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INFLUENCE!OF!MYCOVIRUSES!ON!THE!POPULATION!STRUCTURE!OF!THE!
CHESTNUT!BLIGHT!PATHOGEN,!CRYPHONECTRIA!PARASITICA!AND!RECOVERY!
OF!AMERICAN!CHESTNUT,!CASTANEA!DENTATA!
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By#
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Joshua#Charles#Springer#
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A#DISSERTATION#
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Submitted#to#
Michigan#State#University#
in#partial#fulfillment#of#the#requirements#
for#the#degree#of##
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Plant#Biology—Doctor#of#Philosophy##
Ecology,#Evolutionary#Biology#and#Behavior—Dual#Major#
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2013

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ABSTRACT!
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INFLUENCE!OF!MYCOVIRUSES!ON!THE!POPULATION!STRUCTURE!OF!THE!
CHESTNUT!BLIGHT!PATHOGEN,!CRYPHONECTRIA!PARASITICA!AND!RECOVERY!
OF!AMERICAN!CHESTNUT,!CASTANEA!DENTATA!
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By#
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Joshua#Charles#Springer#
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#
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DoubleMstranded#RNA#(dsRNA)#mycovirus#hyperparasites#can#infect#the#chestnut#
blight#pathogen,#Cryphonectria-parasitica,#causing#a#reduction#in#virulence#and#
inhibition#of#sexual#reproduction#in#C.-parasitica,#which#can#lead#to#recovery#of#the#
blight#pathogen’s#host,#American#chestnut#(Castanea-dentata).#Studies#to#determine#the#
level#at#which#mycoviruses#can#influence#populationMlevel#and#genomic#level#diversity#
of#C.-parasitica#have#previously#not#been#done.#The#main#objectives#of#this#dissertation#
are:#1)#determine#how#mycovirus#hyperparasites#can#alter#vegetative#compatibility#
group#(VCG)#diversity#over#long#time#periods,#2)#evaluate#the#affect#of#mycoviruses#on#
genome#level#diversity#in#C.-parasitica,#3)#test#a#hypothesis#that#mycovirus#biological#
control#agents#would#be#most#successful#on#American#chestnut#trees#in#the#1—10cm#
diameter#at#breast#height#(DBH)#size#class.##
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VCG#studies#suggest#that#the#presence#of#mycoviruses#influence#C.-parasitica#

population#structure#in#Michigan.#In#C.-parasitica#populations#where#mycoviruses#are#
present#VCG#diversity#is#low#and#VCGs#generally#unique#to#individual#sites#and#where#
mycoviruses#are#absent#VCG#diversity#is#higher#and#VCGs#are#shared#across#these#
sexually#reproducing#populations.
#

Similarly,#genomeMwide#diversity#in#C.-parasitica#also#appears#to#be#structured#
according#to#mycovirus#presence#and#absence.#Population#genetic#differentiation#values#
(Φ#PT)#are#higher#for#mycovirusMinfected#C.-parasitica#populations#while#lower#values#
are#found#at#mycovirusMfree#sites.##The#pattern#of#differentiation#does#not#suggest#any#
isolation#by#distance.#The#overall#pattern#suggests#significant#migration#among#
epidemic#populations#of#C.-parasitica#where#mycoviruses#are#absent,#while#pathogen#
populations#with#mycoviruses#are#largely#isolated.##Thus,#C.-parasitica#in#Michigan#is#
composed#of#a#patchwork#of#sites#whose#dynamics#appears#to#be#governed#by#the#
presence/absence#of#mycoviruses.##
Preliminary#results#of#a#longMterm#experiment#using#mycoviruses#as#biological#
control#mechanism#in#C.-parasitica#is#showing#promising#results.#Trees#in#the#1—10cm#
DBH#size#class#were#tested#for#response#to#mycovirus#infected#strains#of#C.-parasitica.#
Annual#survivorship#was#73.6%#across#three#tree#populations.#Persistence#of#mycovirus#
in#treated#cankers#however,#was#only#around#50%#from#year#to#year#suggesting#that#
repeated#introductions#of#mycoviruses#as#biological#control#agents#may#be#necessary#to#
achieve#success.#Further,#local#environmental#conditions,#tree#genotype,#and#amount#of#
competition#from#large#overstory#trees#may#play#a#role#in#American#chestnut#recovery.##
This#study#investigates#both#phenotypic#and#genome#diversity#in#C.-parasitica#
relative#to#the#presence#and#absence#of#naturally#occurring#mycovirus#hyperparasites#
that#are#useful#for#biological#control#of#the#chestnut#blight#pathogen.#

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Dedicated#to#the#memory#of#Bruce#D.#Parfitt#(1952—2009)#
My#undergraduate#mentor,#friend,#and#fellow#birder

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ACKNOWLEDGEMENTS!
#
!
I#am#thankful#and#indebted#to#the#many#people#who#have#guided#and#helped#me#

along#during#my#years#at#Michigan#State#University.##First,#I#thank#my#advisor,#Andy#
Jarosz#for#his#guidance,#patience,#prodding,#encouragement#and#for#always#providing#
challenging#questions#throughout#my#graduate#career.##Andy#also#gets#thanks#for#his#
generous#laboratory#support#and#willingness#to#discuss#science#and#for#helping#me#
discover#the#exciting#world#of#chestnut#research;#I#look#forward#to#future#collaboration.##
Dr.#Dennis#Fulbright#provided#copious#amounts#of#knowledge#and#background#
information,#along#with#great#future#ideas.##I#am#thankful#for#his#amazing#insight#into#
the#American#chestnut#system#and#his#willingness#to#allow#members#of#his#own#lab#to#
help#me#learn#new#techniques#and#for#providing#me#with#summer#salaries#and#for#
inviting#me#to#see#European#chestnuts#in#Italy.##I#also#thank#Dr.#Jeffery#Conner#and#Tao#
Sang#for#serving#on#my#committee#and#providing#helpful#answers#to#questions#I#had#
along#the#way.##Alan#Prather#deserves#a#special#thank#you#for#becoming#a#member#of#my#
committee#at#the#last#minute#and#for#providing#a#summer#salary#for#me#by#allowing#me#
to#work#in#the#MSU#herbarium.##
#

I#also#wish#to#thank#(in#no#particular#order#of#importance)#Sara#Stadt#and#

Carmen#MedinaMMora#from#Dr.#Fulbright’s#lab,#the#Sear’s#lab,#Mark#Double#and#Dr.#
William#MacDonald#from#West#Virginia#State#University,#Dr.#Anita#Davelos#Baines#from#
the#University#of#WisconsinMLaCrosse#for#providing#encouragement#and#for#answering#
my#many#questions#and#requests,#Eleanor#Morgan#at#Curtin#University#(Bentley#
Campus)#in#Perth,#Western#Australia,#and#Cyril#Dutech#at#L'Institut#national#de#la#
recherche#agronomique#(INRA)#in#Bordeaux,#France#who#both#assisted#with#the#
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analysis#of#my#microsatellite#data#in#Chapter#3.##Undergraduates#who#provided#
countless#hours#of#work#include:#Matthew#Kolp,#Reece#Hammer,#and#Deepika#
Kandasamy#and#deserve#many#thanks#for#their#hard#work#and#expert#technical#
assistance#in#the#laboratory.#
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When#it#comes#to#helping#me#complete#my#graduate#work#in#a#timely#fashion,#I#

wish#to#thank#Matthew#Chansler#(Chauncey),#my#fourMyear#undergraduate#employee#
who#began#working#in#the#Jarosz#lab#as#a#freshman#in#2008.#Matt#was#a#dedicated#and#
thorough#employee#who#provided#countless#hours#of#hard,#tedious#work#with#endless#
enthusiasm#for#science.##I#could#not#have#completed#my#graduate#work#as#quickly#
without#his#laboratory#expertise,#enthusiasm,#and#wit.#I#am#forever#indebted#to#him#for#
his#dedication.###
#

The#experience#of#being#a#graduate#student#in#the#department#of#Plant#Biology#

and#EEBB#has#been#made#better#by#discussions#with#many#faculty#and#from#the#help#of#
the#knowledgeable#department#office#staff:#Jan#McGowan,#Tracey#Barner,#Stacy#LaClair,#
and#Adelle#Rigotti.##
#

Fellow#graduate#students#and#research#assistants#in#the#Department#of#Plant#

Biology#provided#immeasurable#amounts#of#advice,#constructive#criticism,#and#laughs#in#
difficult#times#including:#Ellen#Cole,#Colin#Phillippo,#Jason#Martina,#Elizabeth#Schultheis,#
and#Tomomi#Suwa.#
#

The#Michigan#Botanical#Foundation#and#The#Hanes#Trust#of#Kalamazoo,#

Michigan#provided#funding#for#a#portion#of#this#work#(Chapter#3).#I#deeply#thank#those#
organizations#for#providing#the#funding#that#allowed#me#to#expand#my#knowledge#of#
molecular#biology#that#provided#insights#to#the#chestnut#blight#system#in#Michigan.##

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This#work#is#dedicated#to#my#friend#Bruce#D.#Parfitt#who#passed#away#at#the#

beginning#of#my#third#year#at#MSU.#Bruce#helped#further#develop#my#love#for#science#
and#taught#me#to#be#more#inquisitive#of#the#world#around#me.#Were#it#not#for#Bruce#I#
would#never#have#considered#graduate#school#nor#would#I#have#travelled#to#Vermont#to#
experience#an#amazing#state#in#our#union.#Bruce#and#I#became#great#friends#while#
birding#in#Michigan#and#in#New#England.#Our#discussions#on#all#topics#will#never#cease#
to#be#inspiration.##
#

Last#but#not#least#I#thank#my#fiancée#Amanda#M.#Audo#for#never#giving#up#on#me#

and#for#her#continual#support#and#understanding;#for#that#I#love#her#even#more.#
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TABLE!OF!CONTENTS!
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LIST#OF#TABLES#..............................................................................................................................#x#
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LIST#OF#FIGURES#............................................................................................................................#xii#
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CHAPTER#1#.......................................................................................................................................#1#
INTRODUCTION,#Plant#Pathogen#Interactions#
Hyperparasites#and#Hyperparasitism#......................................................#4#
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Chestnuts,#Blight,#and#Mycoviruses#in#Michigan#.................................#8#
DoubleMstranded#(ds)#RNA#mycoviruses#................................................#10#
Vegetative#incompatibility#............................................................................#16#
Mycoviruses#as#biological#control#agents#...............................................#17#
The#chestnut#blight#situation#in#Michigan#.............................................#18#
Structure#of#this#Dissertation#......................................................................#19#
LITERATURE#CITED#.......................................................................................................#22#
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CHAPTER#2#
HYPERPARASITES#INFLUENCE#POPULATION#STRUCTURE#OF#THE#CHESTNUT#BLIGHT#
PATHOGEN,#CRYPHONECTRIA-PARASITICA-..........................................................................-31#
ABSTRACT#..........................................................................................................................#31#
INTRODUCTION#...............................................................................................................#32#
MATERIALS#AND#METHODS#......................................................................................#36#
DATA#ANALYSIS#...............................................................................................................#39#
RESULTS#..............................................................................................................................#41#
Vegetative#compatibility#group#diversity#...............................................#41#
Mating#type#allele#frequencies#....................................................................#47#
DISCUSSION#.......................................................................................................................#50#
LITERATURE#CITED#.......................................................................................................#54#
#
CHAPTER#3#
AN#ANALYSIS#OF#MICROSATELLITE#LOCI#FROM#POPULATIONS#OF#THE#CHESTNUT#
BLIGHT#PATHOGEN,#CRYPHONECTRIA-PARASITICA,#IN#MICHIGAN#.......................#60#
ABSTRACT#..........................................................................................................................#60#
INTRODUCTION#...............................................................................................................#61#
METHODS#...........................................................................................................................#64#
RESULTS#..............................................................................................................................#66#
DISCUSSION#.......................................................................................................................#73#
LITERATURE#CITED#.......................................................................................................#79#
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CHAPTER#4#
BIOLOGICALLY#CONTROLLING#CHESTNUT#BLIGHT#PATHOGEN#POPULATIONS#IN#
MICHIGAN#USING#A#NATIVE#MYCOVIRUS:#INITIAL#RESULTS#OF#A#LONGMTERM#
EXPERIMENT#...................................................................................................................................#83#
ABSTRACT#..........................................................................................................................#83#
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INTRODUCTION#...............................................................................................................#84#
What#trees#to#treat?#.........................................................................................#87#
A#Michigan#mycovirus#used#for#biological#control#.............................#88#
METHODS#...........................................................................................................................#89#
Application#of#GH2#...........................................................................................#90#
Determining#level#of#mycovirus#success#................................................#92#
.
RESULTS#..............................................................................................................................#93#
Mycovirus#prevalence#after#treatments#..................................................#93#
DISCUSSION#.......................................................................................................................#101#
LITERATURE#CITED#.......................................................................................................#105#

CHAPTER(5#.......................................................................................................................................#110#
CONCLUSIONS#...................................................................................................................#110#
LITERATURE(CITED#.......................................................................................................#116#
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APPENDIX#.........................................................................................................................................#117#
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LIST!OF!TABLES!
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Table#2M1:#Summary#diversity#statistics#for#Epidemic#and#Recovering#populations.#VC#
richness#represents#the#total#time#period#1996#to#2009#........................#47#
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Table#2M2:#Distribution#of#mating#types#within#Cryphonectria-parasitica#populations#in#
Michigan.#Mixed#indicates#number#of#samples#in#which#both#mating#types#
were#present.#The#chiMsquare#(X2)#value#tests#whether#the#population#is#at#
the#50:50#mating#frequency#expected#if#sexual#recombination#is#occurring#
regularly#.......................................................................................................................#49#
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Table#3M1:Range#of#allele#sizes#in#base#pairs#and#number#of#alleles#amplified#for#
thirteen#loci#of#C.-parasitica#in#Europe#(Breuillin#et#al#2006)#and#ten#in#
Michigan#.......................................................................................................................#68#
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Table#3M2:#Summary#table#of#population#level#diversity#of#alleles#at#each#of#ten#loci.#
Data#in#gray#boxes#are#shown#for#comparison#and#were#not#included#in#
population#level#analyses#due#to#poor#amplification#and#resulting#small#
sample#size#..................................................................................................................#69#
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Table#3M3:#AMOVA#statistics#for#tests#of#genetic#differentiation#within#and#among#
populations#of#C.-parasitica#in#Michigan.#Data#were#calculated#without#site#
MH,#locus#CPG5#and#any#sample#where#no#loci#amplified#......................#70#
#
Table#3M4:#Average#number#of#alleles#per#locus,#effective#number#of#alleles#per#
locus,#percentage#of#loci#that#were#polymorphic#and#Shannon#
diversity#for#each#population.#Standard#errors#of#the#mean#are#in#
parentheses#................................................................................................................#71#
#
Table#3M5:#Pairwise#population#differentiation#values#(ϕPT;#below#the#main#
diagonal)#based#on#nine#loci#across#six#C.-parasitica#populations;#all#
probabilities#of#obtaining#values#were#significant#at#p<0.05#except#LE#x#
MD#(p=0.096).#StraightMline#distances#between#populations,#in#km,#are#
shown#above#the#diagonal.#Light#shading#indicates#comparisons#
between#populations#without#mycoviruses#while#darker#shading#
indicates#comparisons#between#populations#with#mycoviruses.#.......#72#
#
Table#3M6:#Average#ϕPT#and#number#of#haploid#migrants#(Nm)#based#on#population#
type,#three#epidemic#sites#and#three#recovering#sites#(described#in#Chapter#2).#
Standard#errors#are#in#parentheses#.................................................................#72#
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Table#3M7:#Average#number#of#migrants#per#generation#of#C.-parasitica#between#six#
Michigan#populations.#Values#are#estimated#based#on#haploid#C.-parasitica#
populations#and#on#ΦPT#values#using#GenAlEx#6.5.#Shading#is#the#same#as#in#
Table#3M5#......................................................................................................................#73#
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Table#4M1:#Percentage#of#treated#cankers#at#three#sites#that#contained#mycovirus.#
Presence#of#mycovirus#was#determined#based#on#culture#morphology#
#..........................................................................................................................................#96#
#
Table#4M2:#Average#size#(DBH#of#trees#that#survived#and#trees#that#died#over#each#time#
step.#Missaukee#Diseased#and#Stivers#North#data#are#control#populations.#
Standard#errors#are#in#parentheses#.................................................................#98#
#
Table#AM1:#Overview#of#isolates#tested#for#resurrection#success#from#six#Michigan#
chestnut#blight#populations.#Totals#are#whole#numbers,#percentages#or#
averages#.......................................................................................................................#120#
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LIST!OF!FIGURES!
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Figure#1M1:#Generalized#pathogenMhost#interaction.#Pathogens#generally#reduce#the#
fitness#of#their#hosts#and#reduce#biomass#of#the#infected#individual#and#
population#.................................................................................................................#2#
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Figure#1M2:#The#three#major#players#in#the#chestnut#triMtrophic#system.#Arrow#labels#
show#general#outcomes#of#interactions#........................................................#9#
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Figure#1M3:#Typical#result#of#Cryphonectria-parasitica#infection#on#American#chestnut.#
Cankers#normally#expand#concentrically#from#a#wound#site#(branch#
point,#left),#collapsing#vascular#tissue#as#they#expand#(right).#Orange#
pigmentation#is#evidence#of#disease.#For#interpretation#of#the#references#
to#color#in#this#and#all#other#figures,#the#reader#is#referred#to#the#
electronic#version#of#this#dissertation##.........................................................#11#
#
Figure#1M4:#Death#of#a#distal#end#of#an#American#chestnut#branch#from#a#girdling#
canker#caused#by#Cryphonectria-parasitica.#Yellow,#wilted#leaves#are#
evidence#of#disease.#...............................................................................................#12#
#
Figure#1M5:#A#mycovirusMfree#(left)#and#mycovirus#infected#(right)#isolate#of#the#
chestnut#blight#pathogen#Cryphonectria-parasitica.#Both#fungal#isolates#are#
the#same#genotype#and#are#both#about#one#week#old.#...........................#12#
#
Figure#1M6:#Examples#of#mycovirus#infected#Cryphonectria-parasitica#cankers#on#
American#chestnut#trees.#Note#swollen#nature#of#wounds.#All#parts#distal#to#
these#cankers#are#alive##.......................................................................................#13#
.
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Figure#2M1:#Vegetative#compatibility#testing#of#Cryphonectria-parasitica#on#
chestnut#stem#pieces.#Compatible#reaction#(dashed#arrow)#between#
sample#226#and#166,#incompatible#reaction#(solid#arrow)#between#
samples#166#and#221#............................................................................................#38#
#
Figure'2M2: Vegetative(Compatibility(diversity(for(the(year(1996(at(five(Michigan(
populations.+Sizes+of+slices$represent$proportion$of$each$VC$type$found$at$
each%site.%A%key%to%Michigan%VC%types%is%provided.%Rare%types%at%recovering%
and$epidemic$populations$are$represented$by$white$and$black$slices,$
respectively,,and,in,some,cases,more,than,one,type,is,represented$by$the$
slice.'*1996%Data%for%Stivers%and%Missaukee%Diseased%represent%only%those%
samples'that'could'be'resurrected'from'storage;'see'text'for'details.'#
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#........................................................................................................................................#43#
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Figure 2-3: Vegetative(Compatibility(diversity(data(for(the(year(2009(at(seven(Michigan(
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populations.'Rare'types'at'recovering'and'epidemic'populations'are'
represented(by(white(and(black(slices,(respectively.(Recovering*sites*are*
denoted&with&leader&lines&ending&in&solid&circles,&epidemic&sites&with&solid&
squares,"respectively ............................................................................45#

#
Figure#4M1:#Representative#mycovirus#transfer#plate:#A#GH2#infected#donor#strain,#
left;#note#flattened#appearance#of#mycelia#with#a#virulent#recipient#strain,#
right;#note#raised#appearance.#Successful#conversion#of#virulent#strain#
occurred#near#arrow.#A#change#in#phenotype#similar#to#the#left#isolate#
shows#successful#transfer#of#GH2#from#donor#to#recipient#strain#....#91#
#
Figure#4M2:#Two#forms#of#treating#cankers#used#in#this#study.#a)#drill#bit#created#
holes#at#canker#margins#where#mycovirus#inoculum#could#be#
introduced#and#b)#"scratcher"#with#lag#screws#to#wound#canker#and#
surrounding#area;#more#surface#area#was#able#to#be#treated#with#this#
method.#Figure#2b#photo#credit:#AM#Jarosz#................................................#92#
#
Figure#4M3:#Survivorship#of#treated#American#chestnut#trees#at#the#Leelanau#site.#Chort#
1#(solid#line)#was#composed#of#13#trees#that#were#treated#initially#in#2009.#
The#second#cohort#(dashed#line)#consisted#of#44#trees#that#were#first#treated#
in#2010#........................................................................................................................#96#
#
Figure#4M4:#Survivorship#of#28#American#chestnut#trees#treated#with#mycovirus#at#
Missaukee#healthy#(solid#line)#and#compared#to#30#untreated#control#
trees#at#Missaukee#Diseased#(dotted#line)#over#the#time#period#2009#to#
2012.#The#MH#and#MD#sites#are#only#300#meters#apart.#Cohort#two#of#five#
trees#at#MH#is#not#shown#....................................................................................#97#
.
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Figure#4M5:#Survivorship#of#87#American#chestnut#trees#at#the#treated#ST#south#
trees(solid#line)#and#30#untreated#control#trees#in#the#northern#portion#
of#the#site#(dotted#line)#from#2009#to#2011#................................................#97#
#
Figure#4M6:#Percentage#annual#change#in#DBH#for#surviving#trees#at#LE#site.#There#
is#no#control#for#the#LE#site.#Diagonal#bars#are#growth#from#2009#to#
2010,#dotted#bars#are#increase#(or#decrease)#from#2010#to#2011,#and#
brick#bars#represent#the#increase#from#2011#to#2012.#At#LE#cohort#2,#
treatment#did#not#begin#until#2010#................................................................#99#
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Figure#4M7:#Percentage#annual#change#in#DBH#for#surviving#trees#at#MH#(treated)#and#
MD#(control)#sites.#Dark#bars#are#growth#from#2009#to#2010,#dotted#bars#are#
increase#from#2010#to#2011,#and#brick#bars#represent#the#increase#from#
2011#to#2012.#At#MH#Cohort#2,#treatment#did#not#begin#until#2010.#
Percentage#change#in#DBH#is#calculated#as#the#average#increase#in#stem#
diameter#over#the#previous#year#for#Figures#6,#7,#and#8.#......................#100#
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#

#

Figure#4M8:#Percentage#annual#change#in#DBH#for#surviving#trees#at#ST#(treated)#and#SN#
(control)#sites.#Diagonal#bars#are#growth#from#2009#to#2010,#dotted#bars#are#
increase#from#2010#to#2011,#Data#for#the#final#period#are#not#available#for#
either#Stivers#site#...................................................................................................#101
.

xiv#

!
CHAPTER!1!
#
INTRODUCTION!
!
Plant!pathogen!interactions!
#
#

Pathogens#can#play#a#major#role#in#structuring#plant#populations#on#both#a#local#

and#regional#scale#by#reducing#survivorship,##growth#and#reproduction#of#infected#hosts#
(Burdon#1987;#Gilbert#2002).#These#interactions#ultimately#affect#the#evolutionary#
dynamics#of#plant#populations#(Price#1980;#Burdon#1987;#Gilbert#2002).#Our#
understanding#of#community#level#effects#is#basic#but#growing#(Dobson#&#Hudson#1986;#
Burdon#et#al.#2006).#Weste#(1980,#1981),#McCormick#&#Platt#(1980),#and#others#have#
reported#that#introduced#plant#pathogens#can#alter#the#species#interactions#within#a#
plant#community.##For#example,#Phytophthora-cinnamomi#Rands#alters#dominance#
relationships#within#Australian#Eucalyptus#forests#resulting#in#a#significant#change#in#
plant#community#structure#(Weste#1980,#1981).##Chestnut#blight#disease,#caused#by#
Cryphonectria-parasitica-(Murrill)#Barr,#eliminated#the#American#chestnut#(Castaneadentata-(Marsh.)#Borkh.#from#the#overstory#of#eastern#hardwood#forests#in#North#
America,#which#allowed#other#species#to#increase#in#prevalence#and#dominance#
(McCormick#&#Platt#1980).##
#

The#fitness#of#plants#after#infection#by#a#pathogen#with#high#virulence#is#greatly#

reduced#(Figure#1M1).#However,#Harper#(1977)#postulated#that#pathogens#with#such#
high#virulence#would#quickly#destroy#their#resourceMbase#so#that#the#evolution#would#
favor#reduced#virulence.##A#reduction#in#pathogen#virulence##
#

#
1#

#

#

#

#
Figure#1.1.#Generalized#pathogenMhost#interaction.#Pathogens#generally#reduce#the#
fitness#of#their#hosts#and#reduce#biomass#of#the#infected#individual#and#population.##
#
#
then,#would#lead#to#recovery#of#host#populations#so#that#the#cycle#of#infection#may#
continue.#A#wellMknown#empirical#example#of#the#evolution#of#reduced#virulence#is#
found#in#the#Myxoma#virusMcommon#rabbit#(Oryctolagus-cuniculus-L.)#system#in#
Australia.#Introduced#for#biological#control#of#destructive#rabbit#populations,#the#
virulent#virus,#causing#myxomatosis,#quickly#reduced#rabbit#populations#but#also#
evolved#reduced#virulence#while#increased#resistance#and#immunity#to#the#virus#
evolved#in#rabbit#populations.#(Fenner#&#Ratcliffe#1965;#Anderson#&#May#1982;#Fenner#
1983;#May#&#Anderson#1983);#the#evolution#of#reduced#virulence#is#not#entirely#
common,#however#(Jarosz#&#Davelos#1995).#A#more#reasonable#model#for#the#evolution#
of#virulence#was#published#by#Lenski#&#May#(1994),#which#predicted#that#intermediate#
levels#of#virulence#should#evolve#over#time#due#to#ecologically#important#factors#that#
affect#densityMdependent#transmission#of#the#pathogen.#The#Lenski#and#May#model#also#
focused#attention#on#individual#selection#rather#than#the#group#selectionist#argument#
hypothesized#by#Harper.##The#evolution#of#reduced#virulence#has#been#of#some#concern#
in#situations#of#biological#control#since#any#loss#of#virulence#in#the#biological#control#
mechanism,#can#lead#to#a#reduction#in#successful#control#of#disease#(Bryner#&#Rigling#
2#

#

#

2011,#2012).##This#is#concerning#since#controlling#invasive#populations#of#plants#with#
pathogens#relies#on#the#success#of#introducing#biological#control#agents#and#realizing#
successful#spread#and#persistence#over#long#periods#of#time.###
#

The#fitness#consequences#of#complex#plantMpathogen#interactions#are#often#

difficult#to#elucidate#(Mordecai#2011)#because#reductions#in#survivorship#and#
reproduction#of#infected#plants#is#often#conditioned#by#abiotic#and#biotic#stresses#that#
are#being#imposed#on#the#plant#(Jarosz#&#Davelos#1995;#Gilbert#2002;#Klironomos#
2003).###In#the#extreme,#fungal#infections#of#a#plant#can#have#negative#fitness#
consequences#under#some#conditions,#but#be#mutualistic#under#others#(Singh#et#al.#
2011).##For#example,#mycorrhizae#may#be#mutualists#in#some#conditions,#but#a#change#
in#the#abiotic#environment,#such#as#temperature#or#amount#of#precipitation,#may#change#
the#interaction#to#parasitism#(Johnson#et#al#1997).#Thus,#hostMpathogen#interactions#can#
have#diverse#consequences#on#both#plant#and#pathogen#populations#but#those#
interactions#can#help#to#maintain#both#species#(Burdon#1987)#and#can#be#quite#variable#
due#to#local#conditions.##
###

#

The#influence#of#native#pathogens#on#plant#populations#is#often#difficult#to#detect#

because#the#interaction#between#the#plant#and#pathogen#has#been#incorporated#into#the#
plant#community#for#a#considerable#time#period.##In#consequence,#the#pathogen’s#
influence#may#be#integrated#into#community#and#its#influence#is#not#obvious.##However,#
the#influence#of#some#native#pathogens#has#been#studied#and#they#have#the#potential#to#
cause#significant#and#wideMranging#effects#on#host#populations#(Burdon#1987).##Native#
pathogens#such#as#fusiform#rust#caused#by#the#pathogen#Cronartium-fusiforme#Hedg.#&#
Hunt#ex#Cumm.#in#the#southeastern#United#States#on#native#pines#increases#in#

3#

#

#

prevalence#when#selective#cutting#of#other#tree#species#occurs#Dinus#(1974).##UromycesariBtriphylli#(Schwein.)#Seeler#and#can#change#the#growth#rates#of#JackMinMtheMpulpit#
(Arisaema-triphyllum#L.)#populations#(Baines#et#al.#in-press)#and#Puccinia-mariaeBwilsonii#
Clinton#can#cause#widespread#disease#epidemics#in#Claytonia#spp.#while#maintaining#
flower#color#variation#in#host#plants#over#time#(Frey#2004).##
The#influence#of#introduced#pathogens#is#often#easier#to#detect#because#their#
invasion#into#plant#community#can#lead#to#a#cascade#of#changes#throughout#the#plant#
community#(Vitousek#at#al.#1996,#1997;#Lovett#et#al.#2006;#Loo#2008).#Fungal#pathogens#
such#as#Ophiostoma-ulmi#(Dutch#Elm#Disease),#Phytophthora-ramorum-(Sudden#Oak#
death),#Nectria#spp.#(Beech#bark#disease),#and#Cryphonectria-parasitica#(Chestnut#
blight),#are#all#examples#of#introduced#pathogens#that#have#negatively#affected#their#
respective#host#species#in#ways#that#have#changed#the#forest#composition#in#areas#
where#they#have#invaded#(McCormick#&#Platt#1980;#Swinton#&#Gilligan#1996;#Paillet#
2002;#Griffin#et#al.#2003;#Garnas#et#al.#2011;#Cobb#et#al.#2012).#In#each#of#the#four#
situations#above,#the#introduced#pathogens#have#greatly#reduced#host#numbers,#and#
caused#disruptions#of#community#composition.##Additionally,#pathogen#effects#can#also#
cascade#down#to#other#members#of#the#community#such#as#birds#and#mammals#by#
reducing#food#resource#availability#(cf.#Monahan#&#Koenig#2006;#Loo#2009).##
#
Hyperparasites!and!Hyperparasitism!
#

#To#make#matters#more#complex,#fungal#pathogens#can#be#infected#with#

hyperparasites#(i.e.,#parasites#of#pathogens#or#parasites#and#referred#to#as#triMtrophic#
interactions),#most#frequently,#viruses#(Nuss#&#Koltin#1990).##Hyperparasitism#is#not#

4#

#

#

confined#to#fungal#pathogens#of#plants#but#is#also#well#documented#across#taxa#such#as#
wasps#and#bacteria#(Sullivan#1987;#Tanaka#et#al#2007;#Johnson#et#al#2010;#Koskella#et#al#
2011)#and#have#farMranging#effects,#many#times#acting#as#biological#control#agents.#Viral#
hyperparasites#of#fungal#pathogens#(hereafter,#mycoviruses)#have#been#documented#to#
decrease#the#virulence#of#fungal#pathogens#they#infect,#allowing#plant#hosts#to#grow#at#
nearly#a#normal#level#(Liesebach#&#Zaspel#2004;#Roossinck#2011).#Mycoviruses#can#
have#other#unexpected#effects#such#as#assisting#fungal#mutualists#to#confer#thermal#
tolerance#to#plants,#which#allows#the#host#plants#to#withstand#higher#temperatures#near#
thermal#pools#in#Yellowstone#National#Park#(Redman#et#al.#2002).##
#

Mycoviruses#have#the#potential#to#act#as#topMdown#regulators#in#plantMpathogen#

systems#(Taylor#et#al.#1998).#The#addition#of#mycoviruses#into#plantMpathogen#
interactions#can#quickly#reduce#pathogen#virulence#ecologically#with#their#invasion;#
mimicking#a#slower#evolutionary#reduction#in#virulence.##Mycovirus#mediated#
reductions#in#virulence#can#often#occur#on#a#shorter#time#scale#than#expected#with#
evolutionary#change#because#mycoviruses#can#be#transmitted#both#vertically#(i.e.,#from#
parent#to#offspring)#and#horizontally#(i.e.,#infectious#spread#to#new#individuals).##
Additionally,#it#appears#that#mycoviruses#are#common#in#nature,#especially#in#fungi#
(Hollings#1982;#Buck#1986;#Roossinck#2011)#and#are#often#unencapsidated#doubleM
stranded#RNA#mycoviruses#that#can#be#found#in#Ascomycetous#fungi#(Oh#&#Hillman#
1995),#Oomycetes#(Nuss#&#Koltin#1990),#and#Basidiomycetes#(Nuss#&#Koltin#1990).#
However,#it#appears#that#mycovirus#infection#of#a#fungus#often#does#not#reduce#
virulence#and#are#quite#benign#(Nuss#2005).#

5#

#
#

#

Ecological#and#evolutionary#outcomes#of#plantMpathogenMmycovirus#triMtrophic#

interactions#may#vary#depending#on#location,#environmental#conditions#within#or#
among#years,#or#the#amount#of#genetic#variation#at#all#levels#of#the#interaction.#
Thompson#(1999)#has#termed#this#the#geographic#mosaic#theory#of#coevolution#(GMTC)#
where#the#interplay#of#organisms#at#differing#hierarchical#scales#can#result#in#vastly#
different#outcomes#at#different#temporal#and#geographic#scales.##For#example,#the#moth#
Greya-politella#and#its#host#plants#Lithophragma#spp.#and#Heuchera#spp.#have#quite#
variable#interactions,#especially#if#competing#pollinators#of#Greya#moths,#Bombyliid#flies,#
are#present#in#local#populations.##Bombyliid#flies#outweigh#the#commensalistic#
interaction#that#Greya#moths#can#develop#with#the#host#plants,#since#the#flies#are#
effective#pollinators#that#do#not#prey#on#developing#seeds#like#Greya#moths#(Thompson#
&#Pellmyr#1992;#Thompson#1999).##Benkman#(1999)#noted#that#red#squirrels#
(Tamiasciurus#spp.)#changed#the#interaction#between#crossbills#(Loxia#spp.)#and#
lodgepole#pines#(Pinus-contorta#Douglas)#that#led#to#development#of#different#
interactions#across#the#geographic#range#of#the#species.##When#present,#red#squirrels#
preempt#crossbills#from#feeding#on#cones#of#lodgepole#pines#and#squirrel#presence#
drives#cone#evolution.#When#red#squirrels#are#absent#from#lodgepole#pine#forests,#
crossbills#drive#cone#evolution#and#these#particular#coevolutionary#hotspots#can#result#
in#divergent#selection,#creating#a#variable#selection#mosaic#across#the#landscape#which,#
if#strong#enough#can#lead#to#speciation#in#both#interacting#members#(Benkman#1999;#
Benkman#et#al.#2011).###Further,#another#type#of#complex#interaction#involves#a#
biological#control#mechanism#of#a#pathogen:#reductions#in#virulence#caused#by#doubleM
stranded#RNA#(dsRNA)#mycovirus#infection#of#C.-parasitica#are#known#to#be#

6#

#

#

temperature#sensitive#(Bryner#&#Rigling#2012)#and#higher#ambient#temperatures#could#
inhibit#the#ability#of#mycoviruses#to#promote#recovery#of#host#plant#populations.#
Complex#interactions#like#these#can#have#farMreaching#effects#for#biodiversity#such#as#
maintaining#disease#at#low#levels#in#plant#populations#and#generating#new#species#
through#tightly#coMevolving#species#driven#by#strong#local#adaptation.##Local,#transient#
maladaptation#may#occur#(Thompson#1999;#Benkman#1999;#Thompson#&#Pellmyr#
1992;#Thompson#2005)#such#as#when#the#evolutionary#forces#genetic#drift,#
recombination,#and#mutation#(Hanski#1999;#Thompson#1999#and#references#therein;#
Smith,#Ericson,#&#Burdon#2003)#can#cause#individuals’#traits#to#be#mixed#within#
populations#and#when#individuals#subsequently#migrate#to#nearby#populations,#thus#
creating#a#geographic#mosaic#of#coevolution#(Thompson#1999,#Thompson#2005).##Local#
populations#of#interacting#species,#as#shown#above,#can#behave#very#differently#in#
isolated#local#populations.#Invasion#by#additional#species#(such#as#dsRNA#mycoviruses)#
may#change#the#original#interactions#and#may#vary#among#sites#within#a#
metapopulation.###!
#

The#question#of#scale#and#local#interactions#between#species#is#becoming#a#major#

topic#in#ecology#where#localized#populations#of#interacting#species#may,#over#short#time#
periods#develop#very#different#dynamics#that#can#change#due#to#varying#biotic#and#
abiotic#conditions#and#outcomes#can#be#quite#different#even#at#small#spatial#scales#
(Hanski#&#Gilpin#1997;#Hanski#1999;#Thompson#1999;#Thompson#2005).#
#

The#GMTC#can#be#applied#to#the#chestnut#blight#triMtrophic#system#discussed#

below.##Gene#flow,#migration,#recombination,#genetic#drift,#and#the#local#abiotic#
environment#(and#other#unknown#factors)#may#continuously#rework#local#landscapes#

7#

#

#

within#a#metapopulation#of#the#interacting#species,#continuously#changing#how#species#
interact.##Additionally,#discussed#in#this#dissertation#is#how#the#size#(diameter)#of#
individual#hosts#may#influence#the#success#of#mycoviruses#on#the#recovery#of#individual#
American#chestnut#trees#(Chapter#4)#and#how#diversity#of#the#blight#pathogen#may#
affect#spread#of#mycoviruses#that#lead#to#host#tree#recovery#in#local#populations.#
#
Chestnuts,!Blight!and!Mycoviruses!in!Michigan!
#

The#chestnut,#chestnut#blight,#and#mycovirus#interaction#is#a#model#triMtrophic#

system#used#for#research#in#this#dissertation.#A#generalized#overview#of#the#major,#
known#players#and#their#effects#in#this#system#is#shown#in#Figure#1M2.#A#historically#
important#forest#species,#Castanea-dentata,#the#American#chestnut#was#an#important#
tree#both#to#the#natural#ecosystem#and#to#people#who#depended#on#the#tree#for#its#many#
uses.##American#chestnut#was#a#tree#that#had#more#uses#than#its#counterparts#in#the#
hardwood#forests#of#the#eastern#United#States#(Griffin#&#Elkins#1986).#Strong,#rotM
resistant#lumber#and#edible#nuts#are#two#products#that#were#important#to#not#only#rural#
families#but#throughout#the#country#(Griffin#&#Elkins#1986).##It#appears#that#eastern#
Asia#is#the#center#of#origin#for#the#genus#Castanea#and#subsequently#spread#to#Europe#
and#then#North#America#(Lang#et#al#2006).##American#chestnuts,#C.-dentata,#are#highly#
susceptible#to#chestnut#blight,#while#European#chestnuts#are#somewhat#less#susceptible#
to#C.-parasitica#than#C.-dentata#(Graves#1950;#Clapper#1952;#Anagnostakis#1992).##Asian#
chestnut#species,#such#as#C.-mollissima-and-C.-davidii#have#much#higher#resistance#to#
blight#than#either#American#or#European#chestnuts.#In#fact,#chestnut#blight#was#not##
#

8#

#

#

#
Figure#1M2.#The#three#major#players#in#the#chestnut#triMtrophic#system.#Arrow#labels#
show#general#outcomes#of#interactions.###
#
discovered#in#Asia#until#1913,#well#after#the#American#epidemic#had#begun#(Fairchild#
1913).#Blight#is#present#in#both#China#and#Japan#but#is#rarely#destructive#(Fairchild#
1913).##Resistance#in#Asian#chestnuts#is#probably#due#to#its#long#coevolutionary#history#
with#blight#(Anagnostakis#1992).#
#

#Cryphonectria-parasitica#(Murrill)#Barr#(previously#Endothia-parasitica)#is#a#

destructive#nonMnative#pathogen#on#chestnuts#in#North#America#and#Europe.#The#blight#
pathogen#decimated#the#onceMcommon#and#beloved#tree#in#the#United#States,#Castaneadentata-(Marsh.)#Borkh.,#the#American#chestnut.##A#wound#pathogen,#C.-parasitica#
quickly#destroys#vascular#tissue#of#American#chestnut,#girdling#branches#and#stems#
(Figure#1M3),#killing#all#parts#of#the#tree#distal#to#the#wound#(Figure#1M4).#The#pathogen#
spread#quickly#throughout#the#range#of#American#chestnut,#just#decades#after#first#being#
introduced#around#New#York#City#(Merkel#1905).##
#

Contrary#to#popular#belief,#C.-parasitica#did#not#cause#the#extinction#of#the#

American#chestnut#but#caused#more#than#3.5#billion#main#trunks#to#die#back#to#ground#
level.##American#chestnuts#once#accounted#for#nearly#25%#of#all#overstory#trees#in#
eastern#hardwood#forests,#but#blight#has#transformed#these#trees#to#understory#

9#

#

#

inhabitants.##American#chestnuts#have#persisted#thanks#to#an#interesting#physiology;#the#
blight#pathogen#does#not#infect#the#root#tissues#of#the#tree,#allowing#stump#or#root#collar#
sprouts#to#grow#for#a#period#of#time#before#becoming#infected,#thus#creating#a#cyclical#
infection#(Griffin#et#al.#1991).#The#only#remaining#diseaseMfree#American#chestnuts#
occur#outside#of#the#native#range##(see#Brewer#1995#for#a#list#of#populations#in#
Michigan).##These#remaining#populations#are#being#colonized#by#blight#as#time#passes.##
Indeed,#the#two#diseaseMfree#sites#we#began#monitoring#in#1996#have#become#infected#
during#the#course#of#our#work#(Davelos#and#Jarosz#2004;#see#also#Chapter#2).##
#
DoubleMstranded!(ds)RNA!mycoviruses!
DoubleMstranded#RNA#(dsRNA)#mycoviruses#(sometimes#referred#to#as#hypoviruses)#
have#been#documented#to#infect#C.-parasitica#(Anagnostakis#&#Waggoner#1981;#
Fulbright#et#al#1988;#Nuss#1990;#MacDonald#&#Fulbright#1991;#Nuss#1992;#Milgroom#&#
Cortesi#2004).#Where#mycoviruses#have#invaded#the#blight#pathogen,#the#tree#
populations#can#exhibit#significant#recovery#and#regain#its#status#as#an#overstory#tree;#
mycovirus#infection#of#C.-parasitica#allows#trees#to#experience#growth,#survivorship,#and#
reproduction#that#mimics#uninfected#trees#(Davelos#&#Jarosz#2004).#Mycovirus#
hyperparasite#presence#within#the#mycelia#effects#a#noticeable#change#in#phenotype#of#
the#fungal#pathogen#(Figure#1M5),#which#most#importantly,#results#in#a#reduction#in#
virulence.#The#decreased#virulence#of#the#fungal#pathogen#thus#allows#the#tree#host#to#
respond#more#effectively#to#this#nowMdebilitated#pathogen#and#by#extension#the#host#
tree#can#lay#down#callus#tissue#to#effectively#prevent#a#blight#canker#from#girdling#a##
#

10#

#

#

#

#

#

#
Figure#1M3.#Typical#result#of#Cryphonectria-parasitica#infection#on#
American#chestnut.#Cankers#normally#expand#concentrically#from#a#
wound#site#(branch#point,#left),#collapsing#vascular#tissue#as#they#expand#
(right).#Orange#pigmentation#is#evidence#of#disease.#For#interpretation#of#
the#references#to#color#in#this#and#all#other#figures,#the#reader#is#referred#
to#the#electronic#version#of#this#dissertation.#

stem#(Figure#1M6).#Mycoviruses#have#no#extracellular#lifeMstage#in#which#to#spread#of#the#
infected#pathogen#(Milgroom#&#Cortesi#2004);#they#spread#through#vertical#
transmission#by#way#of#asexual#conidia#but#not#through#sexually#produced#ascospores#
(Anagnostakis#1988).#Another#way#mycoviruses#spread,#and#important#for#their#
dissemination#is#through#hyphal#fusion#(horizontal#transmission#through#anastomosis#
of#hyphae)#when#one#fungal#isolate#infected#with#mycoviruses#comes#into#contact#with#
an#uninfected#strain#and#subsequently#transmits#mycoviruses#through#the#cytoplasm#

11#

#

#

that#stream#between#the#newly#connected#hyphae.#Huber#(1996)#described#the#
influence#of#individual#vegetative#incompatibility#(vic)#loci#in#C.-parasitica#on#the#

#
Figure#1M4.#Death#of#a#distal#end#of#an#American#
chestnut#branch#from#a#girdling#canker#caused#
by#Cryphonectria-parasitica.#Yellow,#wilted#
leaves#are#evidence#of#disease.##
#

#

#

#
Figure#1M5.#A#mycovirusMfree#(left)#and#mycovirus#infected#(right)#isolate#of#
the#chestnut#blight#pathogen#Cryphonectria-parasitica.#Both#fungal#isolates#
are#the#same#genotype#and#are#both#about#one#week#old.##

#
#

12#

#

#

#

#
Figure#1M6.#Examples#of#mycovirus#infected#Cryphonectria-parasitica#
cankers#on#American#chestnut#trees.#Note#swollen#nature#of#wounds.#All#
parts#distal#to#these#cankers#are#alive.##

#
#
transmission#of#mycoviruses#between#strains;#mismatches#at#any#of#the#seven#known#
vic#loci#between#two#isolates#of#C.-parasitica#can#significantly#influence#anastomosis#
(discussed#later).##
Mycoviruses#are#commonly#found#to#be#infecting#C.-parasitica#in#Europe#but#they#
have#not#spread#to#all#regions#of#the#continent.#Chestnut#blight#is#prevalent#across#the#
European#continent#but#the#condition#of#hypovirulence#is#variable.#Detection#of#
mycoviruses#and#healing#cankers#on#chestnuts#is#normally#found#to#occur#around#10#
years#after#the#initial#blight#epidemic#(Milgroom#&#Cortesi#2004);#hypovirulence#caused#
by#mycoviruses#is#rare#or#absent#in#areas#where#C.-parasitica#has#been#recently#spread#
such#as#northern#Switzerland,#northern#France,#Germany,#and#Turkey#(Robin#&#

13#

#

#

Heiniger#2001;#Milgroom#et#al.#2004;#Prospero#&#Rigling#2012).##Italy,#southern#
Switzerland,#southern#France,#and#Spain#are#areas#where#the#CHV1#type#of#mycovirus#is#
ubiquitous,#where#European#chestnut#trees#are#recovering#and#where#chestnut#blight#is#
no#longer#a#major#concern#(A.#Vannini,#pers.#comm.).##Five#subMtypes#of#CHV1#have#been#
documented#and#in#some#areas,#the#subtype#recovered#after#introduction#of#
mycoviruses#for#biological#control#differs#from#the#naturally#occurring#type,#lending#
evidence#of#natural#spread#of#hypovirulence#in#some#forest#(and#orchard)#situations.#
CHV1#has#been#found#in#Japan,#Korea,#and#China#but#there#is#no#known#active#research#
on#biological#control#of#C.-parasitica#in#Asia#since#blight#does#not#cause#major#problems#
except#rarely#in#orchard#situations#where#trees#could#be#experiencing#environmental#
stress#(Jaynes#1975;#Milgroom#&#Cortesi#2004).##
Mycovirus#induced#hypovirulence#in#the#United#States#is#much#less#common.##
CHV1#has#been#introduced#from#Europe#as#a#biological#control#mechanism#but#has#
failed#to#become#established#and#spread#beyond#initial#introductions#(Peever#et#al.#
1997).##CHV1#causes#female#sterility#in#C.-parasitica,#severely#debilitates#the#pathogen#
while#reducing#sporulation,#pigmentation#and#virulence.#These#characteristics#of#CHV1#
led#to#the#thought#that#this#would#be#a#useful#mycovirus#for#biological#control#on#the#
North#American#populations#of#American#chestnuts#but#was#not#successful.#CHV2#has#
been#found#in#New#Jersey#and#is#severely#debilitating#toward#C.-parasitica,#reducing#
fungal#development#and#fecundity#(Hillman#et#al.#1992;#1994).##Mycovirus#GH2,#a#CHV3#
type,#is#a#naturally#occurring#mycovirus#in#Michigan,#(discussed#in#Chapter#4).##CHV3#
type#viruses#have#been#found#outside#of#Michigan#in#Kentucky#and#West#Virginia#but#
were#most#likely#the#result#of#their#release#for#attempted#biological#control#(Peever#et#al.#

14#

#

#

1997).#Viruses#similar#to#GH2#have#been#found#across#North#America#(Paul#&#Fulbright#
1988;#Peever#et#al.#1997)#and#in#southern#Ontario,#Canada#(Melzer#&#Boland#1999).##
Mycoviruses#of#the#CHV3#type#do#not#change#morphology#of#C.-parasitica#but#still#
reduces#fungal#virulence.#SR2Mtype#viruses,#now#included#in#the#CHV4#type#have#been#
found#in#Maryland,#Michigan#(a#second#mycovirus#type#found#at#the#Frankfort#site),#and#
throughout#the#Appalachian#region#but#has#little#to#no#effect#on#virulence#or#
morphology#of#C.-parasitica-(Enebak#et#al.#1994).##
The#unencapsidated#dsRNA#mycoviruses#that#infect#C.-parasitica#are#members#of#
the#monogeneric#virus#family#Hypoviridae#(Hillman#et#al.#1994,#1995).#All#CHV#
mycoviruses#belong#to#the#genus#Hypovirus#and#are#divided#into#species#(and#subtypes)#
depending#on#how#each#mycovirus#genome#is#organized#such#as:#the#structure#of#its#
genetic#sequence,#and#symptoms#caused#in#infected#isolates#of#C.-parasitica#(Gobbin#et#
al.#2003).#CHV1#contains#a#fragment#12.7kb#long#with#two#open#reading#frames#with#
four#subtypes#that#can#vary#in#sequence#divergence#between#11#and#19%.#CHV1#
mycoviruses#are#similar#to#CHV2#viruses#(Suzuki#et#al.#2003)#with#a#total#length#of#about#
12.5#kb.##CHV3#(specifically,#GH2)#contains#three#to#four#fragments,#the#largest#at#9.8kb#
with#others#at#3.5kb#and#1.0kb#and#a#single#large#open#reading#frame;#smaller#ORFs#are#
possible#(Smart#et#al.#1999).#CHV4/SR2#types,#those#least#effective#at#decreasing#
virulence#of#C.-parasitica#also#contains#one#ORF#(Suzuki#et#al#2003)#and#a#9.1kb#genome#
(LinderMBasso#et#al.#2005).#Many#regions#of#the#genome#of#Cyphonectria#hypoviruses#
have#been#tested#for#the#specific#actions#they#have#on#C.-parasitica#but#it#appears#that#
regions#known#as#p40#and#p29#specifically#affect#blight#virulence#and#transmission#of#
mycoviruses#into#asexual#conidia#(Suzuki#et#al.#2003).#

15#

#

#

#
Vegetative!incompatibility!
Transmission#of#mycovirus#hyperparasites#from#an#infected#fungal#isolate#to#a#
mycovirusMfree#isolate#can#be#limited#through#the#formation#of#barrage#zones#when#
anastomosis#of#fungal#hyphae#fails#(see#Figure#2M3,#Chapter#2).#Failure#of#anastomosis#
occurs#when#two#neighboring#fungal#colonies#of#the#blight#pathogen#meet#and#
subsequently#die#back#due#to#programmed#cell#death#or#apoptosis.##Mycovirus#transfers#
(or#conversions)#occur#when#two#fungal#colonies#meet#and#anastomose#and#
subsequently#transfer#materials#through#cytoplasmic#streaming.##Liu#and#Milgroom#
(1996)#have#shown#that#the#success#of#mycovirus#transfer#is#negatively#correlated#with#
the#number#of#vegetative#incompatibility#(vic)#genes#that#differ#between#isolates#that#
attempt#to#unite.#Huber#(1996)#showed#that#individual#alleles#that#control#vic#gene#loci#
can#have#varying#effects#on#mycovirus#transmission;#some#vic#loci#are#stronger#at#
preventing#mycovirus#transmission#than#others.#In#this#system#there#are#between#six#
and#eight#biMallelic#loci#that#control#anastomosis.#Loci#are#unequal#in#their#effect,#and#
multiple#mismatches#at#weak#loci#may#continue#to#allow#mycovirus#transmission#while#
an#allelic#mismatch#at#one#locus#of#strong#effect#can#totally#inhibit#mycovirus#
transmission#(Huber#1996).##
Vegetative#compatibility#(VC)#is#a#self/nonMself#recognition#system#in#fungi#and#is#
thought#to#help#maintain#the#integrity#of#individuals#(Rayner#1991)#and#prevent#the#
spread#of#alien#nuclei#and#infectious#elements#such#as#mycoviruses.#In#C.-parasitica,#high#
vegetative#compatibility#group#(VCG)#diversity#in#has#been#linked#to#and#blamed#for#the#
failure#of#mycoviruses#to#spread#through#C.-parasitica#populations#in#North#America#
(Anagnostakis#1986;#Anagnostakis#&#Kranz#1987).##Vegetative#compatibility#groups#
16#

#

#

(VCGs)#are#relatively#easy#to#determine#in#the#laboratory#(see#Chapter#2)#and#have#been#
used#to#assess#diversity#within#fungal#populations#(Milgroom#&#Cortesi#1999).#The#use#
of#vegetative#compatibility#(sometimes#referred#to#as#heterokaryon#or#somatic#cell#
incompatibility)#to#determine#populationMlevel#diversity#is#a#common#technique#for#
fungi,#especially#filamentous#Ascomycetes#(Glass#&#Kuldau#1992;#Leslie#1993),#but#
other#fungal#taxa#such#as#Basidiomycetes#(Casselton#&#Economou#1985;#Aimi#et#al.#
2002),#Zygomycetes#(Griffin#&#Perrin#1960;#Bayman#&#Bennett#1998),#and#
Myxomycetes#(Betterley#&#Collins#1984;#Lane#1987;#Clark#&#Haskins#2012)#also#exhibit#
vegetative#incompatibility.#Understanding#fungal#pathogen#diversity#within#host#
populations#is#important#to#understanding#temporal#and#spatial#dynamics#of#the#system#
and#how#the#pathogen#can#affect#host#individuals#and#populations#over#time##(Burdon#
1987).##
!
Mycoviruses!as!biological!control!agents!
A#common#hypothesis#for#the#successful#invasion#and#spread#of#mycoviruses#in#
populations#of#C.-parasitica#is#that#low#diversity#of#vegetative#compatibility#groups#of#
the#blight#pathogen#allows#easier#spread#of#mycoviruses#(Anagnostakis#1977;#
Anagnostakis#et#al.#1986;#Milgroom#et#al.#1991;#Milgroom#and#Cortesi#2004)#since#
mismatches#of#alleles#are#less#common.#If#mycoviruses#can#spread#through#C.-parasitica#
populations,#they#can#have#significant#effects#on#the#fitness#of#the#blight#pathogen#and#
the#host#tree,#Castanea-dentata#(Griffin#&#Elkins#1986).###
Mycoviruses#have#been#championed#as#potential#biological#control#agents#of#C.parasitica,#but,#their#success#in#North#America#has#been#quite#disappointing#(Milgroom#

17#

#

#

&#Cortesi#2004)#while,#in#Europe,#mycovirus#hyperparasites#are#commonly#found#to#be#
infecting#C.-parasitica#on#European#chestnut#host#trees#(Turchetti#1982;#Heiniger#&#
Rigling#1994;#Robin#&#Heiniger#2001;#Milgroom#&#Cortesi#2004).##MycovirusMmediated#
recovery#of#American#chestnut#populations#has#occurred#naturally#only#at#a#number#of#
sites#in#Michigan,#(Fulbright#et#al.#1983;##
MacDonald#&#Fulbright#1991;#Brewer#1995;#Milgroom#&#Cortesi#2004).##At#these#sites,#
American#chestnuts#grow#into#the#forest#canopy,#flower#and#reproduce#at#rates#
comparable#to#diseaseMfree#populations#in#Michigan#(Davelos#&#Jarosz#2004).#
!
The!chestnut!blight!situation!in!Michigan!
!

American#chestnuts#are#native#to#the#southeastern#corner#of#Michigan’s#lower#

peninsula#(Little#1971),#but#early#settlers#planted#them#widely#across#the#lower#
peninsula#of#Michigan#especially#in#areas#where#Lake#Michigan#modulates#temperatures#
(Brewer#1995).##In#many#areas,#these#planted#trees#have#given#rise#to#naturalized#
American#chestnut#populations#once#the#original#homesteads#were#abandoned.##
#

Chestnut#blight#was#discovered#in#Michigan#around#1928#(Baxter#and#Strong#

1931).##As#in#the#native#range,#many#chestnuts#in#northwestern#Michigan#(see#Chapter#
2)#have#succumbed#to#blight.#However,#abnormal#cankers#were#discovered#to#harbor#
mycoviruses#around#1976#(Fulbright#et#al.#1983;#Brewer#1995)#and#were#naturally#
occurring#since#no#mycoviruses#were#introduced#to#Michigan#before#their#discovery#
(Fulbright#et#al.#1983).#The#state#of#Michigan#contains#the#only#known#examples#of#
successful#natural#invasion#of#mycoviruses#in#North#America#(Fulbright#et#al.#1983).#
Trees#with#cankers#that#contained#mycovirus#infected#C.-parasitica#were#discovered#in#

18#

#

#

at#least#seven#locations#in#Michigan.#At#least#three#mycovirus#types#exist#in#Michigan:#
Cryphonectria#hypovirus#3#(CHV3),#CHV4,#and#a#unique#type#termed#SR2#(Fulbright#et#
al#1983;#Fulbright#et#al.#1988;#Paul#and#Fulbright#1988;#Hillman#et#al.#1995;#Peever#et#al.#
1997,#1998)#are#all#found#in#the#state.#CHV3#is#the#most#common#type#that#is#associated#
with#tree#recovery,#being#found#at#Grand#Haven#(since#removed#for#a#housing#
development),#County#Line#and#Frankfort#(Peever#et#al.#1997).#Frankfort#(discussed#in#
Chapters#2#and#3#in#this#dissertation),#is#characterized#by#the#presence#of#both#CHV3#
and#SR2#(Peever#et#al.#1997).##Thus,#mycoviruses#have#naturally#invaded#in#Michigan#
populations#of#C.-parasitica#and#have#allowed#trees#to#survive#there#much#longer#than#
trees#infected#by#only#the#blight#pathogen#(Fulbright#et#al.#1983;#Davelos#&#Jarosz#2004).##
The#extreme#patchiness#of#recovery#in#Michigan#hints#at#the#possibility#that#genetic#
makeMup#of#trees#at#these#sites#influences#the#eventual#success#or#failure#of#mycoviruses#
as#biological#control#agents#(see#Chapter#4).#
#
Structure!of!this!Dissertation!
#

In#the#following#pages#of#this#dissertation,#the#impact#of#mycoviruses#on#

populations#of#C.-parasitica#and#recovery#of#C.-dentata#are#explored.#My#work#was#
designed#to#address#the#following#questions#and#objectives:##
#

A.#How#is#population#structure#of#the#chestnut#blight#pathogen,#C.-parasitica-

influenced#by#the#presence#of#mycoviruses#with#regard#to#vegetative#compatibility#
group#diversity#of#C.-parasitica?#
#

B.#How#does#the#presence#of#mycoviruses#influence#the#population#structure#of#

the#chestnut#blight#pathogen#at#the#molecular#level?###Additionally,#is#the#pattern#of#

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#

molecular#variability#concordant#with#that#observed#for#vegetative#compatibility#group#
diversity?#
#

C.#Implement#a#deployment#strategy#proposed#by#Davelos#(1999)#and#Davelos#&#

Jarosz#(2004),#which#advocated#that#the#best#trees#to#treat#with#mycoviruses#are#subM
adults#with#1—10cm#DBH#(diameter#at#breast#height)##
#

D.#Are#mycoviruses#susceptible#to#degradation#when#stored#as#static#cultures#

over#long#time#periods?#
#

My#aim#is#to#provide#an#understanding#of#the#population#structure#of#the#

chestnut#blight#pathogen#in#Michigan#with#the#ultimate#goal#of#further#understanding#
the#effect#of#mycovirus#hyperparasites#on#fungal#population#structure.##Additionally,#
this#work#aims#to#determine#what#strategies#work#best#for#deploying#mycoviruses#but#
these#are#longMterm#experiments#that#need#longMterm#monitoring.#This#triMtrophic#
system#can#be#used#as#a#model#system#to#understand#of#similar#systems#where#
techniques#of#biological#control#of#pathogenic#fungi#can#be#applied.##
#

In#Chapter#2,#results#from#a#temporal#comparison#of#vegetative#compatibility#

group#(VCG)#diversity#in#the#blight#pathogen#are#presented.#VCG#data#from#1996#
(Davelos#1999)#are#compared#with#fungal#isolates#collected#in#2009#to#elucidate#
changes#in#population#structure#of#C.-parasitica#in#three#Michigan#population#types:#
those#infected#for#extended#time#periods#with#both#C.-parasitica#and#mycoviruses,#those#
which#only#have#had#C.-parasitica#present#for#a#long#time#period,#and#those#populations#
of#chestnut#hosts#recently#infected#by#the#blight#pathogen.#Data#specific#to#the#success#of#
reviving#stored#cultures#from#1996#collections#are#presented#in#Appendix#A#along#with#
data#that#indicate#the#loss#of#mycoviruses#during#long#term#storage.#In#Chapter#3,#I#use#

20#

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#

nine#microsatellite#loci#to#characterize#molecular#diversity#within#C.-parasitica#
populations,#and#compare#the#pattern#of#diversity#using#microsatellites#with#VCG#data#
from#chapter#2.###
Preliminary#results#of#a#longMterm#study#of#mycovirus#introductions#into#three#C.parasitica#populations#are#presented#in#Chapter#4.#Chapter#5#presents#overall#
conclusions#and#discusses#implications#of#the#work#for#successfully#managing#or#
controlling#the#chestnut#blight#pathogen#so#that#American#chestnut#trees#may#be#
restored#to#the#eastern#hardwood#forests#of#the#United#States.##Additionally,#chapter#5#
presents#directions#for#future#work#on#this#important#model#triMtrophic#system.##
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LITERATURE!CITED!
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LITERATURE!CITED!
!

!
Aimi#T,#Yotsutani#Y,#and#Morinaga#T.#2002.#Cytological#Analysis#of#Anastomoses#and#
Vegetative#Incompatibility#Reactions#in#Helicobasidium-monpa.#Current#Microbiology.#
44:148–152.##
#
Anagnostakis#SL.#1977.#Vegetative#Incompatibility#in#Endothia-parasitica.#Experimental#
Mycology.#1:306–316.##
#
Anagnostakis#SL.#1987.#Chestnut#Blight:#The#Classical#Problem#of#an#Introduced#
Pathogen.#Mycologia.#79:3–37.#
#
Anagnostakis#SL.#1988:#Cryphonectria-parasitica:#cause#of#chestnut#blight.#Advances#in#
Plant#Pathology.#6:123–136.#
#
Anagnostakis#SL.#1992.#Chestnuts#and#the#introduction#of#chestnut#blight.#Annual#
Report#of#the#Northern#Nut#Growers#Association.#83:39–42.#
#
Anagnostakis#SL,#Hau#B,#and#Kranz#J.#1986.#Diversity#of#Vegetative#Compatibility#Groups#
in#Cryphonectria-parasitica#in#Connecticut#and#Europe.#Plant#Disease.-70:#536–538.##
#
Anagnostakis#SL,#Waggoner#PE.#1981.#Hypovirulence,#Vegetative#Incompatibility,#and#
the#Growth#of#Cankers#of#Chestnut#Blight.#Phytopathology.#71:1198–1202.##
#
Anderson#RM#and#May#RM.#1982.#Directly#transmitted#infectious#diseases:#control#by#
vaccination.#Science.#215:1053M1060.#
#
Baxter#DV#and#Strong#FC.#1931.#Chestnut#blight#in#Michigan.#Michigan#Agricultural#
Experiment#Station#Circular#Bulletin.#135:1M18.#
#
Bayman#P#and#Bennett#JW.#1998.#NitrateMnonutilizing#Mutants#and#Vegetative#SelfM
incompatibility#in#Cunninghamella-Echinulata#var.#elegans.##Biotechnology#Letters.20:901–903.#
#
Benkman#CW.#1999.#The#Selection#Mosaic#and#Diversifying#Coevolution#Between#
Crossbills#and#Lodgepole#Pine.#The#American#Naturalist.#153:S75—S91.##
Benkman#CW#and#Parchman#TL.#2003.#Reciprocal#Selection#Causes#a#Coevolutionary#
Arms#Race#Between#Crossbills#and#Lodgepole#Pine.#The#American#Naturalist.#162:182–
194.#
Betterley#DA#and#Collins#OR.#1984.##Vegetative#incompatibility#and#myxomycete#biology.#
Mycologia.#76:785–792.#

23#

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#

Biraghi#A.#1946.#Il#cancro#del#castagno#causato#da#Endothia-parasitica.#Italia#Agricola.#
7:1–9.#
Brewer#LG.#1995.#Ecology#of#Survival#and#Recovery#from#Blight#in#American#Chestnut#
Trees#(Castanea-dentata#(Marsh.)#Borkh.)#in#Michigan.#Bulletin#of#the#Torrey#Botanical#
Club.#122:40.##
Bryner#SF#and#Rigling#D.#2011.#TemperatureMdependent#GenotypeMbyMgenotype#
Interaction#Between#a#Pathogenic#Fungus#and#Its#Hyperparasitic#Virus.#The#American#
Naturalist.#177:65–74.##
Bryner#SF#and#Rigling#D.#2012.#Virulence#Not#Only#Costs#but#Also#Benefits#the#
Transmission#of#a#Fungal#Virus.#Evolution.-66:2540–50.##
#
Buck#KW.#1986.#Fungal#VirologyM#an#overview.#Pages#1M84#in:#Fungal#Virology.#K.#W.#
Buck,#ed.#CRC#Press,#Boca#Rotan,#FL,#U.S.A.#
#
Burdon#JJ.#1987.#Diseases#and#plant#population#biology.#Cambridge#University#Press,#
Cambridge.##
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Burdon#JJ,#Thrall#PH,#and#Ericson#L.#2006.#The#Current#and#Future#Dynamics#of#Disease#
in#Plant#Communities.#Annual#Review#of#Phytopathology.#44:19–39.##
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#
Clapper#RB.#1952.#Relative#blight#resistance#of#some#chestnut#species#and#hybrids.#
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#
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Transformation#by#Emerging#Infectious#Disease:#Loss#of#Large#Tanoak#from#California#
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#
Davelos#AL#and#Jarosz#AM.#2004.#Demography#of#American#chestnut#populations:#
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#
Davelos#AL.#1999.#DoubleMstranded#RNA#mediated#recovery#of#American#chestnut#
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Lansing,#Michigan.#
#

24#

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#

Dinus#RJ.#1974.#Knowledge#about#natural#ecosystems#as#a#guide#to#disease#control#in#
managed#forests.#Proceedings#of#the#American#Phytopathological#Society.#1:184—190.##
#
Dobson#AP#and#Hudson#PJ.#1986.#Parasites,#Disease#and#the#Structure#of#Ecological#
Communities.#Trends#in#Ecology#&#Evolution.#1:11–5.##
#
Fairchild#D.#1913.#The#discovery#of#the#chestnutM#bark#disease#in#China.#Science#38:#297M
299.#
#
Fenner#F.#1983.#Biological#control#as#exemplified#by#smallM#pox#eradication#and#
myxomatosis.#Proceedings#of#the#Royal#Society#of#London#B#218:259M285#
#
Fenner#F#and#Radcliffe#FN.#1965.#‘Myxomatosis’.#Cambridge#University#Press:#
Cambridge,#UK.#
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#
#
#
#
#
#
CHAPTER(2!
#
#
HYPERPARASITES*INFLUENCE*POPULATION*STRUCTURE*OF*THE*CHESTNUT*
BLIGHT'PATHOGEN,'CRYPHONECTRIA'PARASITICA'!
#
#
ABSTRACT!
!

Vegetative#compatibility#(VC)#is#commonly#used#to#characterize#structure#and#

diversity#in#fungal#populations.##In#the#chestnut#blight#fungus,#Cryphonectria-parasitica,#
high#VC#diversity#is#hypothesized#to#be#responsible#for#the#failure#of#hyperparasitic#
mycoviruses#to#spread#through#pathogen#populations#in#North#America.#To#test#this#
hypothesis,#we#assessed#VC#diversity#at#three#recovering#sites#in#Michigan#where#
mycoviruses#had#invaded#and#compared#them#with#four#epidemic#populations#where#
mycoviruses#were#absent.#VC#diversity#was#assessed#for#samples#collected#in#1996#and#
2009,#which#allowed#us#to#determine#how#C.-parasitica#populations#changed#with#time.##
Twelve#VC#types#were#found#in#1996#while#thirty#were#found#in#2009;#75%#of#types#
overlapped#between#the#sample#dates.#Sites#where#mycoviruses#were#present#had#
unique#VC#structures#with#the#exception#of#the#recovering#population#site#at#County#
Line#where#the#main#VC#group#was#also#detected#at#two#epidemic#sites.#Cryphonectriaparasitica#populations#at#epidemic#sites#usually#had#more#VC#groups#and#were#more#
diverse.##Mating#type#analyses#of#blight#populations#revealed#that#two#of#three#
recovering#populations#were#significantly#skewed#for#MAT2#suggesting#asexual#
reproduction,#while#epidemic#sites#with#a#long#history#of#blight#infection#had#ratios#near#
31#

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50:50#suggesting#sexual#reproduction.#We#propose#that#selection#in#the#largely#asexual#
C.-parasitica#populations#at#two#recovering#sites#favors#the#mostMfit#fungal#genotype#by#
mycovirus#combination#and#results#in#reduced#diversity#relative#to#the#sexually#
reproducing#pathogen#populations#at#epidemic#sites.#
#
INTRODUCTION!
#
#

Introduced*pathogens*have*a*long*history*of*causing*extensive*destruction*to*

their&plant&hosts&with&trees&being&particularly&vulnerable.&&For&example,&Dutch&elm&
disease&caused&by&Ophiostoma*ulmi*and!O.#novoBulmi!has$changed$the$urban$landscape$
in#the#United#States#and#Europe#by#killing#highly#susceptible#elms#planted#as#shade#trees#
(Brasier(1995,(2001;(Potter(et(al.(2011;(Santini(et(al.(2012).((American(chestnuts(
(Castanea'dentata!L.)$have$suffered$a$similar%fate%in%North%America.%%Prior%to%the%1900s,%
C.#dentata!composed(nearly(25%(of(the(canopy(in(eastern(hardwood(forests,(but(these(
natural'populations'were'decimated'when'Cryphonectria-parasitica-Murrill&(Barr.),&the&
cause&of&chestnut&blight,&was&accidentally%introduced%to%the%United%States%in%the%early%
twentieth'century'near'New'York'City'(Merkel'1906).''Cryphonectria-parasitica-enters&
wounds'on'the'trunk'and'branches'of'susceptible'trees'and'causes'a'canker'that'
expands(in(size(until(the(branch(or(trunk!is#girdled,#killing#all#plant#material#distal#to#the#
canker.((Trees(accumulate(multiple(infections(during(an(epidemic(and(eventually(all(
aboveground+portions+of+the+tree+are+killed.++However,+C.#parasitica!cannot&enter&the&
roots%of%infected%trees%and%chestnuts%can%survive%for%considerable%time%as%a%live%root%
system&that&periodically&produces&root&collar&sprouts.&&These&sprouts&grow&for&several&
seasons&before&being&infected&and&killed&by&C.#parasitica.""Consequently,"C.#dentata!is#still#

32#

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#

a"common,"persistent,"understory"tree"in"several"areas"(Anagnostakis"2001;"Griffin"&"
Elkins'1986;"McCormick"&"Platt"1980).###
Cryphonectria-parasitica!was$also$introduced$in$Europe$in$the$1930s$and$the$
initial&stages&of&the&European&blight&epidemic&were&similar&to&that&in&North"America.""
However,(European(chestnuts((Castanea'sativa)"began"recovering"by"the"1950s"
(Heiniger(&(Rigling(1994).((Canker(expansion(on(recovering(trees(was(contained(by(
wound&callus&tissue,&which&prevented&girdling&of&the&trunk&and&allowed&infected&trees&to#
continue(growing.((Recovery(was(not(due(to(resistance(in(European(chestnuts((although(
their&reduced&susceptibility&relative&to&American&chestnuts&may&play&a&role),&but&was&
caused'by'reduced'pathogen'virulence'induced'by'the'presence'of'a'doubleMstranded!
RNA$mycovirus$hyperparasite$(Grente$1965;$MacDonald$&$Fulbright$1991;$Milgroom$&$
Cortesi(2004).(Mycoviruses(reduce(pathogen(virulence(by(reducing(fungal(growth,(
decreasing*conidia*production,*and*inhibiting*sexual*reproduction*(i.e.,*ascospore*
production)!and$therefore$can$act$as$a$biological$control$mechanism$against$the$blight$
pathogen)(Anagnostakis)1987;)Carbone)et)al.)2004;)Elliston)1985;)McCormick)&)Platt)
1980).''Over'time,'mycoviruses'spread'to'many'areas'in'Europe'and'are'thought'to'be'
responsible*for#the#recovery#of#C.#sativa.""With"the"exception"of"some"isolated"
populations*in*Michigan,*mycoviruses*are*relatively*rare*in*North*America*and*efforts*to*
introduce*them*as*biological*control*have*been*largely*unsuccessful*(Milgroom*&*
Cortesi(2004).###
Considerable)speculation)on)why)mycoviruses)have)not)invaded)North)American)
populations*of*C.#parasitica!has$centered$on$the$higher$diversity$of$vegetative$
compatibility+(VC)+groups+in+North+America+relative+to+Europe+(Anagnostakis+et+al.+

33#

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#

1986).''VC'groups'are"a"consequence"of"a"self/nonMself%recognition%system%found%in%
many%ascomycetes%that%are%controlled%by%several%unlinked%vegetative%incompatibility%
(vic)"or"heterokaryon"incompatibility"(het)"loci"(Glass"et"al."2000).""The"incompatibility"
loci%operate%to%detect$nonMself%hyphae%when%the%hyphal%tips%of%adjacent%colonies%fuse%
(anastomosis).++Mismatches+at+any+incompatibility+locus+trigger+apoptosis+(programmed+
cell$death)$causing$the$connection$between$mismatched$hyphae$to$die$(for$review,$see$
Glass%&%Kaneko%2003).%%%Apoptosis%prevents%alien%nuclei%from%invading%the%fungal%colony%
and$reduces$the$transfer$of$cytoplasmic$elements$such$as$mycoviruses$(Anagnostakis$
2001;%Bertrand%2000;%Huber%1996;%Liu%&%Milgroom%1996;%MacDonald%&%Fulbright%1991;%
Milgroom(&(Cortesi(1999).$Alleles$at$vic!loci%are%thought%to%be%under%negative%frequency%
dependent&or&balancing&selection&which&maximizes&the&ability&of&individuals&to&detect&
nonMself%hyphae%upon%fusion%(Glass&&&Kaneko&2003;&Micali&&&Smith&2006;&MirMRashed'et'
al.$2000;$Wu$et$al.$1998)$and$promotes(high(levels(of(diversity(for(VC(groups(within(
fungal'populations'(see'Table'1'of'Nauta'&'Hoekstra'1994).""However,"theoretical"work"
by#Taylor#et#al.#(1998)#and#Morozov#et#al.#(2007)#suggest#that#the#vic!incompatibility,
system&may&not!be#a#complete#barrier#to#mycovirus#invasion#since#mismatches#at#one#or#
two$vic!loci%reduces%but%does%not%entirely%prevent%transmission%of%mycoviruses%between%
fungal'colonies'(Liu'&'Milgroom'1996).''#
# Here$we$address$a$different$aspect$of$the$interaction$between%the%chestnut%blight%
pathogen)and)its)mycoviruses)by)comparing)seven)C.#parasitica!populations*in*Michigan*
to#determine#how#invasion#by#mycoviruses#influences#the#VC#structure#of#the#pathogen.##
Mycoviruses+have+been+present+for+an+extended+period+of+time%(since%at%least%the%early%
1980s)'at'three'of'our'study'sites:'County'Line,'Frankfort'and'Roscommon'(Brewer'

34#

#

#

1995;%Davelos%1999;%Fulbright%et%al.%1983),%while%they%are%largely%absent%at%the%other%
four%sites%(Davelos%1999).%In%1996%when%this%study%was%initiated,'greater'than'90%'of'all'
C.#parasitica!samples'from'the'three'recovering'sites'contained'mycovirus'(Davelos'
1999).%%%Mycoviruses%have%a%substantial%positive%influence%on%chestnut%trees%at%the%three%
sites%by%increasing%tree%survivorship,%growth%and%reproduction+resulting+in+a+general+
“recovery”)of)these)chestnut)populations)(hereafter)recovering)sites).))Indeed,)the)
population)demographics)at)two)of)the)recovering)sites)are)similar)to)those)of)diseaseM
free$chestnut$populations$in$Michigan$(Davelos$&$Jarosz'2004).''In'contrast,'trees'at'the'
four%sites%without%mycoviruses%have%demographics%similar%to%what%is%found%in%the%main%
range&of&the&American&chestnut&where&all&large&trees&are&dying&back&as&a&result&of&the&C.#
parasitica!epidemics((hereafter(epidemic"sites).""#
What%is%not%known%is%the%extent%to%which%invasions%by%mycoviruses%influence%the%
population)structure)of)the)C.#parasitica!pathogen.***Mycoviruses*would*be*expected*to*
alter&the&population&genetics&of&C.#parasitica!since&they&inhibit&the&pathogen’s$sexual$
cycle%(Gobbin%et%al.%2003).%%The%lack%of%a%sexual%cycle%in%mycovirusMinfected(individuals(
of#C.#parasitica!would&be&expected&to&have&multiple&influences&on&pathogen&population&
structure:((1.(Diversity(of(VC(groups(should(decrease(because(genetic!recombination+
through'sexual'reproduction'does'not'occur'on'a'regular'basis,'2.'Selection'will'operate'
at#the#wholeMgenome&level&in&asexual&populations&of&the&fungal&pathogen&because&of&the&
lack%of%recombination,%3.%Dispersal%of%the%fungal%pathogen%should!be#altered#because#the#
windMdispersed'sexual'ascospores'are'not'produced'and'the'splashMdispersed'asexual'
spores&(i.e.,"conidia)"have"a"more"limited"dispersal"distance."#
# Our$objectives$were$to$compare$the$VC$structure$of$C.#parasitica!found&at&the&three&

35#

#

#

recovering)populations)with)that)of)the)four)epidemic)populations)to)determine)how)
mycoviruses+influence+VC+structure+over+time.+Additionally,+we+investigated+the+
temporal)pattern)at)five)sites)to)determine)if)the)pattern)of)variability)differed)between)
recovering)and)epidemic)populations)over)the)time)period)1996!to#2009."#
#
MATERIALS)AND)METHODS!
!
# Chestnut(blight(cankers(were(sampled(from(five(blighted(American(chestnut(
populations*in*1996*and*seven*in*2009.**Thirty*haphazard*samples*from*each*
population)were#collected#in#2009#while#the#number#of#samples#used#for#comparison#
from%1996!was$variable$since$we$were$unable$to$resurrect$some$samples$from$storage$
(see$Chapter$5).$Three$of$the$blighted$tree$populations,$County$Line$(CL),$Roscommon$
(RC),&and&Frankfort"(FR)"have"been"invaded"naturally"by"doubleMstranded(RNA(
mycoviruses+and+the+American+chestnut+trees+at+those+sites+are+recovering+from+the+
blight'epidemic'(Davelos'&'Jarosz'2004;'AM'Jarosz'and'JC'Springer,'unpublished'data).'
However,(in(recent(years,(many"trees"at"the"Frankfort"site"have"died"due"to"blight"
infections!(AM$Jarosz$and$JC$Springer,$unpublished$data).$The$four$remaining$sites,$
Stivers((ST)(and(Missaukee(Diseased((MD)(Missaukee(Healthy((MH)(and(Leelanau((LE),(
are$experiencing$blight$epidemics$that$have$severely$debilitated$the$chestnut$trees;$
mycoviruses+have+not+been+found+at+these+sites+at+a+level+higher+than+5%+of+cankers+
sampled((Davelos(1999;(JC(Springer,(unpublished(data).(Population(location(coordinates(
can$be$found$in$Davelos$and$Jarosz$(2004)&for&all&sites&except&Roscommon.&&The&
Roscommon'site'is'located'at'44º'31''28"N,'M84º$30'$48"W$(see$Figs.$1$&$2$for$general$
locations).+Blight+was+first+found+at+MH+and+LE+in+1997;+samples+were+obtained+from+

36#

#

#

these%sites%only%in%2009.%Samples%from%1996"were"resurrected"from"sterile"water"
storage(vials(inoculated(with(mycelial(plugs;(200μl"of"water"from"each"vial"were"plated"
onto$potato$dextrose$agar$(PDA)$petri$plates$(Difco,$Becton,$Dickinson$and$Company;$
Sparks,(MD).(For(2009(samples,(2mm(x(2mm(subsamples&of&each&bark&collection&were&
surface(sterilized(in(10%(sodium(hypochlorite(solution(for(20(seconds,(rinsed(in(double(
distilled'H20"twice"for"20"seconds;"clean"isolates"were"plated"onto"PDA."#
Since&the&presence&of&mycoviruses&can&obscure&VC&testing,&(DW#Fulbright,#pers.#
obs.)&mycovirusMfree$isolates$were$obtained$from$all$samples$before$testing$for$
vegetative'compatibility.'''Since,'transmission'of'mycovirus'to'conidia'is'rarely'100%'
(Enebak(et(al.(1994;(Wu(et(al.(1998)(mycovirusMfree$isolates$were$obtained&by&plating&
serially(diluted(single(conidia(onto(PDA(plates(and(an(isolate(with(a(fastMgrowing'
virulent)phenotype)was)chosen.)))Vegetative)compatibility)tests)were)performed)on)deM
barked'chestnut'stem'pieces'embedded'in'PDA.''Chestnut'pieces'were'approximately*
one$centimeter$long,$cut$longitudinally$and$placed$in$standard$100$mm$×!15#mm#petri#
plates'(Medegen;'Gallaway,'TN)'according'to'Huber'(1996).'Isolates'were'allowed'to'
grow%across%the%chestnut%stem%pieces%for%approximately%two%weeks%before%compatibility&
reactions*were*scored*as*either*compatible*or*incompatible*using*evidence*of*a*solid*
mat$of$mycelia$or$barrage$formation$between$isolates,$respectively$(Fig.$3).$$An$internal$
control'of'two'isolates'from'the'same'canker'was'included'on'each'plate.''Fresh&isolates&
of#similar#age#(usually#less#than#one#week#old)#were#used#for#testing#since#they#
exhibited(rapid,(consistent(growth(on(the(petri(plate.((VC(types(were(determined(
visually,)since)the)formation)of)barrage)lines)is)sufficient)to)determine)that)cultures(
differ&for&VC&group.&&#

37#

#

#

For$mating$type$analysis,$isolates$were$grown$on$PDA$plates$overlaid$with$
sterilized)cellophane.)After)approximately)two)weeks,)fungal)mats)were)scraped)off)of)
the$cellophane$and$dried$at$room$temperature$overnight.$Isolates!were$ground$to$a$fine$
powder'in'mortars'with'liquid'nitrogen.'DNA'extractions'were'performed'with'25—
30mg%of%powdered%fungus%with%a%Qiagen%plant%mini%kit%(Qiagen%corporation,%Valencia,%
CA)$according$to$manufacturer$directions.$Primer$sequences$for$MAT!1"and#MAT!2"and"
thermocycler*protocol*follow*previous*work*of*(Marra*&*Milgroom*2001).*GoTaq*Green*
master'mix'(Promega'corporation,'Madison,'WI)'was'used'according'to'manufacturer'
directions*in*place*of*separate*PCR*components.*PCR*products*(5uL*subsample)*were#
run$on$a$1%$agarose$gel$for$35$minutes$at$90v$and$visualized$under$UV$light$and$scored$
according)to)migration)distance)on)the)gel)(Marra)&)Milgroom)2001).)ChiMsquare%tests%#

#

#
Figure#2M1.#Vegetative#compatibility#testing#on#chestnut#stem#
pieces.##Compatible#reaction#(dashed#arrow)#between#sample#226#
and#166,#incompatible#reaction#(solid#arrow)#between#samples#
166#and#221.
38#

were$performed$on$the!frequencies*of*the*two*MAT!alleles%in%each%site%to%determine%if%
populations*diverged*from*the*50:50*ratio*expected$if$the$pathogen$population$was$
sexually(reproducing.#
#
DATA$ANALYSIS!
# The$lack$of$a$sexual$cycle$in$mycovirusMinfected(individuals(of(C.#parasitica!would&be&
expected'to'have'multiple'influences'on'pathogen'population'structure:''1.'Diversity'of'
VC#groups"should"decrease"because"genetic"recombination"does"not"occur"on"a"regular"
basis,&2.&Selection&will&operate&at&the&wholeMgenome&level&in&asexual&populations&of&the&
fungal'pathogen'because'of'the'lack'of'recombination,'3.'Dispersal'of'the'fungal'
pathogen)should&be&altered&because&the&windMdispersed'sexual'ascospores'are'not'
produced(and(the(splashMdispersed'asexual'spores'(i.e.,"conidia)"have"a"more"limited"
dispersal)distance.)#
To#specifically#address#these#predictions,#we#asked#the#following#questions:#1.#!Do#
populations*with*mycovirus*have*lower*VC*diversity*than*populations*without*
mycovirus?+2.+Does+VC+diversity+decrease+over+time+in+populations+with+mycovirus?+If+
yes,%this%result%would%be%consistent%with%selection%for%a%most%fit%fungusMmycovirus*
combination.&&3."Are"population"structures"similar"in"populations"that"are"spatially"
close?''If'yes,'this'pattern'would'be'consistent'with'limited'dispersal'of'spores.'#
To#address#the#first#two#questions,#diversity#measures#were#calculated.##Alpha#diversity,#
used#here#as#population#level#VC#richness,#was#calculated#as#the#total#number#of#VC#
groups#within#a#population.#Strong#weighting#to#dominant#members#of#the#community#

39#

of#interest#is#given#by#Simpson’s#index#(Hill#et#al.#2003).#Simpson’s#diversity#index#is#
based#on#the#formula:#
#DS#=#1M[∑ni(niM1)/N(NM1)],##
where#ni-is#the#number#of#isolates#in#a#VC#group#and#N#is#the#total#number#of#isolates.#A#
moderate#weighting#to#rare#and#intermediate#species#in#comparison#to#dominant#
species#is#provided#by#the#Shannon#index.##Thus,#the#Shannon#index#is#more#sensitive#to#
changes#in#abundance#of#rare#groups#in#comparison#to#other#diversity#indices#(Hill#et#al.#
2003).-The#Shannon#diversity#index#is#based#on#the#formula:#
#H#=#M∑xi/xo)ln(xi/xo),#
-where#xi-is#the#number#of#isolates#in#a#vc#group#and#xo-is#the#total#number#of#isolates.#
Diversity#indices#and#standard#deviations#for#epidemic#and#recovering#populations#
were#calculated#using#EstimateS#(Colwell#2005).#
Morisita’s#index#of#dispersion#measures#overlap#of#VC#types#among#samples#or#
populations#(Magurran#1988)#and#can#be#thought#of#as#a#probability.#Index#values#are#
generated#using#the#formula:##
CD#=#2∑S#i=1#xiyi/(Dx-+-Dy)-XY)#
Where#xi#represents#how#many#instances#species#i#is#in#the#total#X#in#one#population,#yi#
how#many#instances#VC#group#i-is#in#the#total#Y#from#another#population.##Dx#and#Dy#are#
replaced#by#Simpson’s#values#according#to#population#x#and#y#and#S#is#the#number#of#
species#unique#in#a#population.#Morisita’s#index#values#range#from#0#(no#similarity#
between#populations)#to#1#(total#similarity).##The#matrices#of#Morisita’s#index#values#
and#physical#distances#(in#kilometers)#for#the#seven#populations#were#compared#with#

40#

each#other#to#determine#if#they#were#correlated#using#Mantel’s#test#(Rohlf#1998).##A#
positive#correlation#indicates#that#populations#that#are#spatially#close#have#similar#VC#
population#structures.##The#significance#of#the#correlation#was#determined#as#described#
in#Lapointe#and#Legendre#(1992).#
#
RESULTS!
Vegetative(compatibility(group(diversity!
#

Twelve&VC&groups&were&detected&among&the&five&populations&sampled&in&1996&

while&29&VC&groups&were&found&among&seven&populations&in&2009.$$$75%$(9$of$12)$of$the$
VC#groups#found#in#1996#were#detected#again#in#2009.##The#VC#diversity#and#structure#of#
C.#parasitica!populations*was*strongly*influenced*by*the*presence*of*mycovirus.****
Recovering*sites,*where*mycoviruses*are*present,*averaged!2.7$and$3.7$VC$groups$per$
site%in%1996%and%2009,%respectively.%%The%increase%in%the%number%of%VC%groups%at%
recovering)sites)in)2009)was)due)entirely)to)an)increase)in)the)number)of)VC)groups)at)
FR#where#the#number#of#VC#groups#increased#from#four#to#eight,"(Figs."1"and"2).""Further,(
the$frequency$of$the$2$dominant$VC$groups$at$FR$changed$dramatically$between$the$
sampling)dates;)the)most)common)VC)group)in)1996)was)found)at)almost)half)the)
frequency)in)2009)(80%)versus)43%))and)the)second)most)common)VC)group#in#1996#
more%than%doubled%in%frequency%in%2009%(13%%versus%30%)%(Figs.%1%and%2).!CL#and#RC#
were$both$dominated$by$a$single$VC$group$in$1996,$which$remained$dominant$in$2009$
(Figs.'1'and'2).''However,'the'most'striking'feature'of'each'recovering'population#was#
that$all$VC$groups$within$a$site$were$unique$to$that$particular$site$(Figs.$1!and$2)"aside"
from%the%main%group%at%County%Line%(MIM5),#which#was#also#found#at#two#of#the#epidemic#

41#

sites,&MD&and&LE.#
# In#contrast#with#the#pattern#in#recovering#populations,#five#out#of#the#thirty#different#
VC#groups#(17%)#were#found#in#multiple#epidemic#populations.##Group#MIM2,#was#
detected#at#all#four#epidemic#sites#in#2009,#while#MIM1#and#MIM5#were#found#at#three#
sites#and#MIM3#and#MIM4#were#found#at#two#sites#(Figs.#1#and#2).##Despite#these#shared#
VC#groups,#the#overall#pattern#of#VC#diversity#was#not#correlated#with#physical#distance#
between#populations#(r#=#0.29,#P#<#0.25).#The#lack#of#correlation#is#especially#curious#
when#considering#two#epidemic#sites,#MH#and#MD;#the#sites#are#separated#by#less#than#a#
kilometer#but#shared#only#a#single#VC#group,#MIM2#(Fig.#2).#We#have#monitored#the#trees#
at#MD#and#MH#since#1996#(16,#AM#Jarosz#and#JC#Springer,#unpublished#data)#and#the#
first#infected#tree#at#MH#was#detected#in#1997.###In#the#fifteen#years#since#the#first#
infection#at#MH#VC#diversity#has#increased#to#a#level#that#is#comparable#with#the#MD#site#
that#has#been#infected#since#the#late#1980s#(DW#Fulbright,#pers.#obs.).##Infected#trees#
were#also#noted#in#1997#at#LE#and#by#2009#the#number#of#VC#groups#and#overall#VC#
diversity#was#similar#to#that#found#at#MD#and#MH,#sites#that#are#in#the#main#area#of#
Michigan#where#chestnut#blight#is#found.##Generally,#epidemic#sites#had#more#VC#groups#
than#recovering#sites,#6.5#in#1996#and#6.75#in#2009#and,#although#not#statistically#
significant,#trends#indicated#higher#overall#diversity#for#VC#groups#in#epidemic#sites#
(Table#1).##The#number#of#VC#groups#increased#from#9#to#11#at#ST#from#1996#to#2009,#
while#the#number#of#VC#groups#remained#constant#at#MD.

42#

Missaukee(Diseased*(

Roscommon'

Frankfort)

MI#17% MI#1%
MI#2%
MI#16%

County'Line'

MI#15%

S"vers*(

MI#3%

MI#14%

MI#4%

MI#13%

MI#5%

MI#12%

MI#6%

MI#8%
MI#11%
MI#10% MI#9%
#
#
Figure'2$2."Vegetative"Compatibility"diversity"for"the"year"1996"at"five"Michigan"populations."Sizes&of&slices&represent&
43#

Figure'2$2(cont’d).+proportion'of'each'VC'type'found'at'each'site.'A'key'to'Michigan'VC'types'is'provided.'Rare%types%at"#
recovering#and#epidemic#populations#are#represented#by#white#and#black#slices,#respectively,#and#in#some#cases#more#
than#one#type#is#represented#by#the#slice.#*1996#Data#for#Stivers#and#Missaukee#Diseased#represent#only#those#samples#
that#could#be#resurrected#from#storage;#see#text#for#details.##
#

44#

Missaukee(Healthy(

!Leelanau!

Missaukee(Diseased(

Frankfort)

Roscommon''

County'Line'

S"vers'

Figure'2$3."Vegetative"Compatibility"diversity"data"for"the"year"2009!at#seven#Michigan#populations.#Rare#types#at##
45#

#

Figure'2$3!(cont’d).*recovering*and*epidemic*populations*are*represented*by*white*and*black*slices,'respectively.'
Recovering*sites*are*denoted*with*leader&lines&ending&in&solid&circles,&epidemic&sites&with&solid&squares,"respectively.#

46#

Table#2*1.#Summary#diversity#statistics#for#Epidemic#and#Recovering#populations.#VC#
richness#represents#the#total#time#period#1996#to#2009.#
Maximum#
Total#number# Simpson±#
Shannon#±#
Site#Type#
Shannon#(%#
of#VC#groups#
2SE#
2SE#
of#max)#
Epidemic#(LE,#
19#
5.67#±#1.03#
1.99#±#0.36#
2.94#(67.6)#
MH,#ST,#MD)#
Recovering#
11#
3.04#±#0.65#
1.25#±#0.27#
2.39#(52.1)#
(RC,#CL,#FR)#
#
#
Mating'type'allele'frequencies'
# Two#recovering#populations,#CL#and#RC,#had#mating#type#allele#frequencies#that#were#
highly#skewed#toward#the#MAT2#allele#in#both#1996#and#2009#(Table#2).##These#two#
populations#deviated#significantly#from#the#expectation#of#a#50:50#mating#type#ratio#
that#would#be#expected#if#these#C.&parasitica#populations#were#regularly#going#through#a#
sexual#cycle.###The#third#recovering#population,#FR,#was#skewed#towards#the#MAT2#allele#
but#frequencies#were#not#as#extreme#as#found#at#CL#and#RC,#and#allele#frequencies#did#
not#deviate#significantly#from#50:50#in#either#1996#or#2009.##As#explained#below,#we#
suspect#that#the#C.&parasitica#population#at#FR#is#gradually#escaping#mycovirus#infection.##
The#mating#type#allele#frequencies#at#the#epidemic#populations#that#were#infected#
before#1980#(ST#and#MD)#did#not#differ#significantly#from#a#50:50#mating#type#ratio#in#
either#1996#or#2009.##Ratios#were#closer#to#50:50#in#2009#at#both#sites.##Contrary#to#
recovering#populations,#ST#and#MD#displayed#a#slight,#but#persistent#skew#towards#
MAT1#at#both#sampling#periods.##Mating#type#ratios#deviated#significantly#from#50:50#at#
MH#and#LE#in#2009.##Trees#at#the#MH#and#LE#sites#were#disease*free#in#1996#and#C.&
parasitica#was#first#noted#at#both#sites#in#1997.##Despite#the#fact#that#both#MH#and#LE#

47#

deviate#from#a#50:50#mating#type#ratio,#MH#was#skewed#toward#MAT1,#while#the#C.&
parasitica#population#at#LE#was#skewed#toward#MAT2.###The#excess#of#MAT1#at#MH#
could#possibly#be#due#to#its#juxtaposition#to#MD,#which#is#only#300M#away#from#MH#and#
displayed#a#slight#excess#of#MAT1#in#both#1996#and#2009.

48#

#
Table#2*2.#Distribution#of#mating#types#within#Cryphonectria-parasitica#populations#in#Michigan.##Mixed#indicates#number#
of#samples#in#which#both#mating#types#were#present.##The#chi*square#(X2)#value#tests#whether#the#population#is#at#the#
50:50#mating#frequency#expected#if#sexual#recombination#is#occurring#regularly.#
#
1996#
2009#
Population#
MAT1#
MAT2#
Mixed#
MAT1#
MAT2Mixed#
X2#
X2#
Recovering#

#

#

#

#

#

#

#

#

CL#

2#

12#

1#

7.14#*a#

3#

18#

1#

10.71*#

RC#

4#

15#

0#

6.37#*#

5#

21#

0#

9.85#*#

FR#
Epidemic#

9#

15#

3#

1.50#n.s##

12#

17#

3#

0.86#n.s.#

#

#

#

#

#

#

#

#

ST#

10#

5#

0#

1.67n.s.#

15#

13#

1#

0.14#n.s.#

11#
4#
0#
3.27#n.s.#
15#
#
#
#
#
20#
b#
***#
***#
***#
***
MH#
#
#
#
#
9#
LE#
***#
***#
***#
***#
a:#Critical#chi*square#for#p#=0.05#is#3.84,#with#one#degree#of#freedom=1.#
b:#MH#and#LE#were#blight*free#in#1996

11#

7#

0.62#n.s.#

8#

1#

5.14#*#

20#

2#

4.17#*#

MD#

49#

DISCUSSION#
# Vegetative#incompatibility#systems#in#fungi#are#thought#to#operate#as#self/non9self#
recognition#systems#that#function#to#thwart#the#invasion#of#alien#nuclei#and#
mitochondria#as#well#as#parasitic#elements#(e.g.,#mycoviruses)#that#inhabit#the#
cytoplasm#(Anagnostakis#1977;#Nauta#&#Hoekstra#1994;#Lawrence#et#al.#1988;#Rayner#
1991;#Bertrand#2000;#Glass#&#Kaneko#2003;#Glass#&#Dementhon#2006;#Burdon#&#Thrall#
2008;#Roper#et#al.#2011).#To#minimize#the#probability#of#invasion#by#alien#entities,#
negative#frequency9dependent#selection#should#operate#to#maximize#variability#at#loci#
controlling#self9incompatibility#within#fungal#populations#(Nauta#&#Hoekstra#1994;#
Milgroom#&#Cortesi#1999;#Brusini#et#al.#2011).#If#this#hypothesis#is#correct#then#we#
expect#the#number#of#VC#groups#within#a#population#to#increase#over#time#and#overall#
VC#diversity#should#also#increase.##High#VC#diversity#is#expected#to#slow#or#even#stop#the#
spread#of#mycoviruses#within#a#fungal#population.#
##

Patterns#of#VC#diversity#in#C.#parasitica#have#been#used#to#explain#the#success#or#

failure#of#mycovirus#mediated#recovery#in#European#and#American#chestnut.##Mycovirus#
invasion#has#been#common#in#Europe#where#VC#diversity#is#low#(Milgroom#&#Cortesi#
2004),#perhaps#due#to#low#diversity#among#the#founders#of#European#populations.##For#
example,#VC#type#EU91#is#found#at#high#frequency#in#C.#parasitica#populations#on#the#
Black#Sea#coast#of#Turkey#(Akilli#et#al.#2009),#while#EU92,#and#EU95#are#common#in#
northern#Italy,#southern#France,#Switzerland,#and#eastern#Spain#(Robin#&#Heiniger#
2001).#Another#VC#type,#EU912,#is#dominant#in#Bulgaria,#Sicily,#Romania,#southern#Italy,#
and,#Greece#comprising#82%#of#isolates#collected#while#VC#type#EU91#is#present#at#low#
frequency,#around#1%.##Similar#situations#can#be#found#elsewhere#in#Europe#where#it#

50#

appears#that#a#few#founders#at#a#site#results#in#the#proliferation#of#one#to#three#groups#
(Sotirovski#et#al.#2006).#In#contrast#to#the#successful#spread#of#mycoviruses#in#Europe,#
the#phenomenon#is#rare#in#North#America;#the#only#known#natural#invasions#occurring#
in#Michigan#(Fulbright#et#al.#1983;#Peever#et#al.#1997;#Milgroom#&#Cortesi#2004;).##
Several#authors#have#hypothesized#that#the#inability#of#mycoviruses#to#gain#a#foothold#
in#North#American#populations#is#due#to#the#relatively#high#VC#diversity#(Anagnostakis#
1986;#Cortesi#et#al.#2001;#Heiniger#&#Rigling#1994;#Milgroom#&#Cortesi#2004;#Nuss#
1992;#Robin#et#al.#2000).##Our#results#are#superficially#consistent#with#this#this#
hypothesis.##There#was#a#tendency#for#a#greater#number#of#VC#groups#and#higher#VC#
diversity#within#C.#parasitica#populations#where#mycoviruses#were#absent,#although#the#
values#were#not#statistically#significant#(Table#1).##In#addition,#mating#type#allele#
frequencies#suggested#that#the#pathogen#populations#at#the#two#epidemic#populations#
with#a#long#history#of#infection#(MD#and#ST)#were#sexually#reproducing.##However,#if#
strong#negative#frequency#dependent#selection#was#operating,#the#number#VC#groups#
and#evenness#of#their#distribution#should#increase#over#time.##This#was#not#the#case#at#
the#ST#and#MD#epidemic#sites#where#the#number#of#VC#groups#increased#slightly#at#ST#
and#not#at#all#at#MD#(Figs#1#and#2).###Further,#VC#diversity#actually#declined#at#both#ST#
and#MD#between#1996#and#2009.#We#suggest#an#alternate#hypothesis#in#which#
mycovirus#invasion#actually#leads#to#a#reduction#in#C.#parasitica#VC#diversity#over#time.##
In#the#absence#of#mycovirus,#C.#parasitica#would#be#expected#to#regularly#undergo#
sexual#reproduction#resulting#in#recombining#of#genes#controlling#vegetative#
compatibility.#However,#mycoviruses#are#known#to#inhibit#sexual#reproduction#in#C.#
parasitica#(Anagnostakis#1987;#Griffin#&#Elkins#1986;#Milgroom#&#Cortesi#2004).#Work#

51#

over#the#past#25#years#with#mycoviruses#found#at#the#CL#site#and#another#mycovirus#
found#at#Grand#Haven,#Michigan#confirm#that#the#CHV3#mycoviruses#do#reduce#
pathogen#growth,#conidia#production,#and#largely#inhibit#sexual#reproduction#(D.#
Fulbright#and#ML#Double;#personal#observation).##Further,#these#CHV3#mycoviruses#are#
transmitted#by#hyphal#anastomosis#in#a#manner#similar#to#that#of#CHV1#mycoviruses#
from#Europe#(Huber#1996).#The#persistent#and#significant#skewing#of#mating#type#allele#
frequencies#at#CL#and#RC#in#1996#and#2009#also#suggest#that#the#C.#parasitica#
populations#at#these#two#sites#are#not#reproducing#via#sexual#reproduction#(Table#2).##
This#trend#is#important#because#selection#at#these#two#recovering#sites#would#then#
operating#on#asexual#populations#in#which#the#most#fit#pathogen#multilocus#genotype#
by#mycovirus#combination#would#be#favored.##In#consequence,#the#number#of#VC#groups#
should#decline#over#time#due#to#the#combined#processes#of#selection#and#genetic#drift.##
We#also#suggest#that#the#process#is#reversible#and#mycoviruses#can#be#lost#from#C.#
parasitica#populations#when#conditions#are#not#favorable#for#the#maintenance#and#
spread#of#mycoviruses.#We#suspect#that#C.#parasitica#at#FR#may#be#shedding#its#
mycovirus#since#the#percentage#of#isolates#containing#mycovirus#declined#from#greater#
than#90%#in#1996#(Davelos#1999)#to#76%#in#2009#(JC#Springer,#unpublished#data).##The#
decline#in#mycovirus#frequency#is#paralleled#by#a#doubling#in#the#number#of#VC#groups#
from#four#to#eight#between#1996#and#2009#and#an#increase#in#Shannon#diversity#(Table#
1).##Perhaps#in#consequence,#trees#at#FR#have#begun#to#die#back#over#the#past#four#years,#
which#contrasts#to#the#gradual#size#increase#occurring#at#CL#and#RC,#the#other#two#sites#
where#mycoviruses#occur#at#high#frequency#(JC#Springer#and#AM#Jarosz,#unpublished#
data).#

52#

# Results'from'this'study'suggest'that'mycoviruses'have'a'significant'influence'on'the'VC'
structure'of'C.#parasitica!populations.+++When+mycoviruses+attain+high+frequency,%the%
processes'of'sexual'reproduction'and'selection'in'the'C.#parasitica!pathogen)population)
may$be$altered$in$a$manner$that$leads$to$reduced$VC$diversity$over$time.$$$Selection$may$
act$to$favor$a$single$most$fit$mycovirus$x$fungal$genotype$combination,"while"the"
inhibition'of'the'sexual'cycle'reduces'the'formation'of'new'VC'groups.'Thus,'VC'diversity'
within&C.#parasitica!populations*will*be*influenced*by*conditions*that*favor*invasion*of*
mycoviruses+into+pathogen+populations+and+the+concomitant+changes!in#pathogen#
virulence.***What*is*not*known*at*present*is*how*the*underlying*vic!locus&diversity&is&
influenced)by)mycovirus)invasion.))We)hypothesize)that)vic!loci%that%have%a%strong%
influence(on(mycovirus(transmission(will(be(influenced(more(than(vic!loci!that$have$
little%influence%on%mycovirus%transmission%(see%Huber%1996).%%%Based&on&this&hypothesis,&
mycovirus*invasion*may*influence*genome9wide%diversity%within%C.#parasitica!
populations*at"recovering"sites"in"Michigan.

53#

(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
LITERATURE(CITED(
(
(
(
(
(
(
(
(
(
(
#
#
#
#
#
#
#
#

(
(

#

54#

LITERATURE(CITED(
(
(
Akilli#S,#Katircioglu#YZ,#and#Maden#S.#2009.#Vegetative#Compatibility#Types#of#
Cryphonectria#parastica#Causal#Agent#of#Chestnut#Blight,#in#the#Black#Sea#Region#of#
Turkey.#Forest#Pathology.#39:#390–396.##
##
Anagnostakis#SL.#1977.#Vegetative#incompatibility#in#Endothia#parasitica.##Experimental#
Mycology.#1:3069316.##
#
Anagnostakis#SL.##1987.#Chestnut#blight:#the#classical#problem#of#an#introduced#
pathogen.
#Mycologia.#79:23937.#
#
Anagnostakis#SL.#2001.#American#chestnut#sprout#survival#with#biological#control#of#the#
chestnut9blight#fungus#population.#Forest#Ecology#and#Management.#152:225–233.##
#
Anagnostakis#SL,#Hau#B,#and#Kranz#J.#1986.#Diversity#of#vegetative#compatibility#groups#
in#Cryphonectria#parasitica#in#Connecticut#and#Europe.#Plant#Disease.#70:5369538.##
#
Bertrand#H.#2000.#Role#of#Mitochondrial#DNA#in#the#senescence#and#hypovirulence#of#
fungi#and#potential#for#plant#disease#control.#Annual#Review#of#Phytopathology.#38:3979
422.##
#
Brewer#LG.##1995.#Ecology#of#survival#and#recovery#from#blight#in#American#chestnut#
trees#(Castanea#dentata#(Marsh.)#Borkh.)#in#Michigan.#Bulletin#of#the#Torrey#Botanical#
Club.#122:40–57.#
#
Burdon#JJ#and#Thrall#PH.##2008.#Pathogen#evolution#across#the#agro9ecological#interface:#
implications#for#disease#management.#Evolutionary#Applications.#1:57965.##
#
Brasier#CM.##1995.#Episodic#selection#as#a#force#in#fungal#microevolution#with#special#
reference#to#clonal#speciation#and#hybrid#introgression.##Canadian#Journal#of#Botany.#
73:S1213–S1221.##
#
Brasier#CM.##2001.#Rapid#evolution#of#introduced#plant#pathogens#via#interspecific#
hybridization.#Bioscience.#51:123–133.##
#
Brusini#J,#Robin#C,#and#Franc#A.#2011.#Parasitism#and#maintenance#of#diversity#in#a#
fungal#vegetative#incompatibility#system:#the#role#of#selection#by#deleterious#
cytoplasmic#elements.#Ecology#Letters.#14:4449452.##
#
Carbone#I,#Liu#Y,#Hillman#BI,#and#Milgroom#MG.##2004.#Recombination#and#migration#of#
Cryphonectria#hypovirus#1#as#inferred#from#gene#genealogies#and#the#coalescent.#
Genetics.#166:1611–1629.##
#

#

55#

Colwell#RK.##2005.#EstimateS:#Statistical#estimation#of#species#richness#and#shared#
species#from#samples.#Version#7.5.#User’s#Guide#and#Application#published#at:#
http://purl.oclc.org/estimates#
#
Cortesi#P,#McCulloch#CE,#Song#HY,#Lin#HQ,#and#Milgroom#MG.##2001.#Genetic#control#of#
horizontal#virus#transmission#in#the#chestnut#blight#fungus,#Cryphonectria#parasitica.#
Genetics.#159:107–118#
#
Davelos#AL.#1999.#Double9stranded#RNA#mediated#recovery#of#American#chestnut#
populations:#a#demographic#analysis.#PhD#thesis,#Michigan#State#University,#East#
Lansing,#Michigan.#
#
Davelos#AL,#and#Jarosz#AM.##2004.#Demography#of#American#chestnut#populations:#
effects#of#a#pathogen#and#a#hyperparasite.#Journal#of#Ecology.#92:6759685.##
#
Elliston#JE.##1985.#Characteristics#of#dsRNA9free#and#dsRNA9containing#strains#of#
Endothia#parasitica#in#relation#to#hypovirulence.#Phytopathology.#75:151–158.##
#
Enebak#SA,#MacDonald#WL,#Hillman#BI.##1994.#Effect#of#dsRNA#associated#with#isolates#
of#Cryphonectria#parasitica#from#the#central#Appalachians#and#their#relatedness#to#other#
dsRNAs#from#North#America#and#Europe.#Phytopathology.#84:5289534.##
#
Fulbright#DW,#Weidlich#WH,##Haufler#KZ,#Thomas#CS,#Paul#CP.#1983.#Chestnut#blight#and#
recovering#American#chestnut#trees#in#Michigan.#Canadian#Journal#of#Botany.#61:31649
3171.##
#
Glass#NL#and#Dementhon#K.#2006.#Non9self#recognition#and#programmed#cell#death#in#
filamentous#fungi.#Current#Opinions#in#Microbiology.#9:5539558.##
#
Glass#NL,#Jacobson#DJ,#Shiu#KT.#2000.#The#genetics#of#hyphal#fusion#and#vegetative#
compatibility#in#filamentous#ascomycetes.#Annuual#Review#of#Genetics.#34:1659186.#
#
Glass#NL#and#Kaneko#I.#2003.#Fatal#Attraction:#Non9self#recognition#and#heterokaryon#
incompatibility#in#filamentous#fungi.#Eukaryotic#Cell.#2:198.#
#
Gobbin#D,#Hoegger#PJ,#Heiniger#U,#Rigling#D.#2003.#Sequence#Variation#and#Evolution#of#
Cryphonectria#Hypovirus#1#(CHV91)#in#Europe.#Virus#Research.#97:39–46.##
#

Grente#J,#1965.#Les#formes#hypovirulentes#d’Endothia#parasitica#et#les#espoirs#de#lutte#
contre#le#chancre#du#châtagnier.#Academie#de#l’agriculture#de#France.#1033–1037.#
#
Griffin#GJ,#Elkins#JR.##1986.#Chestnut#blight,#other#endothia#diseases,#and#the#genus#
Endothia.#St.#Paul,#Minnesota.#American#Phytopathological#Society#Press.#53#pp.#
#
Heiniger#U#and#Rigling#D.#1994.#Biological#control#of#chestnut#blight#in#Europe.#Annual#
Review#of#Phytopathology.#32:581–599.#
#

56#

#
Hill#TCJ,#Walsh#KA,#Harris#JA,#Moffett#BF.#2003.#Using#ecological#diversity#measures#with#
bacterial#communities.#FEMS#Microbiology#and#Ecology.#43:1911.#
#
Huber#DH.#1996.#Genetic#analysis#of#vegetative#incompatibility#polymorphisms#and#
horizontal#transmission#in#the#chestnut#blight#fungus,#Cryphonectria#parasitica.#PhD#
Dissertation.#Michigan#State#University,#East#Lansing,#Michigan.##
#
Lapointe,#F.#J.#and#Legendre,#P.#1992.#Statistical#significance#of#the#matrix#correlation#
coefficient#for#comparing#independent#phylogenetic#trees#Syst.#Biol.#41:378–384.#
#
Lawrence#GJ,#Boelen#MG,#and#Pryor#A.#1988.#Transmission#of#double9stranded#RNAs#in#
flax#rust,#Melampsora#lini.#Canadian#Journal#of#Botany.#66:61–66.#
#
Liu#YC,#and#Milgroom#MG.#1996.#Correlation#between#hypovirus#transmission#and#the#
number#of#vegetative#incompatibility#(vic)#genes#different#among#isolates#from#a#
natural#population#of#Cryphonectria#parasitica.#Phytopathology.#86:79–86.##
#
MacDonald#WL#and#Fulbright#DW.#1991.#Biological#control#of#chestnut#blight:#use#and#
limitations#of#transmissible#hypovirulence.#Plant#Disease.#75:656–661.##
#
Magurran#A.#1988.#Ecological#diversity#and#its#measurement.#Princeton#University#Press,#
Princeton,#NJ.#
#
Marra#RE#and#Milgroom#MG.#2001.#The#mating#system#of#the#fungus#Cryphonectria#
parasitica:#selfing#and#self9incompatibility.#Heredity.#86:134–143.#
#
McCormick#JF#and#Platt#RB.#1980.#Recovery#of#an#Appalachian#forest#following#the#
chestnut#blight#or#Catherine#Keever9you#were#right!#American#Midland#Naturalist.#
104:264–273.##
#
Merkel#HW.#1906.#A#deadly#fungus#on#the#American#chestnut.#10th#Annual#Report#of#the#
New#York#Zoological#Society.#The#Crow#Press.#New#York,#New#York.##
#
Micali#CO#and#Smith#ML.##2006.#A#nonself#recognition#gene#complex#in#Neurospora#
crassa.#Genetics.#173:#1991–2004.#
#
Milgroom#MG#and#Cortesi#P.#1999.#Analysis#of#population#structure#of#the#chestnut#
blight#fungus#based#on#vegetative#incompatibility#genotypes.#P.#Natl.#Acad.#Sci.#USA.#
96:10518910523.##
#
Milgroom#MG#and#Cortesi#P.#2004.#Biological#control#of#chestnut#blight#with#
hypovirulence:#a#critical#analysis.#Annual#Review#of#Phytopathology.#42:311938.##
#

#

57#

Mir9Rashed#N,#Jacobson#DJ,#Dehghany#MR,#Micali#OC,#Smith#ML.#2000.#Molecular#and#
functional#analyses#of#incompatibility#genes#at#het96#in#a#population#of#Neurospora#
crassa.#Fungal#Genetics#and#Biology.#30:1979205.##
#
Morozov#AY,#Robin#C,#Franc#A.##2007.#A#simple#model#for#the#dynamics#of#a#host9
parasite9hyperparasite#interaction.#Journal#of#Theoretical#Biology.#249:2469253.#
#
Nauta#MJ#and#Hoekstra#RF.#1994.#Evolution#of#vegetative#incompatibility#in#filamentous#
ascomycetes.#I.#Deterministic#models.#Evolution.#48:9799995.##
#
Nuss#DL.#1992.#Biological#Control#of#Chestnut#Blight:#An#Example#of#Virus9Mediated#
Attenuation#of#Fungal#Pathogenesis.#Biological#Control.#56:561–576.#
#
Peever#TL,#Liu#YC,#Milgroom#MG.#1997.#Diversity#of#hypoviruses#and#other#double9
stranded#RNAs#in#Cryphonectria#parasitica#in#North#America.#Phytopathology.#87:10269
1033#
#
Potter#C,#Harwood#T,#Knight#J,#Tomlinson#I.#2011.#Learning#from#history,#predicting#the#
future:#the#UK#Dutch#elm#disease#outbreak#in#relation#to#contemporary#tree#disease#
threats.#Philosophical#Transactions#of#the#Royal#Society#B.#366:196691974.#
#
Rayner#ADM.##1991.#The#phytopathological#significance#of#mycelial#individualism.##
Annual#Rev.#Phytopathology.#29:#3059323.##
#
Robin#C,##Anziani#C,#and#Cortesi#P.#2000.#Relationship#between#biological#control,#
incidence#of#hypovirulence#and#diversity#of#vegetative#compatibility#types#of#
Cryphonectria#parasitica#in#France.#Phytopathology.#90:730–737.#
#
Robin#C.#and#Heiniger#U.#2001.#Chestnut#Blight#in#Europe:#Diversity#of#Cryphonectria#
parasitica,#Hypovirulence#and#Biocontrol.#Forest#Snow#and#Landscape#Research.#
367:361–367.#
#
Rohlf#FJ.#1998.#NTSYSpc.#Numerical#taxonomy#and#multivariate#analysis#system.#
Version#2.#User#Guide.#Applied#Biostatistics,#Inc.,#Setauket,#NY.#
#
Roper#M,#Ellison#C,#Taylor#JW,#and#Glass#NL.#2011.#Nuclear#and#genome#dynamics#in#
multinucleate#ascomycete#fungi.##Current#Biology.##21:R7869R793.##
Russin#JS#and#Shain#L.#1985.#Disseminative#fitness#of#Endothia#parasitica#containing#
different#agents#for#cytoplasmic#hypovirulence.##Canadian#Journal#of#Botany.#63:54957.##
Santini#A,#Pecori#F,#Pepori#A,#Brookes#A.#2012.#Morfeo#Elm:#a#new#variety#resistant#to#
Dutch#elm#disease.#Forest#Pathology.#42:171—176.##
#

#

58#

Sotirovski#K,#Milgroom#MG,#Rigling#D,#and#Heiniger#U.#2006.#Occurrence#of#
Cryphonectria#Hypovirus#1#in#the#Chestnut#Blight#Fungus#in#Macedonia.#Forest#
Pathology.#36:136–143.##
#
Taylor#DR,#Jarosz#AM,#Lenski#RE,#Fulbright#DW.#1998.#The#acquisition#of#hypovirulence#
in#host9pathogen#systems#with#three#trophic#levels.##American#Naturalist.#151:3439355.#
#
Wu#J,#Saupe#SJ,#Glass#NL.#1998.#Evidence#for#balancing#selection#operating#at#the#het9c#
heterokaryon#incompatibility#locus#in#a#group#of#filamentous#fungi.#PNAS.#95:123989
12403.##
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CHAPTER(3(
#
AN(ANALYSIS(OF(MICROSATELLITE(LOCI(FROM(POPULATIONS(OF(THE(CHESTNUT(
BLIGHT(PATHOGEN,(CRYPHONECTRIA,PARASITICA,(IN(MICHIGAN(
ABSTRACT#
#
Vegetative#compatibility#group#diversity#in#populations#of#Cryphonectria#

parasitica,#the#fungus#causing#chestnut#blight,#varies#according#to#mycovirus#presence#
or#absence#in#Michigan.#In#populations#where#mycoviruses#are#present,#VCG#diversity#is#
generally#low#and,#with#one#exception,#each#VCG#is#found#at#only#a#single#site.##In#
populations#where#mycoviruses#are#absent,#VCG#diversity#is#higher#and#several#VCGs#
are#shared#among#sites.##Since#mycovirus#presence#prevents#sexual#reproduction#in#C.#
parasitica,#selection#may#act#to#sort#through#local#combinations#of#mycovirus#and#blight#
pathogen#to#favor#a#single#most9fit,#locally#adapted#combination.#Microsatellite#markers#
developed#for#European#populations#of#C.#parasitica#are#used#here#to#determine#if#
genome9wide#diversity#is#concordant#with#the#pattern#found#for#VCGs.#Analysis#of#
molecular#variance#(AMOVA)#found#significant#differentiation#among#populations,#with#
15%#of#the#total#genetic#variance#being#partitioned#among#the#six#C.#parasitica#Michigan#
population.##Average#ΦPT#values#for#mycovirus9free#sites#was#0.079#while#mycovirus9
infected#sites#was#0.219.###These#values#are#consistent#with#the#idea#that#gene#flow#is#
more#limited#among#mycovirus9infected#C.#parasitica#populations.##In#contrast,#
populations#fell#into#two#broad#categories#for#within#population#genetic#diversity#that#
did#not#correspond#with#presence#or#absence#of#mycoviruses.##The#three#high#diversity#
populations#had#Shannon#values#of#0.37#or#greater#and#a#percentage#of#polymorphic#loci#
of#63%#or#greater,#while#the#three#low#diversity#populations#had#Shannon#values#of#0.26#

#

60#

or#lower#and#percentages#of#polymorphic#loci#of#40%#or#lower.##The#largest,#mycovirus9
free#population#at#Stivers#displayed#low#within#population#diversity,#while#Frankfort,#a#
mycovirus#infected#populations#displayed#high#within#population#diversity.##Recent#
observations#suggest#that#the#mycoviruses#at#the#C.#parasitica#population#at#Frankfort#
may#be#shedding#its#mycoviruses.##
#
INTRODUCTION(
#

In#Chapter#2,#I#investigated#diversity#in#Michigan#populations#of#C.#parasitica#by#

identifying#vegetative#compatibility#groups#within#and#among#seven#populations.#The#
presence#of#mycoviruses#in#some#blight#populations#has#seemingly#influenced#the#
number#and#diversity#of#vegetative#compatibility#groups#(VCGs).#VCG#diversity#is#
generally#lower#in#C.#parasitica#populations#where#mycoviruses#are#present#compared#
to#populations#where#they#are#absent.#Sexual#reproduction#is#known#to#be#inhibited#in#C.#
parasitica#infected#by#mycoviruses#(Bryner#&#Rigling#2012),#which#changes#two#
important#factors#that#would#influence#the#genetic#structure#of#C.#parasitica#populations.##
First,#with#no#recombination#in#the#pathogen,#selection#will#tend#to#favor#the#most9fit#
pathogen#genotype#by#mycovirus#combination.##Second,#pathogen#spread#will#be#limited#
to#asexual,#splash9dispersed#conidia.##These#two#factors#lead#to#the#prediction#that#
populations#with#mycoviruses#should#have#a#different#structure#relative#to#C.#parasitica#
populations#where#mycoviruses#are#absent.##In#the#presence#of#mycoviruses#I#expect#
lower#overall#genomic#diversity#and#the#possibly#high#levels#of#differentiation#among#
mycovirus9infected#populations#because#of#asexually#reproducing#clonal#populations.##
In#contrast,#I#expect#higher#diversity#within#C.#parasitica#populations#where#

#

61#

mycoviruses#are#absent#since#sexual#reproduction#is#most#likely#occurring#at#those#sites#
and#lower#differentiation#among#populations#due#to#increased#gene#flow.#
Although#commonly#used#for#determining#diversity#in#populations#of#C.#
parasitica#throughout#its#range#in#North#America#and#Europe,#VCG#analyses#have#
several#drawbacks#since#the#technique#is#time#consuming#(Breuillin#et#al.#2006)#and#it#is#
difficult#to#score#some#pairings#(see#Chapter#2).###Additionally,#VCG#are#controlled#by#six#
to#eight#vic#loci#that#are#thought#to#be#under#negative#frequency#dependent#selection#
(Huber#1996;#Cortesi#&#Milgroom#1998).##Further,#determining#the#genetic#basis#of#each#
VCG#requires#pairing#to#genetically#characterized#testers#(Cortesi#&#Milgroom#1998).##
Additional#problems#occur#because#the#six#characterized#vic#loci#do#not#encompass#all#of#
the#vic#loci#controlling#VCGs,#and#in#consequence,#many#studies#have#reported#isolates#
that#do#not#pair#with#any#of#the#characterized#testers.#(Milgroom#&#Cortesi#1999;#Robin#
&#Heiniger#2001;#Braganca#et#al.#2007).####The#cumbersome#method#of#pairing#isolates#
together#to#determine#VCG#or#genotypic#information#is#useful#but#time9consuming#and#
interactions#are#difficult#to#understand,#thus#my#use#of#molecular#markers#to#determine#
genomic#diversity.##
Molecular#markers#such#as#microsatellites#can#be#used#to#efficiently#characterize#
population#genetic#structure#of#various#organisms#(Selkoe#&#Toonen#2006).##
Understanding#diversity#at#the#genetic#level#allows#ecologists#to#answer#key#questions#
about#population#structure#of#organisms#across#diverse#taxa#e.g.,#California#Channel#
Island#Foxes,#(Goldstein#et#al.#1999);#Cuban#cattle,#(Acosta#et#al.#2013);#Lemba#fruit,#
(Babaei#et#al.#2012);#Baobabs,#(Munthali#et#al.#2013);#chickpea#blight,#(Peever#et#al.#
2004);#and#the#chestnut#blight#pathogen,#(Breuillin#et#al.#2006).##Microsatellite#markers#

#

62#

are#widely#distributed#throughout#the#genome#of#eukaryotes;#they#are#co9dominant,#and#
extremely#variable#which#makes#them#very#useful#in#genetics#studies#(Shikano#et#al.#
2010).#Microsatellites,#a#type#of#variable#number#tandem#repeats,#are#highly#variable#
short#segments#of#DNA,#(sometimes#called#simple#sequence#repeats#[SSRs]#or#short#
tandem#repeats#[STRs]),#usually#1—6#base#pairs#long,#many#times#repeated#up#to#60#
times#(Goldstein#&#Pollock#1997).##Use#of#these#neutral#markers#can#provide#an#
opportunity#to#determine#population#genetic#structure#of#organisms#in#regions#of#their#
genome#that#are#independent#of#loci#normally#under#selection.##
#

The#use#of#microsatellite#markers#has#become#ubiquitous#as#a#tool#for#

investigating#the#population#structure#of#plant#pathogenic#fungi#(Goldstein#&#Pollock#
1997)#and#can#help#detect#patterns#of#genetic#variability#within#and#among#populations.##
Plant#pathogens#are#great#model#systems#to#study#how#evolutionary#forces#affect#
population#structure#over#short#time#periods#and#at#various#spatial#scales#(Holderegger#
&#Wagner#2008;#Linde#2010;#Rieux#et#al.#2013).#Since#plant#pathogens#(and#other#
organisms)#are#heavily#influenced#by#evolutionary#forces#such#as:#selection,#drift,#
migration,#recombination,#and#mutation#(McDermott#&#McDonald#1993),#we#can#better#
understand#how#these#forces#have#acted#in#the#past#(McDonald#&#Linde#2002)#by#
obtaining#information#about#their#genetic#structure.#Additionally,#spatially#separated#
populations#of#plant#pathogens#can#allow#us#to#infer#how#geographic#scale,#local#
environmental#conditions,#biotic#interactions,#or#in#the#case#studied#below:#how#the#
presence#of#double9stranded#RNA#(dsRNA)#mycovirus#hyperparasites#can#influence#
population#structure#of#Cryphonectria#parasitica.##

#

63#

#

#This#study#is#the#first#to#investigate#how#mycoviruses#influence#the#genetic#

structure#in#C.#parasitica#populations#in#Michigan.###This#chapter#will:#1)#investigate#how#
mycoviruses#alter#the#genetic#variability#of#C.#parasitica,#2)#help#us#further#understand#
the#patterning#of#genetic#differentiation#among#C.#parasitica,#populations,#43#investigate#
potential#factors#that#influence#the#patterning#of#geographic#variation#(e.g.,#isolation#by#
distance#or#the#presence/absence#of#mycoviruses),#and#4)#provide#a#base9line#
population#structure#of#C.#parasitica#that#will#allow#tracking#of#genetic#changes#over#
time#as#three#populations#C.#parasitica#respond#to#the#invasion#of#mycoviruses#(see#
Chapter#4).##
#
METHODS(
#

Isolates#collected#in#2009#and#used#previously#to#characterize#seven#C.#parasitica#

populations#in#Michigan#for#vegetative#compatibility#group#diversity#(Chapter#2)#were#
grown#in#culture#for#microsatellite#analysis.##Thirty#isolates#from#each#of#seven#
populations#were#grown#on#PDA#petri#plates#overlaid#with#uncoated,#sterilized#
cellophane.##After#approximately#three#weeks#of#growth,#fungal#mats#were#scraped#off#
the#cellophane,#dried#in#mortars#at#65oC#and#ground#to#a#fine#powder#with#liquid#
nitrogen.#Whole#DNA#extractions#were#performed#with#the#Plant#Mini#Kit#(Qiagen,#
Maryland,#USA)#with#25—30mg#of#dried#tissue#following#manufacturers’#directions.#
Extracted#DNA#was#stored#in#microcentrifuge#tubes#at#920oC#until#PCR#could#be#
performed.##

#

64#

#

Polymerase#chain#reactions#were#performed#with#GoTaq#Green#Master#Mix#

(Promega#Corporation,#Madison,#WI)#in#a#total#volume#of#25uL#as#recommended#by#the#
manufacturer.#The#thirteen#primers#developed#by#Breuillin#et#al.#(2006)#were#tested#
using#DNA#from#ten#random#isolates#to#determine#if#the#primers#would#amplify#C.#
parasitica#from#Michigan.##Ten#of#the#primers#were#chosen#for#further#analysis#of#the#
seven#Michigan#C.#parasitica#populations#(Table#1).##PCR#was#run#according#to#the#
protocols#of#Breuillin#et#al.#(2006);#9#cycles#at#94oC#for#40s,#annealing#temperature#
decreasing#from#69oC#to#65oC#(0.5oC#per#subsequent#cycle)#for#30s,#and#72oC#for#1min.#
PCR#was#then#run#for#30#cycles#at#94oC#for#40s,#65oC#for#30s#and#72oC#for#1min#and,#a#
final#extension#at#72oC#for#30#min#then#held#at#10oC#until#frozen.#PCR#products#were#
stored#in#96#well#plates#at#920oC#until#fragment#sizes#could#be#analyzed#on#an#Applied#
Biosystems#ABI#3130#genetic#analyzer.#Fragment#peak#sizes#were#called#manually#using#
PeakScanner#v1.0#(Applied#BioSystems).#Fragment#sizes#were#rounded#to#whole#
numbers#using#power#functions#in#the#tandem#software#version#1.09#(Salzburger#2009)#
after#being#converted#to#the#proper#file#format#using#CREATE#(Coombs#et#al#2008).##
Amplification#was#not#consistent#either#within#a#locus#across#isolates#or#across#loci#
for#an#individual#C.#parasitica#isolate.##Second#and#some#third#attempts#to#amplify#these#
missing#data#all#failed.##In#consequence,#the#final#data#set#has#a#considerable#number#of#
missing#data#(approximately#40%#of#data#are#missing#from#2,100#data#points#expected),#
which#reduced#the#power#of#analysis#considerably.##Population#MH#had#the#lowest#
amplification#success#and#was#dropped#from#the#final#analyses.##In#addition,#locus#CPG5#

#

65#

amplified#poorly#across#all#populations,#and#it#was#also#dropped#from#the#final#analyses.#
Finally,#all#loci#that#had#sample#sizes#≤5#for#a#population#were#removed#from#
subsequent#analyses#since#they#provided#little#information#to#the#dataset#and#hindered#
some#population#genetic#tests#in#GenAlEx#6.5.##Finally,#poor#amplification#across#loci#for#
all#samples#precluded#an#analysis#of#multilocus#genotypes#and#level#of#linkage#
disequilibrium#in#the#six#remaining#populations.#
Data#were#then#analyzed#with#GenAlEx#software#version#6.5#(Peakall#&#Smouse#
2006,#2012)#where#frequencies#of#alleles,#private#alleles,#and#pairwise#population#
comparison#tests#across#all#alleles#were#performed.#ΦPT,#an#analogue#of#the#FST#fixation#
index,#was#used#to#characterize#the#level#of#population#divergence#among#the#six#C.#
parasitica#populations.#Pairwise#genetic#difference#between#individuals#within#
populations#was#calculated#in#GenAlEX#6.5#by#counting#the#number#of#genetic#
differences#between#all#combinations#of#individuals#across#populations.##Isolation#by#
distance#was#tested#by#comparing#the#genetic#distance#matrix#with#the#geographic#
distance#matrix#using#a#Mantel#test.##GenAlEX#6.5#was#also#used#to#calculate#the#average#
number#of#migrants#per#generation#(Nm),#number#of#alleles#(Na),#and#number#of#
effective#alleles#in#each#population#(Ne).##
#
RESULTS(
#

Ten#of#the#thirteen#microsatellite#markers#developed#for#European#populations#

by#Breuillen#et#al.#(2006)#successfully#amplified#in#Michigan#samples#of#C.#parasitica#
(Table#391).##The#number#alleles#detected#and#size#of#individual#alleles#differed#between#

#

66#

Europe#and#Michigan.##One#locus#(CPG5)#that#appeared#to#be#fixed#for#one#allele#of#
220bp#in#Europe#was#much#more#variable#in#Michigan#populations#and#had#four#alleles#
(Table#391).#Five#loci#amplified#with#Michigan#isolates#matched#the#number#of#alleles#
found#in#European#populations.#Loci#CPE1#and#CPE8#had#fewer#alleles#in#Michigan#
isolates#with#three#and#two,#respectively,#compared#to#five#each#in#European#
populations.#Allele#sizes#were#similar#between#Michigan#and#European#populations#and#
overlapped#in#number#of#base#pairs#except#for#locus#CPE8#that#was#shifted#to#a#smaller#
size#in#tested#Michigan#isolates#(Table#391).###Table#392#lists#all#alleles#per#locus#for#each#
of#the#seven#C.#parasitica#populations#in#Michigan.##As#explained#above,#the#MH#
population#and#CPG5#locus#were#dropped#from#population#level#analyses#because#of#
poor#amplification#and#the#resultant#small#sample#size#that#precluded#population#level#
analyses.#
A#molecular#analysis#of#variance#(AMOVA)#across#the#nine#microsatellite#loci#for#the#
six#Michigan#C.#parasitica#populations#found#that#15%#of#the#genetic#variation#was#
partitioned#among#populations,#while#85%#was#found#within#populations#(Table#393).##
Populations#fell#into#two#categories#with#regard#to#within#population#variability.###MD,#
LE#and#FR#were#relatively#more#variable#than#ST,#CL#and#RC#(Table#394).##Between#63#
and#70%#of#the#loci#were#polymorphic#in#the#three#more#variable#populations,#which#
also#averaged#between#1.88#and#2.00#alleles#per#locus#and#had#Shannon#diversity#values#
between#0.37#and#0.45.##In#contrast,#only#30#to#40%#of#the#loci#were#polymorphic#in#the#
low#variability#group.##The#average#number#of#loci#at#ST,#CL#and#RC#averaged#between#
1.18#and#1.35#alleles#per#locus#and#Shannon#diversity#were#between#0.16#and#0.26.###
#

#

67#

#
#
#
Table#391.#Range#of#allele#sizes#in#base#pairs#and#number#of#alleles#
amplified#for#thirteen#loci#of#C.#parasitica#in#Europe#(Breuillin#et#al#2006)#
and#ten#in#Michigan.#
#

Locus#
CPG1#
CPG2#

European#
Range#
in#base#pairs#
22699228#
15299156#

European#
Alleles#
2#
3#

Michigan#
Range#
in#base#
pairs#
22599229#
15099156#

CPG3#
CPG4#
CPG5#
CPE1#
CPE2#
CPE3#
CPE4#
CPE5#
CPE6#
CPE7#
CPE8#

1889262#
18899208#
220#
13199149#
14099144#
17699194#
22499280#
25699265#
11099112#
17699191#
10799122#

8#
4#
1#
5#
2#
4#
6#
3#
2#
3#
5#

99a#
18399203#
19899218#
12499140#
99#
18299194#
99#
24899258#
10599107#
18299186#
11199113#

a:#these#primers#were#not#amplified

#

68#

Michigan#
Alleles#
3#
3#
99#
5#
4#
3#
99#
4#
99#
3#
2#
3#
2#

#
#
#
#
#
#
#
#
#
#
#
#
#
#
#

Table!3*2!Summary!table!of!population!level!diversity!of!alleles!at!each!of!ten!loci.!Data!in!gray!boxes!are!shown!for!
comparison!and!were!not!included!in!population!level!analyses!due!to!poor!amplification!and!resulting!small!sample!
size.!
CPG1!

CPG2!
152,!
156!
150,!
152!
150,!
152!
150,!
152!

CPG4!

CPG5!

CPE1!

CPE3!

CPE5!

CPE6!

CPE7!

CPE8!

203!
199,!
203!
187,!
203!
187,!
203!

218!

124!

253!

107!

184!

218!

124!

190!
182,!186,!
190!

253!

105,!107!

184,!186!

111!
111,!
113!

218!
198,!
218!

138!
124,!
138!

190!

107!

186!

111!

107!

184,!186!

111!

227,!229!

152!

!
!
!
225,!227!

!
!
!
152!

203!
183!
199!
203!
187,!
191!

218!
206!
218!

190!
190!

107!
107!

182,!184!
184!
186!

111!
111!

!
214,!
218!

124!
124!
138!
140!

258!
253,!
258!
248,!
253!
253!
258!

3!

2!

5!

4!

!
MH!

227,!229!

MD!

225,!227!

ST!

225,!227!

LE! 225,!227,!229!
CL!
FR!

Total!
alleles!

!
!
RC!

!

190!

138!

!
!
194!

3!

4!

69!

!
248,!
253!

!
!
107!

!
186!

!
!
111!

3!

2!

3!

2!

Within!population!variability!was!not!associated!with!the!presence!or!absence!of!
mycovirus,!since!two!of!three!epidemic!populations,!MD!and!LE!!(i.e.,!those!with!no!
mycovirus)!were!in!the!group!with!higher!variability!and!the!third!population,!ST,!was!
in!the!low!variability!group.!!The!opposite!was!true!for!the!recovering!population!
where!mycoviruses!were!present.!!FR!was!in!the!more!variable!group,!while!CL!and!RC!
were!in!the!low!variability!group.!!As!discussed!below,!the!C.#parasitica!population!at!
FR!appears!to!be!shedding!its!mycoviruses,!which!may!explain!the!higher!variability!at!!
!

Table!3J3.!AMOVA!statistics!for!tests!of!genetic!differentiation!within!and!among!
populations!of!C.#parasitica!in!Michigan.!Data!were!calculated!without!site!MH,!locus!
CPG5,!and!any!sample!where!no!loci!amplified.!!
!
Source!
Among!
Pops.!
Within!
Pops!
Total!
!

df!

SS!

MS!

Molecular!
Var.!

5!

62.31!
351.8
3!
414.1
3!

12.46!

15%!

2.02!

85%!

174!
179!

100%!

ϕPT!
0.147!

Prob.!
<0.00
1!

!
!

!
!

!

this!site.!PairJwise!comparisons!between!populations!indicated!considerable!
differentiation!among!populations.!!ϕPT!values!ranged!over!nearly!an!order!of!
magnitude!from!0.029!to!0.275!(Table!3J5).!!The!only!pairJwise!comparison!that!was!
not!significantly!different!from!zero!was!between!MD!and!LE!(Table!3J5).!!These!two!
epidemic!site!C.#parasitica!populations!are!separated!by!43!kilometers.!!PairJwise!
comparisons!did!follow!a!pattern!with!comparisons!among!C.#parasitica!populations!
from!epidemic!sites!(Comparisons!with!the!diagonal!hatching!in!Table!3J5)!having!!

!

70!

Table!3J4.!Average!number!of!alleles!per!locus,!effective!number!of!alleles!per!locus,!
percentage!of!loci!that!were!polymorphic!and!Shannon!diversity!for!each!population.!!
Standard!errors!of!the!mean!are!in!parentheses.!
!
Average!
Average!number!
number!of!
%!loci!
Population!
of!effective!alleles!
alleles!per!
Polymorphic!
per!locus!
locus!
MD!
2.00!(0.26)!
1.38!(0.15)!
70!
ST!
1.50!(0.22)!
1.22!(0.11)!
40!
LE!
1.90!(0.23)!
1.54!(0.15)!
70!
CL!
1.40!(0.22)!
1.18!(0.12)!
30!
FR!
1.88!(0.29)!
1.49!(0.17)!
63!
40!
RC!
1.40(0.16)!
1.35(0.15)!

Shannon!
Diversity!
0.37!(0.11)!
0.21!(0.09)!
0.45!(0.11)!
0.16!(0.09)!
0.41!(0.13)!
0.26!(0.11)!

!
!
!
!
!
!

!
lower!ϕPT!than!comparisons!among!C.#parasitica!populations!from!recovering!sites!
(Comparisons!with!cross!hatching!in!Table!3J5;!see!also!Table!3J6).!!Indeed,!the!
comparisons!with!the!two!highest!ϕPT!values!involved!comparisons!between!
recovering!sites!(CL!vs.!RC!and!CL!vs.!FR).!!!The!level!of!differentiation!among!
recovering!site!populations!also!tended!to!be!higher!than!the!comparisons!between!
epidemic!and!recovering!sites!(Table!3J6).!The!average!number!of!haploid!migrants!per!
generation!(Nm)!across!all!populations!was!2.91,!but!the!predicted!number!of!migrants!
differed!with!population!type.!!Epidemic!populations!had!predicted!migration!rates!
ranging!from!3.8!to!17!per!generation!with!an!average!of!8.6!(Table!3J7).!!Migration!was!
predicted!to!be!much!lower!between!recovering!populations!with!a!range!from!1.3!to!
2.4!and!an!average!of1.9.!All!sites!except!LE!most!likely!meet!the!assumption!of!large,!
stable!population!size,!the!LE!site!is!experiencing!annual!expansion!of!disease!on!
previously!uninfected!trees!(AM!Jarosz,!ALD!Baines,!and!JC!Springer!pers.!obs.).!!

!

71!

!
!
Table!3J5.!Pairwise!population!differentiation!values!(ϕPT;!below!the!main!diagonal)!
based!on!nine!loci!across!six! C.#parasitica!populations;!all!probabilities!of!obtaining!
ΦPT! values! were! significant! at! p<0.05! except! LE! x! MD! (p=0.096).! StraightJline!
distances!between!populations,!in!km,!are!shown!above!the!diagonal.!Light!shading!
indicates! comparisons! between! populations! without! mycoviruses! while! darker!
shading!indicates!comparisons!between!populations!with!mycoviruses.!
!

!

MD!
ST!
LE!
CL!
FR!
RC!
!
MD!
JJ!
56.7!
76.1!
71.7!
88.2!
51.9!
ST!
0.117!
JJ!
67.2!
15.2!
34.1!
107.7!
LE!
0.029!n.s.!
0.092!
JJ!
69.8!
67.6!
103.4!
CL!
0.104!
0.194!
0.185!
JJ!
20.2!
122.3!
FR!
0.107!
0.155!
0.050!
0.214!
JJ!
136.9!
RC!
0.172!
0.173!
0.124!
0.275!
0.170!
JJ!
n.s.!indicates!the!only!pairJwise!comparison!that!is!not!significantly!different!from!0.!!!

Table!3J6.!Average!!ϕPT!and!number!of!haploid!migrants!(Nm)!
based! on! population! type,! three! epidemic! sites! and! three!
recovering!sites!(described!in!Chapter!2).!Standard!errors!are!in!
parentheses.!
!
!
Epidemic!
Recovering!
Between!pop.!
types!!

Average!ϕPT!
0.079!(0.026)!
0.219!(0.030)!

Nm!
8.560!(4.216)!
1.860!(0.325)!

0.140!(0.017)!

3.700!(0.778)!

!
!
!
!
!

!

72!

Table!3J7.!Average!number!of!migrants!per!generation!of!C.#
parasitica! between! six! Michigan! populations.! Values! are!
estimated! based! on! haploid! C.# parasitica! populations! and!
on!ϕPT!values!using!GenAlEx!6.5.!Shading!is!the!same!as!in!
Table!3J5.!
!
!
ST!
LE!
CL!
FR!
RC!

MD!
3.778!
16.969!
4.306!
4.152!
2.400!

ST!
JJ!
4.946!
2.074!
2.732!
2.392!

LE!

CL!

FR!

RC!

!
JJ!
2.204!
9.516!
3.531!

!
!
JJ!
1.831!
1.321!

!
!
!
JJ!
2.448!

!
!
!
!
JJ!

!
A!test!for!isolation!by!distance!between!populations!was!carried!out!using!a!
Mantel!test!to!determine!if!geographic!distance!(in!kilometers;!Table!3J5)!and!Nei’s!
(1987)!genetic!distance!were!correlated.!!The!R2!=!0.0005,!P=0.09!indicated!little,!if!any,!
geographic!patterning!of!genetic!variation.!
Missing!data!precluded!any!multilocus!genotype!analyses!within!or!among!
populations,!nor!could!linkage!disequilibrium!values!be!calculated;!key!values!
important!for!understanding!the!level!of!asexual!reproduction.!!!
!
DISCUSSION!
!

A!previous!study!that!investigated!the!pattern!of!vegetative!compatibility!group!

(VCG)!diversity!found!that!the!presence!of!mycovirus!affected!the!structure!of!C.#
parasitica!populations!(see!chapter!2).!!Two!populations!with!mycovirus,!CL!and!RC,!
displayed!low!VCG!diversity!while!populations!where!the!mycovirus!was!absent!(i.e.,!
epidemic!populations)!generally!had!more!VCGs,!and!also!displayed!higher!diversity!for!
VCGs.!!Cryphonectria#parasitica!populations!also!shared!several!VCGs!which!suggested!

!

73!

that!C.#parasitica!from!epidemic!sites!experienced!more!gene!flow;!although!the!CL!
recovering!site!shared!a!VCG!with!two!epidemic!sites.!!!Although!commonly!used!to!
detect!patterns!of!C.#parasitica!diversity!in!Europe!and!North!America!(Anagnostakis!et!
al.!1986;!Bissegger!et!al.!1987),!vegetative!compatibility!is!cumbersome!and!time!
consuming.!In!this!study,!microsatellite!markers!were!used!to!investigate!whether!the!
VCG!pattern!extended!across!the!C.#parasitica!genome.!!The!pattern!of!microsatellite!
variability!supported!some!but!not!all!of!the!VCG!findings.!!Three!epidemic!populations,!
ST,!LE!and!MD,!were!less!differentiated!than!the!three!recovering!sites.!!Indeed,!genetic!
distance!among!the!recovering!sites!indicated!they!are!not!closely!related.!!It!is!also!
known!that!the!mycoviruses!at!the!three!recovering!sites!differ!significantly!with!CHV3!
at!CL,!CHV3!and!CHV4!at!FR!and!SR2!at!RC!(see!Chapter!1;!Tartaglia!et!al.!1986;!Paul!&!
Fulbright!1988;!Durbahn!1992;!Smart!et!al.!1994).!!!Taken!together,!this!information!
suggests!that!both!the!mycovirus!and!C.#parasitica!colonized!each!recovering!site!
independently!and!possibly!has!remained!distinct!from!other!C.#parasitica!populations!
in!Michigan!over!the!twenty!plus!years!since!mycoviruses!invaded.!
!

The!contrasting!pattern!for!epidemic!populations!of!C.#parasitica!suggested!more!

gene!flow!among!these!sites.!!This!makes!sense!biologically,!since!frequencies!for!
mating!type!alleles!indicated!that!C.#parasitica!populations!at!two!longJinfected!sites!
(ST!and!MD)!were!sexually!reproducing,!while!C.#parasitica!at!two!recovering!sites!(CL!
and!RC)!displayed!skewed!allele!ratios!that!indicated!asexual!reproduction!(see!Chapter!
2).!!Mycoviruses!are!known!to!inhibit!the!sexual!cycle!in!C.#parasitica!and,!thus,!
reproduction!occurs!via!asexually!produced!conidia!(Zhang!et!al.!1998;!Gobbin!et!al.!

!

74!

2003).!!Sexually!produced!ascospores!are!windJdispersed!and!known!to!be!more!
mobile!than!splash!dispersed!conidia!(Heald!et!al.!1915).!
!

Patterns!of!microsatellite!variation!only!lent!partial!support!to!the!hypothesis!

that!epidemic!populations!are!largely!sexual!and!more!variable!genetically!than!the!
asexually!reproducing!C.#parasitica!populations!at!recovering!sites.!!As!expected,!the!CL!
and!RC!recovering!sites!had!C.#parasitica!populations!with!low!microsatellite!diversity,!
but!the!FR!recovering!site!displayed!relatively!high!genetic!diversity!at!microsatellite!
loci!(Table!3J4).!!High!diversity!at!FR!might!be!due!to!the!fact!that!the!C.#parasitica!
population!appears!to!be!in!the!process!of!escaping!mycovirus!infections,!since!>90%!of!
isolates!obtained!from!the!site!in!1996!harbored!mycovirus!(Davelos!1999)!and!only!
76%!of!isolates!collected!in!2009!were!infected!with!mycoviruses!(JC!Springer,!
unpublished!data).!!!Epidemic!populations!also!provided!only!partial!support!for!the!
hypothesis!that!they!were!sexually!reproducing!and!expected!to!display!more!genetic!
variability.!!The!MD!and!LE!populations!were!both!relatively!diverse,!which!supported!
my!hypothesis.!!However,!the!ST!epidemic!population!had!low!diversity,!despite!the!fact!
that!C.#parasitica!has!been!resident!at!this!site!for!over!20!years!(DW!Fulbright,!pers.!
comm.),!and!with!greater!than!1100!trees,!it!has!the!largest!American!chestnut!
population!of!any!we!have!monitored!since!1996!(Davelos!and!Jarosz!2004).!!!
!

These!results!suggest!that!C.#parasitica!across!Michigan!is!organized!into!a!

patchwork!of!populations!that!differ!in!their!level!of!sexual!reproduction,!level!of!
genetic!variability!and!level!of!migration!among!patches.!!Some!of!this!patchwork!can!
be!explained!by!the!presence!of!mycoviruses!in!some!populations,!but!not!others.!!
However,!we!know!little!about!environmental!conditions!that!led!to!mycoviruses!

!

75!

invading!in!some!populations!and!not!others.!!It!is!tempting!suggest!that!the!presence!of!
mycoviruses!is!simply!the!result!of!colonization!of!events!at!some!locations!and!not!
others.!!However,!attempts!to!introduce!mycoviruses!into!C.#parasitica!populations!
across!North!America!have!generally!met!with!failure,!which!suggests!that!other!factors,!
such!as!abiotic!conditions!or!tree!genetics!are!also!influencing!mycovirus!spread.!!These!
factors!remain!to!be!explored,!but!it!is!becoming!increasingly!evident!that!C.#parasitica!
in!Michigan,!at!least!superficially,!represents!as!geographic!mosaic!of!coevolutionary!
interactions!(see!Thompson!1999).!!There!are!obvious!hotspots!where!the!mycoviruses!
are!important!and!their!presence!has!led!to!recovery!of!chestnut!populations!(Davelos!
&!Jarosz!2004).!!But!interspersed!among!these!recovering!sites!are!populations!where!C.#
parasitica!continues!to!decimate!the!local!C.#dentata!populations.!Many!questions!
remain!to!be!answered!before!we!can!predict!whether!mycoviruses!will!ultimately!
spread!more!widely!and!cause!a!general!recovery!of!American!chestnut!within!
Michigan.!
Four!populations!studied!in!France!to!understand!both!VCG!and!microsatellite!
diversity,!all!infected!with!mycoviruses,!were!considered!to!be!isolated!by!distance!
(Breuillin!et!al.!2006).!Biologically!this!is!a!reasonable!expectation!given!considerations!
of!how!mycoviruses!affect!sexual!reproduction!in!C.#parasitica.!Michigan!C.#parasitica!
populations!although!not!isolated!by!distance!do!however!appear!to!be!structured!
according!to!mycovirus!presence!as!shown!with!VCG!data!in!Chapter!2.!!Further,!
Breuillin!et!al.!(2006)!showed,!based!on!measures!of!genetic!diversity,!that!populations!
in!France!exhibited!reduced!gene!flow!between!populations!probably!since!chestnut!
forests!there!are!fragmented!across!the!landscape!and!are!managed!as!orchards,!similar!

!

76!

to!the!fragmented!patchwork!of!chestnut!populations!in!Michigan.!VCG!diversity!in!
French!populations!was!much!higher!(range!6—16!groups)!compared!to!populations!in!
Michigan!that!are!also!infected!with!mycoviruses!(range!2—8!groups;!see!Chapter!2);!it!
is!unknown!how!long!mycoviruses!have!been!present!in!the!four!French!populations.!!
Shannon!diversity!across!populations!was!much!higher!in!France!than!in!Michigan!
populations,!average!2.32!and!0.31.!Similarly,!the!average!number!of!alleles!per!locus!in!
French!populations!was!higher!than!that!in!Michigan,!average!2.10!and!1.68,!
respectively.!In!C.#parasitica,!VCG!diversity!(and!by!extension!genomic!diversity)!may!
increase!after!colonization!events!by!way!of!sexual!reproduction!(similar!to!
diversification!in!many!other!plant!pathogens!when!they!are!introduced).!!As!
mycoviruses!invade,!VCG!and!genomic!diversity!may!decline!through!the!effects!of!
genetic!drift!and!natural!selection.!!However,!a!reduction!in!diversity!may!not!be!
maintained!given!variable!environmental!conditions,!biotic!interactions,!and!human!
influence!(e.g.,!accidental!introduction!of!new!VCG!types!or!by!changing!of!forest!
structure!due!to!logging)!thus!creating!boom!and!bust!cycles!of!VCG!and!genomic!
diversification,!mycovirus!invasion,!and!eventual!loss!of!mycoviruses!caused!by!sexual!
reproduction!in!rare!individual!VCGs!that!escape!mycovirus!infection!!
!

As!shown!in!Chapter!2!mycoviruses!have!the!ability,!to!alter!natural!selection!by!

favoring!the!most!fit!fungal!genotype!by!mycovirus!combinations,.!!Breuillin!et!al.!
(2006)!suggested!that!further!genetic!analyses!should!be!done!throughout!the!
geographic!distribution!of!C.#parasitica,!especially!where!populations!are!under!the!
influence!of!different!mycovirus!types!which!can!provide!informative!results!regarding!
the!effects!of!mycovirus!hyperparasites!on!the!genetic!structure!of!individual!pathogen!

!

77!

populations.!Although!different!mycoviruses!exist!at!each!of!three!Michigan!sites,!my!
work!demonstrates!that!genomic!diversity!within!mycovirusJinfected!populations!is!
often!low!but!these!same!populations!are!highly!differentiated!from!one!another.!!!!
!

Future!work!on!microsatellite!analyses!in!Michigan!and!throughout!the!range!of!

C.#parasitica!in!North!America!should!first!focus!on!optimizing!PCR!and!amplification!
for!current!primers!and!loci!along!with!developing!markers!unique!to!North!American!
populations!and!second,!further!sampling!determining!structure!of!the!pathogen!
population!across!the!geographic!range!in!North!America.!Phylogenetic!studies!as!
extensions!of!this!work!will!allow!an!understanding!of!how!C.#parasitica!spread!and!will!
help!us!understand!how!mycoviruses!can!influence!blight!pathogen!population!
structure!where!they!have!been!introduced!for!biological!control!of!C.#parasitica!(e.g.,!
West!Salem,!WI!and!the!three!populations!discussed!in!Chapter!4).!Since!we!have!
isolates!of!C.!parasitica!from!populations!where!I!have!introduced!dsRNA!mycoviruses!
(MH,!ST,!and!LE!discussed!in!Chapter!4),!studies!of!how!genetic!structure!changes!in!
response!to!mycovirus!invasion!might!be!possible!in!the!future.!!
!

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LITERATURE!CITED!
!
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79!

LITERATURE!CITED!
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Acosta!AC,!Uffo!O,!Sanz!A,!Ronda!R,!Osta!R,!Rodellar!C,!MartinJBurriel!I,!and!Zaragoza!P.!
2013.!Genetic!Diversity!and!Differentiation!of!Five!Cuban!Cattle!Breeds!Using!30!
Microsatellite!Loci.!Journal!of!Animal!Breeding!and!Genetics.#130:79–86.!!
Anagnostakis!SL,!Hau!B,!and!Kranz!J.!1986.!Diversity!of!Vegetative!Compatibility!Groups!
in!Cryphonectria#parasitica!in!Connecticut!and!Europe.!Plant!Disease.!70:536–538.!!
Babaei!N,!Abdullah!NAP,!Saleh!G,!and!Abdullah!TL.!2012.!Isolation!and!Characterization!
of!Microsatellite!Markers!and!Analysis!of!Genetic!Variability!in!Curculigo#latifolia!
Dryand.!Molecular!Biology!Reports.!39:9869–9877.!!
Bissegger!M,!Rigling!D,!and!Heiniger!U.!1997.!Population!Structure!and!Disease!
Development!of!Cryphonectria#parasitica!in!European!Chestnut!Forests!in!the!Presence!
of!Natural!Hypovirulence.!Phytopathology!87:50–59.!
Breuillin!F,!Dutech!C,!and!Robin!C.!2006.!Genetic!Diversity!of!the!Chestnut!Blight!Fungus!
Cryphonectria#parasitica!in!Four!French!Populations!Assessed!by!Microsatellite!
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Bryner!SF!and!Rigling!D.!2012.!Virulence!Not!Only!Costs!but!Also!Benefits!the!
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Goldstein!DB!and!Pollock!DD.!1997.!Launching!Microsatellites:!A!Review!of!Mutation!
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Goldstein!DB,!Roemer!GW,!Smith!DA,!Reich!DE,!Bergman!A,!and!Wayne!RK.!1999.!The!
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in!a!Natural!Model!System.!Genetics.!151:797–801.!!
Heald!FD,!Gardner!MW,!and!Studhalter!RA.!1915.!Air!and!wind!dissemination!of!
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CHAPTER!4!
!
BIOLOGICALLY!CONTROLLING!CHESTNUT!BLIGHT!PATHOGEN!POPULATIONS!IN!
MICHIGAN!USING!A!NATIVE!MYCOVIRUS:!INITIAL!RESULTS!OF!A!LONG8TERM!
EXPERIMENT!
!
!
ABSTRACT!
!
!
Mycovirus!hyperparasites!were!introduced!over!a!threeJyear!period!into!three!
populations!of!American!chestnuts!(Castanea#dentata#(Marsh.)!Borkh.)!infected!with!
chestnut!blight!caused!by!Cryphonectria#parasitica#(Murrill)!Barr.).!The!GH2!mycovirus,!
which!is!native!to!Michigan,!was!used!to!determine!whether!American!chestnuts!in!the!
size!class!1—10cm!diameter!at!breast!height!(DBH)!would!provide!population!level!
response!to!mycovirus!treatment!of!blight!cankers.!Persistence!of!mycovirus!after!
introduction!was!slightly!over!50%!for!all!three!sites.!!Treated!trees!did!not!survive!any!
better!than!untreated!control!trees!(74%!versus!73%,!respectively,!over!the!three!year!
period).!Survivorship!did!differ!among!sites!with!87%!of!the!treated!trees!at!Stivers!
surviving,!followed!by!Leelanau!(72%),!and!Missaukee!Healthy!(65%).!!!Survivorship!of!
treated!trees!with!a!DBH!less!than!3cm!DBH!was!very!low!(40%).!!Future!work!should!
consider!only!treating!trees!with!DBHs!above!3cm!DBH.!Growth!of!treated!trees!was!
low!at!Leelanau!but!treated!trees!grew!better!than!untreated!trees!at!both!Stivers!and!
Missaukee.!Environmental!conditions!or!tree!genetics!may!play!a!role!in!the!recovery!of!
treated!trees!at!each!site.!!
!
!
!
!
!
!
!
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!

INTRODUCTION!
!
Biological!control!of!nonJnative!and!invasive!species!has!become!an!important!

management!tool!in!ecological!and!agricultural!systems!(McEvoy!&!Coombs!1999!and!
references!therein)!especially!since!the!use!of!biological!control!agents!to!control!nonJ
native!species!may!be!less!harmful!to!the!environment!and!human!health!than!chemical!
control!(Simberloff!&!Stiling!1996)!and!may!be!more!stable!evolutionarily!than!other!
methods!of!control!since!invasive!species!may!not!evolve!resistance!as!quickly!as!with!
chemical!means!(Holt!&!Hochberg!1997).!The!management!of!nonJnative,!invasive!pests!
and!pathogens!with!biological!control!organisms!(sometimes!in!combination!with!other!
control!techniques)!has!become!a!common!method!to!preserve!biodiversity!(deBach!
1974;!Jezorek!et!al.!2012).!Biological!control!organisms!must!meet!rather!specific!needs!
because!they!must!control!the!target!organism!while!at!the!same!time!avoid!being!a!
pest!in!their!own!right.!!Examples!of!biological!control!schemes!failing!are!common!(e.g.,!
parasitoid!wasps!failed!to!spread!in!Florida!to!control!invasive!bromeliadJeating!
weevils:!Cooper!et!al.!2011;!failure!of!pathogens!to!control!termites:!Chouvenc!et!al.!
2011),!and!there!are!also!examples!where!the!biological!control!agent!have!become!a!
dangerous!pests!!(e.g.,!cane!toads,!Bufo#marinus!L.!in!Australia;!Shanmuganathan!et!al.!
2010).!!The!spread!of!biological!control!organisms!can!also!be!detrimental!when!they!
begin!to!affect!native!species!(a!host!shift),!especially!those!considered!federally!
threatened!e.g.,!the!seedJeating!weevil!Larinus#planus!Fabricius!becoming!a!seed!
predator!of!Pitcher’s!Thistle,!Cirsium#pitcheri!(Torr.!ex!Eat.)!Torr.!&!A.!Gray!in!Michigan!
and!Tracy’s!thistle,!Cirsium#undulatum!(Nutt.)!Spreng.!var.!tracyi!(Rydbg.)!S.L.!Welsh!in!
Colorado!after!it!was!introduced!to!control!invasive!thistles!elsewhere!(Louda!&!

!

84!

O’Brien!2002;!Havens!et!al.!2012).!!Additionally,!biological!control!organisms!may!
compete!with!native!species!leading!to!changes!in!community!structure!(Simberloff!&!
Stiling!1996).!Simberloff!and!Stiling!(1996)!provide!a!detailed,!useful!review!of!the!risks!
of!biological!control!such!as!instances!of!predation,!herbivory,!and!parasitism!on!nonJ
target!species!across!diverse!taxa.!!
!

The!success!of!biological!control!schemes!is,!however,!sometimes!difficult!to!

ascertain!even!with!repeated!introductions!of!the!control!organism!(e.g.,!Milgroom!&!
Cortesi!2004).!!In!at!least!one!instance!(Chondrilla#juncea#L.,!Skeletonweed!in!Australian!
agricultural!fields),!biological!control!was!successful!against!one!genotype!of!the!
invasive!plant!but!has!failed!to!control!other!genotypes!of!C.#juncea#that!increased!in!
frequency!when!the!common!form!of!the!weed!was!controlled!by!the!pathogen,!
Puccinia#chondrillina#Bubak!&!Syd.!(Burdon!et!al.!1981).!!However,!examples!of!
successful!biological!control!can!also!be!found.!Two!popular!examples!of!success!are!the!
control!of!lepidopterans!affecting!agricultural!crops,!using!endoparasitoid!stingless!
wasps,!Trichogramma!spp.!(Smith!1996)!and!control!of!invasive!Purple!Loosestrife,!
Lythrum#salicaria#L.!by!using!Galerucella!spp.!leaf!beetles!(Grevstad!2006!and!
references!therein;!Yeates!et!al.!2012).!Mixed!success!of!biological!control!are!also!quite!
common!(cf.!Murdoch!et!al.!1985).!!
!

Potential!difficulties!in!using!biological!control!include!understanding!which!size!

of!organisms!to!treat!or!protect!to!increase!population!size,!timing!of!treatment!or!
transmission!of!control!mechanisms!(Chua!1978;!Reilly!&!Hajek!2012),!and!how!often!
to!treat!!(Crow!2005)!which!could!all!be!contributing!factors!to!the!success!or!failure!of!
biological!control!!(Milgroom!&!Cortesi!2004).!Additionally,!longJterm!monitoring!of!

!

85!

systems!where!biological!control!has!been!implemented!is!essential!to!determining!
longJterm!quantitative!success!but!has!rarely!been!done!(McEvoy!&!Coombs!1999;!
Barton!et!al.!2007;!Grevstad!2006).!!It!is!also!difficult!to!evaluate!the!success!of!a!
biological!control!program!because!most!studies!do!not!incorporate!comparison!control!
sites!where!biological!control!agents!are!not!released!(Morin!et!al.!2009).!
!

Biological!control!of!chestnut!blight!in!Europe!has!been!successfully!managed!by!

naturally!occurring!and!deliberately!introduced!mycoviruses!that!infect!the!mycelium!
of!the!blight!fungus,!Cryphonectria#parasitica#(Murrill)!Barr.!The!success!of!mycoviruses!
in!Europe!has!led!to!attempts!to!use!mycoviruses!to!control!blight!on!American!
chestnuts,!Castanea#dentata#(Marsh.)!Borkh.,!but!these!efforts!have!been!largely!
unsuccessful.!!Why!mycoviruses!control!blight!in!Europe!but!not!North!America!
remains!an!open!question,!but!researchers!have!proposed!several!explanations!
including!the!complex!vegetative!compatibility!structure!of!North!America!C.#parasitica!
populations!(Anagnostakis!et!al.!1986;!Anagnostakis!&!Kranz!1987),!differences!in!the!
susceptibility!of!chestnuts!in!Europe!and!North!America!(Griffin!1986),!and!treating!
trees!inefficiently!(Davelos!and!Jarosz!2004).!
!

Blight!cankers!quickly!girdle!American!chestnut!trees!infected!with!C.#parasitica.!

Girdling!results!in!the!death!of!all!tree!material!distal!to!the!canker.!!Naturally!occurring!
doubleJstranded!(ds)!RNA!mycoviruses!first!discovered!in!blighted!populations!of!
European!chestnuts!with!abnormal!cankers!(Biraghi!1953;!Grente!1965)!were!later!
found!to!be!naturally!occurring!in!some!Michigan!populations!of!the!chestnut!blight!
pathogen!(Elliston!et!al!1977;!Fulbright!et!al.!1983).!Mycoviruses!alter!the!virulence!of!C.#
parasitica!in!such!a!way!that!can!allow!the!host!trees!to!tolerate!infection!by!blight!(see!

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86!

Chapter!1).!This!condition,!known!as!hypovirulence,!is!a!textbook!example!for!biological!
control!and!has!been!known!in!the!chestnutJchestnut!blight!system!for!over!60!years!
(Milgroom!&!Cortesi!2004).!!!The!success!of!biological!control!in!Europe!has!escalated!
interest!in!using!mycoviruses!to!mange!blight!in!North!America!(Van!Alfen!et!al.!1975;!
Van!Alfen!1982;!Anagnostakis!1987;!Fulbright!&!Paul!1988;!MacDonald!&!Fulbright!
1991).!!A!goal!in!chestnut!blight!research!is!to!successfully!control!or!manage!the!
disease!of!American!chestnuts!with!mycoviruses!(cf.!MacDonald!&!Fulbright!1991)!so!
they!might!again!attain!significant!stature!in!the!eastern!hardwood!forests!of!the!United!
States!and!in!areas!where!they!and!their!hybrids!have!been!introduced!for!nut!crop!
production!(DW!Fulbright!pers.!comm.).!!
!
What!trees!to!treat?!
!

Demographic!analyses!have!been!used!often!to!determine!population!viability!

and!to!identify!critical!lifeJhistory!stages!important!for!protecting!atJrisk!populations!of!
endangered!or!threatened!organisms!(Silvertown!et!al.!1993;!Crowder!et!al.!1994,!
1995;!Kephart!&!Paladino!1997;!Fujiwara!&!Caswell!2001;!Andrieu!et!al.!2013).!This!
information!can!be!used!when!designing!conservation!schemes!!(Caswell!2010).!
!

A!demographic!analysis!of!American!chestnut!populations!in!Michigan!proposed!

that!treating!the!largest!blightJinfected!trees!is!not!the!most!efficient!way!to!achieve!
recovery!of!American!chestnuts!(Davelos!&!Jarosz!2004).!!Matrix!projection!models!and!
sensitivity!analyses!proposed!that!treating!subJadult!trees!with!a!diameter!at!breast!
height!(DBH)!between!1!and!10cm!would!be!the!most!efficient!way!of!introducing!

!

87!

mycovirus!since!these!trees!contribute!significantly!to!the!overall!chestnut!population’s!
growth,!leading!to!faster!recovery!(Davelos!1999;!Davelos!&!Jarosz!2004).!!
!
A!Michigan!mycovirus!used!for!biological!control!
!

The!naturally!occurring!Cryphonectria!mycovirus,!a!CHV3!type!(hereafter!GH2,!

see!Chapter!1)!used!in!this!study!was!discovered!near!Grand!Haven,!Michigan!in!the!late!
1970s!(Elliston!et!al.!1977;!Fulbright!et!al.!1983).!Chestnut!trees!at!the!site!were!
recovering!from!blight!until!the!trees!were!removed!for!a!housing!development!(DW!
Fulbright,!pers.!comm.).!!GH2!is!of!moderate!virulence!regarding!its!effect!on!the!blight!
pathogen!compared!to!similar!mycoviruses!found!in!Michigan!and!those!in!Europe!
(Fulbright!et!al.!1983;!Enebak!et!al.!1994);!the!average!size!of!cankers!produced!by!GH2!
infected!C.#parasitica!are!approximately!oneJthird!the!size!of!cankers!produced!by!C.#
parasitica!that!is!not!infected!with!GH2!(Fulbright!et!al.!1983).!Other!mycoviruses!may!
debilitate!the!pathogen!so!much!that!asexual,!conidia!production!nearly!ceases!while!
other!mycoviruses!have!little!affect!on!canker!expansion!(Fulbright!et!al.!1983;!
MacDonald!&!Fulbright!1991;!Dawe!&!Nuss!2001;!Milgroom!&!Cortesi!2004).!It!should!
also!be!noted!that!since!mycoviruses!inhibit!sexual!reproduction!of!C.#parasitica,!they!
only!spread!via!asexual!conidia!that!can!cause!new!cankers!(vertical!transmission)!or!
converting!existing!cankers!via!hyphal!anastomosis!(horizontal!transmission)!
(Anagnostakis!1984;!Fulbright!&!Paul!1988;!Zhang!et!al.!1998;!Chapters!1!&!2).!!
!

My!reasons!for!using!GH2!were!twofold:!1)!slow!canker!expansion!so!that!

cankers!do!not!girdle!stems!or!branches!and!b)!allow!the!pathogen!to!grow!and!
produce!mycovirus!containing!asexual!conidia,!to!promote!mycovirus!spread.!!The!ideal!

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88!

mycovirus,!which!effectively!reduces!symptoms!of!chestnut!blight!while!still!spreading!
efficiently!through!the!blight!population,!might!be!considered!a!“Goldilocks!mycovirus”!
(see!Chapter!1!for!a!discussion!of!how!different!mycoviruses!affect!C.#parasitica).!!
!

This!study!details!results!for!the!first!three!years!of!implementing!Davelos!and!

Jarosz’s!assertion!that!mycovirus!introductions!should!concentrate!on!trees!between!1!
and!10cm!DBH!and!investigates!how!trees!in!that!size!class!have!performed!at!three!
sites!since!mycovirus!introduction.!Success!or!failure!of!this!biological!control!
mechanism!cannot!be!fully!determined!in!three!years;!a!longJterm!approach!to!
recovery!is!imperative!as!is!other!biological!control!situations!(Havens!et!al!2012).!!
!
METHODS!
!

Three!blightJinfected!populations!of!American!chestnuts!were!used!for!

mycovirus!introductions.!!Site!locations!and!descriptions!of!Missaukee!Healthy,!Stivers,!
and!Leelanau!(MH,!ST,!and!LE,!respectively)!can!be!found!in!Davelos!&!Jarosz!(2004).!
Two!populations!(MH!and!LE)!became!infected!with!blight!in!1997!(Chapter!2;!AL!
Davelos!Baines!and!AM!Jarosz,!unpublished!data)!while!the!ST!site!has!been!infected!by!
the!blight!pathogen!since!the!1980s!(DW!Fulbright!pers.!comm.).!The!presence!of!
mycoviruses!at!these!sites!prior!to!my!mycovirus!introductions!was!less!than!5%!
(Davelos!1999,!and!this!study).!!!
!

Bark!samples!from!thirty!cankers!in!each!population!were!collected!early!in!

2009!and!the!three!most!common!vegetative!compatibility!groups!(VCGs)!were!
determined!using!the!techniques!of!Huber!(1996;!and!see!Chapter!2).!!A!representative!

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89!

from!each!VCG!at!each!site!was!paired!with!a!GH2!infected!C.#parasitica!isolate!revived!
from!filter!paper!longJterm!storage.!!

!

Mycovirus!transfer!to!the!uninfected!isolate!was!evident!by!a!change!in!phenotype!
(Figure!4J1)!including!slowed!growth!rate!relative!to!uninfected!isolate!and!scalloped!
margin!of!the!newly!infected!isolate.!For!each!VCG,!a!slurry!of!each!mycovirus!
containing!isolate!was!created!using!one!liter!of!water!agar,!one!liter!sterile!water,!and!
20—25!petri!plates!containing!the!GH2!infected!isolate!and!blending!it!into!a!
consistency!similar!to!applesauce.!!
Trees!in!the!1—10cm!DBH!size!class!were!chosen!randomly!from!a!pool!of!available!
trees!in!each!population;!comparative!performance!of!trees!above!10cm!was!not!
possible!since!there!are!few!trees!available!at!the!three!sites!in!that!size!class.!Total!
numbers!of!trees!and!cankers!treated!varied!across!the!threeJyear!mycovirus!
deployment!period.!!Both!infected!and!diseaseJfree!tree!were!inoculated!with!
mycovirus.!!A!mixture!of!mycovirus!infected!isolates!representing!the!three!most!VCGs!
(see!Chapter!2)!were!used!to!inoculate!trees.!!At!the!ST!population,!cankers!were!
treated!with!the!VC!type!matching!the!VCG,!or!with!a!mixture!of!VC!types!if!canker!VCG!
was!unknown!or!if!new!cankers!were!created.!!
!
APPLICATION!OF!GH2!!
!

For!the!2009!and!some!2010!applications,!a!cordless!drill!with!a!0.6!or!1!cm!drill!

bit!was!used!to!create!holes!around!the!canker!circumference!in!uninfected!tissue!(the!
smallest!trees!were!injured!with!the!smaller!drill!bit!and!vice#versa,#Figure!4J2a).!Canker!
conversion!and!canker!initiation!rates!were!very!low!using!this!method!so!a!scratching!

!

90!

method!was!used!in!subsequent!applications.!In!May!of!2010!and!2011!the!margins!of!
existing!cankers!were!scratched!with!a!small!wooden!paddle!with!lag!screws!attached!
at!one!end!(Figure!4J2b).!!!Threads!of!the!lag!screws!allowed!the!bark!to!be!wounded!
sufficiently!to!expose!both!the!phloem!and!cambium!leading!to!a!larger!amount!of!
surface!area!available!to!infect!than!drill!bits.!A!thick!slurry!of!mycovirus!infected!
mycelia!‘applesauce’!was!applied!with!a!laboratory!squirt!bottle!and!paintbrush.!In!
2009!the!treated!and!created!cankers!were!covered!with!laboratory!bench!paper!for!
two!weeks!to!maintain!a!humid!environment!but!was!not!used!in!subsequent!treatment!
years.!

!
Figure! 4J1.! Representative! mycovirus! transfer! plate.! A! GH2! infected! donor!
strain,! left;! note! flattened! appearance! of! mycelia! with! a! virulent! recipient!
strain,! right;! note! raised! appearance.! ! Successful! conversion! of! virulent!
strain!occurred!near!arrow.!A!change!in!phenotype!similar!to!the!left!isolate!
shows!successful!transfer!of!GH2!from!donor!to!recipient!strain.!!

!

91!

a

b

!

Figure!4J2.!Two!forms!of!treating!cankers!used!in!this!study.!a)!drill!bit!created!holes!at!
canker!margins!where!mycovirus!inoculum!could!be!introduced!and!b)!"scratcher"!with!
lag!screws!to!wound!canker!and!surrounding!area;!more!surface!area!was!able!to!be!
treated!with!this!method.!Figure!2b!photo!credit:!AM!Jarosz.!
!
!
DETERMINING!MYCOVIRUS!PREVALENCE!
!

Trees!were!visited!during!August!of!each!year!to!determine!the!level!of!

mycovirus!transfer!to!existing!cankers!and!creation!of!hypovirulent!cankers!on!
uninfected!trees.!Each!treated!tree!was!measured!at!DBH!(at!a!height!of!approximately!
1.3!meters).!In!2011!before!mycoviruses!were!applied!and!in!June!of!2012,!previously!
treated!cankers!were!sampled!by!removing!bark!tissue!at!canker!margins.!
!

Subsamples!of!2x2mm!were!removed!from!the!larger!bark!sample!and!surface!

sterilized!in!10%!sodium!hypochlorite!for!15s,!repeated,!then!washed!in!ddH20!for!15s.!
Bark!subsamples!were!placed!onto!petri!plates!with!potato!dextrose!agar!(PDA).!When!
chestnut!blight!fungus!began!to!grow,!cultures!were!hyphal!tipped!and!placed!onto!new!
PDA!plates.!After!2!weeks,!isolates!were!scored!for!the!presence!of!mycovirus!based!

!

92!

culture!morphology.!!A!scalloped!margin!on!the!culture!indicated!that!mycoviruses!
were!present.!
!

Average!DBH!for!treated!trees!was!calculated!for!surviving!trees!at!each!annual!

time!step!between!2009!and!2012!(Table!4J2).!!Diameter!at!breast!height!was!used!as!a!
proxy!for!tree!size;!growth!from!year!to!year!was!calculated!only!for!trees!that!
remained!alive!during!each!timeJstep.!!The!survivorship!of!main!stems!across!the!time!
period!was!also!determined!at!both!treated!and!control!populations.!
!

Two!control!populations,!Missaukee!Diseased!(MD),!a!population!300!meters!

north!of!MH!and!Stivers!North!(SN)!did!not!receive!mycovirus!treatments.!!Cankers!
were!sampled!in!2012!at!these!two!control!sites!to!determine!the!prevalence!of!
naturally!occurring!mycovirus.!!Additionally,!a!cohort!of!30!trees!in!the!1—10cm!
diameter!at!breast!height!(DBH)!size!class!was!tracked!through!the!time!period!2009—
2012!for!comparison!with!the!mycovirus!treated!populations!
!

These!initial!studies!focus!on!survivorship!and!growth!of!the!main!stem,!future!

studies!will!consider!the!fate!of!individual!genets!and!whether!the!GH2!mycovirus!has!
spread!to!other!cankers!on!inoculated!trees!and!to!new!trees!within!the!populations.!!
!
RESULTS!
Mycovirus!prevalence!after!treatments!
!

Greater!than!50%!of!the!mycovirus!treated!cankers!had!mycovirus!in!May!2011!

and!June!2012!(Table!4J1).!!Mycovirus!presence!increased!slightly!at!LE!from!2011!to!
2012,!which!is!curious!given!the!performance!of!treated!trees!at!that!site,!but!decreased!
at!the!two!remaining!sites.!!Only!one!of!19!cankers!contained!mycovirus!at!the!

!

93!

Missaukee!Diseased!control!site,!is!similar!to!the!prevalence!reported!by!Davelos!
(1999)!in!the!1990s.!!
!

Annual!survivorship!of!all!1!to!10cm!DBH!trees!monitored!across!all!five!sites!

was!74%.!!However,!annual!survivorship!varied!among!treated!sites!with!average!
survivorship!of!treated!trees!being!highest!at!ST!(87%),!intermediate!at!LE!(72%),!and!
MH!(65%)!having!the!lowest.!!Control!sites!MD!and!SN!had!survivorship!annual!
survivorship!at!77%!and!69%,!respectively.!!!Nearly!half!of!the!first!cohort!of!13!trees!
treated!at!LE!in!2009!died!by!2010!(Fig.!4J3).!!A!second!cohort!of!44!trees!was!treated!
at!LE!in!2010!and!monitored!until!2012.!!!Survivorship!trends!for!both!cohorts!from!
2010!to!2012!were!roughly!similar!(Figure!4J3),!suggesting!that!survivorship!may!vary!
from!year!to!year!due!to!environmental!conditions—see!discussion.!!TwentyJeight!trees!
treated!at!MH!beginning!in!2009!were!compared!to!a!similar!number!of!untreated!trees!
of!approximately!the!same!size!from!MD!site,!which!is!only!300M!to!the!northwest.!!!
Treated!trees!at!MH!displayed!lower!survivorship!from!2009!to!2011!compared!to!MD,!
but!survived!much!better!from!2011!into!2012!(Fig.!4J4).!!!The!trend!was!more!
consistent!at!Stivers!where!the!cohort!of!87!treated!trees!survived!better!than!
untreated!trees!from!2009!to!2011.!!Unfortunately,!I!lost!access!to!this!site!in!2012!and!
was!unable!to!continue!monitoring.!!
Davelos!(1999)!found!that!the!success!of!mycovirus!treatment!was!dependent!
on!the!size!of!the!treated!branch!with!small!branches!succumbing!to!disease!at!a!much!
higher!rate!than!larger!branches.!!In!consequence,!I!expected!a!pattern!where!larger!
trees!would!survive!better!after!treatment!than!smaller!trees.!!This!pattern!was!
observed!only!at!Leelanau!where!treated!trees!that!survived!were!larger,!when!

!

94!

measured!as!DBH,!than!trees!that!died!(Table!4J2).!!There!was!no!pattern!among!the!
treated!trees!at!MH!with!surviving!trees!having!a!larger!DBH!in!2009J10,!smaller!in!
2010J11!and!similar!in!2011J12.!!Somewhat!surprisingly,!surviving!treated!trees!at!ST!
had!smaller!diameters!than!trees!that!died!in!both!2009J10!and!2010J11.!
Trees!at!LE!displayed!little!to!no!growth!over!the!course!of!the!experiment!with!
the!exception!of!trees!in!cohort!2!in!from!2010!to!2011!(Figure!4J6)!which!is!also!
evident!in!the!average!increase!in!diameter!over!all!time!periods!and!cohorts!at!that!site.!!
In!contrast,!trees!at!MH,!MD!and!ST!exhibited!strong!growth!over!the!course!of!my!
monitoring!and!this!represents!a!possible!positive!effect!of!mycovirus!treatment!
(Figures!4J7and!4J8).!Aside!from!trees!at!the!LE!site,!which!performed!poorly!
throughout!the!course!of!the!experiment!(Figure!4J6),!treated!trees!at!MH!and!ST!
increased!in!diameter!faster!than!trees!at!control!sites!except!for!the!period!2011J2012!
at!the!MD!site.!The!second!cohort!at!MH!consisted!of!only!five!trees,!four!of!which!
survived!to!2012.!!There!was!also!suggestion!of!a!significant!positive!effect!of!mycovirus!
treatment!on!growth!at!ST,!where!treated!trees!in!the!southern!portion!of!the!
population!grew!faster!than!untreated,!control!trees!from!the!northern!portion!in!both!
2009J10!and!2010J11!(Figure!4J8).!
!
!
!
!
!

!

95!

Table! 4J1.! Percentage! of! treated! cankers! at! three! sites! that! contained!
mycovirus.! ! Presence! of! mycovirus! was! determined! based! on! culture!
morphology.!
!
Missaukee!Healthy!
!
2011!
2012!
Leelanau!
2011!
2012!
Stivers!
2011!
2012!
Missaukee!Diseased!

Percentage!with!
mycovirus!

N!
!
27!
38!
!
73!
64!
!
42!
69!
!

0.67!
0.55!
!
0.75!
0.8!
!
0.57!
0.55!
!

19a!
2012!
0.05!
a:!Cankers!were!not!treated!with!mycovirus!at!Missaukee!Diseased.!!Thus,!
values!represent!natural!levels!of!mycovirus!prevalence!at!this!site.!
!

!!

Survivorship!(%)!

100!
80!
60!
40!
20!
0!
2009!

2010!

Year!

2011!

2012!

!

Figure!4J3.!Survivorship!of!treated!American!chestnut!trees!at!the!Leelanau!
site.! Cohort! 1! (solid! line)! was! composed! of! 13! trees! that! were! treated!
initially!in!2009.!!The!second!cohort!(dashed!line)!consisted!of!44!trees!that!
were!first!treated!in!2010.!
!

!

96!

Survivorship!(%)!

100!
80!
60!
40!
20!
0!
2009!

2010!

2011!

2012!

Year!

!
Figure! 4J4! Survivorship! of! 28! American! chestnut! trees! treated! with!
mycovirus!at!Missaukee!healthy!(solid!line)!and!compared!to!30!untreated!
control!trees!at!Missaukee!Diseased!(dotted!line)!over!the!time!period!2009!
to!2012.!!!The!MH!and!MD!sites!are!only!300!meters!apart.!Cohort!two!of!
five!trees!at!MH!is!not!shown.!
!

Survivorship!(%)!

100!
80!
60!
40!
20!
0!
2009!

2010!

2011!

Year!

!

!
!
!
!
!

Figure!4J5.!Survivorship!of!87!American!chestnut!trees!at!the!treated!ST!south!
trees!(solid!line)!and!30!untreated!control!trees!in!the!northern!portion!of!the!
site!(dotted!line)!from!2009!to!2011.!!

97!

!
!
Table!4J2.!Average!size!(DBH)!of!trees!that!survived!and!trees!that!died!over!each!
time! step.! Missaukee! Diseased! and! Stivers! North! data! are! control! populations.!
Standard!errors!are!in!parentheses.!!
2009-10
Alive
Dead
Leelanau–LE
(treated)
Missaukee
Healthy–MH
(treated)
Missaukee
Diseased–
MD (control)
Stivers–ST
(treated)
Stivers
North-SN
(control)

2010-11
Alive
Dead

2011-12
Alive
Dead

4.7 (1.7)

4.8 (1.9)

5.4 (0.8)

4.3 (1.9)

5.6 (0.8)

4.3 (1.1)

3.3 (0.7)

2.6 (0.9)

3.9 (0.9)

4.2 (1.5)

3.9 (0.8)

3.9 (2.3)

2.5 (0.5)

2.4 (1.2)

2.9 (0.6)

2.6 (0.8)

3.1 (0.8)

3.2 (0.8)

2.3 (0.4)

4.4 (1.6)

3.9 (0.9)

4.2 (1.3)

na*

na

3.0 (0.7)

2.3 (0.7)

3.1 (0.9)

4.1 (1.0)

na

na

*Data!for!this!time!step!are!not!available!for!these!sites.!!

!

98!

!
!
4!
3!
2!
1!
0!
J1!
J2!
LE!Cohort!1!

!

!
!
!
!
!

!

LE!Cohort!2!

Figure!4J6.!Percentage!annual!change!in!DBH!for!surviving!trees!at!LE!
site.!There!is!no!control!for!the!LE!site.!Diagonal!bars!are!growth!from!
2009!to!2010,!dotted!bars!are!increase!(or!decrease)!from!2010!to!2011,!
and!brick!bars!represent!the!increase!from!2011!to!2012.!At!LE!Cohort!
2,!treatment!did!not!begin!until!2010.!

!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
99!

!

!
!
!
30!
25!
20!
15!
10!
5!
0!
MH!Cohort!1!

MH!Cohort!2!

!

!

!

MD!

!

Figure! 4J7.! Percentage! annual! change! in! DBH! for! surviving! trees! at!
MH!(treated)!and!MD!(control)!sites.!Dark!bars!are!growth!from!2009!
to!2010,!dotted!bars!are!increase!from!2010!to!2011,!and!brick!bars!
represent!the!increase!from!2011!to!2012.!At!MH!Cohort!2,!treatment!
did! not! begin! until! 2010.! Percentage! change! in! DBH! is! calculated! as!
the! average! increase! in! stem! diameter! over! the! previous! year! for!
Figures!6,!7,!and!8.!!
!

100!

!
!
!
!
!
!
30!
25!
20!
15!
10!
5!
0!
ST!

SN!

!

!
!

!

Figure! 4J8.! Percentage! annual! change! in! DBH! for! surviving! trees! at!
ST! (treated)! and! SN! (control)! sites.! Diagonal! bars! are! growth! from!
2009!to!2010,!dotted!bars!are!increase!from!2010!to!2011,!Data!for!
the!final!period!are!not!available!for!either!Stivers!site.!!!!
!
!
DISCUSSION!
!
Biological!control!of!chestnut!blight!has!received!intense!interest!since!

mycoviruses!were!discovered!in!this!system!(Van!Alfen!1982;!Fulbright!&!Paul!1988;!
MacDonald!&!Fulbright!1991).!However,!the!persistence!and!spread!of!mycoviruses!has!
been!difficult!in!North!America,!while!mycovirus!induced!recovery!in!European!
chestnut!populations!has!been!more!successful!(Robin!&!Heiniger!2001;!Milgroom!&!
Cortesi!2004;!Chapters!1!&!2).!Failure!in!North!America!has!been!attributed!to!the!high!

!

101!

VC!diversity!within!C.#parasitica!populations!(see!Chapters!1!&!2)!but!may!also!be!
blamed!on!the!fact!that!mycoviruses!might!not!succeed!in!areas!far!beyond!where!they!
are!found!to!be!naturally!occurring!(MacDonald!&!Fulbright!1991),!thus!my!choice!to!
use!GH2!for!this!study.!!
!

Survivorship!may!vary!from!year!to!year!due!to!environmental!conditions!at!

individual!sites.!This!is!evident!when!cohorts!that!began!treatment!in!different!years!
perform!similarly!(e.g.,!the!LE!site)!across!years.!!Differences!among!sites!and!among!
years!may!also!be!due!to!biotic!interactions!at!individual!sites.!!Performance!at!each!
treated!site!may!be!linked!to!community!structure.!Both!ST!and!SN!have!little!
competition!from!other!tree!species!and!are!nearly!chestnut!monocultures!of!small!to!
medium!sized!trees.!The!MH!site!has!few!chestnut!overstory!trees!and!other!tree!
species!(Red!Maple!and!American!Beech)!may!be!outcompeting!infected!American!
chestnuts.!Where!large!overstory!chestnuts!have!been!harvested!at!the!MH!site,!small!
chestnuts!have!begun!to!grow!rapidly!(JC!Springer!pers.!obs.).!The!MD!site!has!many!
large!chestnut!trees!in!the!overstory!that!have!died,!allowing!small,!understory!
chestnuts!to!be!released!into!the!canopy!at!an!increased!rate!probably!due!to!increased!
light!availability.!The!LE!site!is!similar!to!the!MH!site!in!that!many!large!nonJchestnuts!
are!in!the!overstory!but!there!are!some!very!large!chestnuts!succumbing!to!chestnut!
blight.!!
!

Data!in!this!chapter!should!be!interpreted!as!preliminary.!The!ultimate!success!

or!failure!of!using!the!native!mycovirus,!GH2,!as!a!biological!control!may!not!be!realized!
until!a!much!longer!time!period!has!elapsed.!Although!trees!were!treated!for!three!
years,!success!of!mycoviruses!being!transferred!to!cankers!has!been!disappointingly!

!

102!

low.!!Future!work!should!explore!alternate!methods!of!inoculating!mycoviruses!onto!
cankers!that!may!prove!to!be!more!efficient!at!transferring!the!mycovirus!
Based!on!stageJbased!demographic!analyses,!Davelos!(1999)!and!Davelos!&!
Jarosz!(2004)!proposed!that!treatment!of!American!chestnuts!in!the!size!class!of!1—10!
centimeters!DBH!would!be!the!most!efficient!at!initiating!population!level!recovery!
from!blight.!A!clear!trend!of!larger!trees!in!the!1—10!cm!DBH!size!class!surviving!better!
than!smaller!trees!in!that!category!was!not!found!with!my!introduction!of!mycovirus!
GH2.!It!would!be!expected!that!treated!trees!above!3cm!DBH!would!have!better!
survivorship!since!smaller!trees!succumb!to!girdling!cankers!much!faster!than!larger!
trees.!!Low!survivorship!of!treated!trees!in!the!1—10cm!size!class!was!not!considered!
by!Davelos!&!Jarosz!(2004),!although!their!data!indicated!the!potential!for!poor!
performance!for!the!smallest!trees.!!The!smallest!trees!in!that!category!(1—2cm!DBH)!
may!actually!perform!most!poorly!since!blight!cankers!may!grow!faster!than!trees!
expand!even!when!mycoviruses!are!present.!Davelos!Baines!et!al.!(in#press)!found!
similar!results!in!their!treatment!of!varying!branch!sizes!with!mycovirus;!branches!
above!2cm!diameter!performed!well!while!those!less!than!2cm!diameter!did!not!see!
enhanced!growth.!Jarosz!et!al.!(in#press)!also!found!that!larger!trees!outperformed!
smaller!trees!when!mycovirus!treatments!were!applied!to!main!stems!in!West!Salem,!
WI.!More!treatment!may!be!needed!at!these!Michigan!sites!and!should!focus!on!trees!
that!have!diameters!greater!than!3cm!DBH.!!Ultimately!however,!more!time!is!needed!
to!determine!how!treated!trees!will!perform!at!each!treated!sites.!!Additionally,!it!may!
be!prudent!to!test!success!of!mycovirus!introduction!in!a!managed!forest!situation!
where!competing!hardwoods!are!cut.!!Griffin!et!al.!(1991)!suggested!that!American!

!

103!

chestnut!may!grow!rapidly!when!released!from!or!competition!from!other!species!is!
lowered,!especially!on!mesic!sites.!!
!

Other!important!aspects!of!this!interaction!that!should!be!studied!in!detail!are:!

a)!the!influence!of!tree!genotype!on!recovery!since!some!trees!that!survive!may!be!
better!able!to!tolerate!infection!by!chestnut!blight!than!their!congeners,!b)!how!
individual!chestnut!blight!VCGs!and!mycovirus!GH2!interact!may!also!be!crucial!to!slow!
canker!expansion,!c)!influence!of!the!abiotic!environment,!timing!of!mycovirus!
application,!and!how!often!to!apply!mycoviruses,!and!d)!how!the!mycovirus!evolves!
may!also!be!critical!to!mycovirus!longJterm!persistence!and!spread.!Additionally,!the!
type!of!site!may!influence!recovery,!i.e.,!monoculture!of!chestnut!versus!a!mixed!forest!
situation.!The!LE!and!MH!sites!could!be!considered!a!mixed!forest!where!a!canopy!of!
other!species!such!as!American!Beech,!Red!Maple,!and!Sugar!Maple!may!be!suppressing!
the!growth!of!understory!American!chestnuts.!The!Stivers!sites!are!essentially!a!
monoculture!of!American!chestnut!with!other!species!rarely!scattered!throughout!the!
community.!Reduced!competition!may!have!allowed!trees!at!the!ST!site!to!grow!larger!
after!mycovirus!GH2!was!introduced!to!chestnut!blight!cankers!in!that!population.!!!!!
!

Managing!the!chestnut!blight!fungus!with!mycoviruses!and!realizing!the!goal!of!

broadJscale!reintroduction!of!American!chestnuts!to!our!hardwood!forests!may!require!
our!persistence!and!patience.!Many!unanswered!questions!remain!regarding!our!ability!
to!use!native!mycoviruses!as!a!biological!control!mechanism!but!progress!is!being!made.!!
!
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104!

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LITERATURE!CITED!
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105!

LITERATURE!CITED!
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Barton!J,!Fowler!SV,!Gianotti!AF,!Winks!CJ,!de!Beurs!M,!Arnold!GC,!and!Forrester!G.!
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Biraghi!A.!1953.!Possible!active!resistance!to!Endothia#parasitica!in!Castanea#sativa.!
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Rome,!Italy.!643J645.!
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Burdon!JJ,!Groves!RH,!and!Cullen!JM.!1981.!The!Impact!of!Biological!Control!on!the!
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Caswell!H.!2010.!Life!Table!Response!Experiment!Analysis!of!the!Stochastic!Growth!
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Chouvenc!T,!Su!NY,!and!Grace!JK.!2011.!Fifty!Years!of!Attempted!Biological!Control!of!
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patterns,!field!experiments,!and!structured!population!models.!Ecological!Applications.!
9:387–401.!!
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Milgroom!MG!and!Cortesi!P.!2004.!Biological!Control!of!Chestnut!Blight!with!
Hypovirulence:!a!Critical!Analysis.!Annual!Review!of!Phytopathology.!42:311–38.!!
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Morin!L,!Reid!AM,!SimsJChilton!NM,!Buckley!YM,!Dhileepan!K,!Hastwell!GT,!Nordblom!
TL,!and!Raghu!S.!2009.!Review!of!Approaches!to!Evaluate!the!Effectiveness!of!Weed!
Biological!Control!Agents.!Biological!Control.!51:1–15.!!
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Reilly!JR!and!Hajek!AE.!2012.!PreyJprocessing!by!Avian!Predators!Enhances!Virus!
Transmission!in!the!Gypsy!Moth.!Oikos.!121:1311–1316.!!
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Robin!C!and!Heiniger!U.!2001.!Chestnut!Blight!in!Europe:!Diversity!of!Cryphonectria#
parasitica,!Hypovirulence!and!Biocontrol.!Forest!Snow!and!Landscape!Research.!
367:361–367.!
Shanmuganathan!T,!Pallister!J,!Doody!S,!McCallum!H,!Robinson!T,!Sheppard!A,!Hardy!C.!
2010.!Biological!Control!of!the!Cane!Toad!in!Australia:!a!Review.!Animal!Conservation.!
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Silvertown!J,!Franco!M,!Pisanty!I,!and!Mendoza!A.!1993.!Comparative!plant!
demography:!relative!importance!of!lifeJcycle!componenets!to!the!finite!rate!of!increase!
in!woody!and!herbaceous!perennials.!Journal!of!Ecology.!81:465J476.!!
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Simberloff!D!and!Stiling!P.!1996.!How!Risky!Is!Biological!Control?!Ecology.!77:1965–
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Smith!SM.!1996.!Biological!Control!with!Trichogramma:!Advances,!Successes,!and!
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Van!Alfen!NK.!1982.!Biology!and!potential!for!disease!control!of!hypovirulence!of!
Endothia#parasitica.!Annual!Review!of!Phytopathology.!20:349–362.!
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Van!Alfen!NK,!Jaynes!RA,!Anagnostakis!SL,!and!Day!PR,!1975.!Chestnut!blight:!biological!
control!by!transmissible!hypovirulence!in!Endothia#parasitica.!Science.!189:890–891.!
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Yeates!AG,!Schooler!SS,!Garono!RJ,!and!Buckley!YM.!2011.!Biological!Control!as!an!
Invasion!Process:!Disturbance!and!Propagule!Pressure!Affect!the!Invasion!Success!of!
Lythrum#Salicaria!Biological!Control!Agents.!Biological!Invasions.14:255–271.!
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Zhang!L,!Baasiri!RA,!and!Van!Alfen!NK.!1998.!Viral!suppression!of!fungal!pheromone!
precursor!gene!expression.!Molecular!and!Cellular!Biology.!18:953J!959.!
!
!
!
!
!
!

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109!

!

CHAPTER!5!
!
CONCLUSIONS!
!
!
When!dsRNA!mycovirus!hyperparasites!invade!populations!of!the!chestnut!

blight!pathogen,!Cryphonectria#parasitica,!they!appear!to!change!the!structure!of!the!
blight!population.!Where!mycoviruses!are!present,!they!prevent!sexual!reproduction!of!
C.#parasitica!and!reduce!blight!pathogen!virulence!on!American!chestnut,!allowing!
American!chestnuts,!Castanea#dentata,!to!tolerate!infection.!!Since!sexual!reproduction!
is!likely!not!occurring!in!populations!of!C.#parasitica!where!mycoviruses!are!present,!
fungal!reproduction!is!limited!to!asexual,!splashJdispersed!conidia,!which!limits!
dispersal!and!by!extension!migration!relative!the!more!mobile,!windJdispersed,!
sexually!produced!ascospores.!Further,!as!presented!in!Chapter!2,!vegetative!
compatibility!group!(VCG)!diversity!of!C.#parasitica!is!low!in!populations!where!
mycoviruses!are!present.!Populations!with!mycovirus!presence!generally!have!a!unique!
VCG!signature!while!populations!of!C.#parasitica!without!mycoviruses!have!higher!VCG!
diversity!and!those!VCGs!are!often!shared!between!populations.!Mating!type!analyses!
presented!in!Chapter!2!lent!further!support!that!mycovirus!infected!blight!populations!
are!clonal!since!mating!type!allele!frequencies!were!skewed!for!the!presence!of!one!
mating!type.!!
Natural!selection!may!play!a!role!in!generating!the!unique!VCG!signatures!found!
at!mycovirusJinfected!C.#parasitica!populations!in!Michigan;!populations!are!clonal!and!
selection!may!act!on!all!possible!combinations!of!mycovirus!and!fungal!genotype!to!find!
the!best!combination!for!a!particular!location,!thus!reducing!VCG!diversity!over!time.!!It!
has!been!hypothesized!that!mycoviruses!would!only!invade!populations!of!C.#parasitica!
!

110!

if!VCG!diversity!is!low,!since!closely!related!VCGs!may!allow!easy!spread!of!mycoviruses!
through!populations.!I!suggest!that!mycoviruses!may!invade!all!VCGs!in!a!blight!
population!and!natural!selection!will!operate!to!reduce!VCG!diversity!and!potentially!
reduce!genomeJwide!diversity!within!a!local!population.!This!may!be!one!of!the!reasons!
why!we!find!a!unique!VCG!signature!AND!unique!mycoviruses!at!each!Michigan!site!
studied!in!Chapters!2!and!3.!!
Microsatellite!analyses!from!Chapter!3!provided!some!support!to!the!hypothesis!
that!mycovirus!presence!in!populations!of!C.#parasitica!would!reduce!overall!genomic!
diversity.!In!populations!where!dsRNA!mycoviruses!have!been!present!for!a!long!time!
(CL!and!RC),!genomic!diversity!does!appear!to!be!much!lower!relative!to!C.#parasitica!
populations!not!infected!with!mycoviruses!(LE!and!MD).!FR,!although!infected!with!
mycoviruses!has!high!VCG!and!also!high!genomic!diversity!indicating!that!sexual!
reproduction!may!be!occurring!there.!Conversely,!ST!a!mycovirus!free!site!exhibits!low!
diversity!but!high!VCG!relative!to!other!sites!in!Michigan;!it!is!unclear!why!diversity!at!
that!site!may!be!low.!Sexual!reproduction!at!mycovirus!free!sites!may!likely!increase!
diversity!and!allow!migration!of!ascospores!between!these!sites;!there!is!evidence!of!
high!migration!rates!between!mycovirus!free!sites!relative!to!mycovirus!infected!sites.!
Together!with!higher!number!of!migrants!predicted!for!epidemic!populations!and!low!
migration!observed!between!recovering!populations!these!generalizations!makes!sense!
given!the!biology!of!the!system.!The!influence!of!mycoviruses!on!the!whole!genome!of!C.#
parasitica!is!only!partially!understood!at!this!point.!!More!work!should!be!done!in!this!
area!to!develop!a!full!dataset!of!genomic!diversity!by!optimizing!primers!developed!for!

!

111!

C.#parasitica!populations!in!Europe!or!by!developing!new!molecular!markers!unique!to!
populations!in!North!America.!!!
Mycovirus!introduction!into!C.#parasitica!populations!has!been!shown!to!help!
American!chestnut!trees!recover!or!at!least!tolerate!blight!infection.!A!test!of!a!
hypothesis!developed!by!Davelos!(1999)!and!Davelos!&!Jarosz!(2004)!was!conducted!to!
understand!whether!mycovirusJmediated!biological!control!of!C.#parasitica!would!be!
most!successful!on!American!chestnut!trees!in!the!1—10cm!diameter!at!breast!height!
(DBH)!size!class!rather!than!focusing!on!the!largest!trees!in!a!population.!!My!
preliminary!results!for!this!longJterm!study!show!that!the!smallest!trees!in!that!size!
class!may!not!be!the!best!choice!for!treatment!and!should!instead!focus!on!the!trees!
between!3—15cm!DBH.!!Annual!survivorship!of!treated!trees!varied!from!87%!to!65%.!
Cohorts!that!began!treatment!in!different!years!responded!similarly!to!mycovirus!
treatment,!suggesting!that!tree!survivorship!may!vary!from!year!to!year!due!to!
environmental!conditions.!Competition!from!other!tree!species!may!also!play!a!role!in!
mycovirus!success;!trees!at!ST!responded!better!to!mycovirus!treatment,!possibly!
because!this!site!is!a!monoculture!of!chestnuts!with!little!competition!or!overstory!
compared!to!the!LE!and!MH!sites.!Mycovirus!GH2!may!hold!some!promise!for!allowing!
recovery!of!American!chestnuts!in!Michigan:!it!has!a!moderate!virulence!on!the!
chestnut!blight!pathogen!relative!to!other!mycoviruses!while!still!allowing!mycovirusJ
containing!asexual!spores!of!C.#parasitica!to!be!produced!that!could!ideally!spread!
naturally!throughout!a!local!blight!population.!One!important!issue!not!to!be!ignored!
when!introducing!mycoviruses!for!biological!control!is!their!persistence!in!C.#parasitica!
cankers!over!time.!Treated!blight!cankers!in!my!study!were!examined!one!year!after!

!

112!

each!of!three!successive!treatments!which!revealed!an!average!of!on!50%!of!cankers!
containing!mycoviruses.!!Repeated!introductions!of!mycoviruses!into!blight!cankers!
may!be!necessary!to!achieve!recovery!in!American!chestnut!populations.!!Future!work!
in!this!area!should!also!explore!whether!tree!genotype!may!play!a!role!in!the!success!or!
failure!of!biological!control!of!C.#parasitica!with!mycoviruses.!Individuals!or!populations!
of!American!chestnut!that!are!less!susceptible!may!be!predisposed!to!tolerating!blight!
infection,!thus!work!that!unites!moderate!virulence!mycoviruses!and!known!genotypic!
effects!of!American!chestnuts!would!be!a!great!addition!our!understanding!of!this!triJ
trophic!interaction.!I!suggest!testing!other!mycoviruses!from!Michigan!for!their!
effectiveness!as!biological!control!agents.!!It!is!possible!that!effective!biological!control!
will!involve!matching!a!specific!mycovirus!with!the!C.#parasitica!and!American!chestnut!
populations!found!at!a!given!sites.!!
The!longJterm!storage!of!isolates!of!C.#parasitica,!especially!those!infected!by!
mycoviruses!is!important!for!future!work!that!considers!how!mycoviruses!have!evolved!
over!short!time!period!with!regard!to!their!effect!on!the!fitness!of!the!fungal!host.!Work!
in!Appendix!A!examined!effectiveness!of!resurrecting!isolates!from!sterile!water!longJ
term!storage.!However,!isolates!that!were!infected!when!stored!in!the!mid!to!late!1990s!
were!not!found!to!be!harboring!mycoviruses!when!they!began!growing!again!for!this!
study;!only!two!of!119!isolates,!originally!identified!at!mycovirusJinfected!could!be!
confirmed!to!still!contain!dsRNA.!!A!stable,!reliable!storage!method!for!mycovirus!
infected!isolates!needs!to!be!found.!!!I!have!initiated!an!experiment!that!will!test!
mycovirus!retention!under!two!different!storage!methods.!

!

113!

My!studies!on!VCG!and!microsatellite!diversity!suggest!that!C.#parasitica!
population!structure!will!change!as!mycoviruses!invade.!!Monitoring!the!LE!and!MH!
sites!where!I!have!been!introducing!mycoviruses!since!2009!may!provide!an!
opportunity!to!follow!this!expected!structural!change.!!Studies!that!try!to!understand!
how!VCG!diversity!change!over!time!in!these!populations!would!be!important!to!
understand!my!hypothesis!presented!at!the!end!of!Chapter!2!stating!that!VCG!diversity!
may!actually!decrease!as!natural!selection!works!to!find!the!best!adapted!combination!
of!C.#parasitica!genotype!by!mycovirus!for!a!local!population.!Moreover,!expansion!of!
microsatellite!work!in!Chapter!3!should!include!further!comparisons!of!populations!of!C.#
parasitica!long!infected!by!mycoviruses!(such!as!those!treated!multiple!times!with!
mycoviruses!as!part!of!a!biological!control!scheme,!i.e.,!the!West!Salem,!WI!site)!and!
those!where!mycoviruses!have!not!been!successful!(i.e.,!the!natural!range!of!American!
chestnut!in!the!eastern!United!States).!A!comparison!of!C.!parasitica!genomic!diversity!
in!Europe!and!the!U.S.!to!that!in!Asia,!the!purported!native!range!of!the!blight!pathogen,!
would!lead!to!a!better!understanding!of!expansion!of!pathogen!diversity!after!
colonization!to!its!nonJnative!ranges.!!
!
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LITERATURE!CITED!
!
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115!

LITERATURE!CITED!
!
Davelos!AL!and!Jarosz!AM.!2004.!Demography!of!American!chestnut!populations:!
effects!of!a!pathogen!and!a!hyperparasite.!Journal!of!Ecology!92:675J685.!
!
Davelos!AL.!1999.!DoubleJstranded!RNA!mediated!recovery!of!American!chestnut!
populations:!a!demographic!analysis.!PhD!thesis,!Michigan!State!University,!East!
Lansing,!Michigan.!!
!

!

116!

APPENDIX

!

117!

EVALUATING THE LONG-TERM STORAGE OF CRYPHONECTRIA
PARASITICA

!

ABSTRACT!
!
Isolates!of!the!Chestnut!blight!pathogen,!Cryphonectria#parasitica,!from!six!

populations!in!Michigan,!were!stored!in!the!late!1990s!as!agar!plugs!of!mycelium!in!
vials!of!sterile!water!held!at!room!temperature.!!Approximately!29%!of!the!fungal!
isolates!were!infected!with!mycoviruses!at!the!time!of!storage.!!!Each!isolate!was!tested!
for!revivification!effectiveness!by!taking!aliquots!from!vials!filled!with!agar!plugs!of!C.#
parasitica!and!sterile!water!and!plating!onto!potato!dextrose!agar.!!Average!
revivification!success!was!70.5%!across!populations!with!a!range!of!33—84%!within!
populations!.!In!situations!where!vials!had!dried!out!during!storage,!success!was!low!
(4%),!while!success!for!vials!that!retained!sterile!water!averaged!90%.!!Most!
importantly!however,!is!the!loss!of!mycoviruses!from!stored!isolates;!only!2!of!119!
stored!mycovirus!infected!isolates!still!contained!mycoviruses!after!storage,!suggesting!
that!the!doubleJstranded!RNA!mycoviruses!are!degraded!during!storage.!!

!

118!

INTRODUCTION
!!
!

A!wellJknown!example!of!longJterm!storage!of!living!organisms!is!the!work!of!

Richard!Lenski!and!colleagues!(Lenski!&!Travisano!1994).!!In!their!work,!aliquots!of!
Escherichia#coli!bacteria!have!been!stored!every!500!generations!for!greater!than!20!
years,!creating!a!frozen,!revivable!equivalent!to!a!fossil!record!(Woods!et!al.!2011;!
Cooper!&!Lenski!2000).!!In!this!way,!baseline,!ancestral!clones!have!been!available!for!
direct!comparison!with!evolved!lines!at!any!point!their!evolutionary!history!(Lenski!et!
al.!1991)!especially!to!determine!average!changes!in!fitness!over!time!(Woods!et!al.!
2011).!!!
Collecting!and!storing!living!isolates!from!diverse!taxa!can!allow!temporal!
changes!in!both!phenotype!or!genotype!to!be!readily!assessed.!!!Storage!of!fungal!
cultures,!for!example,!in!a!viable!and!stable!state!is!important!for!future!studies!that!
relate!to!pathogen!identification,!disease!control,!quarantine,!and!breeding!resistant!
plants!(AbdJElsalam!et!al.!2010)!and!for!detecting!any!changes!in!virulence.!!!
!

Fungal!isolates!are!commonly!stored!for!short!or!long!time!periods!by!subJ

culturing!onto!new!media!filled!petri!plates,!silica!gel,!or!water!suspension!at!5oC!
(Richter!and!Bruhn!1989),!or!the!use!of!organic!substrates!such!as!wood!chips!or!filter!
paper,!or!freezing!(Nakasone!et!al!2004).!!Isolates!were!stored!originally!using!a!
method!developed!by!Jones!et!al.!(1991)!for!use!in!storing!taxonomically!diverse!fungal!
species.!!The!method!is!simple,!economical,!and!does!not!require!freezing!or!
refrigeration.!!It!has!not!been!tested!previously!with!C.!parasitica,!the!pathogen!
!

119!

responsible!for!chestnut!blight!disease!in!American!chestnuts,!Castanea#dentata!
(Marsh)!Borkh..!!Cultures!for!this!study!have!been!in!storage!since!being!collected!from!
six!populations!in!Michigan!between!1996!and!2000!(Davelos!1999;!AL!Davelos,!
unpublished!data).!!!This!study!reports!on!my!attempts!to!revive!these!samples!for!use!
in!a!temporal!study!of!vegetative!compatibility!group!patterns!across!C.#parasitica!
populations!in!Michigan!(Chapter!2).!!
!
MATERIALS!AND!METHODS!
INITITAL!STORAGE!
Storage!in!oneJdram!vials!followed!the!technique!of!Jones!et!al.!(1991).!!!After!growing!
for!about!two!weeks!on!Potato!Dextrose!agar!(PDA,!Difco:!Becton,!Dickinson!and!
Company.!Sparks,!MD).!!two!to!three!plugs!of!C.#parasitica!mycelia!made!using!a!3J
millimeter!cork!borer!or!sterilized!end!of!a!glass!pipette!and!placed!into!15!x!45!
millimeter!1!dram!glass!vials!(Kimble!Glass,!Inc.),!filled!with!approximately!2mL!of!
sterile!water!and!then!sealed!with!parafilm.!!The!isolates!were!originally!collected!from!
six!Michigan!populations!of!C.#parasitica:!Roscommon!(RC),!Stivers!(ST),!County!Line!
(CL),!Frankfort!(FT),!Missaukee!Diseased!(MD)!and!Missaukee!Healthy!(MH).!!All!
populations!are!located!in!the!northern!half!of!Michigan’s!lower!peninsula!(see!Davelos!
and!Jarosz!2004!for!detailed!locations!and!see!Chapter!2!for!a!map).!Numbers!of!
samples!saved!and!year!of!storage!were!variable!for!each!population!(Table!1).!Isolates!
from!three!populations!(RC,!CL,!and!FT)!were!infected!with!doubleJstranded!RNA!
(dsRNA)!mycoviruses,!which!reduce!both!growth!rates!and!conidia!production!of!

!

120!

infected!C.#parasitica!cultures.!!Mycoviruses!were!not!present!in!isolates!from!the!
remaining!three!sites!(ST,!MD,!and!MH).!!
REVIVIFICATION!TESTS!
!

In!2010,!vials!were!inspected!for!water!content!and!filled!with!sterile!water!if!

they!had!dried!out.!!Sterile!water!was!added!to!dry!vials!along!the!vial!shoulder!in!order!
to!attempt!to!rehydrate!spores!or!mycelia.!!All!vials!were!vortexed!to!homogenize!the!
sample!and!allowed!to!rest!for!approximately!one!hour!before!200µL!aliquots!were!
removed!and!spread!onto!PDA!plates!with!a!sterile!glass!hockey!stick.!!Growth!of!C.#
parasitica!or!contaminants!such!as!bacteria!or!other!fungal!species!was!noted!after!
approximately!two!weeks;!successfully!revived!isolates!were!placed!back!into!longJ
term!storage!in!new!vials!using!initial!storage!methods.!!Samples!were!only!tested!once.!!
!
RESULTS!
!

Stored!cultures!were!considered!revived!if!C.!parasitica!grew!from!the!plated!

sample.!!Lack!of!growth!or,!growth!of!bacterial!or!fungal!colonies!other!than!C.#
parasitica!were!noted!as!unsuccessful!or!contaminated,!respectively.!!In!rare!cases,!tiny!
subJsamples!or!hyphal!tips!of!contaminated!C.#parasitica!could!be!taken!from!a!
contaminated!plate!and!successfully!plated!onto!fresh!PDA!media.!!Cryphonectria#
parasitica!was!successfully!revived!from!70.5%!of!the!stored!vials,!but!populations!
differed!in!their!success!rate!ranging!from!a!low!of!33.3%!for!MD!samples!to!84.3%!for!
CL!samples!(Table!1).!!!The!success!rate!for!vials!that!had!dried!out!was!very!low!(3!of!
73!=!4%),!while!89.7%!of!the!samples!with!water!remaining!were!successfully!revived.!
Additionally,!there!was!no!significant!difference!in!the!revivification!success!for!isolates!!

!

121!

!
Table!A*1.!Overview!of!isolates!tested!for!resurrection!success!from!six!Michigan!chestnut!blight!populations.!
Totals!are!whole!numbers,!percentages!or!averages.!

Population!
Roscommon!
Frankfort!
County!Line!
Stivers!
Missaukee!Diseased!
Missaukee!Healthy!
Totals/Averages!

!

Total! wet! dry!
%!
Wet! dry!
%!
tested! alive! alive! alive! dead! dead! dead! Contaminated! %!contaminated!
140!
111!
1! 80.0%!
8!
18! 18.6%!
2!
1.4%!
86!
52!
0! 60.5%!
2!
30! 37.2%!
2!
2.3%!
89!
73!
2! 84.3%!
2!
11! 14.6%!
1!
1.1%!
32!
18!
0! 56.3%!
8!
4!
37.5%!
2!
6.3%!
36!
12!
0! 33.3%! 12!
2!
38.9%!
10!
27.8%!
27!
20!
0! 74.1%!
1!
5!
22.2%!
1!
3.7%!
410!
286!
3! 70.5%! 33!
70! 25.1%!
18!
4.4%!

122!

infected!with!mycoviruses!(71.8%)!and!isolates!not!infected!with!mycovirus!(81.1%).!Most!
importantly!however,!mycoviruses!were!recovered!from!only!two!of!119!stored!cultures!
known!to!be!infected!with!mycoviruses!at!the!time!of!storage.!!Finally,!only!4.4%!of!the!
vials!were!contaminated!with!other!microbes.!!!!
!
DISCUSSION(
Storing!of!biological!samples!for!long!time!periods!is!an!important!aspect!of!scientific!
record!keeping.!!Voucher!samples!of!fungi!should!be!stored!in!a!viable!state!,!so!they!can!be!
used!for!future!use!in!pathogen!identification,!disease!control,!quarantine,!and!breeding!
resistance.!!Work!by!Richter!and!Bruhn!(1989)!has!shown!that!viability!among!fungal!
species,!even!after!a!few!months,!can!vary!from!0!to!100%!for!their!5oC!refrigerated!water!
storage!technique.!!Additionally,!Borman!et!al.!(2006)!have!shown!that!revival!of!fungal!
isolates!averages!90%!for!isolates!stored!since!1983!but!that!a!species!effect!is!seen!
(Hartung!de!Capriles!et!al.!1989).!Techniques,!such!as!mineral!oil!and!silica!gel!were!a!less!
successful!and!freezing!at!T80oC!was!the!worst!!(Pumpyanskaya!1964).!!My!work!reviving!
isolates!of!Cryphonectria-parasitica!indicates!that!there!are!differences!in!revival!success!
even!among!populations.!For!instance,!at!the!Missaukee!Diseased!population,!stored!
isolates!that!remained!wet!were!still!unable!to!be!resurrected!50%!of!the!time.!The!County!
Line!population!however,!had!very!good!resurrection!success:!97%!of!revivable!isolates!
were!in!tubes!that!still!contained!sterile!water.!(
!

Reviving!stored!Cryphonectria-parasitica!samples!was!effective!as!long!as!some!

distilled!water!remained!in!the!glass!vial.!!If!the!water!evaporated!entirely,!allowing!spores!
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and!mycelia!to!desiccate,!reTgrowth!of!the!culture!could!not!be!obtained!even!when!dry!
material!in!the!tubes!was!immersed!in!fresh!sterile.!!Thus,!a!tight!seal!of!the!cap!and!
wrapping!with!parafilm!are!vital!for!water!retention!in!the!vials.!!
!

Most!important!from!this!study,!however,!may!be!the!fact!that!only!two!isolates!out!

of!119!isolates!infected!with!mycoviruses!maintained!their!mycovirus!until!2010.!This!has!
important!implications!for!the!longTterm!storage!of!isolates!that!are!used!for!biological!
control!of!C.-parasitica,!especially!if!isolates!are!stored!from!year!to!year!and!are!revived!to!
continue!treatment!of!blight!cankers!during!subsequent!field!seasons.!!
!

If!presence!of!mycovirus!in!C.-parasitica!mycelia!is!not!important,!periodic!

inspection!of!water!levels!in!glass!vials!must!be!done!to!determine!if!additional!sterile!
water!should!be!added!to!maintain!isolate!viability.!!Alternatively,!if!water!has!begun!to!
evaporate!from!tubes,!cultures!can!be!grown!again!and!stored!immediately!in!a!new!waterT
filled!tube.!!Duplicate!or!triplicate!tubes!of!isolates!should!be!kept!so!that!the!chances!of!
future!resurrection!are!increased,!especially!if!there!are!interTtube!differences.!!!
!

Although!it!is!not!my!intent!to!completely!review!techniques!for!storing!and!

reviving!fungal!cultures,!these!data!on!the!chestnut!blight!fungus,!C.-parasitica,!can!be!
added!to!the!list!of!studies!documenting!sample!storage!methods!and!success!of!reviving!
after!a!relatively!long!time!period.!!This!simple!method!of!storing!C.-parasitica!requires!
minimal!effort,!is!economical,!and!easy!to!maintain!with!no!input!of!electricity!and!
therefore!avoids!the!potential!hazard!of!an!electrical!outage!destroying!isolates.!
!

The!ability!to!effectively!store!and!revive!a!fungal!isolate!is!important!for!

comparative!purposes!such!as!confirming!the!identification!of!a!unknown!quarantine!
isolate,!using!isolates!for!comparative!purposes!in!taxonomic!studies,!determining,!
!
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determining!changes!in!virulence!over!time!(if!the!culture!stored!has!remained!static!
during!storage),!and!simply!good!recordTkeeping!of!biological!samples.!!!
!

At!present,!a!test!of!longTterm!persistence!of!mycoviruses!is!being!done.!Chestnut!

blight!isolates!known!to!contain!mycoviruses!have!been!stored!in!sterile!water!and!PDA!
slants!at!ambient!temperature!and!on!filter!paper!at!T20!degrees!Celsius.!This!longTterm!
experiment!will!test!mycovirus!persistence!over!30!years!tested!at!designated!intervals.!!
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LITERATURE(CITED(
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LITERATURE(CITED(
(
(
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Stephenson!SL,!Cai!L,!and!Hyde!KD.!2010.!Culture!Collections,!the!New!Herbaria!for!Fungal!
Pathogens.!Fungal!Diversity!45:21–32.!!
!
Borman!AM,!Szekely!A,!Campbell!CK,!and!Johnson!EM.!2006.!Evaluation!of!the!Viability!of!
Pathogenic!Filamentous!Fungi!After!Prolonged!Storage!in!Sterile!Water!and!Review!of!
Recent!Published!Studies!on!Storage!Methods.!Mycopathologia!161:361–368.!!
!
Cooper!VS!and!Lenski!RE.!2000.!The!population!genetics!of!ecological!specialization!in!
evolving!E.!coli!populations.!Nature.!407:736T739.!
!
Davelos!AL.!1999.!DoubleTstranded!RNA!mediated!recovery!of!American!chestnut!
populations:!a!demographic!analysis.!PhD!thesis,!Michigan!State!University,!East!Lansing,!
Michigan.!
!
Hartung!de!Capriles!C,!Mata!S,!and!Middelveen!M.!1989.!Preservation!of!fungi!in!water!
(Castellani):!20!years.!Mycopathologia.!106:73—79.!!!
!
Jones!RJ,!Sizmur!KJ,!and!Wildman!HG.!1991.!A!Miniaturised!System!for!Storage!of!Fungal!
Cultures!in!Water.!Mycologist.!5:184–186.!
!
Lenski!RE,!Rose!MR,!Simpson!SC,!and!Tadler!SC.!1991.!LongTterm!Experimental!Evolution!
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Lenski!RE.!and!Travisano!M.!1994.!Dynamics!of!adaptation!and!diversification.!A!10000!
generations!experiment!with!bacterial!populations.!Proceedings!of!the!National!Academy!
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Nakasone!KK,!Peterson!SW,!Jong!SC.!2004.!Preservation!and!distribution!of!fungal!cultures.!
In:!Foster!MS,!Bills!GF,!Mueller!GM!(eds)!Biodiversity!of!fungi:!Inventory!and!monitoring!
methods.!Elsevier!Academic,!USA,!pp!37–47!
Pumpyanskaya!LY!1964.!Storage!of!microorganisms!under!oil.!Mikrobiologiya.!33:!1065–
1070.!
Richter!DL!and!Bruhn!JN.!1989.!Revival!of!saprotrophic!and!mycorrhizal!basidiomycete!
cultures!from!cold!storage!in!sterile!water.!Canadian!Journal!of!Microbiology.!35:1055—
1060.!!
Woods!RJ,!Barrick!JE,!Cooper!TF,!Shrestha!U,!Kauth!MR,!and!Lenski!RE.!2011.!SecondTOrder!
Selection!for!Evolvability!in!a!Large!Escherichia!Coli!Population.!Science!331:!1433–1436.!!
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