates under frmgicides
pressure, are sufficiently taken into account (69).

Preserving earlier fungicides for use on plant pathogens is very important to
mitigating the development of fungicide resistance to new chemistries. Preserving earlier
fungicides provides a greater number of rotation partners for use within the season. With
bans on certain residual partners, the selection of alternative fungicides is becoming
insufficient for many pathogens (4). Abandoning conventional, protectant fungicides
would therefore be in poor judgment and new fungicide groups are needed (69).
Management strategies such as tank mixing fungicides and rotating the mode of action
have successfully delayed or contained resistance in the past (13, 69), and is a current
resistance management strategy used today. Additionally, resistance management could
be potentiated by collecting baseline fungicide sensitivity data in populations of plant
pathogens of particular commodities. If pathogen populations were monitored for small
changes in sensitivity levels, preemptive management strategies could be adopted to

avoid the loss of efficacy (69).

17

SUMMARY

Resistance to DMI fungicides has recently been confirmed in B. jaapii
populations in Michigan (62). With the loss in efficacy of DMI fungicides, it has become
important to determine the current sensitivity status of B. jaapii isolates to dodine, which
is a probable and available alternative. Establishing baseline sensitivity levels may aid in
predicting practical field resistance, and assist in evaluating the value of the firngicide
resistance management strategies being employed.

DMI fungicides are currently the most frequently utilized fungicides for
controlling M. fiuctz’cola in Michigan. Resistance to DMI fungicides is well documented
in various fimgal pathogens of fi'uit, including M. fructicola populations in stone fruit
orchards from New York and Georgia (20, 72, 81). Fenbuconazole (a DMI fiingicide) is
being applied extensively in Michigan cherry orchards, and because of the single-site
mode of action, is at risk for resistance development. Determining the status of M.
fructicola sensitivity to fenbuconazole in Michigan is an important facet of managing
firngicide resistance. Tracking changes in fungicide sensitivity over time can aid in
predicting practical field resistance, and assist in evaluating the value of current fungicide

resistance management strategies.

18

10.

ll.

12.

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25

CHAPTER 1

SENSITIVITY OF BLUMERIELLA JAAPII TO DODINE

INTRODUCTION

Cherry leaf spot (CLS), caused by the ascomycete Blumeriellajaapii (Rehm) Arx,
is the most damaging fungal pathogen of tart cherry trees (Prunus cerasus L.). After a
severe outbreak in 1993, thousands of tart cherries died during the following winter (11).
Cherry leaf spot also affects sweet cherry (P. avium L.) production. The impact on the
Michigan cherry industry is exacerbated by the fact that the Montrnorency cultivar, which
constitutes more than 90% of tart cherry production in Michigan, is highly susceptible to
CLS. With Michigan producing upwards of 80% of the national tart cherry crop (19),
losses due to CLS not only affect Michigan, but the national tart cherry market as well.

Cherry leaf spot causes the formation of small, purple lesions on the leaf that
reduces the photosynthetic ability of the tree. As leaves accumulate CLS lesions, they
turn yellow and on tart cherry trees leaves prematurely defoliate. When tart cherry trees
become severely defoliated before harvest, fruit is soft and immature, has low soluble
solids, and ripens unevenly (11). During seasons with high disease pressure CLS can
cause defoliation as early as midsummer. In the years following a severe outbreak with
early defoliation, bud formation and fruit set is significantly reduced (8). Trees
defoliated extremely early (J uly-August) are also less able to cope with cold winters and
may be killed by freezing temperatures.

All commercially cultivated cherry cultivars are susceptible to cherry leaf spot,

(11) most notably Montrnorency. Currently, management of CLS relies heavily on

26

fungicides and requires five to seven applications per year, depending on disease pressure
(18). The extensive use of sterol demethylation inhibitor (DMI) fungicides has led to
newly documented resistance in B. jaapii populations (20), and has increased the need for
alternative fungicides to control CLS, such as dodine.

Although the mode of action of dodine is unknown, it was an effective and widely
utilized asset for controlling CLS before the introduction of DlVII fungicides. After the
introduction of DMI fungicides, the use of dodine was greatly reduced in Michigan.

With DMI resistance in Michigan B. jaapii populations (20) new options for CLS control
are needed. Dodine should be reevaluated for potential widespread application to control
CLS in Michigan. Baseline sensitivity levels of B. jaapii need to be established to aid in
monitoring B. jaapii for the development of dodine resistance under more widespread
use.

Dodine was introduced to the US. fungicide market in 1959 and was used
intensively in northeastern North America to control economically important pathogens
of cherry and apple, particularly Venturia inequalis, the causal agent of apple scab (5).
Dodine is a guanidine-based, protectant fungicide classified as having multi-site activity
(4). Dodine also has post-infection properties. Generally, multi-site fungicides are
thought to have a lower probability of developing resistance issues than single-site
fungicides (1). Multi-site fungicides have broad fimgicidal activity and target metabolic
pathways or processes; versus a single enzyme as is the case in single-site fungicides.
Despite expectations of low risk, resistance to dodine was found in Michigan V. inequalis
isolates in 1981 (9), and then internationally shortly thereafter (17). Resistance in V.

inaequalis appears to be genetically stable and has been documented in orchard

27

populations from sites where dodine has not been used for 20 years (14). The potential
use of dodine for the control of CLS and the risk of resistance development in B. jaapii
populations, needed to be carefully evaluated before it is recommended.

DMI fimgicides have been a major part of most management strategies in
commercial cherry orchards in Michigan. Dodine may become a more popular control
method for CLS because of the development of resistance in B. jaapii populations to
DMI fungicides (20). Monitoring pathogen populations for changes in fungicide
sensitivity can aid in detecting the early stages of resistance, before it becomes severe
enough to cause practical field resistance (2). Determining the sensitivity of B. jaapii
populations in Michigan now will contribute to monitoring dodine for resistance
development in subsequent seasons by allowing for comparison of current sensitivity
levels to future measurements. The objectives of this study were to determine the efficacy
of dodine in controlling CLS in Michigan orchards, and determine the in vitro sensitivity

of B. jaapii populations to dodine.

MATERIALS & METHODS

Dodine field trials

During the 2005 and 2006 growing seasons experiments to determine the field efficacy of
dodine for the control of CLS were conducted at the Michigan State University
Northwest Horticultural Research Station (NWMHRS) located in Leelanau County,
Michigan. Fungicide sprays were applied to a block of 12-yr-old Montrnorency and 11-
yr-old Balaton tart cherry trees (0.99 hectares) or a 26-year-old Montrnorency block

(0.56) hectares. Sprays were applied at 2,809 liters of water per hectare, using a custom

28

built research plot sprayer with a JBS model 705 hand gun (John Bean Sprayer Co., AR);
(Figure LA). The experimental design was a randomized complete block with single-tree
plots, replicated four times per treatment. Randomization occurred each year. Twenty
terminal branches from each tree were rated and disease incidence was recorded as the
percentage of leaves infected or defoliated. Fisher's Protected Least Significant
Difference test was used to determine the significance of variability in disease incidence
between treatments.

In addition to dodine (Syllit 65W; Platte Chemical Company, Geeley, CO), other
fungicides commonly used to control CLS were tested for comparison, including a
triflurnizole (Procure 4SC; Uniroyal Chemical Company, Middlebury, CT), boscalid
(Endura 70 WDG; BASF, Germany), trifloxystrobin (Flint; Syngenta Crop Protection,
Switzerland), chlorothanlonil (Bravo Ultrex 82.5 WDG; Syngenta Crop Protection,
Switzerland), and two elemental copper materials (Cuprofix Disperss 40DF; Cerexagri
Incorporated, 402 Wihnington, DE; and Kocide 2000, DuPont, DuPont, WA).

2005 trial

The focus of the 2005 field trial was to compare the efficacy of dodine and copper
firngicides in controlling CLS. These fungicides were integrated into spray programs
based on the resistance management concept of rotating the mode of action within the
season (Table 1.3). Spray dates, respective cherry growth stages and CLS infection
periods were recorded (Table 1.1). From June 1 to 16, rainfall totaled 2.6 centimeters
(Appendix A, Table A1). Cherry leaf spot disease data was not collected until
September 12, after sufficient infection had occurred. Five terminals from each tree

quadrant (20 terminals per tree) were evaluated.

29

 

Figure 1.A. Custom built research plot sprayer with a J BS model 705 hand gun, used to
apply fungicides for efficacy trials at the Northwest Horticultural Research Station.

30

2006 trial

Fungicide rotations were evaluated in a 0.56 hectare block, 27-yr-old
Montrnorency cherry trees at the NWMHRS. The block contained a total of 88 trees.
This experiment examined chemicals commonly recommended for the management of
CLS. Individual fungicides were applied for all applications during the growing season
(5 cover sprays); (Table 1.4). Fungicides were applied with a handheld applicator to run-
off (equivalent to 2,809 liters/hectare). Treatments were arranged in a randomized
complete block using four replications of single-tree plots. Spray dates, respective
growth stages and cherry leaf spot infections were recorded (Table 1.2). There were 10
days (May 9-18) with measurable rainfall totaling 5.77 centimeters. Mid-summer was
dry; June had 7 days with rainfall that totaled 4.22 cm, and July had 8 days of rainfall
totaling 4.09 centimeters (Appendix A, Table A2). CLS disease assessment was
conducted on July 28; a second assessment was performed on September 18. Leaf spot

foliar incidence and defoliation were calculated fi'om observations on 20 shoots per tree.

31

Table 1.1. The 2005 spray dates (in bold), respective
growth stages, and cherry leaf spot infections periods“
as recorded at the Northwest Horticultural Research

 

 

Station.

Date Event

9-May Low disease severity

10-May Bloom

19-May Low disease severity

24-May Low disease severity, Shuck Split
3-Jun First cover
8-Jun Low disease severity
13-Jun Second cover
23-Jun Moderate disease severity, Third cover
4-Jul Low disease severity

5-Jul Fourtha and final cover
21-Jul Harvest
24-Ju1 Low disease severity
25-Jul Moderate disease severity
28-Jul Low disease severity
4-Aug Low disease severity
10-Aug Low disease severity
12-Aug Low disease severity
18-Aug High disease severity
12% Disease rating

 

*Cherry leaf spot infection periods as defined by
www.cnviroweather.msu.edu.

32

Table 1.2. The 2006 spray dates (in bold), respective
growth stages, and cherry leaf spot infections periods“
as recorded at the Northwest Horticultural Research

 

 

Station.
Date Event
2-May Low disease severity
10-May Petal fall
1 1-May Low disease severity
12-May Moderate disease severity
19-May Shuck split
25-May High disease severity
28-May Low disease severity
30-May First cover
31-May High disease severity
6-Jun Moderate disease severity
9-Jun Second cover
18-Jun High disease severity
19-Jun Third cover
26-Jun Moderate disease severity
28-Jun Moderate disease severity
l-Jul Moderate disease severity
3-Jul Fourth and final cover
9-Ju1 Moderate disease severity
17-Jul Moderate disease severity
21-Jul Harvest
26-Jul Low disease severity
28—Jul Disease rating
l-Aug High disease severity
9-Aug Low disease severity
l4-Aug Low disease severity
23-Aug Moderate disease severity
24-Aug Low disease severity
26-Aug Moderate disease severity
18-Sep Disease rating

 

*Cherry leaf spot infection periods as defined by
www.cnviroweather.msu.edu.

33

B. jaapii isolate collection

The eight hundred and sixty B. jaapii isolates used in the in vitro dodine assay
were fiom the DMI survey collection of B. jaapii, established by T. Proffer (20). In
2003-2004, Proffer collected B. jaapii isolates from commercial orchards in all four of
the major tart cherry producing areas of Michigan: Traverse City, Benzie, Hart/ Shelby,
and the Southwest region. Proffer also collected isolates fiom a cormnercial orchard in
eastern Michigan; research orchards from Michigan State University; dooryard trees near
Salem, OH; and an abandoned sweet cherry orchard in Berlin Center, OH. Additional B.
jaapii isolates were from black cherry (P. serotina), choke cherry (P. virginiana), pin
cherry (P. pensylvanica), mahaleb (P. mahaleb), and dwarf cherry (P. fiuticosa) trees in
Michigan and Ohio. Cherry leaves were selected at random to be used as the source of
monoconidial isolates. Individual germinated conidia were transferred from 2% water
agar to modified malt extract agar (MMEA) plates and maintained on MMEA (recipe in
Appendix A) slants at 5°C (20).
Determination of in vitro sensitivity of B. jaapii isolates to dodine

Previous in vitro fungicide sensitivity studies have used reduced mycelial grth
to measure differences in sensitivity (6, 15, 16, 22). There are two methods to measure
mycelial response to fungicides; quantitative and qualitative. The quantitative method is
based on the fimgicide concentration at which mycelial growth is reduced 50%, relative
to growth in unarnended medium; this is referred to as the Effective Concentration 50
(ECso). The alternative, qualitative method involves determining the minimum
concentration at which colony growth is completely inhibited, commonly referred to as

the Minimum Inhibitory Concentration (MIC) (6, 15, 16, 22). It is important to note that

34

B. jaapii isolates are very slow growing and monoconidial isolates often take two weeks
or more to produce colonies of only a few millimeters in diameter. Due to slow growth,
the qualitative MIC method was used to measure dodine sensitivity.

The sensitivity of eight hundred and sixty B. jaapii isolates to dodine was tested
during 2006. Dodine was dissolved in 95% ethanol to prepare stock solutions.
Quantities of these stock solutions were added to modified malt extract agar (MMEA)
after it had been autoclaved for sterilization and cooled to 50°C. The concentration of
active ingredient (a.i.) was adjusted to 0.05, 0.1, 0.5, l, 5, 10, 25, 50, 100, 200, and 400
ug/ml. MMEA amended with 125 pl of ethanol (equal to the highest concentration of
ethanol in the treatments) was used as a control. Mycelial plugs, 1.0 mm in diameter,
were transferred to MBA plates amended with the previously mentioned 11 levels of
dodine concentrations and the control; this was replicated three times for each isolate.
After 21 days of incubation at 23 to 25°C, the plates were examined for colony expansion
and the MIC of fungicide for each isolate was recorded (Figure 1.B). The MIC reported
is the concentration at which the growth of at least 2 of 3 isolate replicates was inhibited.
The MIC was defined as no mycelial expansion of B. jaapii from the inoculation plug

onto the amended MMEA agar plate.

35

 

Figure 1.B. Blumeriellajaapii isolates (16) growing on dodine
amended modified malt extract agar to determine the minimum
inhibitory concentration.

36

RESULTS
Dodine efficacy field trials
2005

Treatments utilizing dodine were as effective as those utilizing copper hydroxide
but were also comparable to infection levels in trees not treated with fungicides (Table
1.3). Treatments with copper sulfate were more effective at 2 of the 3 spray rates
screened than any other treatment and disease levels were significantly lower than in the
unsprayed control, most likely due to the long residual action associated with copper
sulfate. No significant defoliation occured. Trees sprayed with four sprays of copper
hydroxide exhibited some darkening between the veins (phytotoxicity) on the underside
of the leaf, but defoliation was not a problem despite of temperatures up to 34 °C in early
August (Appendix A, Table AD.
2006

The 2006 growmg season was dry (Appendix A, Table A2) and slowed the
development of CLS during midsummer, resulting in an unusually long interval between
the last spray on July 3 and the final leaf spot disease rating on September 18. Under
these dry conditions, control of CLS early in the season was outstanding with all products
except triflumizole. The boscalid maintained the best late control of CLS with dodine
and chlorothalonil showing similarly effective potency for disease suppression and
acceptable levels of defoliation (Table 1.4). On the September 18 more than 95% of the
remaining leaves on unsprayed control trees were infected with CLS and more than 90%

had been defoliated.

37

Table 1.3. Incidence of cherry leaf spot infection during the 2005 growing season based on
use of copper and dodine in a resistance management spray regime.

 

Fungicide and ratefir hectare Spray Timing Incidence (% leaves)
Chlorothalonil (3.4 kg) Bloom (5/ 10); Shuck split (5/24)
Trifloxystrobin (183.4 ml) First cover (6/3)
Dodine (2.3 kg) Second (6/13); Third cover (6/23)
Tebuconazole (440.6 ml) Fourth cover (7/5) 55.1 bcd
Chlorothalonil (3 .4 kg) Bloom (5/10); Shuck split (5/24)
Trifloxystrobin (183.4 ml) First cover (6/3)
Copper sulfate (4.0 kg) Second (6/ 13); Third cover (6/23)
Tebuconazole (440.6 ml) Fourth cover (7/5) 33.5 ef
Chlorothalonil (3.4 kg) Bloom (5/10); Shuck split (5/24)
Trifloxystrobin (183.4 ml) First cover (6/3)
Copper sulfate (2.0 kg) Second (6/ 13); Third cover (6/23)
Tebuconazole (440.6 ml) Fourth cover (7/5) 55.9 bc
Chlorothalonil (3.4 kg) Bloom (5/ 10); Shuck split (5/24)
Trifloxystrobin (183.4 ml) First cover (6/3)
Copper sulfate (1 kg) Second (6/ 13); Third cover (6/23)
Tebuconazole (440.6 ml) Fourth cover (7/5) 34.1 ef
Chlorothalonil (3.4 kg) Bloom (5/10); Shuck split (5/24)
Trifloxystrobin (183.4 ml) First cover (6/3)
Copper hydroxide (2.3 kg) Second (6/13); Third cover (6/23)
Tebuconazole (440.6 ml) Fourth cover (7/5) 50.9 bcd
Chlorothalonil (3 .4 kg) Bloom (5/ 10); Shuck split (5/24)
Trifloxystrobin (183.4 ml) First cover (6/3)
Copper hydroxide (4.5 kg) Second (6/ 13); Third cover (6/23)
Tebuconazole (440.6 ml) Fourth cover (7/5) 53.6 bcd
Untreated control 64.5 ab

 

* Means followed by the same letter are not significantly different according to Fisher’s
Protected LSD (a = 0.05).

38

Table 1.4. Cherry leaf spot incidence during the 2006
growing season based on season long applications of

fungicides commonly used for control.

 

 

 

Infected leaves Defoliation

Treatment and product per (%) (°/0)
hectare 28-Jul l8-Sep 18-Sep
Trifloxystrobin (219.6 ml) 2.9 b 97.7 a 49.1 c
Triflumizole (585.6 ml) 54.1 a 100.0 a 90.5 a
Triflumizole (1171.2 ml) 30.7 c 100.0 a 78.5 b

Boscalid (585.6 ml) 3.9 b 16.0 b 3.4 d
Chlorothalonil (3.2 kg) 0.7 b 79.7 c 11.3 d
Dodine (1976.4 ml) 0.7 b 75.9 c 32.6 e
Untreated control 61.5 a 95.1 a 90.9 a

 

39

In vitro sensitivity of B. jaapii isolates to dodine

The in vitro MIC of the 860 B. jaapii isolates ranged from 0-400 ppm dodine
(Figure 1.C). The overall population profile formed a bell shaped curve, with most of the
isolates having a moderate MIC value (Figure l.C). The difference in MIC values
between isolates from dodine-exposed, and unexposed sources was significant according
to a Chi-square test (P = 0.1). B. jaapii isolates from sites that had likely been exposed to
dodine required a higher dosage of dodine to inhibit mycelial growth than isolates fi'om
sites with limited or no exposure to dodine (Figure 1D).

Additionally, one set of isolates (AH), were collected from an orchard that was
abandoned in the early 1980’s. The source of the AH isolates was probably only exposed
to dodine for 30 years, versus the more than 50 years of possible exposure in
conventional orchards. The isolates from the abandoned orchard had intermediate MIC
values when compared with those from exposed and unexposed sites (Figure 1D).

Greater than 69% of the isolates fiom sources not exposed to dodine were
controlled at a dodine concentration of less than or equal to 10 ppm. Conversely, almost
20% of B. jaapii isolates fi'om sources exposed to dodine required greater than 100 ppm
of dodine to inhibit mycelial expansion. Figure 1D illustrates a clear the shift in the bell-
shaped, population profiles. Respective curves move fi'om lower MIC values in B. jaapii
isolates from unexposed sources, to the higher MIC values in isolates from sources

exposed to dodine.

4O

 

250.0

N
O
p
O

. 150.0

Frequency of B. jaapii Isolateses
S 8
O O

 

0.0

0 0.05 0.1 0.5 1 5 10 25 50 100 200 400
Dodine MIC (ppm)

 

 

 

Figure 1.C. Distribution of dodine minimum inhibitory concentration values for all
Blumeriellajaapii isolates.

41

 

 

 

 

 

 

Relaive Frequent

 

 

 

 

 

Figure 1.D. Relationship of dodine exposure of the isolate source to in vitro
sensitivity of Blumeriellajaapii isolates, as measured by the minimum inhibitory
concentration. Unexposed isolate sources included dooryard trees, wild cherry, and
black cherry. Exposed isolates were collected fi'om commercial cherry orchards.
The abandoned orchard isolates source has not been exposed to dodine since the
1980s. A Chi-square test was performed and indicated a significant difference
between the exposed and unexposed sources (P = 0.1).

42

DISCUSSION

The management of CLS relies heavily on firngicides and requires five to seven
applications per year, beginning at petal fall and continuing into late summer (18). The
most recent firngicide recommendations include an additional post harvest spray to
reduce initial inoculum the following year. The fungicides used to control B. jaapii
include chlorothalonil, captan, strobilurins and several sterol demethylation inhibitors
(fenbuconazole, tebuconazole, myclobutanil, and fenarimol) (19). More recently dodine
has been recommended (21); dodine use had decreased after the introduction of DMI
fungicides.

Each of the currently recommended frmgicides has potential problems, limiting
use. Chlorothalonil has serious residue concerns because it is carcinogenic (3), as a result
applications are limited to before shuck-split and after harvest. F enbuconazole has been
used extensively because of low cost, season long availability, and action against multiple
pathogenic fungi such as brown rots and powdery mildew (10,12,19). This extensive use
of the DMI fungicides has led to newly documented resistance in B. jaapii populations
(20). Strobilurin fungicides were registered for use on tart cherry in 2002 and have
proven effective in controlling CLS (12, 19). Strobilurins are prone to fungicide
resistance because they have a single-site mode of action, which may be overcome by a
single pathogen mutation (1, 7). Due to the previously listed issues with common
fungicides used to control CLS, it is critical to evaluate all available and effective
treatments. It is also imperative that an integrated program is developed to minimize the

development of fungicide resistance.

43

Spray trial results confirmed that dodine is as effective as many recommended
fungicides currently used to control CLS (Table 1.4). The 2005 growing season was the
driest since weather data recording began at the NWT-IRS in 1982 (Appendix A, Table
A.1). Dry conditions slowed CLS development and disease pressure was low, pushing the
efficacy evaluation date back to September. At harvest (July 21), few leaves were
infected and CLS lesions were hard to find, even on the unsprayed control trees. The
length of time between final spray (July 5) and evaluation (September 12) was unusually
long. The 2005 spray trial suggests that dodine could control CLS as well as most copper
products. This is impressive as copper had been highly effective in the control of CLS in
past seasons (13) and has also been proven effective under current conditions (18). Low
disease pressure during the 2005 growing seasons (Table 1.3) did not allow conclusive
data on the overall efficacy of dodine in the field. The 2006 growing season did produce
significant disease pressure (Table 1.4), due to substantial rainfall in the northwest region
of the state (Appendix A, Table A2). Dodine was shown to suppress CLS as effectively
as representatives fiom many popular fungicides including, strobilurins, boscalids and
chlorothalonil (Table 1.4). Dodine was also effective at reducing defoliation (Table 1.4).
Dodine has proven a viable firngicide for the control of CLS in Michigan cherry orchards,
but additional efficacy trials are needed.

The overall baseline dodine sensitivity data was variable, with in vitro
sensitivities ranging fi'om 0.05 to more than 400 ppm of dodine. Wide variability in
baseline data (8,000-fold) may be an indicator of a higher risk for resistance
development. It is thought that the broader the base-line range is, the greater the

proclivity of the population for subsequent population shifts towards lower levels of

sensitivity under fungicide selection pressure (1). The in vitro assay also showed a
population shift towards reduced dodine sensitivity in B. jaapii isolates when evaluated
based on their previous exposure to dodine. The population shift (Figure 1D) is a
common precursor of reduced fungicide sensitivity (2). To slow the reduction in B.
jaapii sensitivity to dodine, fungicide resistance management strategies should be
utilized. Rotating fungicide classes within the growing season, and tank mixing with an
appropriate broad spectrum fungicide, are critical facets of resistance management (1).
Further tracking of the sensitivity to dodine in Michigan B. jaapii populations will be
critical in determining the long term use of dodine, and in amending firture

recommendations based on subsequent findings.

45

LITERATURE CITED

. Brent, K. J. 1995. Fungicide resistance in crop pathogens: How can it be managed?
FRAC Monograph No. 1, GIFAP. Brussels. <www.fi‘ac.info/frac/index.htm>.

. Brent, K.J., and Hollomon, D.W. 2007. Fungicide Resistance, the Assessment of
Risk, Monograph 2. Accessed October 2007. <http://www.fi'ac.info/frac/index.htm>.

. Environmental Protection Agency. Federal Register: March 12, 2001. Federal
registration, rules and Regulations, Volume 66, Number 48. Page 14330-342.
Accessed October 2007. <wais.access.gpo.gov>.

. Fungicide Resistance Action Committee. 2006. Fungicide Code List 2. Accessed
September 2007. <http://www.frac.info/fi‘ac/index.htm>.

. Gilpatrick, J. D. 1982. Case study 2: Venturia on pome fi'uits and Monilinia on stone
fi'uits. Pages 195-206 in: Fungicide Resistance in Crop Protection. J. Dekker and S.
G. Georgopoulos. Centre for Agricultural Publishing and Documentation,
Wageningen.

. Golembiewski, R.C., Vargas, J .M., Jones, A.L., and Detweiler, AR. 1995. Detection
of demethylation inhibitor resistance in Sclerotinia homoeocarpa populations. Plant
Disease 79:491-93.

. Heaney, S.P., Hall, A.A., Davies, SA, and Olaya, G. 2000. Resistance to fungicides
in the QoI—STAR cross-resistance group: current perspectives. Plant Disease 2:755-
62.

. Howell, GS, and Stackhouse, SS. 1973. The effect of defoliation time on
acclimation and dehardening in tart cherry (Prunus cerasus L.). Journal of the
American Society for Horticultural Science 98: 132-36.

. Jones, A. L. 1981. Fungicide resistance: Past experience with benomyl and dodine
and future concerns with sterol inhibitors. Plant Disease 65:990-92.

10. Jones, A. L., Ehret, G. R., Garcia, S. M., Kesner, C. D., and Klein, W. M. 1993.

Control of cherry leaf spot and powdery mildew on sour with alternate-side
applications of fenarimol, myclobutanil, and tebuconazole. Plant Disease 77:703-06.

11. Jones, A. L., and Sutton, TE. 1996. Diseases of tree fruits in the East. NCR

Publication 45. 95 pp. Michigan State University Extension, East Lansing, MI.

46

12. Jones, A.L., and Ehret, GR. 2000. Evaluation of strobilurin firngicides for cherry leaf
spot and brown rot control F ungicide and Nematicide Tests 55:53. The American
Phytopathological Society, St. Paul, MN.

13. Keitt, G.W. 1937. The epidemiology and control of cherry leaf spot. Wisconsin
Agricultural Experiment Station Research Bulletin 132.

14. Kdller, W. 1994. Chemical control of apple scab — status quo and future. Norwegian
Journal of Agricultural Science 17: 149—70.

15. Keller, W., Wilcox, W. F ., Barnard, J ., Jones, A. L., and Braun, P. G. 1997. Detection
and quantification of resistance of Venturia inaequalis populations to sterol
demethylation inhibitors. Phytopathology 87: 1 84-90.

16. Ma, Z., Morgan, P., Felts, D., and Michailides, T.J. 2002. Sensitivity of
Botryosphaeria dothidea from California pistachio to tebuconazole. Crop Protection
21 :829-835.

17. McKay, M. C. R., and MacNeill, B. H. 1979. Spectrum of sensitivity to dodine in
field populations of Venturia inaequalis. Canadian Journal Plant Pathology. 1:76-78.

18. McManus, P.S., Proffer, T.J., Berardi, R., Gruber, B.R., Nugent, J .E., Ehret, G.R.,
Ma, Z., and Sundin, G. W. 2007. Integration of copper-based and reduced-risk

fungicides for control of Blumeriellajaapii on sour cherry. Plant Disease 91 :294-
300.

19. McManus, P. S., Stasiak, M., Schauske, B., and Weidman, R. 2003. Evaluation of
fungicides for control of sour cherry diseases in Wisconsin, 2002. Fungicide and
Nematicide Tests Report 56:STF1. DOI: 10.1094/FN56. The American
Phytopathological Society, St. Paul, MN.

20. Proffer, T.J., Berardi, R., Ma, Z., Nugent, J .E., Ehret, G.R., McManus, P.S, Jones,
A.L., and Sundin, G.W. 2006. Occurrence, distribution, and polymerase chain
reaction-based detection of resistance to sterol demethylation inhibitor fungicides in
populations of Blumeriellajaapii in Michigan. Phytopathology 96:709-717.

21. Rothwell, N., and Sundin, G. 2006. Integrated Pest Management Resources. Fruit
Crop Advisory Team Alert Newsletter, Vol. 21, No. 6.

22. Stanis V.F., and Jones AL. 1985. Reduced sensitivity to sterol-inhibiting fungicides
in field isolates of Venturia inaequalis. Phytopathology 75:109-101.

23. US. Department of Agriculture. 2006 Statistics. National Agricultural Statistics
Service. Accessed Oct. 2007. <http://www.nass.usda.gov>.

47

24. Yoder, KS, and Klos, E.J. 1976. Tolerance to dodine in Venturia inaequalis.
Phytopathology 66:918-923.

48

CHAPTER 2

SENSITIVITY OF MONILINA FRUCTICOLA TO FENBUCONAZOLE

INTRODUCTION

Monilinia species cause a wide range of stone fruit diseases worldwide, including
fruit rots and blossom blight. Only M. laxa and M. fi-ucticola are currently found in the
United States (11). Moniliniafiucticola, is the causal agent of American brown rot
(ABR), and is an important pathogen on cherry, particularly varieties of sweet cherry.
ABR also affects apricot, peach, nectarine and plum fruit. ABR reduces cherry yield by
causing fruit rot before and after harvest. Direct infection through the fruit cuticle occurs
during a 2-3 day period of warm, wet and humid weather. The entire secondary infection
cycle takes only three days to complete, allowing for epidemic outbreaks in a matter of
days. The fungus attacks fruit, blossoms, spurs, and shoots, with infection occurring
more readily on mature fi'uit as the season progresses. The most distinctive sign of the
disease is the gray/tan conidia that appear on the surface of the fi'uit. Infected fi'uit may
rot and abscise or persist through the winter in a mummified form. Mumrrrified cherry
fruit provide the primary source of conidia that act as the primary inoculum in the spring.
In rare cases M. fructicola form apothecia (sexual fi'uiting structures) on the orchard
floor, producing ascospores in the spring. (13).

Sterol demethylation inhibitor frmgicides (DMI fungicides) are currently the most
common chemistries used to control ABR. Cultural practices such as the removal of
mummies and blighted twigs are recommended to reduce inoculums sources, but are not

feasible in commercial orchards. Avoiding damage to the fruit is critical to minimizing

49

disease. Cultivars currently in production have exhibited no resistance to the pathogen
and ftmgicides are the primary strategy for disease control (13).

Prior to 1970, most fungicides had multi-site modes of action, and interfered with
multiple metabolic pathways, inhibited spore germination, and acted as protectants. The
development of resistance to these fungicides was rare, but problems with toxicity to non-
target organism, such as insects and humans, were considerable. Conversely, in the late
1960’s, DMI fungicides were introduced and disrupted only a single metabolic process or
structural site in the fimgus (21). The use of DMI fungicides has typically involved
multiple applications during a growing season, and provides conditions for intense
selection of fungicide resistant isolates. Genetic mutations are the most prevalent cause
of firngicide resistance in fungi and resistant isolates exist at very low fi'equencies within
most pathogen populations (3). When fungicide pressure is present, as is the case in
conventionally managed cherry orchards, isolates with fungicide resistance are selected.
The population size of resistant isolates may then increase disproportionately to the
normal population structure, when compared to population structure in the absence of
fimgicides. The increase in less sensitive individuals may ultimately lead to a lack of
disease control in the field, or practical field resistance (15).

DMI fungicides specifically inhibit the sterol C-l4 alpha-demethylation of the 24-
methylenehydrolanosterol, a precursor of ergosterol; a critical fungal cell membrane
component (3). Resistance in M. fi-ucticola populations to DMI fimgicides was first
documented in a laboratory study (18). Currently, DMI resistance has been confirmed in
field populations of M. fiucticola in peach orchards (27), specifically in Georgia (23) and

New York (19). Resistance of V. inequalis isolates to DMI fungicides in experimental

50

apple orchards has also been reported (2, 10, 24). In 2004, cross-resistance of B. jaapii
isolates to four DMI fungicide groups was reported in Michigan’s commercial cherry
orchards (21). Three major mechanisms for resistance to DMI fungicides have been
previously described, including: mutations in the target enzyme, 14 alpha-demethylase
(CYP51), that leads to a decreased affinity of DMI fungicides to the target protein (1, 5,
6); overexpression of the CYP51 gene leading to increased production of the target
enzyme (8, 17, 22); and overexpression of the ATP-binding cassette (9, 28).

The objectives of this study were to: (i) determine the current sensitivity levels of
Michigan M. fiucticola populations to fenbuconazole (a DMI commonly used in
Michigan cherry orchards); (ii) evaluate the significance of these sensitivity levels with
regard to disease control; and (iii) establish whether the most and least sensitive isolates

showed significant genetic variations in the CYP51 gene.

MATERIALS AND METHODS
Monilinia fiucticola isolate collection

Stone fi'uit infected with M. fi-ucticola were randomly collected fi'om 24
commercial cherry or peach orchards from around Michigan, including Grand Traverse,
Leelanau, Benzie, Berrien, Montrnorency, and Oceana County. Infected cherries were
pressed gently on the surface of a water agar plate, transferring conidia fi'om the fruit’s
surface to the agar plate. These conidia were then spread with a sterile glass rod. Water
agar plates were used to reduce bacterial growth. Conidia were incubated for 24 hours at

temperatures between 23 and 25 °C, and single conidial isolates were transferred to potato

51

dextrose agar (Appendix B, Table B. l). Isolates were maintained on potato dextrose agar
(PDA), between 23 and 25°C.
PCR-based species confirmation

Monilinia laxa and M. fructicola are both found in Michigan cherry orchards and
have similar morphology so PCR was used to confirm that the isolates tested for DMI
sensitivity were M. fructicola. Tissue for PCR analysis was prepared using a bead-beater
centrifuge to disrupt the fungal cell membrane. The protocol section of Appendix B
describes the bead-beater procedure. Actively grong mycelium (0.1 g) was collected
fiom two randomly selected representative isolates fiom each sampling location (Table
2.1). The ITSlecl and ITS4Mfc1 primers were used (12). A Qiagen, DNeasy Plant
Mini Kit was used for DNA extraction; the mini protocol was followed. Electropheresis
was then used to confirm the amplification of the ITS region. M. laxa primers (ITSMlx
and ITS4Mlx) were also used to exclude cross-amplification as a possibility (12). The
thermocycler pro gram and template preparation are described in the protocol section of
Appendix B. The PCR products were run on a 1% agarose gel in 1x sodium borate (SB)
buffer, at 94 volts for 45 minutes. KBPlus (LI-COR) was used to determine product size
and one well was loaded with master mix and sterile water to serve as a negative control.
Additionally, one M. fiucticola isolate (DRAD-29) was randomly selected and
sequenced; identification was then confirmed through comparison with GenBank

sequences of M. fructicola.

52

Table 2.1. List of suspected Moniliniafi-ucticola isolates screened to
determine species, including the source, locus and associated PCR code.

 

 

Gel
Isolate Code Code Source Locus"
500-4 5 Tart Cherry NWHRS
500-5 8 Tart Cherry NWHRS
BRC-15 10 Tart Cherry NW MI
BRC-7 4 Tart Cherry NW MI
BRR-l 6 40 Tart Cheny NW MI
BRR-l9 39 Tart Cherry NW MI
DRA-6 14 Sweet Cherry NW MI
DRA-9 13 Sweet Cherry NW MI
DRAD-14 38 Sweet Cherry NW MI
BRAD-29 37 Sweet Cherry NW MI
EMP-l6 16 Sweet Cherry NW MI
EMP-Zl 15 Sweet Cherry NW MI
ENT1000-15 1 l Tart Cherry NWHRS
ENT1000-1 8 6 Tart Cherry NWHRS
ENT450-10 1 Sweet Cherry NWHRS
ENT450-7 12 Sweet Cherry NWHRS
HH-26 18 Sweet Cherry NW MI
HH-27 17 Sweet Cherry NW MI
HRG-l 8 43 Tart Cherry Hart, MI
HRG-4 44 Tart Cherry Hart, MI
KHG-l6 41 Sweet Cherry Gillspier, MI
KHG-l8 42 Sweet Cherry Gillspier, MI
LAB-l4 2 Tart Cherry Benzie Co.
LAB-4 7 Tart Cherry Benzie Co.
LAR-l 3 Tart Cherry Montrnorency Co.
LAR-2 9 Tart Cherry Montrnorency Co.
MRC-Z 27 Sweet Cherry Coopersville, MI
MRC-4 28 Sweet Cherry Coopersville, MI
PF R-6 26 Peach Benton Harbor, MI
PFR-8 25 Peach Benton Harbor, MI
RBT-24 30 Tart Cherry NW MI
RBT-26 29 Tart Cherry NW MI
RES-20 33 Sweet Cherry NW MI
RPS-21 34 Sweet Cherry NW MI
VNL-12 20 Tart Cherry Hart, MI
VNL-6 l9 Tart Cherry Hart, MI
VPD-3 22 Sweet Cherry NW MI
VPD-5 21 Sweet Cherry NW MI
VPDS-2 32 Sweet Cherry NW MI
VPDS-8 31 Sweet Cherry NW MI
WIL-13 23 Peach Benton Harbor, MI
WIL-2 24 Peach Benton Harbor, MI
YBR-20 36 Tart Cherry NW MI
YBR-21 35 Tart Cherry NW MI

 

* NWHRS is the Northwest Horticultural Research Station and NW MI
stands for Northwest Michigan.

53

Moniliniafi-ucticola in vitro, fenbuconazole assay

Fungicide resistance studies often use reduced mycelial growth to measure
sensitivity (7, 15 , 16, 24). There are two acceptable methods of measuring mycelial
response to a firngicide; quantitative and qualitative. The quantitative method is based on
the fungicide concentration at which mycelial growth is reduced 50%, relative to normal
growth, this is referred to as the effective concentration 50 (ECso). The alternative,
qualitative method involves determining the minimum concentration at which colony
growth is completely inhibited, commonly referred to as the minimum inhibitory
concentration (MIC) (7, 15, 16, 24). M. fiucticola is fast growing, only taking seven days
to reach colony diameters of 8 cm, for this reason the quantitative ECso value was used
for this study.

In 2007, the sensitivity of .M. fiucticola isolates to fenbuconazole was measured.
Fenbuconazole (Indar WSP; Dow AgroSciences, Indianapolis, IN) was dissolved in
sterile water to prepare stock solutions. After autoclaving the PDA for sterilization and
allow it to cool to 50°C, quantities of the stock solutions were added. The fenbuconazole
concentrations were selected based on a prior subsampling of the collection. The
preliminary assay screened five M. fiucticola isolates at fenbuconazole concentrations of
0.01, 0.05, 0.1, 0.5, 1.0, 5.0, and 10.0 rig/ml. The preliminary isolates were all controlled
by less than 0.1 rig/ml of fenbuconazole so the concentrations for the assay were adjusted
to below 0.1 rig/ml. Fenbuconazole concentrations used for the assay were 0, 0.001,
0.005, 0.01, 0.05, and 0.1 11ng fenbuconazole. A total of one hundred and forty-eight
M. fi-ucticola isolates were tested. Mycelial plugs one millimeter in diameter from each

isolate were transferred to PDA plates amended with fenbuconazole or an unarnended

54

control (Figure 2.A). Each treatment was replicated three times. After five days of
incubation at 23 and 25°C the plates were examined for colony expansion, two diameter
measurements were recorded for each isolate and the diameters for the three replicates of
each treatment were averaged. The ECso values were calculated using a regression
analysis frmction included in Si gmaPlot (Systat Software Incorporated) statistical
software. The ECso reported is the fungicide concentration at which the growth of the

colony would be reduced by 50%.

55

 

 

Figure 2.A. Monliniafiucticola isolates growing on PDA plates amended with six
concentrations of fenbuconazole (0, 0.001, 0.005, 0.01, 0.05, and 0.1 rig/ml).

56

Inoculation trial

A fi'uit assay was performed to establish if there is a difference in the ability of M.
fructicola isolates to cause disease in the presence of fenbuconazole based on the
previously determined in vitro ECso values. The assay was designed to test the ability of
the four isolates with the lowest ECso values (LAB-3, MRC-20, WIL-3 and LAR-3) and
highest ECso values (RF S-lO, VNL-l8, BRC-l6, and KHG-l) to produce infections on
fi'uit that had been dipped in a range of fenbuconazole concentrations. Peaches were
inoculated with the isolates to confirm pathogenicity. The isolates were then recovered
from the peaches and plated onto three types of agar to induce conidial production. The
agars included acidified PDA, cherry concentrate agar, and V8 agar (Appendix B, Table
B1). The fungi were grown for a minimum of seven days at temperatures between 23
and 25°C. The colony surfaces were washed twice with 3 ml of sterile water to dislodge
propagules. A hemacytometer was used to determine propagule concentration, which
was adjusted to a concentration of 105/ml by dilution in sterile water. Each suspension
was amended with one drop of 10%, Tween 20 Solution (Sigma, St. Louis, MO).

Sweet and tart cherry fruit were randomly collected from untreated trees at the
NWHRS approximately 2 weeks prior to their respective harvest dates. The cherries were
prepared by briefly dipping them in one of three concentrations of fenbuconazole.
Fenbuconazole concentrations were based on the recommended field rate of 146 ml of
fenbuconazole/ 2,810 liters water/ ha, and represented rates equivalent to 25, 50, and
100% of the labeled spray rate. A control set of cherries were also dipped in sterile
water. Thirteen sweet cherries and twenty tart cherries were tested for each isolate, at

each fimgicide concentration. One 20 ul droplet of M. fructicola suspension was placed

57

on each sweet cherry. In the case of the smaller tart cherries, firngal inoculum was
sprayed until runoff using an atomizer. One set of cherries was not inoculated to
determine if latent M. fructicola infections were present at the time of collection.
Cherries were suspended on hardware cloth over water in aluminum trays and covered
with plastic wrap. These trays were placed in a 23°C incubator for 14 days. The number
of cherries with ABR infections was then recorded.
Propiconazole calibration

M. fructicola populations in Georgia have established ECso propiconazole (DMI)
levels from baseline M. fructicola populations and ECso values associated with practical
field resistance (Figure 2G). Michigan M. fiucticola isolates with known fenbuconazole
sensitivites (LAB-3, MRC-ZO, WIL-3, LAR-3, RFS-lO, VNL-18, BRC-l6, and KHG-l)
were tested for propiconazole sensitivity. Comparison of the sensitivity of Michigan M.
fructicola populations to fenbuconazole and propiconazole were measured in an attempt
to develop a means of calibrating the differences in reported ECso values.

Propiconazole (Orbit; Syngenta Crop Protection, Switzerland) was
dissolved in sterile water to prepare stock solutions. Quantities of these stock solutions
were added to PDA after it had been autoclaved for sterilization and cooled to 50°C. The
propiconazole concentrations were selected based on discriminatory dose information
from Georgia (Schnabel, in publication). Five isolated were screened at propiconazole
concentrations of 0.005, 0.01, 0.05, 0.1, 0.15, 0.2 and 0.25 ug/ml.

Mycelial plugs 1.0 mm in diameter from each isolate were transferred to PDA
plates amended with propiconazole. Each treatment was replicated three times for each

isolate. After five days of incubation at temperatures between 23 and 25°C, the plates

58

were examined for colony expansion and the diameters of the replicates were measured
twice to account for colonies not forming perfect circles, these values were then
averaged. The ECso values were calculated using a regression analysis function included
in SigmaPlot (Systat Software Inc) statistical software. The ECso reported is the
firngicide concentration at which the growth of the colony was reduced by 5 0%.
CYP51 sequencing

Tissue for PCR analysis was prepared by using a bead-beater centrifuge to disrupt
the fungal cell wall, as described in the protocol section of Appendix B. Actively
growing mycelitun (0.1g) was collected from isolates with the highest and lowest ECso
values determined in the fenbuconazole sensitivity screening (LAB-3, MRC-20, WIL-3,
LAR-3, RPS-10, VNL-18, BRC-l6, and KHG-l). The MfCYP51-F and MfCYP51-R '
primers (23) were used. A Qiagen, DNeasy Plant Mini Kit was used; the mini protocol
was followed. The thermocycler program and template preparation are described in the
protocol section of Appendix B. The PCR products were run on a 1% agarose gel in 1x
SB buffer at 94 volts for 45 minutes. KBPlus (LI-COR) was used to determine product
size. The PCR product was then sequenced at the Michigan State University Research

and Technology Support Facility.

RESULTS
PCR-based species confirmation

Of the forty-four isolates tested, PCR testing confirmed that all, except 500-4,
were M. fi-ucticola. Isolate 500-4 was also not amplified by the M. laxa primers. No

cross amplification with the M. laxa primer was observed (Figures 2.B-2.D).

59

b
i
a
2

 

Figure 2.B. PCR amplification of the ITS regions using (A) Monilinia laxa ITS
primers 1 and 4, or (B) Moniliniafructicola ITS primers 1 and 4 for suspected

Moniliniafiucticola isolates 1-6.

60

KB+C-le(» 17 18 19 20

400Kb ---------

 

Figure 2.C. PCR amplifications of the ITSl and IT S4 regions using
Moniliniafructicola ITS primers 1 and 4, for suspected Monilinia
fructicola isolates 7-24

61

kb+ C’— 10 13 27 2s :0

“0*.--- ..

34 35 ,36 37 38 39 40 41 4243 44
0’ ‘

poi-‘00:" oo-

 

Figure 2.D. PCR amplifications of the ITSl and ITS4 regions
using Moniliniafructicola ITS primers 1 and 4, for suspected
Moniliniafructicola isolates 13, 19 and 27-44.

62

Moniliniafructicola in vitro, fenbuconazole assay

A greater than 10-fold difference in fenbuconazole sensitivity was found among
M. fructicola populations when comparing isolates from different orchards (Table 2.2).
Overall ECso values were extremely low when compared to the average fenbuconazole

field rate of 37.45 ppm; indicating the probability of highly sensitive isolates.

63

Table 2.2. In vitro, mean fenbuconazole ECso value
and standard error based on location, and isolate
source of Monilinia fi-ucticola isolate.

 

 

Farm Code Source Mean Std Error
PF R Peach 0.0061 0.0029
PRL Peach 0.0045 0.0018
WIL Peach 0.0045 0.0008
DRA Sweet cherry 0.0038 0.0010

DRAD Sweet cherry 0.0049 0.0010
EMP Sweet cherry 0.0228 0.0047
ENT450 Sweet cherry 0.0156 0.0026
HH Sweet cherry 0.0243 0.0049
KHG Sweet cherry 0.0344 0.0085
MRC Sweet cherry 0.0057 0.0009
RFS Sweet cherry 0.0379 0.0117
VPD Sweet cherry 0.0113 0.0019
VPDS Sweet cherry 0.0286 0.0070
500 Tart cherry 0.0123 0.0007
BRC Tart cherry 0.0318 0.0087
BRR Tart cherry 0.0165 0.0026
ENT1000 Tart cherry 0.0182 0.0035
HRG Tart cherry 0.0351 0.0092
LAB Tart cherry 0.0049 0.0020
LAR Tart cherry 0.0050 0.0004
RBT Tart cherry 0.0101 0.0035
VNL Tart cherry 0.0245 0.0073
YBR Tart cherry 0.0049 0.0008

 

64

Moniliniafructicola fruit assay

The overall trend for M. fructicola fruit infections in inoculated tart and sweet
cherries showed a decrease in infection as fenbuconazole concentration increased,
regardless of isolate sensitivity (Tables 2.3 and 2.4). The infection rates of all of the
isolates were relatively low in the inoculated cherries dipped in water. In both fi'uit
assays, there were no latent M. fiucticola infections on uninoculated fi'uit, establishing
that the infections on inoculated fruit likely occurred as a direct result of inoculation. The
results of the tart cherry assay (Figure 2E) showed that as a group, the isolates least
sensitive to fenbuconazole in the in vitro study were more able to produce infections on
the inoculated cherries at all fenbuconazole concentrations including the water-dipped
cherries. The sweet cherry assay (Figure 2.F) produced a pattern similar to the tart cherry
assay. Infection of the sweet cherries with M. fructicola decreased as fenbuconazole
concentrations increased and the least fenbuconazole-sensitive isolates caused
significantly more infection than the most sensitive isolates. Sweet cherries had a lower
overall infection rate than tart cherries, and the percentage of ABR infections on the
sweet cherries dropped more drastically as the concentration of fenbuconazole increased.
Propiconazole calibration

No direct association could be determined between the fenbuconazole and
propiconazole sensitivity of M. fiucticola isolates from Michigan (Table 2.5). The data
did show Michigan isolates are as sensitive to propiconazole as baseline, unsprayed M.

fructicola populations fi'om Georgia (Figure 2G).

65

Table 2.3. Percent tart cherries infected, fifteen days after inoculation with a 105
/ml Moniliniafiucticola propagules. Cherries were dipped in three concentration

of fenbuconazole“.
Most Sensitive Isolates

Fenbuconazole rate LAB-3 MRC-20 WIL-3 LAR-3 Mean Std Error
Control (0 til/ml) 95.0 57.0 30.0 50.0 58.0 0.0304
Quarter (9.36 til/ml) 20.0 25.0 5.0 85.0 34.0 0.03936
Half (18.45 ul/ml) 5.0 0.0 15.0 5.0 6.0 0.00703
Full (37.45 ul/ml) 0.0 0.0 5.0 0.0 1.0 0.0028
Least sensitive isolates
Fenbuconazole rate RFS-lO VNL-18 BRC-16 KHG-1 Mean Std Error
Control (0 111/1111) 50.0 86.0 80.0 80.0 74.0 0.01809
Quarter (9.36 ul/ml) 29.0 70.0 55.0 55.0 52.0 0.01927
Half (18.45 til/ml) 0.0 50.0 50.0 25.0 31.0 0.02676
Full (37.45 ul/ml) 0.0 75.0 48.0 0.0 31.0 0.0415
Uninoculated Control
Fenbuconazole rate Percent infection
Control (0 ul/ml) 0.0
Quarter (9.36 ul/ml) 0.0
Half (1 8.45 Ill/ml) 0.0
Full (37.45 til/mp 0.0

 

*Fenbuconazole rates of quarter, half, and full concentration are based on the
reccommended field rate of 146 ml fenbuconazole/2,810 liters water/hectare.

66

Table 2.4. Percent sweet cherries infected, fifteen days after inoculation with a 105
/ml Monilinia fi'ucticola propgaules. Cherries were dipped in three concentration

of fenbuconazole“.
Most Sensitive Isolates
F enbuconazole rate LAB-3 MRC-20 WIL-3 LAR-3 Mean
Control (0 ule) 36.0 43.0 7.0 50.0 33.9

Quarter (9.36 Ill/ml) 0.0 14.0 0.0 0.0 3.6
Ha1f(18.45 u1/m1) 0.0 0.0 0.0 7.0 1.8
Full (37.45 til/ml) 0.0 0.0 7.0 7.0 3.6

Least sensitive isolates
Fenbuconazole rate RPS-10 VNL-18 BRC-16 KHG-1 Mean
Control (0 ul/ml) 93.0 71.0 93.0 29.0 71.4
Quarter (9.36 ul/ml) 36.0 14.0 93.0 0.0 35.7
Half (18.45 1.1le) 50.0 21.0 64.0 7.0 35.7
Full (37.45 111/1111) 38.0 21.0 14.0 0.0 18.3

Uninoculate Control

Fenbuconazole rate Percent infection
Control (0 ul/ml) 0.0
Quarter (9.36 ul/ml) 0.0
Half (18.45 ul/ml) 0.0
Full (37.45 ul/ml) 0.0

Std Error
0.021
0.00799
0.00399
0.00461

Std Error
0.03388
0.04564
0.02916
0.01743

 

*Fenbuconazole rates of quarter, half, and full concentration are based on the
reccommended field rate of 146ml fenbuconazole/2,810 liters water/hectare.

67

 

I Least sarsitive
CI Most sensitive

Percent ABR Infection

 

Control Quarter Half Full
Fenbuconazole rate

 

 

 

Figure 2.E. Average incidence of tart cherries infected with Moniliniafi‘ucticola, based
on isolate sensitivity, and fenbuconazole rate (Full rate is 146 ml/2,810 liter water/

hectare).

68

 

I Least sensitive
D Most sensitive

Percent ABR Infection

 

Control Quarter Half Full

 

Fenbuconazole rate

 

Figure 2.F. Average incidence of sweet cherries infected with Monilinia fructicola,
based on isolate sensitivity, and fenbuconazole rate (Full rate is 146 m1/2,810 liter water/
hectare).

69

 

Table 2.5. Sensitivity of Michigan Monilinia

fructicola isolates to fenbuconazole and

 

 

 

propiconazole.
ECso (Its/ml)
Fenbuconazole Propiconazole
LAB-3 0.0014 0.0108
LAR-3 0.0034 0.0169
RFS-lO 0.0723 0.0191
WIL-3 0.0018 0.0113
KHG-1 0.1024 0.0098
VNL-18 0.0447 0.0133
MRC-20 0.0033 0.0069
BRC-16 0.0562 0.0079

 

7O

 

Calibration of Fenbuconazole to Propiconazole

 

LAR-3

3
S

RFS—1 0
WIL-3
KH G-1
VN L-1 8
MRC-20
BRC-16

El Fenbuconazole
M. fructicola Isolate I Propiconazole

Figure 2.G. Comparison of fenbuconazole and propiconazole sensitivity of Michigan
Moniliniafructicola isolates (LAB-3, LAR-3, RFS-lO, WIL-3, KHG-1, VNL-18,
MRC-20, BRC-16) with baseline propiconazole levels fi'om Georgia (DL), and an
isolate resistant to propiconazole (AP).

 

 

 

71

CYP51 sequencing

Variability was found in the CYP51 gene sequences of the M. fructicola isolates
tested (LAB-3, MRC-20, WIL—3, LAR-3, RPS-10, VNL-18, BRC-16, and KHG-l).
However, the base pair differences in sequence did not differentiate isolates based on
fenbuconazole sensitivity. No consistent differences in sequence were found between the

groups of the most and least sensitive B. jaapii isolates.

DISCUSSION

Historically two brown rot frmgi (M. laxa, and M. fructicola) have been identified
based on colony morphology and germ tube growth when grown in vitro (4, 26). This
method of identification, based on morphological characteristics, is not always reliable
enough to be used when accurate speciation is needed (20, 25). For the purposes of this
paper accurate species identification was critical. Recent developments in molecular
techniques now allow for quick and reliable PCR-based testing (12). This molecular
technique confirmed that all but one of the isolates screened were M. fi-ucticola (Figure
2.B-2.D). Isolate 500-4 was neither M. laxa nor M fructicola.

Relating the in vitro fungicide sensitivity levels of a pathogen, to efficacy in the
field can be difficult. Practical field resistance to DMI frmgicides in M. fiucticola
populations has not occurred in Michigan to date, so the in vitro sensitivity of field
resistant isolates remains unknown. M. fructicola populations in Georgia have
established in vitro ECso propiconazole levels associated with both baseline sensitivities
practical field resistance (Figure 2.G). If a direct correlation between the in vitro efficacy

of propiconazole and fenbuconazole on Michigan isolates could have been determined,

72

then an ECso value of fenbuconazole at which practical field resistance would occur could
be estimated. The sensitivity of Michigan M. fructicola populations to fenbuconazole
were compared with propiconazole sensitivity in an attempt to develop a means of
calibrating the differences in ECso values. No direct relationship could be determined
(Table 2.5). In some cases more propiconazole than fenbuconazole was required for the
MIC, in others more fenbuconazole than propiconazole was required (Figure 2.G). The
baseline propiconazole data did show Michigan isolates are as sensitive to propiconazole
as baseline M. fructicola populations from Georgia. This development is surprising as
both fungicides are triazoles (sterol demethylation inhibitors) and thought to have a
similar mode of action, making them susceptible to the development of cross resistance.
In this case it appears propiconazole could be used in Michigan to control ABR, but
larger samplings are needed to be sure.

Molecular techniques were used to look for a genetic difference in M. fructicola
isolates, based on their in vitro fenbuconazole sensitivity. Three major mechanisms for
resistance to DMI frmgicides have been previously described, including: mutations in the
target enzyme, 14 alpha-demethylase (CYP51), leading to a decreased affinity of DMI
fungicides to the target protein (1, 5, 6); overexpression of the CYP51 gene leading to
increased production of the target enzyme (8, 18, 23); and overexpression of the ATP-
binding cassette (9, 28). In Michigan, M. fructicola populations are sensitive to
fenbuconazole in the field. Moreover, differences in the CYP51 gene sequence between
isolates, failed to correlate to specific isolate sensitivities. Base pair changes were seen in
both the most and least fenbuconazole-sensitive isolates, none of the base pair differences

were exclusively found in sensitive or less sensitive M. fructicola isolates. The

73

differences found in the base pair sequence of the CYP51 gene do not affect
fenbuconazole sensitivity in a significant way.

Characterizing the CYP51 genes of inherently sensitive isolates will be useful in
future endeavors. Ifa significant change in DMI sensitivity occurs in M. fructicola
populations in Michigan and a genetic cause is suspected, this screening will provide
baseline sequence data for comparison. Sequencing the CYP51 gene may also aid in
identifying the mechanism of resistance being utilized.

The overall in vitro fenbuconazole sensitivity data was variable, with M.
fructicola sensitivity varying up to 10-fold between orchards with ECso values ranging
fi‘om 0.0038-0.0379 rig/ml (Table 2.2). Variability in baseline data may be an indicator
of a population’s proclivity for shifting towards reduced sensitivity, however a 10-fold
difference is considered only moderately variable (3).

The fruit assay demonstrated a significant difference in the ability of the most
and least fenbuconazole-sensitive isolates to cause infection in both sweet and tart cherry
fi'uit (Figure 2E and 2.F). Sweet cherries had a lower overall infection rate than tart
cherries and the percentage of ABR infections on the sweet cherries dropped more
drastically as the concentration of fenbuconazole increased. This is likely due to the
differences in inoculation techniques. Sweet cherries received one droplet of inoculum,
while the entire surface of the tart cherries was rrristed with inoculum using an atomizer
that covered more of the surface with inoculum and increasing the chance of infection.
Overall, the fruit assay illustrated that the M. fructicola sensitivity levels, determined by
the in vitro fenbuconazole screening, do affect the ability of the pathogen to infect fruit in

the presence of fenbuconazole.

74

Rotating fungicide classes within the growing season, and tank mixing with an
appropriate broad spectrum fungicide, are vital facets of managing for resistance (3).
Establishing baseline fenbuconazole sensitivity levels was critical to monitoring the
sensitivity of Michigan’s M. fiucticola populations in subsequent years. Monitoring
fungicide sensitivity will be vital to the development of subsequent recommendations that

may help prolong the useful lifespan of DMI fungicides against CLS.

75

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78

APPENDIX A

MMEA Recipe

Combine 20.0g malt extract, 1.0g yeast extract, 20.0g agar, and 1.0 liter DI water. The

mixture needs to be autoclaved (20 minutes) for sterilization.

79

Table A.1. Weather conditions during the 2005 growing season in Suttons Bay,
Michigan, including; temperature, wetting period duration and rainfall.

 

Start of Wetting End of Wetting Duration Avg. Temp Rainfall Cherry Leaf
Period Period (Hrs) (C) (cm) Spot
Wet: 13
5/9 4-5PM 5/10 8-9AM Span: 16 16.9 0.23 Moderate
Wet: 14
5/10 9-10PM 5/1 1 Noon-1PM Span: 15 6.9 0.03 None
Wet: 51
5/ 13 7-8AM 5/ 16 8-9AM Span: 73 6.1 1.04 None
Wet: 24
5/19 8-9AM 5/20 8-9AM Span: 24 9.1 1.37 None
Wet: 21
5/22 7-8AM 5/23 Noon-1PM Span: 29 1 1.2 0.76 Low
Wet: 10
5/23 ll-lZPM 5/24 9-10AM Span: 10 9.8 0.05 None
Wet: 4
5/26 9-10AM 5/26 1-2PM Span: 4 11.9 0.05 None
Wet: 3
5/27 5-6AM 5/27 8-9AM Span: 3 9.5 0.08 None
Wet: 2
6/5 8-9AM 65 10-1 1AM Span: 2 20.7 0.05 None
Wet: 2
6/5 11-12PM 6/6 l-ZAM Span: 2 20.6 0.05 None
Wet: 7
6/8 1-2AM 6/8 8-9AM Span: 7 16.7 0.30 Low
Wet: 23
6/14 2-3PM 6/15 9-10PM Span: 31 15.8 0.74 High
Wet: 1
6/23 l-ZPM 6/23 2-3PM Span: 1 17.6 0.08 None
Wet: 5
6/26 3-4AM 6/26 9-10AM Span: 6 17.6 0.03 None
Wet: 1
6/30 7-8AM 6/30 8-9AM Span: 1 20.8 0.03 None
Wet: 5
7/1 8-9AM 7/1 2-3PM Span: 6 14.7 0.05 None
Wet: 6
7/4 6-7AM 7/4 Noon-1PM Span: 6 19.0 1.22 Low
Wet: 3
7/13 6-7AM 7/13 9-10AM Span: 3 21.4 0.03 None
Wet: 1
7/17 3-4PM 7/ 17 4-5PM Span: 1 28.2 0.84 None
Wet: 2
7/18 6-7AM 7/18 8-9AM Span: 2 24.4 0.05 None
Wet: 9
7/24 3-4AM 7/24 Noon-1PM Span: 9 21.0 0.64 Low
Wet: 15
7/25 11-12PM 7/26 2-3PM Span: 15 20.3 2.21 Moderate
Wet: 12
7/28 9-10PM 7/29 9-10AM Span: 12 16.1 0.53 Low
Wet: 9
8/4 3-4AM 8/4 2-3PM Span: 11 23.1 0.89 Low
Wet: 11
8/10 1-2AM 8/ 10 Noon-1PM Span: 11 21.0 0.05 Low
Wet: 12
8/12 Midnight-1AM 8/ 12 Noon-1PM Span: 12 18.6 0.76 Moderate
Wet: 40
8/18 Noon-1PM 8/20 Noon-1PM Span: 48 19.0 3.81 High
Wet: 7
8/21 10-11PM 8/22 9-10AM Span: 11 14.0 0.13 None
Wet: 7
8/27 5-6AM 8/27 Noon-1PM Span: 7 18.5 0.84 Low

 

80

Table A.2. Weather conditions during the 2006 growing season in Suttons Bay, Michigan,
including; temerature, wetting period duration and rainfall.
End of Wetting Duration Avg. Temp Rainfall Cherry Leaf

 

Start of Wetting Period Pen od (Hrs) (, C) (cm) Spot
Wet: 13
5/9 4-5PM 5/ 10 8-9AM Span: 16 16.9 0.23 Moderate
Wet: 14
5/10 9-10PM 5/11 Noon-1PM Span: 15 6.9 0.03 None
Wet: 51
5/13 7-8AM 5/16 8-9AM Span: 73 6.1 1.04 None
Wet: 24
5/19 8-9AM 5/20 8-9AM Span: 24 9.1 1.37 None
Wet: 21
5/22 7-8AM 5/23 Noon-1PM Span: 29 11.2 0.76 Low
Wet: 10
5/23 11-12PM 5/24 9-10AM Span: 10 9.8 0.05 None
Wet: 4
5/26 9-10AM 5/26 l-2PM Span: 4 11.9 0.05 None
Wet: 3
5/27 5-6AM 5/27 8-9AM Span: 3 9.5 0.08 None
Wet: 2
6/5 8-9AM 6/5 10-1 1AM Span: 2 20.7 0.05 None
Wet: 2
6/5 11-12PM 6/6 1-2AM Span: 2 20.6 0.05 None
Wet 7
6/8 l-ZAM 6/8 8-9AM Span: 7 16.7 0.30 Low
Wet: 23
6/14 2-3PM 6/15 9-10PM Span: 31 15.8 0.74 High
Wet: l
6f23 l-2PM 6/23 2-3PM Span: 1 17.6 0.08 None
Wet: 5
6/26 3-4AM 6/26 9-10AM Span: 6 17.6 0.03 None
Wet: 1
6/30 7-8AM 6/30 8-9AM Span: 1 20.8 0.03 None
Wet: 5
7/1 8-9AM 7/1 2-3PM Span: 6 14.7 0.05 None
Wet: 6
7/4 6-7AM 7/4 Noon-1PM Span: 6 19.0 1.22 Low
Wet: 3
7/13 6-7AM 7/13 9-10AM Span: 3 21.4 0.03 None
Wet: l
7/ 17 3-4PM 7/17 4—5PM Span: 1 28.2 0.84 None
Wet: 2
7/18 6-7AM 7/ 18 8-9AM Span: 2 24.4 0.05 None
Wet: 9
7/24 3-4AM 7/24 Noon-1PM Span: 9 21.0 0.64 Low
Wet: 15
7/25 11-12PM 7/26 2-3PM Span: 15 20.3 2.21 Moderate
Wet: 12
7/28 9-10PM 7/29 9-10AM Span: 12 16.1 0.53 Low
Wet: 9
8/4 3-4AM 8/4 2-3PM Span: 11 23.1 0.89 Low
Wet: ll
8/ 10 l-2AM 8/10 Noon-1PM Span: 11 21.0 0.05 Low
Wet: 12
8/12 Midnight-1AM 8/ 12 Noon-1PM Span: 12 18.6 0.76 Moderate
Wet: 40
8/18 Noon-1PM 8/20 Noon-l PM Span: 48 19.0 3.81 High
Wet: 7
8/21 10-11PM 8/22 9-10AM Span: 11 14.0 0.13 None
Wet: 7
8/27 5-6AM 8/27 Noon-1PM Span: 7 18.5 0.84 Low

 

81

APPENDIX B

PROTOCOLS
Bead-beater protocol

One hundred milligrams of fresh mycelium was aseptically placed in shatter-
resistant, 2ml centrifuge tubes with two glass beads. Centrifuge tubes are then
submerged in liquid nitrogen for 30 seconds and placed in the centrifuge. Short intervals
(30 seconds) at moderate RPMs are used until the mycelia become a chalk-like powder.
PCR reaction formula for all reactions

PCR reaction volumes were 5011] and included: 5 ul of Invitrogen PCR Buffer,
1.5ul Invitrogen MgCl Buffer, .4111 dNTPs, 1.25 ul Taq DNA polymerase, 41.1ul sterile
DI water, lul of each primer and 5111 template from each isolate.
Thermocycler program for PCR confirmation of M. fructicola species

Amplifications were performed with the following thermocycler program: 94°C
for 3 minutes; 36-cycles of 94°C for 30 seconds, 65°C for 30 seconds, and 72°C for 1.5
minutes; 10 minutes at 72°C; and finally held at 4°C.
Thermocycler program for PCR amplification of the CYP51 gene

Amplifications were performed with the following thermocycler program: 94°C
for 3 minutes; 30-cycles of 94°C for 1 minute, 55°C for 1 minute, and 72°C for 2

minutes; followed by a final extension step of 5 rrrinutes at 72°C.

82

Table B.1. Culture media for inducing conidia formation in
Moniliniafiucticola

 

Acidified PDA agar: 39g of potato dextrose agar with 1 liter
deionized water and autoclave for 20 minutes. Allow agar to
cool to 50°F and add 1.3m] of 25% lactic acid, pour plates.

Cherry concentrate agar: Mix 12g agar, 400ml deionized water,
and 60ml commercially available cherry concentrate diluted
per the labeled instructions. Autoclave (20 minutes) and then
adjust the pH to 5 (should take around 40ml of 10% KOH).

V8 agar: Mix 125ml V8, 375ml deionized water, and 15g of
agar, then autoclave Q0 minutes).

 

83

 

MIC V

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