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Genetic diversity and variation in Michigan isolates
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Christine L. Snyder

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1m animus-p.14

GENETIC DIVERSITY AND VARIATION IN MICHIGAN ISOLATES OF
MONILINIA FR UC TIC OLA AND MONILINIA LAXA

By

Christine L. Snyder

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

MASTER OF SCIENCE

Department of Botany and Plant Pathology

1997

III} I t

GENETIC DIVERSITY AND VARIATION IN MICHIGAN ISOLATES OF
MONILINIA FR UC TIC OLA AND MONILINIA LAXA

By

Christine L. Snyder

One hundred and seventeen vegetative compatibility groups (VCGs) were
identified among 169 isolates of M. fructicola collected from Michigan cherry orchards.
Compatibility varied between three individual orchards with 70 isolates forming 25
VCGs, 61 isolates forming 54 VCGs, and 38 isolates forming 38 VCGs. PCR-mediated
analysis of DNA from M. fructicola and M. laxa revealed internal transcribed spacer 1
(ITSl) regions that were 146 bp in length and 98% similar. A base pair change at position
105 created a Mse I restriction site in M. fructicola. A 421-bp region present in the SSU
rDNA of 32 isolates of M. fructicola but absent from eight isolates of M. laxa was
identified as a group I intron. PCR amplification of the intron-containing region yielded
a product of approximately 940 bp from M. fructicola and 520 bp from M. laxa.
Arbitrarily primed-PCR with primers (GACA)4 and (GTG)5 each yielded distinct multiple

banding patterns between the species.

For my parents, George and Yvonne Snyder,
for all of their love and support

iii

ACKNOWLEDGMENTS

I would like to express my appreciation to those people who have aided me
throughout my masters program. First to Dr. Alan Jones, my major professor, for
providing guidance and support throughout my research. To Dr. Gerard Adams for
expanding my knowledge of mycology and providing input and evaluation of my
program.

And to Dr. Andrew J arosz for his input concerning population genetics and his critical
evaluation of my research.

I would also like to thank Elise Palmer for sharing her knowledge of molecular
techniques and providing critical input regarding these techniques. And to Jon Powell for
sharing ideas, providing invaluable guidance on some of the basics of graduate student
life, and for his friendship.

And, of course, I would like to thank my parents for their constant support and

encouragement.

iv

TABLE OF CONTENTS

LIST OF TABLES .............................................................................................................. vi
LIST OF FIGURES ........................................................................................................... vii
INTRODUCTION ............................................................................................................... 1
REVIEW OF LITERATURE

Brown rot ................................................................................................................. 4

Vegetative compatibility .......................................................................................... 7

Molecular Methods in Mycology ........................................................................... 10
CHAPTER 1

VEGETATIVE COMPATIBILITY IN MICHIGAN POPULATIONS OF

MONILINIA FRUCTICOLA .................................................................................. 14
CHAPTER 2

VARIATION IN INTERNAL TRANSCRIBED SPACER SEQUENCES,
ARBITRARILY PRIIVIED-PCR PRODUCTS, AND GROUP I INTRON

PRESENCE BETWEEN MONILINIA FR UCTICOLA AND M LAXA ............... 23
CHAPTER 3

FINAL CONCLUSIONS ....................................................................................... 46
RECOMMENDATIONS FOR FUTURE STUDY ........................................................... 52
LIST OF REFERENCES ................................................................................................... 55
APPENDICES ................................................................................................................... 62

LIST OF TABLES

Table 1 - Frequency of vegetative incompatibility among isolates of Monilim'a

fiucticola collected from Michigan cherry orchards .......................................... 19
Table 2 - Isolates of Moniliniafructicola and M. laxa used in PCR-mediated

analysis and the results of analysis ..................................................................... 30
APPENDICES

Appendix A - Vegetative compatibility groups (VCGs) containing more than
one isolate of Moniliniafiucticola ............................................................. 62

vi

LIST OF FIGURES

Figure l - Cultural interactions of isolates of Mom'linia fructicola ................................... 17

Figure 2 - Alignment of the ITS] sequences of isolates of Monilinia fi'ucticola
and M. laxa determined in this study ................................................................ 32

Figure 3 - Alignment of the consensus sequences of the ITSI region of Monilim'a
fi'ucticola and M. laxa determined in this study with Sclerotinia
sclerotiorum (U21810) ..................................................................................... 34

Figure 4 — Restriction fragment length polymorphism in Mse I-digested PCR-
amplified ITSI DNA from Monilinia laxa isolates DG-8, NP-2, WI-2,
and Grants (lanes 1-4) and M. fructicola isolates SC-4l, KK-20,
JB-13, and CB-2 (lanes 5-8). .......................................................................... 35

Figure 5 - Products of PCR amplification of a region of the SSU rRNA gene
of Monilim'a fructicola and M. laxa with primers A1139 and A1140 ............... 37

Figure 6 - Alignment of the SSU rDNA sequences amplified with primers AJ139
and AJ14O from Moniliniafi-ucticola strain KK—ZO and M. laxa strain
DG-7 ................................................................................................................. 38

Figure 7 - Alignment of the conserved sequence elements of the group I intron
within the nuclear SSU rDNA of M. fructicola with introns from and
five other fungi ................................................................................................. 40

Figure 8 - Arbitrarily primed-PCR amplification of genomic DNA from M.
fructicola and M. laxa using microsatellite primers (GACA)4 in A

and primer (GTG), in B .................................................................................... 41
APPENDICES
Appendix B - Sequences and positions of oligonucleotide primers in the rDNA ............. 63

vii

INTRODUCTION

Brown rot, caused by Moniliniafiucticola (Winter) Honey, is the most
economically important disease of stone fruits in the United States. Within Michigan
orchards, M. fructicola is a serious concern for the cherry industry, causing blossom
blight, twig cankers and significant pre- and post-harvest fi'uit rot (Jones and Sutton,
1996). A second brown rot fungus, M. laxa Aderhold and Ruhland, is common along the
Pacific coastal regions and has caused minor outbreaks in Wisconsin, Michigan, and New
York (Cation et al., 1949; Kable and Parker, 1963; Keitt et al., 1943; Jones and Sutton,
1996)

The impact of brown rot on stone fruit industries indicates the need for study of
the exact nature of the disease cycle and the genetic diversity that exists within field
populations of the pathogen. While limited study of the genetics and diversity of M.

fiucticola has been performed in California populations of the pathogen (Sonoda et al. ,
1990; Free at al. , 1996), little has been done to examine the diversity that exists within
Michigan populations. The level of diversity that exists within populations of the
pathogen and the role of sexual sporulation in the disease cycle are all important factors
in understanding the spread of the disease and implementing the most effective and
economical control measures. With the occurrence of M. laxa and M. fi-ucticola together

in some regions, it is also important to understand the relationship between these two

1

2

species and to develop methods for distinguishing between hard to identify isolates of the
two species.

The purpose of this study was to began an assessment of the level of diversity
which exists within populations of M. fiucticola and M. laxa. This study focused
specifically on diversity within Michigan populations of these fungi, and the results found
from these local populations may not indicate the diversity of the global population.
However, this study had the additional purpose of identifying techniques useful for
revealing diversity within the species and which can be applied to future studies on a
broader geographic scale.

Vegetative compatibility and a variety of molecular techniques have been used by
mycologists and plant pathologists to examine diversity within species as well as to
determine the relationship between different species. Many of these studies have also
uncovered rapid means of distinguishing between species and important sub-specific
groups.

This study of Michigan field populations utilized vegetative compatibility to
initiate an examination of the diversity of M. fructicola in Michigan. As will be
discussed in chapter 1, the frequency of vegetative compatibility may directly reflect the
level of genetic diversity within a population. Molecular methodology was employed to
obtain a more accurate view of the genetic diversity that exists within M. fi‘ucticola, and
to develop an understanding of the relationship between M. fiucticola and M. laxa.
Nucleotide sequence analysis of the internal transcribed spacer 1 (ITSI) region provided

evidence of the diversity within M. fructicola and also revealed the apparent close

3

relationship between M. fructicola and M. laxa. Examination of the small subunit (SSU)
ribosomal DNA for the presence of an intron and random amplification of microsatellite
DNA provided data regarding the apparent level of diversity within M. fructicola and M.
laxa. Each of the these three methods also identified differences which may serve as
useful markers for the rapid differentiation of Michigan isolates of M. fi-ucticola and M.

laxa.

REVIEW OF LITERATURE
Brown Rot

Brown rot is one of the most economically important diseases of stone fi'uit
throughout the temperate regions of the world (Byrde and Willletts, 1977). The brown
rot fimgi include three species of Discomycetes within the Family Sclerotiniaceae:
Moniliniafructicola (Winter) Honey, M. laxa Aderhold & Ruhland, and M. fructz'gena
Honey. These fungi cause symptoms ranging from fruit rot to blossom blight, stern and
leaf blight, and stem cankers. Economic losses result primarily from the rotting of fruit in
the orchard before harvest, but losses also occur during transport of harvested fruit and
from yield reduction by destruction of blossoms early in the season (Jones and Sutton,
1996).

The earliest description of a brown rot fungus was Persoon’s 1796 description of a
European fungus with buff-colored conidial pustules (then T urola fiuctigena). In 1818,
Ehrenberg described a second brown rot fungus with grey conidial pustules on apricot .
(Oidiium laxum). Both species were eventually transferred to the genus Monilia, and a
third name, M. cinerea, was proposed for the brown rot on chenies. In 1886 Saccardo
and Voglino listed the three brown rot species of Europe as Moniliafructigena, M. laxa,
and M. cinerea, but the latter two were eventually found to be conspecific. In 1893,
Schrtiter transferred the brown rot fungi to the genus Sclerotinia based on the

morphology of their conidial stages.

5

The first description of a brown rot fungus in America was Peck’s 1881
description of a fungus which he identified as Moniliafructigena. Two years later,
Winter made the first known record of the perfect stage of any brown rot fungus. He
named this fungus, found on mummified peaches in Pennsylvania, Ciboriafi-ucticola
(later changed to Sclerotiniafructicola). In 1902, Norton made a description of the
perfect stage of a second brown rot fungus in North America which he called S.
fiuctigena. However, Reade later determined that S. fructicola and S. fi-uctigena were
synonymous when referring to the common American brown rot fungus and Pollack
suggested the use of the species epithet fi-ucticola. In 1928, Honey proposed the generic
name Monilinia to include those members of the Sclerotiniaceae which produce moniloid
conidia.

M. fiucticola, commonly called the American brown rot fimgus, is widespread
throughout stone fruit growing regions of the United States. A European brown rot
fungus, later identified as M. laxa, was discovered in Oregon in 1915 and since its
discovery has become common along the Pacific coastal regions, and also appears to be
endemic on sour cherry (Prunus cerasus L.) in Michigan (Cation et al., 1949), New York
(Kable and Parker, 1963), and Wisconsin (Keitt etal., 1943). However, M. fructicola
remains the species of greatest economic importance throughout the United States. The
third brown rot pathogen, M. fructigena is widely distributed in Europe, but is currently
not found in North America.

The primary role of M. fiucticola is that of a fruit rot pathogen. Fruit is rotted in

the orchard, but significant post-harvest fruit rot also occurs during transportation and

6

storage. M. fructicola infects a wide range of stone fruits, including apricot, peach, plum,
nectarine, and cherry. Within Michigan orchards, M. fiucticola is the primary, and often
only, brown rot fungus evident, causing significant pre- and post harvest fruit rot on both
sweet and sour cherry. M. laxa, unlike M. fiucticola, is a blossom and twig blight
pathogen and rarely causes fruit rot. The destruction of blossom and fruit spurs results in
a reduced fruit yield latter in the season. Within the Great Lakes region, including
Michigan, M. laxa rarely causes significant levels of disease, although a serious outbreak
occurred in Michigan and Wisconsin in 1993 (Jones and Sutton, 1996). When M. laxa
does occur in Michigan it is only found on sour cherry.

The brown rot fimgi overwinter as mycelium in infected peduncles, twig cankers,
and mummified fruit on the tree, or as pseudosclerotia in mummified fruit on the ground.
Primary inoculum for infection of blossoms in the spring are conidia produced on
infected peduncles, cankers, and mummies on the tree, and, for M. fructz‘cola, ascospores
produced within apothecia that arise from mummies buried in the ground. However,
while apothecia have been noted in California orchards (Hewitt and Leach, 1939, Free et
al., 1996), they are rarely evident in Michigan orchards, and no apothecia were observed
in New York orchards over a three year period (Wilcox, 1989). Because of the apparent
absence of apothecia from orchards of the Midwest and East coast, the importance of the
sexual stage of M. fi-ucticola to spread of the disease in these regions remains unclear and
conidia are considered the most important inoculum for fruit infection and spread of the

disease throughout the growing season (Byrde and Willets, 1977).

7

A few studies regarding the genetics of M. fructicola have been conducted.
Ezekial (1924) and Harada (1977) reported that M. fiucticola is homothallic and
additional studies have described the fimgus as heterokaryotic and capable of out-
crossing (Byrde and Willetts, 1977; Sanoamuang et al., 1995; Thind and Keitt, 1949;
Sonoda et al., 1982). However, a recent study suggests that M. fructicola may actually be
heterothallic (Free et al., 1996). The study of single ascospore progeny revealed the
segregation of fungicide resistance genes (Sanoamuang et al., 1995) and that the genes

for vegetative compatibility segregate independent of one another (Free et al., 1996).

Vegetative Compatibility

Fungal vegetative compatibility refers to the formation of stable heterokaryons via
anastomosis (fusion) of the vegetative hyphae of fungi. Isolates which anastomose to
form stable heterokaryons are considered to be vegetatively compatible and are assigned
to a single vegetative compatibility group (V CG). Hyphal fusion between some isolates
cannot occur or results in the death of the cells involved. These isolates, between which
stable heterokaryons cannot form, are vegetatively incompatible and are placed in
different VCGs.

Several methods have been developed to test for compatibility, including
auxotrophic mutants, morphological mutants, and barrage formation (Anagnostakis,
1977; Puhalla, 1979; Sonoda et al., 19823; Puhalla, 1985; Correll et al., 1987). However,
the method employed typically depends on the growth habits of the fungus being studied

and must also be carefully evaluated to ensure that the chosen method offers the most

8

accurate results. Studies of Verticillium dahliae using microsclerotial color mutants
revealed sixteen VCGs among 86 isolates (Puhalla, 1979; Puhalla and Hummel, 1983).
However, in later work, representatives from fifteen of the known VCGs were found to
form only four VCGs when tested using nitrate-nonutilizing (nit) mutants (J oaquim and
Rowe, 1990). The disparity between the results fiom these two methods may reflect the
occurrence of weakly compatible reactions (Joaquim and Rowe, 1990). While some
interactions were compatible, the interaction was too weak to generate the expected
microsclerotial color, but strong enough to be detected with nit mutant testing. The
conflicting results fi'om the studies of V. dahliae also indicate the need to exercise caution
when interpreting VCG data.

Within many species of fungi, vegetative hyphae will undergo anastomosis
regardless of their natural compatibility or incompatibility. Anastomosis of vegetative
hyphae which contain vegetatively incompatible nuclei results in a killing reaction which
ultimately leads to the death of the newly formed heterokaryotic cell. The killing reaction
leads to the formation of a region of dead and dying cells between the isolates, and a layer
of dark pigment is often deposited between the isolates. The region of dead and dying
cells and dark pigment is commonly referred to as the barrage zone. Barrage zone
formation has become one to the primary means by which vegetative incompatibility is
scored in ascomycetes.

Vegetative compatibility testing within M. fructicola has utilized the formation of
barrage zones between incompatible isolates (Sonoda et al. , 1982a; Sonoda et al., 1990;

Free et al. , 1996). The production of dark lines between some isolates of M. f ructicola

9

when grown together in culture was first described by Thind and Keitt (1949). This
barrage formation has since been identified as characteristic of vegetative incompatibility
in Ascomycetes. Sonoda et al. determined that these interactions could be used to
genetically group isolates of M. flucticola (1982a). Vegetative compatibility studies
involving both M. fiucticola and M. laxa also suggested that these interactions might be
useful in distinguishing cultures of M. laxa from M. fiucticola (Sonoda et al., 1982b).
Vegetative compatibility has been examined in California populations of M. fructicola
(Sonoda et al., 1990; Free at al., 1996), but has never been examined in Michigan
populations.

Vegetative compatibility reactions have been used for a number of purposes in the
study of other fungi. Vegetative compatibility has been described as a potential
diagnostic tool on that basis that strains of the same sub-specific group, such as a
pathogenicity group, may be members of only one or a few VCGs (Puhalla, 1985). The
pathogen could then be identified rapidly by placement into a VCG rather than the more
time consuming method of pathogenicity testing. However, this use of vegetative
compatibility as a diagnostic tool has displayed only limited success (Elias and
Schneider, 1991; Jacobson and Gordon, 1988; Ploetz and Correll, 1988).

Vegetative compatibility has also been used in the study of diversity and
epidemiology in a variety of plant pathogens (Puhalla, 1986; Kohn et al., 1990; Chacko et
al., 1994; Daayf et al., 1995). As will be discussed in chapter 1, the frequency of VCG
diversity may reflect the level of genetic diversity within a population (Leslie, 1993).

Vegetative compatibility may also provide insight into the mode of reproduction and

10

spread of a pathogen (Nalim et al., 1995). The occurrence of VCGs, their location
relative to one another within a field, and the location of isolates of the same VCG may
establish a pattern which would provide insight into the role of ascospores verus conidia
in the spread of disease as well as revealing the general pattern of spread (Adams et al.,

1990; Sonoda et al., 1990; Nalim et al, 1995).

Molecular Methods in Mycology

While vegetative compatibility may be used as an initial step to identify the
potential level of diversity that exists within populations, molecular methodology must be
employed to obtain the most accurate view of genetic diversity. Molecular methodology
and DNA analysis have provided useful tools for phylogenetic and taxonomic studies, as
well as studies of population genetics and the analysis of genetic variability within
species. Studies of variability within species have also often revealed methods for
distinguishing important sub-specific groups, such as pathogenicity groups, and have
revealed the nature of mutations causing a variety of phenomena, such as fungicide
resistance.

One of the commonly used techniques in both population and phylogenetic
studies is sequence analysis of the internal transcribed spacer (ITS) regions of the nuclear
ribosomal DNA. The ITS regions are non-coding sequences and are thought to be less
subject to selection and may therefore display random genetic drift (Morton et al., 1995).
The ITS regions are generally conserved at the species level but are variable at higher

taxonomic levels (Bruns et al., 1991). ITS analysis has therefore become a useful tool for

11

species differentiation and phylogenetic studies (Chen et al., 1992; Lee and Taylor, 1992;
Carbone and Kohn, 1993). However, the ITS regions of several firngi have been shown
to be highly variable at the species level, providing a means to identify subspecific
variation and also aiding in phylogenetic analysis (O’Donnell, 1992; Morton et al., 1995;
Kusaba and Tsuge, 1995).

The ITSI region of M. fiucticola and three related species, M. fructigena, M.
megalospora (Woronin) Whetzel, and M. oxycocci (Woronin) Honey, were examined in a
study of diversity within the Sclerotiniaceae (Carbone and Kohn, 1993). The results of
the study suggested that sequence variation within the ITSI region could be used to
distinguish M. fructicola from other Monilim'a species. The study also revealed that M.
fiucticola and M. fructigena appear to be more closely related to one another and to
various Sclerotinia species, than either is to the other Monilim'a species examined.
However, the ITSI sequence of M. laxa was not included in this study, and its
relationship to the other Monilinia species is uncertain.

The small subunit (SSU) ribosomal DNA is a conserved region that has also been
utilized in various phylogenetic and taxonomic studies (Hendriks et al., 1992; Wilrnotte
et al., 1993; Liu et al., 1995; Morales et al., 1995). The SSU gene is highly conserved at
the species levels and is primarily usefirlness for phylogenetic studies of higher
taxonomic levels, such as Family and Order. In recent years, several studies have
revealed the presence of group I introns within the SSU gene of Ustilago maydis (De
Watcher et al., 1992), Pneumocystis carinii (Sogin and Edman, 1989), Protomyces

inouyei (N ishida et al., 1993), and several other fungal species. The presence of introns

12

in the SSU gene of widely divergent species is thought to be due to lateral gene transfer
(Sogin et al., 1986; Dujon, 1989). Introns present in pathogenic species may also provide
a means of identifying an intron-containing organism within host tissue (Gargas et al.,
1995)

Sclerotinia sclerotiorum, a close relative of M. fiucticola and M. laxa, was shown to
possess group I introns in both the nuclear SSU and the mitochondrial SSU (Wilrnotte et
al., 1993; Carbone et al., 1995). The SSU genes of M. fi-ucticola and M. laxa have not
been examined, but isolates of one or both of these species may possess one or more
introns. The identification of an intron within either species, and the location of the
intron, may provide further insight into the relationship between these species and may
also provide a rapid means of distinguishing isolates of the two species.

A third molecular method used in mycology involves the detection of microsatellite
DNA. Microsatellites, or short tandem repeats, are stretches of tandem one to four
nucleotide repeats, of varying length. These repeats are widespread in the genome of
eukaryotes, including fungi (Bruford and Wayne, 1993; Rosewich and McDonald, 1994).
The loci containing microsatellites may be useful for studies of genetic variation because
they are typically highly polymorphic and usually undergo Mendelian inheritance
(Groppe et al., 1995).

Different alleles of a microsatellite-containing locus have been identified by size
variation when amplified with oligonucleotides corresponding to the sequences flanking
the microsatellite. A study of Epichloé' species identified five alleles of a single

microsatellite locus from a field of 91 isolates (Groppe et al., 1995). Arbitrarily primed-

13

PCR (ap-PCR), using oligonucleotides corresponding directly to the microsatellites, has
also been used to identify polymorphisms between species and among individuals within
a species (Freeman and Rodrigez, 1995; Groppe et al., 1995; Freeman et al., 1996;
Longato and Bonfante, 1997). However, success in identifying intraspecies
polymorphisms has varied. Ap-PCR of Colletotrichum gloeosporioides isolates revealed
no polymorphisms between 57 Israeli almond isolates, while 57 Israeli avocado isolates
revealed eight distinct phenotypes (Freeman et al., 1996).

Each of the three techniques described above may be useful to identify diversity
within populations of M. fi-ucticola and M. laxa. These techniques might also reveal
differences between the two species which will provide a rapid means of distinguishing

the two species from one another.

Chapter 1

VEGETATIVE COMPATIBILITY IN MICHIGAN POPULATIONS OF
MONILINIA FRUCTICOLA

Introduction

Monilim'a fructicola is an ascomycetous fungus with a known teleomorph.
Apothecia arise from mummified fruit on the orchard floor early in the spring and the
ascospores may serve as inoculum for initial blossom infection. Conidia are considered
the most important inoculum for fruit infection and spread of the disease throughout the
growing season (Byrde and Willetts, 1977). While apothecia have been noted in
California orchards (Hewitt and Leach, 1939; Free et al., 1996), they are rarely evident in
Michigan orchards, and no apothecia were observed in New York orchards over a 3 year
period (Wilcox, 1989). Because of the apparent absence of apothecia from orchards of
the Midwest and the East coast, the importance of the sexual stage of M. fiucticola to
spread of the disease in these regions remains unclear.

The level of genetic diversity that exists within field populations of M. fructicola
is also uncertain. Studies have been made of the spread and persistence of fungicide
resistance in field populations of M. fiucticola, but little has been done to examine the
general diversity that exists within populations of this pathogen. Knowledge of the level

of genetic diversity within populations of a species can provide insight into the

14

15

population structure, adaptability, and potential migratory history of the species. One
approach to the study of genetic diversity in firngal populations utilizes the phenomenon
of vegetative compatibility. Thind and Keitt (1949) first described the production of dark
lines between some isolates of M. fiucticola when these isolates were grown together in
culture. This barrage formation has since been identified as a common characteristic of
vegetative compatibility in Ascomycetes. Vegetative compatibility has been used in the
study of diversity and epidemiology in a variety of plant pathogens (Chacko et al., 1994;
Kohn et al., 1990; Puhalla, 1986; Daayf et al., 1995). Sonoda et al. (1982a) determined
that these interactions could be used to genetically group isolates of M. fiucticola.

Examination of vegetative compatibility among California isolates of M.
fructicola has been conducted (Sonoda et al., 1990; Free et al., 1996). Seventy four
isolates collected from individual lesions on nectarines formed 38 VCGs, revealing 51%
incompatibility (Sonoda et al., 1990). Fifty four of the 74 isolates came from three trees,
while the remaining twenty were collected one each from twenty additional trees. The
same study revealed that while individual lesions were colonized by only one VCG,
neighboring lesions may contain different VCGs. The study of single ascospore progeny
from 82 apothecia, found that individual apothecia typically produced progeny which fell
into eight or more VCGs (Free et al., 1996). The study further revealed that the genes for
mycelial compatibility segregated independent of one another.

The purpose of this study was to examine the occurrence of vegetative incompati-
bility within Michigan field populations of M. fiucticola. These data may then be used to

begin an evaluation of the genetic diversity of Michigan field populations of the fungus.

16

Material and Methods

One-hundred sixty nine isolates of M. fructicola were collected from two cherry
orchards (designated JB and KK) near Traverse City, Michigan, and one orchard (SC)
near Paw Paw, Michigan, in summer 1993. Seventy and 61 isolates from orchards JB
and KK, respectively, were collected at random throughout the orchard from individual
sweet cherry fruit. Thirty-eight isolates from orchard SC were similarly collected from
individual sour cherry fruit. Cultures from the fi'uit were then isolated by hyphal tipping.

Methodology for compatibility testing was adapted from Sonoda et al. (1982) and
Kohn et al. (1990). Hyphal tipped Isolates were cultured on Difco potato-dextrose agar
(PDA) at 25 °C. After five days, 4 mm plugs were taken from the growing margin of
cultures. To test for compatibility, mycelial plugs of selected isolates were placed 3 cm
apart on PDA agar in 9 cm diameter plates. Compatibility test plates were incubated in
the dark at 25 °C. Each isolate was tested against itself and against all other isolates.

Tests were initially performed on half strength PDA amended with six drops/L of
McCormick’s red food color (Kohn et al., 1990), but growth was very slow. The media
was therefore amended to full strength PDA with six drops/L of red food color. Previous
study of vegetative incompatibility in Sclerotinia sclerotiorum (Kohn et al., 1990)
revealed that red pigment from the food color was deposited in the barrage zone between
incompatible isolates, thus providing an addition character for scoring interactions.
However, deposition of red pigment was not detected in this study of M. fi-ucticola.
Replicates of each test were performed on full strength PDA without red food color.

Cultures were examined at 7, 10, 14 and 21 days to identify compatible versus

 

Figure 1. Cultural interactions of isolates of Mom'lim'a fructicola. Interactions were
more easily scored as compatible or incompatible when viewed from the underside of the
plate (left). The top three isolates which grew together with no distinct interaction zone
were scored as compatible. The remaining isolates between which dark barrage zones
formed were scored as compatible.

18

incompatible reactions. Pairings were scored as incompatible if a dark barrage zone
formed between neighboring isolates, or as compatible when isolates grew together with
no distinct interaction zone (Figure 1), based upon the criterion outlined by Kohn et al.

(1990)

Results

All isolates of M. fructicola examined in this study were found to be self-
compatible. Vegetative compatibility was identified among the isolates collected from
orchard JB and among the isolates collected from orchard K, but no vegetative
compatibility was identified among the 38 isolates collected from orchard SC (Table 1).

The 70 isolates from orchard JB formed 25 VCGs. Three of these groups
contained 28, 16, and 4 isolates, respectively. The remaining 22 groups each contained
one isolate. The 61 isolates from orchard KK formed 54 VCGs. Three of these groups
contained 5, 3, and 2 isolates, respectively. The remaining 51 groups each contained one
isolate. No compatibility was identified among the isolates from orchard SC. These
results show 36, 89, and 100 % vegetative incompatibility within orchards JB, KK, and
SC respectively (Table l). Vegetative compatibility groups containing more than one
isolate are given in Appendix A.

No compatibility was identified between isolates collected from different
orchards. The 169 isolates examined formed 117 VCGs, 111 of which contained only

one isolate each. This revealed 69 % incompatibility for all isolates examined (Table 1).

19

Table 1. Frequency of vegetative incompatibility among isolates of Moniliniafi'ucticola
collected from Michigan cherry orchards.

 

 

Number of Number of Percent
Orchard Location Isolates VCGsa Incompatibilityb
SC Paw Paw 38 38 100
K Kewadin 61 54 89
JB Empire 70 25 36
All isolates 169 1 17 69

 

“ Vegetative compatibility groups
b Number of VCGs/ Number of isolates X 100

20

Discussion

The examination of vegetative compatibility among Michigan isolates of M.

fi—ucticola revealed an 69 % incompatibility among all field isolates collected. However,

this value may underestimate the actual level of incompatibility. All isolates fiom
orchard SC were collected from different trees and it is unlikely that the sample included
any fruit infected through contact with adjacent fi'uit. However, infected fruit from
orchards JB and K were collected by local extension personnel who may have taken
less care in obtaining single fruit from different trees in the orchard. While isolations
were made from a random sample of infected fruit received from these orchards, it is
possible that isolations were made from two or more fi'uit infected with the same strain.
In such an instance, the reactions recorded as compatible may reflect self-compatibility.

The frequency of vegetative compatibility varies widely among fungi. Podospora
anserina has 17 identified genetic loci affecting vegetative compatibility (Glass and
Kuldau, 1992). This would suggest a minimum of 217 possible VCGs. Verticillium
dahliae, however, has only four identified VCGs (Joaquim and Rowe, 1990). Previous
study of M. fructicola (Sonoda et al., 1990) identified 38 VCGs among 74 isolates of M.

fructicola collected fi'om within a single nectarine orchard in California.

The frequency of vegetative compatibility within a population may correlate
directly with the degree of genetic variability within the population and may provide
insight into the relative significance of sexual and asexual reproduction (Leslie, 1993). In
a predominantly asexually reproducing population, only a small number of VCGs would

be expected. Offspring of a given parent would share a common genotype, including

21

compatibility genes, and would therefore be compatible with one another. Alternatively,
the presence of a large number of vegetative compatibility groups within a population
suggests the predominance of sexual reproduction. With each generation a variety of
genotypes, and of compatibility gene combinations, would be generated thus increasing
the likelihood of incompatibility. The increased genetic variability associated with sexual
versus asexual reproduction would be translated to a higher number of VCGs.

The frequency of vegetative incompatibility revealed in this study of M. fiucticola
collected from Michigan cherry orchards suggests a high degree of genetic variability
within populations of M. fiucticola. This also suggests that the parasexual phenomenon
is an inadequate model to account for the fi'equency of genetic recombination. The large
number of VCGs revealed here would inhibit genetic exchange and recombination via
fusion of vegetative hyphae, indicating that genetic variability results primarily from
sexual reproduction.

The predominance of sexual reproduction may play a crucial role in the spread of
fungicide resistance within populations of M. fi-ucticola. In an asexual population,
resistance genes must correlate with other fitness characters in order to be maintained in
the population (Sanoamuang et al., 1995). However, in a sexually reproducing
population resistance genes may be spread, via recombination, regardless of VCG or
other characteristics. The segregation of carbendazim resistance and benomyl resistance
has been shown to occur among sexual progeny of M. fiucticola (Sanoamuang et al.,
1995; Free et al., 1996). Free et al. (1996) also showed that the segregation of benomyl

resistance is independent of the segregation of vegetative compatibility.

22

However, vegetative compatibility alone does not accurately reflect the genetic
diversity within a population, and cannot identify a predominantly sexual verus asexual
population. A study of a population of Aspergillus flavus, a fungus known to only
reproduce asexually, was found to have a large number of VCGs relative to the other
local field populations of Aspergillus (Baymen and Cotty, 1991). The authors of the
study suggested that the source of the high number of VCGs may be the influx of spores
from external sites. Similarly, the VCG diversity revealed in this study of M. fructicola
may reflect massive migration of isolates into the orchards: VCGs would be introduced
to the orchards from external populations, rather than arising independently within the
orchards.

Alternatively, VCG diversity may be favored by selection. In a population in
which VCG is under frequency dependent selection, VCG diversity could be maintained
in the absence of diversity in other, non-selected portions of the genome. Therefore,
VCG diversity may not give an accurate representation of the genetic diversity within a
population or of the predominance of sexual versus asexual reproduction.

While the vegetative compatibility data presented here appear to suggest that field
populations of M. fiucticola possess a high level of genetic diversity. The examination

of regions of the nuclear rDNA will clarify the true nature of diversity within this species.

Chapter 2
VARIATION IN INTERNAL TRANSCRIBED SPACER SEQUENCES,

ARBITRARILY PRIMED-PCR PRODUCTS, AND GROUP I INTRON
PRESENCE BETWEEN MONILINIA FRUCTICOLA AND M LAXA

Introduction

Brown rot, caused by various Mom'linia species, is an economically important
disease of stone fruit crops worldwide. In North America the brown rot fungus Monilinia
fructicola (Wint.) Honey is common in all areas where stone fruits are grown while the
European brown rot fungus M. laxa Aderhold & Ruhland is common primarily on stone
fruit crops in the West. Minor outbreaks of M. laxa have been reported on sour cherry
(Prunus cerasus L.) from Michigan (Cation et al., 1949), New York (Kable and Parker,
1963), and Wisconsin (Keitt et al., 1943). After the 19605 the disease was quiescent in
the Great Lakes region of eastern North America until 1993 when an outbreak occurred
on sour cherry in Michigan and Wisconsin (Jones and Sutton, 1996). A third brown rot
pathogen, M. fiuctigena Honey, is widely distributed in Europe but eradicated from North
America (Batra, 1979).

An examination of cultural characteristics on potato dextrose agar (PDA) is the
primary means of distinguishing isolates of M. laxa fiom those of M. fructicola. M. laxa
generally produces lobed, slow growing colonies with poor conidial production, while M.

fructicola generally produces colonies with smooth margins and abundant conidial

23

24

production (Hewitt and Leach, 1939). The similarity in colony morphology of some
isolates of M. fructicola and M. laxa often requires the use of other characteristics to
distinguish hard to identify isolates. Electrophoretic patterns (Penrose et al., 1976), germ
tube branching (Jenkins, 1965), and vegetative compatibility interactions (Sonoda et al. ,
1982) have been used to distinguish M. laxa from M. fiucticola where identifications
based on cultural characteristics are uncertain. A study of the internal transcribed spacer
(ITS) region in several species within the Sclerotiniaceae by Kohn and Carbone (1993)
suggested that sequence variation in the ITSI region could be used to distinguish M.
fiucticola from other Monilinia species. However, no M. laxa and only a single isolate of
M. fiucticola were included in their study and no sequence data for the ITSI region of M.
laxa or additional sequences for M. fiucticola were found in a search of GenBank.

Other potential methods of distinguishing the two species may include length
variation of the small subunit (SSU) rRNA gene directly preceding the ITSI region and
arbitrarily primed PCR (ap-PCR) of genomic DNA. Wilrnotte et al. (1993) identified a
group I intron in the SSU rDNA of Sclerotinia sclerotiorum, however, neither this intron
nor any other has been reported in Mom'lz'nia. Arbitrarily primed-PCR (ap-PCR) analysis
may be used to distinguish isolates of a single species (Freeman et al., 1995;
Sreenivasaprasad et al., 1992). Ap-PCR analysis of isolates of Colletotrichum
gloeosporioides from avocado with four primers led to the identification of eight distinct
phenotypes (Freeman et al. , 1996). Ap-PCR analysis may also be useful for
distinguishing M. laxa from M. fructicola.

The objective of this study was to examine the genetic diversity within

25

populations of M. fructicola and M. laxa. An additional objective was to determine if
isolates of M. fiucticola could be distinguished from isolates of M. laxa on the basis of
sequence differences in their ITSl region. In addition, the length of the SSU rRNA gene
which directly precedes the ITSI region and the pattern of ap-PCR products were
examined to determined whether there were differences between M. laxa and M.

fi-ucticola

Materials and Methods

Isolates, growth conditions, and DNA extraction. All isolates of M. fiucticola were
collected fi'om sweet and sour cherry orchards in Michigan (Table 2). One isolate was
collected in 1975 (Jones and Ehret, 1976), the others were collected in 1993 from three
geographically isolated orchards. These isolates were a subgroup of 50 to 75 isolates per
orchard, each from a separate fi'uit. Isolates used in this study were selected to include
members of common vegetative compatibility groups (V CGs) as well as members of
different VCGs within an orchard. The isolates of M. laxa were fi'om infected spurs of
sour cherry collected at three sites in Michigan and one site in Wisconsin in either 1993
or1994.

Each isolate was grown in 125 ml of potato dextrose broth (Difco Laboratories,
Detroit, M1) at room temperature for 7-10 days. Using sterile forceps, mycelium was
transferred from liquid culture to a 1.5 m1 rnicrocentrifuge tube and centrifuged for 1
minute. After the supernatant was poured off, additional mycelium was transferred to the

tube and it was centrifuged again. This process was repeated until the tube contained

26

approximately 500 u] of mycelium. The mycelium was washed with 600 ul of TE buffer,
centrifirged for 5 min, and the supernatant poured off (Cenis, 1992). Then 600 it] of
extraction buffer (200 mM Tris-HCI, pH 8.5; 200 mM NaCl; 25 mM EDTA; 0.5% SDS;
Reader and Broder, 1985) was added and the mycelium was macerated using a disposable
pellet pestle. DNA was purified by phenol/chloroform extraction, precipitated with two
volumes ethanol and 1/10 volume 3M sodium acetate overnight at —20°C, pelleted,
rinsed twice with 70% ethanol, vacuum dried, and suspended in 100 #1 of TE buffer
(Maniatas et al., 1982). DNA was stored at -20°C.
Amplification and Sequencing. All primers were synthesized by the Macromolecular
Structure Facility at Michigan State University. Primers for the ITSI region (ITSlF and
ITS2) and for the 3' end of the SSU rDNA (N S7 and NS8) were taken from Gardes and
Bruns (1993) and White et al. (1990). Primers AJ 139 and AJ 140 were developed using
Oligo primer analysis software (National Biosciences, Inc., Plymouth, MN) based on the
SSU rDNA sequence of S. sclerotiorum (GenBank accession number X69850). These
primers were used to amplify the part of the SSU rDNA just upstream from primer NS7.
The sequences and positions of these primers in the rDNA are given in Appendix B.
Microsatellite primer (GACA)4 for arbitrarily primed-amplification of genomic DNA was
taken from Freeman et al. (1995) and microsatellite primer (GTG)5 was taken fi‘om
Longato and Bonfante (1997). All polymerase chain reactions (PCR) were performed in
MJ Research thermocycler model PTC-150 (MJ Research, Inc., Watertown, MA).

PCR reactions for amplification of the ITSI region and the SSU rRNA gene were

carried out in a 50 ,ul volume containing 20 mM Tris-HCl pH 8.4, 50 mM KCl, 1.5 mM

27

MgClz, 0.1% Triton X-100, 160 uM each dNTP, 50 pmols of each primer, 2 U T aq DNA
polymerase, and 10-100 ng of template DNA and overlaid with two drops of mineral oil
(Kohn et al., 1991). A negative control containing no template was also included. The
thermal program consisted of an initial denaturation at 94°C for 3 min, followed by 35
cycles of denaturation at 94°C for 1 min, annealing at 50°C for 1 min, and extension at
72°C for 3 min, followed with a final extension at 72°C for 7 min. PCR amplifications
were performed two or three times from each isolate.

PCR products were observed by electrophoresis on a 1% agarose gel (SeaKem
LE, F MC BioProducts, Rockland, ME) in IXTAE buffer. Products were purified from
the PCR reaction mix using a DNA binding resin (Promega Wizard PCR Preps DNA
purification system, Promega, Madison, WI). Automated flourescent sequencing of PCR
amplification products from each primer set was performed by the MSU-DOE-PRL Plant
Biochemistry Facility using the ABI Catalyst 800 for Taq cycle sequencing and the ABI
373A Sequencer for the analysis of products. All products were sequenced from both
strands. Sequences of the two strands were used to generate a consensus strand for each
isolate. Ambiguities revealed by alignment of the two strand sequences were resolved by
sequencing the region of DNA of the given isolates again. Sequences obtained from
additional sequencing were added to the original two to generate a consensus. Identity of
the ITSI sequence was confirmed by aligning the consensus sequence from PCR products
with ITSI sequences retrieved from GenBank: M. fructicola (Accession number
U21815), M. fiuctigena (1121825), M. oxycocci (U21833), and M. megalospora

(U21834), Sclerotinia sclerotiorum (U21810), and S. minor (U21818). The SSU

28

sequence was confirmed by aligning the consensus sequence from PCR products with the
SSU sequence for Sclerotinia sclerotiorum (X69850) retrieved from GenBank. Sequence
comparison was performed by MegAlign (DNASTAR, Inc., Madison, WI) using the
Clustal V method to examine sequence distances for all sequence pairs and then align
sequence groups (Higgins and Sharp, 1989).

Ap-PCR amplification of genomic DNA. Random amplifications using microsatellite
primers (GACA), and (GTG)5 were performed on 36 isolates following the protocol of
Freeman et al. (1995). Each reaction was performed in a 50 #1 volume containing 20
mM Tris-HCl pH 8.4, 50 mM KCl, 1.5 mM MgC12, 0.1% Triton X-100, 160 MM each
dNTP, 50 pmols of primer, 2 U T aq DNA polymerase, and 10-100 ng of template DNA
and overlaid with two drops of mineral oil. A negative control containing no template
was also included. The thermal program consisted of an initial denaturation at 95 °C for 5
min, followed by 30 cycles of denaturation at 95 °C for 30 sec, annealing at 48 °C for 30
sec, and extension at 72°C, for 1.5 min, with a final extension at 72°C for 4 min. PCR
amplifications were performed two or three times per isolate. PCR products were
separated on a 1.5% TAE agarose gel and electrophoresed at 80 V for 2 h.

Restriction digestion. The ITSl regions from twelve isolates of M. fi-ucticola and eight
isolates of M. laxa were amplified and purified as described above. The purified product
from each reaction was digested with 5 U of Mse I (ICN Pharmaceuticals Inc., Costa
Mesa, CA) at 37°C for 3 h. Restriction fiagments were separated on a 2% TAE agarose
gel.

Nucleotide sequence accession numbers. The nucleotide sequences of the ITSl regions

29

of M. laxa and M. fructicola are available in Genbank under accession numbers AF 10503
and AF 10500, respectively. ITS sequences showing sequence divergence are available
under accession number AF 10540 for M. laxa and AF 10501 and AF 10502 for M.
fructicola. The nucleotide sequence of the group I intron of M. fructicola including the
flanking SSU rRNA gene sequences is available under number AF 10505. The sequence

of the 3' end of the SSU rRNA gene of M. laxa is available under number AF 010506.

Results

Sequence analysis of the IT S1 region. PCR with primers ITSlF and ITSZ yielded a
product of approximately 250 bp from each of 30 isolates of M. fructicola and eight
isolates of M. laxa (Table 2). The nucleotide sequence of the 250-bp PCR fragment from
each of 12 isolates of M. fructicola and four isolates of M. laxa revealed the 3' end of the
SSU rRNA gene, a 146-bp ITSI region, and the 5' end of the 5.8S rRNA gene (Figure 2).
The partial SSU rDNA and 5.8S rDNA sequences of all 16 isolates were identical. These
flanking sequences also exhibited sequence identity with sequences surrounding the ITSI
region in M. fructicola, M. fructigena, M. oxycocci, M. megalospora, S. sclerotiorum,
and S. minor obtained from GenBank.

Alignment of the ITSI sequences from the 12 isolates of M. fi-ucticola revealed
only minor nucleotide differences among the isolates (Figure 2). No correlation was
found between ITSI sequence variation and VCG. The consensus ITSI sequence for
isolates of M. fructicola from Michigan matched the M. fructicola U21815 ITSI

sequence. Alignment of the ITSI sequences from the four isolates of M. laxa revealed

30

Table 2. Isolates of Moniliniafiucticola and M. laxa used in polymerase chain reaction
(PCR)-mediated analysis and the results of analysis.

PCR-ITSI PCR-SSU AP-PCR

 

Year of fragment fragment band
Isolate isolation Locality Host (bp)' (bp)" patternsc
Moniliniafructicola
SC-5 1993 Paw Paw, MI Montrnorency sour cherry 250 nd 2, 4
S041 1993 Paw Paw, MI Montrnorency sour cherry 250 (146) 940(421)“ nd
SC-44 1993 Paw Paw, MI Montrnorency sour cherry 250 (146) 940 nd
SC-49 1993 Paw Paw, MI Montrnorency sour cherry 250 (146) 940 nd
SC-55 1993 Paw Paw, MI Montrnorency sour cherry 250 940 nd
SC-57 1993 Paw Paw, MI Montrnorency sour cherry 250 (146) 940 2, 4
SC-59 1993 Paw Paw, MI Montrnorency sour cherry nd 940 2, 4
SC-70 1993 Paw Paw, MI Montrnorency sour cherry 250 940 2, 4
SC-75 1993 Paw Paw, MI Montrnorency sour cherry nd 940 2, 4
KK-ll 1993 Kewadin, MI Sweet cherry 250 940 2, 4
KK-15 1993 Kewadin, MI Sweet cherry 250 940 2, 4
KK~20 1993 Kewadin, MI Sweet cherry 250 (146) 940 (421)“ 2, 4
KK-22 1993 Kewadin, MI Sweet cherry 250 940 2, 4
KK-27 1993 Kewadin, MI Sweet cherry nd 940 nd
KK-29 1993 Kewadin, MI Sweet cherry nd nd 2, 4
KK-31 1993 Kewadin, MI Sweet cherry 250 940 2, 4
KK-33 1993 Kewadin, MI Sweet cherry nd 940 (421)“ n
KK-45 1993 Kewadin, MI Sweet cherry 250 940 2, 4
KK-48 1993 Kewadin, MI Sweet cherry 250 (146) 940 2, 4
KK—49 1993 Kewadin, MI Sweet cherry 250 nd 2, 4
KK-SO 1993 Kewadin, MI Sweet cherry nd nd 2, 4
KK-54 1993 Kewadin, MI Sweet cherry 250 (146) 940 2, 4
JB-2 1993 Empire, MI Sweet cherry 250 940 2, 4
JB-4 1993 Empire, MI Sweet cherry 250 940 2, 4
JB-6 1993 Empire, MI Sweet cherry 250 940 n
JB-8 1993 Empire, MI Sweet cherry 250 940 2, 4
JB-9 1993 Empire, MI Sweet cherry 250 (146) 940 2, 4
JB-lO 1993 Empire, MI Sweet cherry 250 940 2, 4
JB-ll 1993 Empire, MI Sweet cherry 250 (146) 940 2, 4
JB-12 1993 Empire, MI Sweet cherry 250 940 2, 4
JB-13 1993 Empire, MI Sweet cherry 250 (146) 940 2, 4
JB-14 1993 Empire, MI Sweet cherry 250 940 2, 4
JB-26 1993 Empire, MI Sweet cherry 250 (146) 940 (421)“ n
JB-62 1993 Empire, MI Sweet cherry 250 940 2, 4
JB-65 1993 Empire, MI Sweet cherry 250 940 2, 4
CB-2 1975 Hart, MI Montrnorency sour cherry 250 (146) 940 3, 4

 

31

Table 2. Continued

 

Monilinia laxa

DG-7 1993 Suttons Bay, MI Meteor sour cherry 250 520 l, 5
DG-8 1993 Suttons Bay, MI Meteor sour cherry 250 (146) 520 1, 5
NP-l 1993 Northport, MI Montrnorency sour cherry 250 520 1, 5
NP-2 1993 Northport, MI Montrnorency sour cherry 250 (146) 520(460)° l, 5
WI-2 1993 Sturgeon Bay, WI Montrnorency sour cherry 250 (146) 520(460)° 1, 5
WI-3 1993 Sturgeon Bay, WI Montrnorency sour cherry 250 520 1, 5
Grants 1994 Shelby, MI Erdi Boterrno sour cherry 250 (146) 520 1, 5
Meteor 1994 Suttons Bay, MI Meteor sour cherry 250 520 1, 5

 

‘ Length of the amplification fragments obtained with primers ITSlF and ITS2. Number in
parentheses indicates the actual length of the ITS] region alone, determined by sequence
analysis and confirmed by comparison to sequences retrieved from GenBank.

b Length of the SSU DNA amplified with primers A1139 and A1140.

° Band patterns 1,2, and 3 were obtained using microsatellite primer (GACA),. Pattern 3
shares six bands in common with pattern 2 and one band in common with pattern 1. Band
patterns 4 and 5, obtained with primer (GTG),, share no bands in common.

“ Number in parentheses indicates the length of the intron determined by sequence analysis.
‘ Number in parentheses indicates the region of the SSU rDNA confirmed by sequence
analysis.

nd = not done

32

Figure 2.

~ATGCCCGAAAGGGTAGACC‘I‘CCCACCC‘l‘TGTGTA‘I‘TA
I fifi

mjoriCYCATTACAGAGTTC
I
40 50
l
“720 o o o o o o o o o o o I o o a o o o a o o o o o o o
“-48 o o c o o o c o o o o o o o o c o a o o o a o o o
“.54 . . . . . O C . . . . . . . . . . . . . . . . . . . . . . . . .

”-9 oooooooouoooOOOOOOCOOOOCOIOOOOO

........ 50
.....50
.......SO
..........50

Ja-rr . . .. . .. . .. . .. . .. . .. . .. . .. .. . .. . .. . .. . . ..
33-13 . . . . . . . . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . so
JB-26 . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . . .. . . .. . . .. . . so
sc-41 . . .. . .. . .. . .. . .. . .. . .. . .. .. . .. . .. . .. . . .. . . .. . . .. . . so
SC-41 . . .. . .. . .. . .. . .. . .. . .. . .. .. . .. . .. . .. . . .. . . .. . . .. . . so
sc—49 . .. . .. . .. . .. .r.. . .. . .. . .. . .. . .. . .. . .. . . .. . . .. . . .. . . so

. .. .. . .. . .. . .. . .. . .. . so

SC-57 . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
CB-Z . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
DG-B . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
GRANTS . . . . . . . . . . . . . . . . . . . . . . . . . . .
WI-Z . . . . . . . . . . . . . . . . . . . . . . . . . . .

NP-z C O O O O O O O I I I O O O O O I O O C I C O D I O I D C

......50
...............SO
.......50
..........50

Hajority'!‘TACTTTGTTGCTTTGGCGAGCTGCCTTC
r

ICK-ZO . . . . . . . . . . . . . . . . . . . . .
100-48 . . . . . . . . . . . . . . . . . . . . . . . . . . .
100-51 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
JB-9 . . . . . . . . . . . . . . . . . . . . . . . . . . . .
JB-ll . . . . . . . . . . . . . . . . . . . . . . . . . .
JB-13 . . '. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
JB-26 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
SEC-41 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
SC-44 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
SC-49 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
SC—57 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
. . . . . 100.

. . . . . . . . . . 100
. . . . 100
. . . . . . . . . . 100
. . . . . . . . . . 100

CB-Z . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
DG-B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
GRANTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
WI-Z . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C 100

NP-Z ..................

HajorityGGATAATTAAACTCTTTTTATTAATGTCGTCTGAGTACTATAT
r I

iCK-ZO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
KK-48 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
KX-54 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

JB-9 ............................................
Js-rr . . . . . . . . . . . . . . . . . . . . .. L . . . . . . . . . . . . . . . . . . . . . . . . . . rso
JB-13 . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . rso
JB-26 . .. . .. . .. . .. . .. . .. . .. .. . .. .. . .. . .. . .. . .. . .. . .. . .. . rso
sc-41 . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . . .. . . .. . . .. . . 150
SC-41 . . .. . . .. . .. . . .. . .. . .. . .. . .. . .. . .. . .. . . .. . . .. . . .. . . 150
sc-49 . . . .. . . .. . . .. . . .. . .. . .. . .. . .. . .. . . .. . . . .. . . .. . . A . . 150
sc-57 . . .. . . .. . .. . . .. . . .. . .. . .. . .. . .. . .. A . .. . . . . . .. . . A . . 150
cs-z . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . 150
oc-a . . .. . . . .. . .. . .. . .. .. . .. .. . .. . .. . .. . .. . .. . .. . .. . . 150

.. . .. . .. . .. . .. . .. . . .. . . .. . . .. . . 150

GRANTS .

>>>)-
nann-

NP-Z .

33

Figure 2 (cont’d).

HajorityAAAACTTTCAACAACGGA

 

160

r
KK-ZO .................. 168
KK-48 ........ ' .......... 168
KK-54 .................. 168
33-9 .................. 168
33-11 .................. 168
JB-13 .................. 168
JB-26 .................. 168
SC-41 .................. 168
SC-14 .................. 168
SC-49 .................. 168
SC-57 G ................. 168
CB-Z .................. 168
DG-B .................. 168
GRANTS .................. 168
WI-Z .................. 168
NP-Z .................. 168

Alignment of the ITSl sequences of isolates of Monilinia fructicola and M. laxa
determined in this study. Positions 1-5 represent the 3' end of the 18S (SSU) rRNA gene.
Positions 151-168 represent the 5' end of the 5.8S rRNA gene. A dot (.) indicates a
match and a letter indicates a mismatch. Letters and numbers to the left of sequences
indicate the isolate from which DNA was purified for PCR amplification. The first
twelve sequences are from isolates of M. fructicola. The last four isolates, DG-8, Grants,
WI-2, and NP-2, are from isolates of M. laxa.

34

 

 

 

 

 

 

 

 

 

Majority CAGAGTTCATGCCCGAAAGGGTAGACCTCCCACCCTTGTG
T’ r r r
10 20 3O 40
I g 1 l
M. frucitcola ........................................ 40
M. laxa ........................ G ............... 40
S. sclerotiorum ........................................ 40
Majority TATTATTACTTTGTTGCTTTGGCGAGCTGC-CTTCGGG-C
I r . I T
50 60 7O 80
r r l JL
M. frucitcola .............................. — ....... - 78
M. laxa .............................. - ....... - 78
S. sclerotiorum .............................. T ....... G . 80
Majority CTTGTATGCTCGCCAGAGAATAATXAAACTCTTTTTATTA
f I I I
90 100 110 120
I I L I
M. frucitcola .................. G ..... T ............... 118
M. laxa ........................ C ............... 118
S. sclerotiorum ...................... T C.A ............... 120
Majority ATGTCGTCTGAGTACTATATAATAGTTA
r r
130 140
L l
M. frucitcola ............................ 146
M. laxa ............................ 146
S. sclerotiorum ............................ 148

Figure 3. Alignment of the consensus sequences of the ITSI region of Monilinia
fructicola and M. laxa determined in this study with Sclerotinia sclerotiorum (U21810).
A dot (.) Indicates a match, a dash (-) indicates an insertion/deletion event, and a letter
indicates a mismatch.

35

IM 1 2 3 4 5 6 7 8

600-
400-

200-

 

Figure 4. Restriction fragment length polymorphism in Mse I-digested PCR-amplified
ITSl DNA from Monilinia laxa isolates DG—8, NP-2, WI-2, and Grants (lanes 1-4) and
M. fructicola isolates SC-41, KK-20, JB-13, and CB-2 (lanes 5-8). Lane M: 100 bp size
marker.

36

only minor sequence variation among isolates of M. laxa (Figure 2.). Alignment of the
consensus ITSl sequences for each species revealed three nucleotide differences between
M. laxa and M. fructicola (Figure 3). M. fructicola showed transition mutations at
positions 25, 99, and 105 (Figure 3). The base pair change at position 105 created a Mse
I restriction site in M. fi-ucticola, creating a restriction fragment 14 bp smaller than the
corresponding fragment in M. laxa (Figure 4). Comparison of the consensus nucleotide
sequences for the ITSl region of M. laxa and of M. fiucticola, M. fiuctigena U21825, M.
megalospora U21834, M. oxycocci U21833, S. sclerotiorum U21810, and S. minor
U218l8 showed 98, 96, 83, 74, 97, and 97 % similarity, respectively.

Identification of an intron in M. fi'ucticola. PCR amplification of the 3' end of the SSU
rDNA with primers NS7 and NS8 yielded a product of approximately 380 bp from both
M. fructicola and M. laxa. Amplification using primers A1139 and A1140 yielded a
product of approximately 940 bp from 32 isolates of M. fructicola and of 520 bp from
eight isolates of M. laxa (Table 2, Figure 5). Sequence analysis of the PCR products
from four isolates of M. fructicola and two of M. laxa revealed the presence of a 421 bp
region in M. fructicola that was absent in M. laxa (Figure 6). However, the flanking
sequences (460) from both species exhibited 99 % similarity. Given the large size of the
SSU rRNA fragment, the entire region could not be accurately sequenced from one
strand. Approximately 30 bp, including the primers and a few adjacent nucleotides, could
not be obtained for either end. Alignment of the SSU rDNA sequences for M. fructicola
and S. sclerotiorum (X69850) showed that the insert in M. fructicola was located at the

same position as a group I intron in S. sclerotiorum. The flanking regions of the SSU

37

M1234567

940-
520-

 

Figure 5. Products of PCR amplification of a region of the SSU rRNA gene of Monilinia
fi-ucticola and M. laxa using primers A1139 and A1140. Lane M, 100-bp ladder; lanes 1-
3, M. fructicola isolates SC-41, KK-20, JB-13; lanes 4-7, M. laxa isolates DG-8, NP-2,
WI-2, Grants.

fructicola
laxa

fructicola
laxa

fructicola
laxa

fructicola
laxa

fructicola
laxa

fructicola
laxa

fructicola
laxa

fructicola
laxa

fructicola
laxa

fructicola
laxa

fructicola
laxa

fructicola
laxa

fructicola
laxa

fructicola
laxa

38

CATTAATCAGTGAACGAAAGTTAGGGGATCGAAGACGATCAGATACCGTCGTAGTCTTAA
CATTAATCAGTGAACGAAAGTTAGGGGATCGAAGACGATCAGATACCGTCGTAGTCTTAA

CCATAAACTATGCCGACTAGGGATCGGGCGATGTTATCTTTTTGACTCGCTCGGCACCTC
CCATAAACTATGCCGACTAGGGATCGGGCGATGTTATCTTTTTGACTCGCTCGGCACCTC

ACGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAG
ACGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAG

AAATTGACGGAAAGGCACCACCAGGCGTTAACTGCAGTAACTCTGCGCCGAAAAGCAGCC
AAATTGACGGAAAGGCACCACCAGGCGT --------------------------------

CGTAAGGGTGAGGTGGTTCGCCTTCAACTTAAATGCTAGTCTATTATAAGGCTACATTCC

----—------—-----------------------------------------—----—-

ACTCAATGCTTTCGAGTGAATATAAAACGGGAGCCTGCGGCTTAATTTGACTCAACACGG
----------------------------- GGAGCCTGCGGCTTAATTTGACTCAACACGG

GGAAACTCACCAGGTCGAGACACAATAAGGATTGACAGATTGAGAGCTCTTTCTTGATTT
GGAAACTCACCAGGTCCAGACACAATAAGGATTGACAGATTGAGAGCTCTTTCTTGATTT

TGTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGCTTAATTGCGA
TGTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGCTTAATTGCGA

TAACGAACGAGACCTTAACCTGCTAAATAGCCCGGCTAGCTTTGGCTGGTCGCTGGCT
TAACGAACGAGACCTTAACCTGCTAAATAGCCCGGCTAGCTTTGGCTGGTCGCTGGCT

60
60

120
120

180
180

240
208

300
208

360
208

420
208

480
208

540
208

600
208

660
239

720
299

780
359

839
417

Figure 6. Alignment of the SSU rDNA sequences amplified with primers A1139 and
A1140 from Moniliniafructicola strain KK-20 and M. laxa strain DG—7. The M.
fiucticola sequence contained an 421 bp insertion (indicated by dashes(-)) relative to the
sequenceirffil.hzna.

39

rDNA of M. fructicola and S. sclerotiorum showed 99% similarity, however, the intron
from M. fructicola showed only 54% similarity to the intron from S. sclerotiorum.
Analysis of the sequence and secondary structure of the 421-bp insertion sequence
from M. fructicola demonstrated that it possessed the characteristic features known to be
conserved among group I introns (Cech, 1988). The conserved GU immediately
preceding the 5' splice site and the G at the 3' splice site were present. The sequence also
contained the four conserved sequence elements P, Q, R, and S and the order of their
occurrence in the sequence (5'- P-Q-R-S-3') necessary for the formation of the group I
intron core structure. Conserved sequence elements P and Q base-paired to form helix
P4, and elements R and S paired to form helix P7. Also, the 3' end of element Q was
base-paired with the 5' end of element R to begin helix P6. Helices P1 to P9, including a
pseudoknot formed by P3 and P7, were also identified. Five fungi containing introns in
the SSU rDNA at the same location as the insert in M. fructicola were selected from
GenBank for comparison of the intron region (Figure 7). All contained the conserved
sequence elements characteristic of group I introns, but the sequences varied in length
from 310 to 453 bp and showed little sequence similarity outside of the conserved
sequence elements.
Comparison of M. fructicola and M. laxa by ap-PCR. From M. fructicola DNA the
microsatellite primer (GACA), generated six PCR products that ranged in length from
200 to 1500 bp (Figure 8). Isolate CB-2 showed a distinct seventh band of approximately
1500 bp that was not observed for any of the other 27 isolates of M. fi'ucticola. Ap-PCR

amplification of DNA from eight isolates of M. laxa generated three PCR products of

Mf
Ss
Urn
By
Pi
Pl

Mf
Ss
Um
By
Pi
P1

Figure 7. Alignment of the conserved sequence elements of group I intron within the

40

5' splice
Intron site
size SSU gene 1 P Q

420 5' - - - ACCACCAGGCQQ- - - -AAUUGCGGG—AA- - - -UAUCCGCAUC- - — -
310 5' - — - ACCACCAGGCQLJ- - - -AACUGCGGGGAA- - - -AAUCCGCAUC- - - -
410 5'- — ~ACCACCAGGA911- - - -AAUUGCGGGAAA- - - -AAUCCGCAGC- - - -
453 5'- - -ACCACCAGGU§LJ- - - -AACUGCGGGAAA- - - -UAUCCGCAUC- - - -
339 5' - - -ACCACCAGGA§_U- - - -AAUUGCGGGAAA- - - -AAUCCGCAGC- - - -
339 5'- - - ACCACCAGGAGLI- - - - AAUUGCGGGAAA- - - -AAUCCGCAGC- - - -

3' splice
site
R s I ssu gene
- — - GUUCAGAGACUAAA— - - - AAGAUAUAGUCC— - - -AC§ GGAGCCUGCG- - — 3'
— - — GUUCAGAGACUAAA— - - - AAGAUAUAGUCC- - - -AU§ GGAGCCUGCG- - - 3'
- - — GUUCAGAGACUAAA- - - —AAGAUAUAGUCC- — - -UCQ GGAGCCUGCG- - - 3'
— - — GUUCAGAGACUAGA— - - - AAGGUAUAGUCC- - - -AA§ GGAGCCUGCG- - - 3'
- - - GUUCAGAGACUAGA— - - - AAGAUAUAGUCC- - - -AUG GGAGCCUGCG- - - 3'
- — — GUUCAGAGACUAGA— - - — AAGAUAUAGUCC— - - - AUG GGAGCCUGCG- - - 3'

nuclear SSU rDNA of Moniliniafructicola with introns fiom five other firngi. The

conserved GU immediately preceding the 5' splice site and the conserved G preceding the
3' splice site are underlined. Fungi (and GenBank accession number) are abbreviated:

Mf, Moniliniafi'ucticola; Ss, Sclerotinia sclerotiorum (X69850); Urn, Ustilago
maydis(X62396); By, Bensingtonia yamatoana (D38239); Pi, Protomyces inouyei
(D11377), and P1, P. lactucae-debilis (D14164).

41

AA 1 2 3 4 5 6 7’ 8

 

B 12345678

1500-

1200-
800-

 

Figure 8. Arbitrarily primed-PCR amplification of genomic DNA from Monilinia
fiucticola and M. laxa using microsatellite primers (GACA)4 in A and (GTG), in B. Lane
M, 100-bp ladder of empty; lanes 1-4, M. fructicola isolates SC-4l, KK-20, JB-13, CB-2;
and lanes 5-8, M. laxa isolates DG—8, NP-2, WI-2, Grants. PCR products in A and B
were separated by electrophoresis through 1.5% agarose gels.

42

approximately 410, 1150, and 1500 bp in length. The patterns for isolates of the two
species did not share any bands in common except the 1500 bp product in M. laxa which
was shared by M. fructicola isolate CB-2.

Amplification of M. fructicola DNA with the microsatellite primer (GTG)5 generated
six PCR products that ranged in length from 300 to 1500 bp (Figure 8). Amplification of
DNA fiom eight isolates of M. laxa generated four PCR products of approximately 350,
500, 900, and 1000 bp in length. No common bands were revealed between the two

species.

Discussion

M. fructicola and M. laxa both cause brown rot of stone hit and are of considerable
economic importance in the United States. Traditionally, M. fructicola was considered
the American brown rot fungus and M. laxa the European brown rot fungus. With the
occurrence of M. laxa in stone fruit growing regions of the United States it has become
necessary to develop an understanding of these two pathogens and the relationship
between them. The examination of the ITSl region from a large number of isolates of M.
fructicola and M. laxa from different geographic areas of Michigan and Wisconsin
suggests that each- species has a highly conserved ITSl region. Sequence analysis of the
ITSI region also revealed only three nucleotide differences between the two species,
suggesting that M. laxa is more closely related to M. fructicola than to other Monilinia
species. Comparison of the M. laxa ITSl sequence with the previously reported sequence

of .M. fructigena (U21825) suggests that M. fiuctigena is also more closely related to M.

43

laxa than to those Monilinia species which do not cause brown rot. These results are
consistent with the report that several species in the Sclerotiniaceae have highly
conserved ITSl regions and that the number of nucleotide differences between some
members of the Sclerotiniaceae is quite limited (Kohn and Carbone, 1993). The limited
number of nucleotide differences between the M. laxa and M. fructicola, and the limited
diversity within each species, suggests the relatively recent divergence of these species.

While M. laxa and M. fructicola both occur on stone fruit crops, they are often
identified based on colony morphology when grown on PDA (Hewitt and Leach, 193 9)
and a few other criteria (Jenkins et al., 1965; Penrose et al., 1976; Sonoda, 1982).
However, accurate identification based on colony morphology is not always possible
given the similarity in morphology of some isolates. The occurrence of morphologically
similar and therefore hard to identify isolates requires the development of alternative
means of distinguishing the species. Although the ITSl sequences for M. laxa and M.
fructicola are very closely related, it was possible to differentiate them based on Mse I
restriction digestion of PCR amplification products of the ITSl region. The apparent
highly conserved nature of the ITSl region supports the efficacy of this method to
distinguish the species. Restriction digestion relies on single base pair changes that in
more variable species may change quickly. The highly conserved nature of the ITSl
regions of M. laxa and M. fi-ucticola indicate that the sequence is relatively static in these
species.

The detection of the same group I intron in the SSU rRNA gene of all isolates of M.

fructicola regardless of their geographic origin in Michigan indicates that this is also a

44

highly conserved character of M. fructicola. Presence of the intron in an isolate collected
in 1975 further suggests that this intron is a stable character within Michigan populations.
It was possible to differentiate M. laxa from M. fiucticola on the basis of length variation
in PCR-amplifications products of the intron-containing region of the SSU rRNA gene.
As with the ITSl region, the highly conserved nature of the intron indicates that it is a
stable character for differentiation of M. laxa from M. fructicola.

Ap-PCR of genomic DNA also provided a rapid means of distinguishing between the
species. Ap-PCR arbitrarily amplifies genomic DNA and the source of DNA in each
band of the pattern is unknown. Patterns based on DNA of unknown origin are
consequently of unknown stability within the populations. However, with primer
(GACA), all but one isolate of M. fi'ucticola yielded the same band pattern and all isolates
of M. laxa yielded a common pattern. With primer (GTG)s all isolates of M. fructicola
yielded the same band pattern and all isolates of M. laxa yielded a common band pattern.
This consistency of banding patterns within each species suggests that this is also a stable
character for differentiation of the two species.

Data from chapter 1 indicates a relatively high level of vegetative compatibility
group (V CG) diversity among Michigan field populations of M. fructicola. Such
diversity was interpreted as a possible indication of a corresponding high level of genetic
variability. However, the data presented here indicates the M. fructicola possesses little
intra-species genetic variability. If the VCG loci are under frequency dependent
selection, VCG diversity could be maintained even in the absence of diversity in

unselected regions of the genome. The results of this study, combined with the data

45

acquired from the study of VCG diversity among the same isolates, indicate that VCG
diversity is highly selected for in M. fructicola.

The results of this study also demonstrate that while M. laxa and M. fructicola are
closely related they can be separated by the restriction digestion of the ITSl sequence,
length variation in the SSU rRNA gene due to the presence of an intron, and in ap-PCR
band patterns. Isolates that are difficult to identify on the basis of colony morphology can
be positively identified with these PCR-mediated techniques. However, this study
utilized only isolates from Michigan and a few fi'orn Wisconsin. Further research is
necessary to determine if these techniques are applicable to the global populations of M.

laxa and M. fructicola.

Chapter 3

FINAL CONCLUSIONS

Molecular data from Chapter 2 identified three PCR-mediated techniques for
rapidly distinguishing between isolates of M. fructicola and M. laxa. Isolates of M.
fructicola used in this study were collected from three geographically isolated orchards,
and while most isolates were collected in 1993, one isolates was originally collected in
1975. Isolates of M. laxa were collected from three geographically isolated orchards in
Michigan and from one orchard in Wisconsin. All isolates within a species possessed the
same molecular characteristics regardless of geographic origin or the year of collection.
All isolates of M. fructicola possessed an Mse I restriction site in the ITSl region which
was not present in the ITSl region of M. laxa. Amplification of the intron containing
region of the SSU gene resulted in a product of approximately 940 bp from M. fructicola,
but only 520 bp from M. laxa. All isolates within a species also shared a common ap-
PCR band pattern. The occurrence of these common characters across geography and
time suggests the stability of these characters and indicates that they will be useful
characteristics for distinguishing between Michigan isolates of the two species.

While providing tools for distinguishing between the two species, sequence
analysis of the ITSl region, the presence of an intron in the SSU rDNA, and ap-PCR

patterns also revealed limited genetic diversity within M. fructicola and M. laxa.

46

47

However, the high fi'equency of vegetative incompatibility revealed in Chapter 1
suggested that M. fructicola populations are very genetically diverse. The high frequency
of VCG diversity was also interpreted to indicate that sexual reproduction may occur
more frequently in Michigan than was previously believed. The data from the two
previous chapters appear to be in direct conflict with one another, but there are possible
resolutions to this apparent conflict. Two points must be kept in mind when interpreting
the data presented here: 1) VCG diversity may not reflect genetic diversity within M.
fructicola and 2) the regions of the genome utilized in this study may not be the most
appropriate regions for study of genetic diversity within M. fructicola.

In Chapter 1, it was stated the VCG diversity reflects genetic diversity within a
species. However, this statement is an oversimplification of the actual events involved in
establishing and maintaining diversity within a population. The suggestion that VCG
diversity reflects genetic diversity may be a generalization to which M. fructicola is an
exception. Clearly, fi'om the data presented here, VCG diversity reflects genetic diversity
in neither nuclear ribosomal DNA nor in microsatellite regions with the GACA or GTG
motif. It is, therefore, also possible that VCG diversity in M. fructicola does not reflect
diversity in other region of the genome.

Furthermore, vegetative compatibility is governed by multiple loci (referred to
here are vegetative incompatibility, or vic, loci) within the genome. Only a small change
at a single vic locus is required to generate an incompatible reaction. A single amino acid
change at one locus has been shown to be sufficient to elicit vegetative incompatibility in

the fungus Podospora anserina (Deleu et al., 1993). The ability of a small change at any

48

one of several loci to elicit vegetative incompatibility indicates the relative ease with
which new VCGs could arise in a population. With various regions of the genome under
different selection pressures, it is not unreasonable to consider that vic loci might be
under higher selection pressure than other regions of the genome. Frequency dependent
selection, for example, acting on via loci could select for the maintenance of a large
number of different VCGs. A high level of VCG diversity could be maintained in the
absence of an equivalent amount of diversity in other regions of the genome, and
regardless of the role of sexual or asexual reproduction in the population.

While VCG diversity may not reflect genetic diversity within M. fi-ucticola, it is
also possible that the regions of the genome selected for study may not be the most
appropriate regions for the intended purpose. This study utilized only nuclear rDNA and
microsatellite regions with GACA or GTG motif. As with diversity at the vic loci,
diversity in these portions of the genome may not accurately reflect diversity in other
regions of the genome. The examination of different regions or the use of different
molecular techniques may have revealed greater diversity than that which was found in
this study of rDNA and microsatellites.

A final analysis of this research, keeping the above points in mind, suggests that
M. fructicola is a species of limited genetic diversity, but that VCG diversity is highly
selected for in Michigan populations of this pathogen.

The data revealed in this study has implications beyond an assessment of genetic
diversity within M. fructicola and M. laxa. Sonoda et al. (1982b) suggested the use of

vegetative compatibility reactions to distinguish between isolates of M. fructicola and M.

49

laxa: The formation of barrage zones (incompatible reactions) between isolates of the
two species could serve as a means of separating M. laxa from M. fructicola. However,
this study revealed a large number of incompatible reactions among isolates M. fructicola
alone. While an incompatible reaction may indicate isolates of different species, as
suggested by Sonoda et al. (1982b), it may also indicate incompatibility between isolates
of one species. Incompatibility reactions will therefore be inadequate for distinguishing
M. laxa from M. fructicola.

Molecular data clearly indicate high conservation of the ITSl regions of Michigan
isolates of M. fructicola and M. laxa, suggesting that the ITS] sequences determined in
this study have become the fixed ITSl types for these populations. Concerted evolution
during meiotic recombination would act to homogenize the ITS region (Dover, 1982),
leading to this fixation of ITS types within the population (O’Donnell, 1992).

Previous studies of the ITS sequences of Monilinia species also suggested the
highly conserved nature of the ITS sequences within the Sclerotiniaceae, including the
Monilinia species (Carbone and Kohn, 1993; Holst-Jensen et al., 1997). Comparison of
ITSl sequences determined in this study with those from previous studies further
indicated the high conservation of the ITSl region within the two species. Two
previously examined isolates of M. fructicola from Ontario, Canada, (Carbone and Kohn,
1993; Holst-Jensen et al., 1997) showed 100% nucleotide sequence similarity to the
Michigan isolates examined here. Three isolates of M. laxa from Norway and one
isolate from Italy (Holst-Jensen et al., 1997) showed greater than 99% nucleotide

sequence similarity (one bp difference) to the Michigan isolates studied here. This

50

comparison clearly indicates the highly conserved nature of the ITS] region of M.
fructicola and M. laxa, and further indicates that ITSl sequence variation will not be a
useful technique for identifying diversity within the global population.

The vegetative compatibility data and the molecular data also have implications
for control of the disease. A high number of VCGs present within an orchard will limited
the ability of advantageous characters, such a fungicide resistance, to be spread
throughout the population. The apparent insignificant level of sexual reproduction within
Michigan populations limits genetic recombination during meiosis as a source of genetic
variation and spread of advantageous characters. The high number of different VCGs
within an orchard also limits genetic exchange via vegetative hyphal firsion, and therefore
inhibits the spread of characters.

The limited genetic diversity suggested by the molecular data also has
implications for control. Limited genetic diversity within any species restricts the ability
of that species to rapidly adapt to a changing environment. A more genetically diverse
population is more able to rapidly adapt to changes. If M. fiucticola is as limited in
genetic diversity as the data suggest, then the species is also less able to rapidly adapt to
changing environmental conditions, such as the application of fungicides. The high
frequency of VCG diversity and the limited genetic diversity within M. fructicola,
therefore, suggest that the spread of characters such as fungicide resistance will occur at a
slower rate than if the species had fewer VCGs and more genetic diversity.

As indicated above, there are several implications of the data presented in this

thesis. However, the basic findings of the research remain: 1) M. fiucticola possesses a

51

group I intron in the SSU rDNA, 2) both M. fructicola and M. laxa possess limited
intraspecies diversity, and 3) VCG diversity is highly selected for in M. fructicola.
However, further research is needed to develop a more accurate view of the genetic

diversity within M. fructicola.

RECOMMENDATIONS FOR FUTURE STUDY

The data presented in this thesis clearly indicate the limited nuclear rDNA and
microsatellite DNA diversity of Michigan populations of M. fructicola and M. laxa.
Further research is necessary to identify a marker more suited to the identification of
genetic diversity within these species, and to determine if the results found among
Michigan populations reflect the characteristics of the global population.

Future research must first expand the geographic range of sampling. Isolate
collection should be made from locations throughout the world where M. fructicola is a
significant pathogen. These locations should include Califonria, Canada, South America,
South Afiica, and Japan. A sampling of approximately twelve to fifteen isolates from
each of these locations will provide a sufficient set of isolates to began analysis of
diversity within the global population. In regions where M. fructicola and M. laxa
coexist, a similar number of isolates of M. laxa should also be collected.

To determine if the tools for distinguishing Michigan isolates of M. laxa from M.
fructicola can be applied to isolates throughout the world, DNA from isolates of both
species should undergo the same molecular analysis as the Michigan isolates in this
study. However, as these characters are to be used simply to distinguish between isolates
of the two species, it is unnecessary to sequence the ITS] and SSU rRNA gene regions

for this purpose.

52

53

The ITSl region and the group I intron of the SSU rRNA gene of one to two
isolates from each location should be sequenced. This will verify the high conservation
of these regions as suggested by this, and previous, study (Carbone and Kohn, 1993;
Holst-Jensen et al., 1997). Given the highly conserved nature of these regions, the
examination of genetic diversity within the species does not require the sequencing of all
isolates collected.

A potentially useful tool for analyzing genetic diversity within M. fructicola is
that of amplified fragment length polymorphism (AF LP). This technique involves the
selective PCR amplification of restriction fragments of genomic DNA, and has been
shown to be a useful tool for identifying genetic diversity within a variety of organisms
(Vos et al., 1995; Janssen et al., 1996; Majer et al., 1996). AP LP analysis is capable of
detecting polymorphisms within species where little diversity was revealed by RF LP
analysis (Majer et al., 1996).

Analysis of genomic DNA of M. fructicola and M. laxa via AF LP analysis may
be a useful method by which to identify diversity within these species. All isolates
collected from each area, including Michigan, should be examined via AF LP analysis.

An additional PCR-mediated technique would employ the intergenic spacer (IGS)
region of the nuclear ribosomal DNA. The IGS region, unlike the ITS regions, is not
transcribed and is the most rapidly evolving of the rDNA spacers (Hillis and Dixon,
1991). This region may therefore present more variability than other regions of the
rDNA. Sub-repeat elements occur in the IGS region of many species, and these sub-

repeats may vary in sequence and the number of repeat elements between individuals of a

54

species (Morton et al., 1995). Difference in the number of repeat elements creates length
variation which may be detected by simply PCR amplification and gel analysis. Primers
for amplification of the IGS region are available in the literature (Anderson and
Stasovski, 1992; Appel and Gordon, 1996).

Attempts to PCR-amplify the IGS region in this study were unsuccessful. The
development of better primers in the future and improved long-distance PCR may resolve
some of the difficulties found in initial attempts to amplify the IGS region of M.

fructicola and M. laxa. While other regions of the rDNA show very limited diversity, the
IGS region may display greater diversity given its more rapid rate of evolution. All
isolates should be examined for length variation in the PCR product. If length variation
is revealed, at least two products of each length should be sequenced for further analysis.
In the absence of length variation, one to two isolates from each region could be
examined for sequence variation.

In addition to increasing the geographic rang from which isolates are collected, it
may also be beneficial to include isolates collected in previous years. The inclusion of
isolates fiom other years, such as isolate CB-2 collected in 1975, may provide insight into

changes in population structure and diversity over time.

LIST OF REFERENCES

LIST OF REFERENCES

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APPENDICES

APPENDIX A

Vegetative compatibility groups (VCGs) containing more than one isolate of Monilinia
fructicola.

 

 

Number of
VCG Isolates Isolates within VCG‘

1 5 KK-25, KK-44, KK-48, KK-52, KK-54

2 2 KK-49, KK-SO

3 3 KK-IS, KK-35, KK-40

4 4 JB-16, JB-22, JB-25, JB-35

5 28 JB-l, JB-2, JB-lO, JB-13, JB-14, JB-17, JB-l9,
JB-20, JB-24, JB-26, JB-29, JB-30, JB-31, JB-
32, JB-34, JB-50, JB-51, JB-52, JB-54, JB-56,
JB-58, JB-60, JB-63, JB-64, JB-65, JB-66, JB-
67, JB-68

6 l6 JB-8, JB-9, JB-l 1, JB-15, JB-l8, JB-21, JB-27,

1B-33, JB-36, JB-37, JB-38, JB-44, 1B-47, JB-
61, 1B-69, JB-70

 

“ KK isolates were collected from Kewadin, MI. JB isolates were collected fiom Empire,
MI. Isolates within each group did not form mycelial interaction zones with members
of the same group, but did form lines of interaction with members of all other groups.

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Sequences and positions of oligonucleotide primers in the rDNA.

APPENDIX B

 

 

 

 

A1160_‘ N81; All39_‘ NS7_‘ l'I‘SlI-LJ
ITSl
[ ssu (18S) 1:21] 5.85
rum r1134 rum Wis: rrrsz

approximate product length
Primer sequence M. fructicola M. laxa
ITSlF CTT GGT CAT TTA GAG GAA GTA A
ITSZ GCT GCG TTC TTC ATC GAT GC 250 250
N53 GCA AGT CTG GTG CCA GCA GCC
N54 CTT CCG TCA ATT CCT TTA AG 600 600
N57 GAG GCA ATA ACA GGT CTG TGA TGC
N58 TCC GCA GGT TCA CCT ACG GA 380 380
AJ139 GAA GAC TAA CTA CTG CGA AAG C
AJ140 ACC TGT TAT TGC CTC AAA CTT 930 510
AJ160 GTC ATA TGC TTG TCT CAA AGA T
AJ161 CCA AGG TTC AAC TAC GAG 620 620

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