APPLICATION
FOR
PERMIT
OR
COURTESY
PERMIT
UNDER
7
CFR
340
 
NAME
AND
ADDRESS
OF
APPLICANT
Dr.
William
L.
MacDonald
Division
of
Plant
and
Soil
Sciences
401
Brooks
Hall,
P.
O.
Box
6058
West
Virginia
University
Morgantown,
WV
26506­
6058
 
PERMIT
REQUEST
Limited­
Interstate
Movement
Limited­
Importation
X
Release
into
the
Environment
Courtesy
Permit
 
THIS
REQUEST
X
New
Renewal
Supplemental
 
Telephone
Number
304­
293­
3911
ext.
2236
 
MEANS
OF
MOVEMENT
Mail
Common
Carrier
X
Baggage
of
Handcarried
(
by
whom:
Dr.
William
MacDonald)

 
GIVE
THE
FOLLOWING
Scientific
Name
Common
Name
Trade
Name
(
1)
Donor
Organism
Cryphonectria
parasitica
CHV1­
Euro7
Chestnut
blight
(
hypovirulent)
(
2)
Recipient
Organism
Cryphonectria
parasitica
Ep
155/
Ep
146
Chestnut
blight
(
virulent)
(
3)
Vector/
Vector
Agent
PLASMID
pXHE7
(
4)
Regulated
Organism
or
Product
Transgenic
hypovirulent
Cryphonectria
parasitica
strains
containing
hypovirus
cDNA
and
expressing
hygromycin
resistance
(
5)
If
product,
list
names
of
constituents
 
QUANTITIY
OF
REGULATED
ARTICLE
TO
BE
INTRODUCED
AND
PROPOSED
SCHEDULE
AND
NUMBER
OF
INTRODUCTIONS
Approximately
4­
5
liters
of
the
genetically
engineered
organism,
mixed
with
water
agar
will
be
distributed
over
the
surface
of
naturally
occurring
or
artificially
established
C.
parasitica
cankers
three
times
per
year
(
June­
August)
for
three
years.

 
DATE
(
or
inclusive
dates
of
period)
OF
IMPORTATION,
INTERSTATE
MOVEMENT,
OR
RELEASE
March
2004
 
August
2006
 
COUNTRY
OR
POINT
OF
ORIGIN
OF
THE
REGULATED
ARTICLE
The
genetically
engineered
inoculum
will
be
prepared
in
Morgantown,
WV
APHIS
PERMIT­
West
Virginia
University,
Dr.
William
MacDonald
2
 
PORT
OF
ARRIVAL
DESTINATION
OR
MOVEMENT,
OR
SPECIFIC
LOCATION
OF
RELEASE
The
specific
release
site
is
in
the
Monongahela
National
Forest
near
Circleville,
WV
(
Pendleton
County)
off
US
Forest
Service
Road
#
48
(
38
º
35'
39"
N,
79
º
34'
16"
W;
elevation
3627
feet).

 
ANY
BIOLOGICAL
MATERIAL
(
e.
g.
culture
medium,
or
host
material)
ACCOMPANYING
THE
REGULATED
ARTICLE
DURING
MOVEMENT
Conidia
of
the
genetically
engineered
(
transgenic)
isolates
will
be
mixed
with
4%
water
agar
to
the
consistency
of
applesauce.

 
APPLICANTS
FOR
A
COURTESY
PERMIT­
STATE
WHY
YOU
BELIEVE
THE
ORGANISM
OR
PRODUCT
DOES
NOT
COME
WITHIN
THE
DEFINITION
OR
A
REGULATED
ARTICLE
 
SEE
REVERSE
SIDE
 
SIGNATURE
OR
REPSONSIBLE
PARTY
 
PRINTED
NAME
AND
TITLE
 
DATE
APHIS
PERMIT­
West
Virginia
University,
Dr.
William
MacDonald
3
APPLICATION
FOR
PERMIT
(
APHIS
FORM
2000)

ENCLOSURE
13a.
NAMES
OF
PERSONS
INVOLVED
IN
THIS
PROJECT
The
transformed
organism
was
developed
by:
Dr.
Donald
L.
Nuss,
Director
Center
for
Biosystems
Research
University
of
Maryland
Biotechnology
Institute
5115
Plant
Sciences
Building
College
Park,
MD
20742
Telephone:
301­
405­
0334
E­
mail:
nuss@
umbi.
umd.
edu
Deployment
tests
will
be
conducted
by:
Dr.
William
L.
MacDonald
Division
of
Plant
and
Soil
Sciences
Department
of
Plant
Pathology
and
Environmental
Microbiology
401
Brooks
Hall,
P.
O.
Box
6058
West
Virginia
University
Morgantown,
WV
26506­
6058
Telephone:
304­
293­
3911
extension
2236
Email:
macd@
mail.
wvu.
edu
APHIS
PERMIT­
West
Virginia
University,
Dr.
William
MacDonald
4
ENCLOSURE
13b.
DESCRIPTION
OF
REGULATED
ARTICLE
A
cDNA
copy
of
CHV1­
Euro7
hypovirus
RNA,
present
in
the
cytoplasm
of
hypovirulent
(
less
virulent)
strains
of
the
chestnut
blight
fungus,
has
been
genetically
engineered
into
the
nucleus
of
the
fungus
to
produce
transgenic
hypovirulent
fungal
strains.

PROBLEM:
Cryphonectria
parasitica
was
responsible
for
the
destruction
of
several
billion
mature
American
chestnut
trees
early
in
the
20th
century
and
remains
a
serious
problem
in
North
America
(
Anagnostakis,
1982;
Griffin,
1986;
MacDonald
and
Fulbright,
1991).
Natural
viruscontaining
hypovirulent
strains
of
C.
parasitica
can
serve
as
biological
control
agents
by
virtue
of
their
ability
to
convert
virulent
strains
to
the
hypovirulent
phenotype.
Introduction
of
the
viral
cDNA
into
the
nucleus
of
virulent
fungal
strains
by
DNA­
mediated
transformation
resulted
in
the
generation
of
engineered
(
transgenic)
hypovirulent
strains.
Since
the
transgenic
hypovirulent
strains
contain
a
chromosomally
integrated
cDNA
copy
of
the
viral
genome
as
well
as
the
normal
cytoplasmically
replicating
viral
RNA
form,
the
hypovirulent
phenotype
can
be
transmitted
to
progeny
of
a
sexual
cross
between
a
transgenic
strain
and
a
sexually
compatible
virulent
strain
(
a
transmission
pathway
not
available
to
natural
hypovirulent
strains).
Thus,
transgenic
hypovirulent
strains
are
predicted
to
be
superior
biological
control
agents
because
of
their
enhanced
dissemination
properties.
We
have
constructed
a
full­
length
cDNA
clone
of
the
dsRNA
of
European
hypovirus,
CHV1­
Euro
7
(
Chen
and
Nuss,
1999).
A
similar
hypovirulenceassociated
viral
RNA
[
CHV1­
EP713],
Choi
and
Nuss,
1992b)
was
field
tested
in
CT
and
WV
between1996­
1999
Anagnostakis
et
al.
1998)..
The
Euro
7
hypovirus
is
less
debilitating
to
the
fungal
host
and
thus,
it
should
be
a
more
effective
biological
control
agent
that
than
the
highly
debilitating
CHV1­
EP713
hypovirus.

BACKGROUND:
Cryphonectria
parasitica
was
introduced
into
North
America
at
the
end
of
the
19th
century
on
imported
Japanese
chestnut
trees,
and
spread
initially
on
nursery
stock
sold
locally
and
shipped
by
mailorder.
Additional
spread
was
effected
by
insects,
gastropods,
birds,
and
animals
that
moved
across
the
bark
cankers,
picking
up
asexual
spores.
In
addition,
winddisseminated
sexual
spores
also
contributed
to
the
epidemic.
The
native
chestnuts,
Castanea
dentata
(
Marsh.)
Borkh.,
and
chinquapins,
Castanea
pumila
(
Linn.)
Mill.
proved
to
be
very
susceptible
to
this
pathogen,
and
massive
loss
of
timber
resulted
(
Anagnostakis,
1982a;
Griffin,
1986;
MacDonald
and
Fulbright,
1991).
The
native
ranges
of
C.
dentata
and
C.
pumila
are
shown
in
Figure
1.
The
fungus
is
a
wound
pathogen,
causing
cankers
that
kill
the
bark
and
cambium
of
American
chestnut
and
chinquapin
distal
to
an
infection.
Sprouts
from
the
root
collar
grow
until
wounded
and
infected,
and
the
cycle
is
repeated.
All
other
species
of
Castanea
are
hosts
of
Cryphonectria
sp.
(
Asian
species,
C.
mollissima,
crenata,
seguini
and
henryi
are
rarely
killed
by
infections).
Other
hosts
with
either
a
parasitic
or
saprophytic
habit
include:
Quercus
alba,
coccinea,
falcata,
ilex,
marcrocarpa,
montana,
muhlenbergi,
petraea,
prinus,
pubecens,
robur
and
rubra,
Eucalyptus
camaldulensis,
haemastoma,
microcorys,
punctata
and
robusta
Acer
palmatum,
pensylvanicum
and
rubrum,
Carpinus
carolinia,
Carya
ovata,
Fagus
sylvatica,
and
Ostrya
virginiana
(
Stipes
et
al.
1978).

Figure
1.
Maps
showing
the
native
ranges
of
the
American
species
of
Castanea.
(
A)
American
chestnut,
Castanea
dentata;
(
B)
Allegheny
chinquapin,
Castanea
pumila,
var.
pumila,
APHIS
PERMIT­
West
Virginia
University,
Dr.
William
MacDonald
5
Natural,
virus­
containing,
virulence­
attenuated
(
hypovirulent)
strains
of
C.
parasitica
can
serve
as
biological
control
agents
by
virtue
of
their
ability
to
convert
virulent
strains
to
the
hypovirulent
phenotype.
French
mycologist,
Jean
Grente,
reported
in
his
1981
thesis
that
chestnut
trees
in
French
orchards
treated
in
1967
were
surviving
well
by
1974,
and
that
spread
of
hypovirulent
strains
spread
was
detected
at
a
rate
of
about
a
meter
per
year.
Such
viruscontaining
strains
have
been
in
use
in
Connecticut
field
tests
since
the
spring
of
1973
when
permission
was
received
from
R.
W.
Beardmore
(
USDA,
APHIS).
A
strain
with
French
hypovirus
CHV1­
EP713
was
sent
to
West
Virginia
for
MacDonald's
use
in
1975
to
initiate
biological
control
studies.
Since
then,
native
hypoviruses
of
C.
parasitica
have
been
discovered
in
West
Virginia.
Field
tests
of
hypovirulent
C.
parasitica
strains
have
been
conducted
at
various
sites
West
Virginia
nearly
continuously
for
the
past
25
years.
Choi
and
Nuss
(
1992a)
developed
a
transgenic
hypovirulent
strain
that
contained
a
cDNA
copy
of
the
RNA
of
the
French
virus,
CHV1­
EP713
(
Choi
and
Nuss,
1992a).
Permission
was
granted
in
1996
(
APHIS,
EPA,
WV
Department
of
Agriculture)
for
field
release
of
two
genetically
engineered
strains
(
containing
CHV1­
EP713),
Ep
146/
pXH9
and
Ep
155/
pXH9
in
Tucker
County,
WV.
These
strains
included
C.
parasitica
strains
Ep146
and
Ep155,
both
containing
a
cDNA
copy
of
hypovirus
CHV1­
Ep713
on
transformation
plasmid
pXH9
and
designated
Ep146/
pXH9
and
Ep155/
pXH9.
Findings
from
the
1996
study
indicated
that
CHV1­
EP713
is
a
very
debilitating
virus
and
disseminated
poorly.
Consequently,
efforts
were
initiated
to
identify
a
less
debilitating
hypovirus
that
could
be
used
to
contruct
a
transgenic
hypovirulent
strain
with
enhanced
properties
of
persistence
in
and
dissemination
through
the
C.
parasitica
population.
CHV1­
Euro7
was
the
identified
hypovirus.
Nuss
and
Chen
(
1999)
constructed
a
full­
length
clone
of
hypovirus
CHV1­
Euor7
RNA.
Introduction
of
the
viral
cDNA
into
virulent
fungal
strains
by
DNA
mediated
transformation
resulted
in
the
generation
of
engineered
hypovirulent
strains
(
transgenic).
Since
the
engineered
by
hpovirulent
strains
contain
a
chromosomally
integrated
cDNA
copy
of
the
viral
genome
as
well
as
the
normal
cytoplasmically
replicating
viral
RNA
form,
the
hypovirulence
phenotype
can
be
transmitted
to
progeny
of
sexual
crosses
between
an
engineered
strain
and
a
sexually
compatible
virulent
strain
(
a
transmission
pathway
not
available
to
natural
cytoplasmic
hypovirulent
strains).
Reversion
(
i.
e.
loss
of
cDNA
information)
of
transgenic
strains
generated
from
virulent
C.
parasitica
strains,
e.
g.,
Ep146
and
Ep155,
would
result
in
virus­
free
strains,
which
already
exist
in
West
Virginia
forests.
The
transgenic
hypovirulent
CHV1­
Euro
7
strains
should
be
superior
biological
control
agents
because
of
their
enhanced
dissemination
properties
and
greater
biological
control
potential,
relative
to
transgenic
CHV1­
Ep713
strains.
The
performance
of
these
strains
in
the
environment
can
be
monitored
by:
(
1)
cultural
screening
of
reisolated
C.
parasitica
strains;
(
2)
testing
for
the
presence
of
the
transformation
plasmid
by
screening
for
hygromycin
resistance;
(
3)
PCR
identification
of
the
inserted
hypovirus
cDNA;
and,
(
4)
identification
of
cDNA­
derived
viral
RNA
by
RealTime
PCR.

Genetically
engineered
(
transgenic)
hypovirulent
strains
of
the
chestnut
blight
fungus,
C.
parasitica
have
been
developed.
These
transgenic
strains
are
likely
to
be
more
effective
biological
control
agents
than
naturally
occurring
hypovirulent
strains
for
the
reasons
indicated
below.
A.
American
chestnut
B.
Allegheny
chinquapin
APHIS
PERMIT­
West
Virginia
University,
Dr.
William
MacDonald
6
HYPOVIRUS
BACKGROUND:
Hypovirulent
strains
of
C.
parasitica,
first
discovered
in
Europe
and
subsequently
in
the
United
States,
can
have
a
curative
effect
when
inoculated
onto
existing
chestnut
cankers.
Provided
that
the
resident
virulent
C.
parasitica
strain
is
of
the
same
or
closely
related
vegetative
compatibility
group
as
the
applied
hypovirulent
strain,
the
two
strains
can
undergo
anastomosis
(
fusion
of
hyphae)
resulting
in
the
transmission
of
the
hypovirus
and
conversion
of
the
resident
virulent
strain
to
the
hypovirulent
phenotype.
We
now
know
with
certainty
that
the
hypovirulence
factor
is
a
cytoplasmically
replicating
virus
found
predominantly
in
a
double­
stranded
(
ds)
RNA
form
(
Choi
and
Nuss,
1992b).
Natural
hypovirulent
C.
parasitica
strains
have
had
a
very
significant
effect
in
reducing
the
severity
of
the
blight
epidemic
in
Italy
and
have
been
used
by
the
French
and
Italian
governments
as
effective
biological
control
agents
(
Robin
and
Heiniger,
2001;
Turchetti,
1982;
Grente
and
Berthelay­
Sauret,
1978).
Natural
hypovirulent
C.
parasitica
strains
also
have
been
commercialized
as
part
of
a
biological
control
service
provided
to
chestnut
growers
by
an
Italian
firm
(
f.
lli.
S.
p.
A.,
Division
Applicazioni
Biologiche
Milano)
to
control
blight
on
European
chestnut
trees.
European
chestnuts
have
recovered
in
many
western
European
countries
because
of
the
natural
spread
of
hypovirulence
(
Turchetti
and
Maresi,
1998).
Hypovirulent
strains
are
reported
to
be
spreading
naturally
in
eastern
Europe
as
well
(
Celiker
and
Onogur,
2001;
Juhásová
and
Bernadovicová,
2001;
Uscuplic
and
Testic,
1998).
With
the
resurgence
of
European
chestnuts,
the
production
of
nuts
has
increased
dramatically
since
1990
(
Bounous,
1999).
With
the
spread
of
hypovirulence,
coupled
with
cultivar
selection
and
cultural
practices,
chestnut
production
has
increased
in
Europe
from
338,000
quintals
(
hundredweight:
1
quintal
=
100
pounds)
in
1983
to
669,000
quintals
in
1997.
These
numbers
still
pale
in
comparison
to
the
8.3
million
quintals
recorded
in
1911
(
Adua,
1998a;
Adua,
1998b;
Buonous,
1999).
The
current
sense
is
that
chestnut
blight
is
no
longer
a
serious
problem
for
European
chestnut;
ink
disease
(
Phythopthora
sp.)
has
supplanted
chestnut
blight
as
the
most
significant
cultural
problem
(
Adua,
1998a).
Reports
of
the
successful
control
of
chestnut
blight
in
Europe
resulting
from
the
natural
dissemination
or
artificial
application
of
natural
hypovirulent
C.
parasitica
strains
stimulated
efforts
to
examine
whether
transmissible
hypovirulence
might
be
effective
in
controlling
blight
in
North
America.
Investigators
at
The
Connecticut
Agricultural
Experiment
Station
confirmed
that
European
hypovirulent
strains
could
cure
cankers
incited
by
North
American
virulent
strains
under
controlled
conditions
(
Van
Alfen
et
al,
1975;
Anagnostakis
and
Jaynes,
1973).
Native
North
American
hypovirulent
strains
were
subsequently
found
in
several
different
geographic
locations
beginning
in
1976
with
the
isolation
of
a
hypovirulent
strain
from
the
bark
of
surviving
American
chestnut
trees
located
in
Michigan
(
Anagnostakis,
1982a;
MacDonald
and
Fulbright,
1991).
Fulbright
and
coworkers
confirmed
the
presence
of
native
hypovirulent
strains
in
various
locations
throughout
the
state
of
Michigan
and
provided
evidence
for
ongoing
biological
control
(
Fulbright
et
al.
1983;
Garrod
et
al.
1985).
However,
initial
attempts
to
control
blight
in
the
eastern
forest
ecosystem
by
the
artificial
introduction
of
hypovirulent
strains
provided
less
encouraging
results
and
have
been
discussed
in
detail
(
Anagnostakis,
1982a;
Griffin,
1986;
Anagnostakis,
1990;
MacDonald
and
Fulbright,
1991).
Although
clear
evidence
for
the
conversion
of
virulent
cankers
was
obtained,
biological
control
by
the
introduced
hypovirulent
strains
was
not
always
sustainable.
There
are
numerous
opinions
as
to
why
biological
control
by
introduced
transmissible
hypovirulence
has
been
less
successful
in
North
America
than
in
Europe
(
Anagnostakis
et
al.
1986).
One
clear
contributing
factor
to
the
efficient
spread
of
introduced
hypovirulent
strains
is
the
vegetative
incompatibility
system
that
controls
the
ability
of
different
C.
parasitica
strains
to
undergo
anastomosis.
Based
on
genetic
analysis,
it
is
estimated
that
7
nuclear
genes
determine
vegetative
compatibility
(
Cortesi
and
Milgroom,
1998;
Huber,
1996).
Two
strains
are
APHIS
PERMIT­
West
Virginia
University,
Dr.
William
MacDonald
7
vegetatively
compatible,
and
thus
freely
able
to
undergo
anastomosis,
if
they
have
the
same
alleles
at
each
of
these
loci.
Viable
anatomosis
between
two
isolates
decreases
as
the
number
of
dissimilar
alleles
increases.
Since
the
natural
hypovirulence­
associated
virus
is
not
transmitted
to
sexual
spores
of
virulent
spores
during
mating
(
Anagnostakis
1982a;
1982b;
1988;
Fulbright
et
al.,
1988),
hypovirulence­
associated
viruses
are
limited
to
the
vegetative
compatibility
groups
of
the
input
hypovirulent
strains.
This
limiting
factor
restricts
the
virus
to
a
very
narrow
number
of
vegetative
compatibility
groups,
thereby
reducing
the
potential
for
virus
movement
and
consequent
biological
control.
Anagnostakis
et
al.
(
1986)
reported
that
vegetative
compatibility
diversity
was
significantly
higher
in
North
American
than
in
European
C.
parasitica
populations,
providing
further
evidence
that
virus
introduction
into
numerous
vegetative
compatibility
groups
is
essential
for
the
success
of
biological
control.
Choi
and
Nuss
(
1992b)
reported
the
development
of
a
full­
length
infectious
cDNA
clone
of
the
hypovirulence­
associated
viral
RNA
present
in
hypovirulent
C.
parasitica
strain
713
(
family
Hypoviridae,
genus
Hypovirus,
species
CHV1­
EP713L).
Transformation
of
virulent
C.
parasitica
strains
with
this
full­
length
cDNA
clone
conferred
the
complete
hypovirulent
phenotype.
Cytoplasmic
dsRNA
was
resurrected
from
the
chromosomally
integrated
cDNA
copy
and
was
able
to
convert
compatible
virulent
strains
to
hypovirulent.
These
results
established
that
viral
dsRNA
is
indeed
the
causal
agent
of
hypovirulence
and
demonstrated
the
feasibility
of
engineering
hypovirulent
fungal
strains.
Since
the
transgenic
hypovirulent
C.
parasitica
strains
contain
a
chromosomally
integrated
viral
cDNA
copy,
it
was
predicted
that,
unlike
the
situation
with
natural
hypovirulent
strains,
viral
genetic
information
would
be
inherited
by
the
progeny
of
a
sexual
cross
between
an
engineered
hypovirulent
strain
and
a
sexually
compatible
virulent
strain.
This
prediction
has
been
confirmed
in
field
tests
(
1997­
1999)
conducted
in
West
Virginia
(
unpublished
data)
and
Connecticut
with
Ep155/
pXH9,
and
the
ascospore
progeny
were
shown
to
contain
the
integrated
viral
cDNA
copy
as
well
as
a
resurrected
cytoplasmically
replicating
ds
form
of
the
viral
RNA
(
Anagnostakis
et
al.
1998).
Unlike
the
vegetative
incompatibility
system
in
C.
parasitica,
sexual
compatibility
is
controlled
by
a
single
mating
type
locus
involving
two
alleles
designated
MAT­
1
and
MAT­
2
(
Anagnostakis,
1988)
and
would
be
expected
to
pose
few
barriers
to
the
spread
of
the
integrated
viral
cDNA
copy
through
a
virulent
fungal
population
by
sexual
crossing.
Due
to
allelic
rearrangement,
the
majority
of
progeny
of
a
sexual
cross
are
of
different
vegetative
compatibility
groups
than
the
parental
strains.
Consequently,
resurrection
of
the
cytoplasmic
viral
RNA
form
in
the
progeny
representing
new
vegetative
compatibility
groups
should
then
result
in
expanded
vegetative
dissemination
of
the
viral
genetic
information.
Stable
transmission
of
a
virus
at
a
high
frequency
(>
99%)
to
asexual
spores
of
engineered
hypovirulent
strains
also
has
been
demonstrated
in
the
laboratory
(
Chen,
Choi
and
Nuss,
1993).
These
combined
properties
should
result
in
significantly
increased
dissemination
and
sustainability
of
the
hypovirulence
phenotype
after
field
introduction
of
a
transgenic
hypovirulent
C.
parasitica
strain.
The
trangenic
hypovirulent
C.
parasitica
strains
containing
the
nuclear
hypovirus
cDNA
copy
appear
identical
to
the
corresponding
hypovirus
transfected
hypovirulent
strains
in
terms
of
morphology,
growth
characteristics,
production
of
the
extracellular
enzyme
laccase,
ability
to
sporulate
and
reduced
virulence
to
chestnut
(
Choi
and
Nuss,
1992;
Chen
and
Nuss,
1999
and
unpublished
data).
In
addition,
the
viral
dsRNA
present
in
the
engineered
hypovirulent
strain
gave
the
same
pattern
as
hypovirus
RNA
when
analyzed
by
terminal
labeling
and
partial
ribonuclease
T1
digestion
(
unpublished
data)
indicating
that
the
viral
dsRNA
that
is
resurrected
from
the
integrated
cDNA
copy
does
not
contain
any
foreign
(
non­
natural)
nucleotides
(
Chen
et
al.
1994).
The
engineered
hypovirulent
fungal
strains
do,
however,
contain
the
gene
for
E.
coli
APHIS
PERMIT­
West
Virginia
University,
Dr.
William
MacDonald
8
hygromycin
phosphotransferase
that
confers
resistance
to
the
antibiotic
hygromycin
B
as
part
of
the
integrated
transformation
plasmid
vector.
APHIS
PERMIT­
West
Virginia
University,
Dr.
William
MacDonald
9
13c.
DESCRIPTION
OF
MOLECULAR
BIOLOGY
Comparison
of
CHV1­
Euro7
and
CHV1­
EP713
hypoviruses.

Cryphonectria
parasitica
strains
infected
with
hypovirus
CHV1­
EP713
form
small
superficial
cankers
on
American
chestnut
trees
that
are
generally
devoid
of
spore­
bearing
stromal
pustules.
In
contrast,
infection
of
chestnut
tissue
by
C.
parasitica
strains
infected
with
hypovirus
CHV1­
Euro7
is
characterized
by
aggressive
canker
formation
early
after
inoculation
that
eventually
slows
or
ceases,
concomitant
with
heavy
callus
formation
at
the
canker
margins.
Moreover,
these
cankers
are
covered
by
stromal
pustules
containing
viable
asexual
spores
(
Chen
and
Nuss,
1999).
MacDonald
and
Fulbright
(
1991)
have
discussed
the
view
that
successful
hypovirulencemediated
biological
control
is
likely
to
require
a
balance
between
ecological
fitness
and
virulence
attenuation.
In
order
to
persist
and
spread,
a
hypovirulent
isolate
must
be
able
to
effectively
colonize
and
produce
spores
on
chestnut
bark.
CHV1­
Euro7­
infected
C.
parasitica
strains
differ
from
strains
infected
with
the
more
severe
hypovirus
CHV1­
EP713
in
precisely
those
properties.
The
ability
to
construct
transgenic
hypovirulent
C.
parasitica
strains
containing
nuclear
copies
of
the
CHV1­
Euro7
infectious
cDNA
provides
the
opportunity
to
combine
properties
of
enhanced
colonization
and
spore
production
with
a
novel
mode
of
virus
transmission
to
ascospore
progeny.
The
CHV1­
Euro7
coding
strand
RNA
is
11
nucleotide
residues
shorter
that
that
of
CHV1­
EP713:
12,701
nucleotides
(
excluding
the
poly(
A)
tail)
versus
12,712
nucleotides
(
Chen
and
Nuss,
1999)
(
Figure
2).
The
difference
relative
to
the
CHV1­
EP713
sequence
include
two
single
nucleotide
deletions
and
one
nucleotide
insertion
within
the
5'
noncoding
region,
the
deletion
within
ORF
B
of
one
codon
that
corresponds
to
CHV1­
EP713
leucine
residue
1400
(
nucleotides
6561­
6563),
and
seven
nucleotide
deletions
within
the
3'
noncoding
region
upstream
from
the
poly(
A)
tail.
Four
of
the
differences
within
the
3'
noncoding
region
occur
adjacent
to
the
poly(
A)
tail:
5'­
GAACAACAAG­
poly(
A)
for
CHV1­
EP713
versus
5'­
GAACAAC­
poly(
A)
for
CHV1­
Euro7.
Thus,
this
four­
base
difference
could
result
from
a
simple
G­
to­
A
transition
at
CHV1­
EP713
map
position
12712.
CHV1­
Euro7
and
CHV1­
EP713
are
very
similar
in
nucleotide
and
amino
acid
sequence
and
have
been
classified
as
isolates
within
the
same
species
(
Chen
and
Nuss,
1999).
Identity
at
nucleotide
level
ranges
from
a
low
of
87%
for
the
helicase
region
to
a
high
of
93%
for
the
noncoding
terminal
regions.
The
identity
at
the
predicted
amino
acid
level
is
90%
for
p40
and
98%
for
the
region
between
the
polymerase
and
helicase
coding
domains
(
Figure
3).
APHIS
PERMIT­
West
Virginia
University,
Dr.
William
MacDonald
10
Figure
2.
The
genetic
organization
of
the
coding
or
sense
strand
of
CHV1­
Euro7
dsRNA
includes
the
495
nt
non­
coding
leader
sequence,
the
1869
nt
ORF
A,
the
5'­
UAAUG­
3'
pentanucleotide
ORF
A/
ORF
B
junction,
ORF
B
consisting
of
9494
nt,
the
844
no
non­
coding
3'­
terminal
domain
and
the
3'­
poly(
A)
tail.

A
B
5'
3'
A
n
RIG
/
NRL
p69
p29
p40
AUTOCATALYTIC
p48
LVG
/
AEE
TAA
ATG
POLYMERASE
MOTIF
HELICASE
MOTIF
494
nt
1869
nt
9494
nt
844
nt
CHV1­
Euro7
APHIS
PERMIT­
West
Virginia
University,
Dr.
William
MacDonald
11
Figure
3.
Comparison
of
the
CHV1­
Euro7
cDNA
sequence
information
with
that
of
a
previously
reported
full­
length
hypovirus
cDNA
sequence:
CHV1­
EP713
(
EP155
from
CT
with
a
hypovirus
from
France).
(
A)
Similarities
at
the
nucleotide
levels.
Previously
identified
protein
coding
regions
are
noted
within
the
open
boxes
representing
the
viral
genome.
The
lengths
in
nucleotides
for
the
5'
and
3'
noncoding
(
nc)
regions
and
ORFs
A
and
B
for
CHV1­
EP713
are
indicated
at
the
top.
The
numbers
of
nucleotides
for
comparable
regions
of
the
other
hypovirus
were
494,
1,869,
9,494,
and
844,
respectively
for
CHV1­
Euro7.
The
percent
nucleotide
identity
for
different
coding
and
noncoding
regions
is
indicated
between
the
different
viral
genome
diagrams
being
compared.
(
B)
Similar
information
at
the
deduced
amino
acid
levels.

Comparison
of
transformation
plasmids
pXHE7
and
pXH9.
Construction
of
an
infectious
full­
length
CHV1­
Euro7
cDNA.
The
general
protocol
previously
used
for
construction
of
a
full­
length
infectious
cDNA
clone
of
CHV1­
EP713
dsRNA
(
pLDST)
(
Choi
and
Nuss,
1992b;
Chen
et
al.,
1994)
was
adapted
for
construction
of
a
CHV1­
Euro7
infectious
cDNA
clone
(
Chen
and
Nuss,
1999).
Early
in
the
construction
process,
cDNA
clones
of
the
terminal
domains
were
modified
through
the
use
of
PCR
to
incorporate
a
unique
Not
I
site
followed
by
a
T7
polymerase
promoter
fused
to
the
5'­
terminus
of
the
CHV1­
Euro7
coding
strand
and
the
addition
of
a
unique
Spe
I
site
following
the
CHV1­
Euro7
3'­
terminal
poly(
A).
Several
large
intermediate
clones
were
generated
from
overlapping
partial
cDNA
clones
utilizing
common
endonuclease
restriction
sites
contained
within
neighboring
clones.
The
full­
length
cDNA
was
obtained
by
ligating
two
terminally
modified
large
cDNA
clones
that
spanned
CHV1­
Euro7
map
positions
1­
5389
and
5220­
to
the
3'­
terminus
at
a
common
Nar
I
site
(
map
position
5310)
and
cloning
into
plasmid
vector
pCRScript
SK(+)
(
Stratagene)
to
form
plasmid
pTE7.
Transcripts
corresponding
to
the
CHV1­
Euro7
coding
strand
were
synthesized
from
Spe
I­
digested
pTE7
in
a
T7
polymerase
reaction
and
used
to
transfect
C.
parasitica
spheroplasts
as
described
by
Chen
et
al.
(
1994).
The
full­
length
CHV1­
Euro7
cDNA
in
pTE7
also
was
used
to
construct
the
transformation
plasmid
pXHE7
used
to
make
the
transgenic
CHV1­
Euro7
C.
parasitica
strains
as
indicated
below.
APHIS
PERMIT­
West
Virginia
University,
Dr.
William
MacDonald
12
Construction
of
transformation
plasmid
pXHE7.
Plasmid
pXHE7
(
Figure
4)
differs
from
the
transformation
plasmids
pXH9
and
pXH103
described
in
the
previous
permit
request
only
in
the
nature
of
the
hypovirus
cDNA
portion
of
the
plasmid;
it
contains
the
CHV1­
Euro7
cDNA
instead
of
the
wild­
type
(
pXH9)
or
tagged
(
pXH103)
CHV1­
EP713
cDNA.
Plasmid
pXHE7
was
constructed
by
liberating
the
CHV1­
Euro7
cDNA
from
plasmid
pTE7
(
see
below)
by
Not
I/
Spe
I
digestion
and
ligating
that
fragment
(
the
CHV1­
Euro7
cDNA)
into
the
base
vector
pCPXHY1
that
had
been
modified
at
the
multiple
cloning
site
to
contain
a
unique
Not
I
site.
The
base
vector
pCPXHY1
also
was
described
in
detail
in
the
previous
request.

Figure
4.
Description
of
tranformation
plasmid
used
to
engineer
hypovirulent
C.
parasitica
strains.
Plasmid
pXHE7
was
used
to
introduce
a
full­
length
copy
of
CHV1­
Euro7
L­
dsRNA
into
virulent
C.
parasitica
strains
by
DNA­
mediated
transformation
(
Choi
and
Nuss,
1992b).
The
plasmid
contains
the
entire
L­
dsRNA
sequence
fused
upstream
to
the
C.
parasitica
glyceraldehyde­
3­
phosphate
gene
(
gpd­
1)
promoter
and
fused
downstream
to
the
gpd­
1
terminator.
Plasmid
pXHE7
also
contains
the
Escherichia
coli
hygromycin
B
phosphotransferase
gene
as
a
selectable
marker
flanked
by
the
Aspergillus
nidulans
trpC
promoter
and
terminator
domains.
APHIS
PERMIT­
West
Virginia
University,
Dr.
William
MacDonald
13
13d.
LOCALITY
OF
DONOR
AND
RECIPIENT
ORGANISMS
AND
VECTOR
STRAINS
USED
Recipient
Strains
strain
155
ATCC
#
38755
isolated
in
1977
from
a
natural
canker
on
an
American
chestnut
tree
on
Sperry
Road
in
Bethany,
Connecticut
vegetative
compatibility
type
40
Mating
type
allele,
MAT­
2
strain
146
ATCC
#
64671
isolated
in
1977
as
isolate
5­
9­
1
in
the
George
Washington
National
Forest
(
Forest
Service
access
road
#
61),
near
Franklin,
West
Virginia
vegetative
compatibility
type
9
Mating
type
allele,
MAT­
1
Donor
Strain
strain
Euro­
7*
ATCC
#
66021
This
strain
is
the
source
of
CHV1­
Euro7
hypovirus
RNA
introduced
in
the
transgenic
and
transfected
strains.
It
was
isolated
in
1978
from
a
natural,
superficial
infection
on
a
European
chestnut
tree
approximately
30
km
north
of
Florence,
Italy.

Wild­
type
Strains
from
Pendleton
County,
WV
(
These
strains
will
be
used
to
initiate
virulent
infections
at
the
onset
of
the
experiment
if
the
number
of
wild­
type
infections
is
not
sufficient
to
promulgate
sexual
spores).
PC17
Virus­
free,
vegetative
compatibility
type
29,
mating
type
allele,
MAT­
1
PC5
Virus­
free,
vegetative
compatibility
type
24,
mating
type
allele,
MAT­
2
PC23
Virus­
free,
vegetative
compatibility
type
49,
mating
type
allele,
MAT­
1
PC48
Virus­
free,
vegetative
compatibility
type
31,
mating
type
allele,
MAT­
2
PC37
Virus­
free,
vegetative
compatibility
type
2,
mating
type
allele,
MAT­
1
PC53
Virus­
free,
vegetative
compatibility
type
26,
mating
type
allele,
MAT­
2
*
C.
parasitica
strain
Euro­
7
is
in
the
Family
Hypoviridae,
Genus
Hypovirus,
Species
Cryphonectria
Hypovirus
1
as
defined
by
Bradley
I.
Hillman,
Dennis
W.
Fulbright,
Donald
L.
Nuss
and
Neal
K.
Van
Alfen
at
the
summer,
1993
International
Virology
Congress
(
Hillman,
et
al.
1995).
APHIS
PERMIT­
West
Virginia
University,
Dr.
William
MacDonald
14
ENCLOSURE
13e.
DESCRIPTION
OF
THE
PURPOSE
OF
THE
INTRODUCTION
AND
FIELD
DESIGN
PURPOSE:
The
objective
of
this
field
trial
is
to
test
the
ability
of
the
transgenic
isolates
to
effectively
disseminate,
become
established
and
initiate
biological
control
of
chestnut
blight.

FIELD
SITE
LOCATION:
American
chestnut
primarily
exists
as
an
understory
shrub
on
sites
where
it
once
grew.
When
the
overstory
is
removed
or
thinned
by
cutting,
insect
defoliation
or
severe
weather
events,
the
sprouts
can
be
released.
Sprouts
generally
grow
vigorously
for
7­
10
years,
reaching
a
diameter
of
8­
12
cm;
this
rapid
growth
is
followed
by
the
blight
epidemic
which
results
in
sprout
death.
A
suitable
field
site
has
been
identified
in
the
Monongahela
National
Forest
in
Pendleton
County,
West
Virginia
(
Figure
5).
The
site
is
accessible
from
USDA­
Forest
Service
Access
Road
#
48
with
coordinates
(
38
º
35'
39"
N,
79
º
34'
16"
W;
elevation
3627
feet;
UTM
coordinates:
17S0624317,
UTM4272289).
The
area
has
been
cut­
over
but
a
partial
canopy
remains
along
with
a
significant
number
of
American
chestnut
sprouts
and
low­
to­
moderate
levels
of
chestnut
blight.
The
site,
beyond
a
locked
USFS
gate,
is
in
a
nonpopulated
area
of
the
county
thus
reducing
the
chance
of
human
intervention
(
Figure
6).

Figure
5.
Map
of
proposed
release
site
of
transgenic
C.
parasitica.
The
location
is
off
USFS
Road
#
48
(
Snowy
Mountain
Quadrangle).
Arrow
indicates
site
location.

Figure
6.
Detailed
map
of
field
plots
in
relation
to
WV
State
Route
28.
To
WV
State
Route
33
APHIS
PERMIT­
West
Virginia
University,
Dr.
William
MacDonald
15
WV
State
Route
28
USFS
Access
gate
To
Durbin
USFS
Road
#
48
Log
Road
Field
Site
FIELD
SITE
SUPERVISOR:
Dr.
William
MacDonald,
Professor,
304­
293­
3911
ext.
2236.

DEPLOYMENT
OBJECTIVE:
To
evaluate
the
ability
of
transgenic
strains
(
Ep146/
pXHE7
and
Ep
155/
pXHE7)
of
Cryphonectria
parasitica
to
become
established,
interact
with
virulent
strains,
disseminate
and
initiate
biological
control
in
a
small
forest
opening.

EXPECTED
STARTING
DATE:
May
15,
2004
FIELD
DESIGN:
Split­
plot
design
with
three
treatments
as
main
plots
and
three
replications.

TREATMENTS:
(
1)
Transgenic
 
CHV1­
Euro7
(
Ep146/
pXHE7
and
Ep155/
pXHE7)
(
2)
Non­
transgenic
 
CHV1­
Euro
7
(
Ep146/
cytoplasmic
and
Ep155/
cytoplasmic)
(
3)
Virulent
 
Ep146
and
Ep155
(
virus­
free)

GENERAL
PLAN
FOR
FIELD
SITE:
Transgenic
C.
parasitica
strains
containing
CHV1­
Euro
7
cDNA
(
Ep146/
pXHE7
and
Ep155/
pXHE7)
will
be
established
in
a
forest
setting
to
determine
their
potential
for
dissemination.
The
performance
of
transgenic
CHV1­
Euro7
stains
will
be
monitored
by
determining
the
frequency
of
recovery
of
hypovirulent
strains
from
newly
formed
and
established
cankers,
in
addition
to
the
production
of
hypovirulent
spores.
Similar
measurements
will
be
performed
in
plots
treated
with
the
corresponding
non­
transgenic
hypovirulent
strains
containing
only
the
cytoplasmicly
replicating
CHV1­
Euro7
RNA
to
test
for
advantages
that
the
transgenic
hypovirulent
strains
provide
to
dissemination.
The
non­
transgenic
CHV1­
Euro7­
infected
strains
used
in
this
study
were
generated
by
transfection
with
infectious
CHV1­
Euro7
synthetic
transcripts
derived
from
the
same
CHV1­
Euro7
cDNA
sequence
(
in
transcription
plasmid
pTE7)
used
to
construct
the
transformation
plasmid
pXH7
as
described
by
Chen
and
Nuss,
1999.
Thus,
the
hypovirus
RNA
in
the
non­
transgenic
hypovirulent
strains
designated
EP155/
tpTE7
and
EP146/
tpTE7
and
in
the
transgenic
hypovirulent
strains
EP155/
pXHE7
and
EP146/
pXHE7
are
derived
from
an
identical
primary
nucleotide
sequence.
Dissemination
of
hypovirulence
is
expected
in
plots
treated
with
transgenic
and
non­
transgenic
cytoplasmic
CHV1­
strains;
however,
only
the
plots
treated
with
transgenic
CHV1­
Euro7
strains
should
produce
hypovirulent
sexual
spores
(
ascospores).
The
control
plots
treated
with
virulent
C.
parasitica
are
expected
to
yield
no
hypovirulent
conidia
or
ascospores.

FUNGAL
STRAINS:
In
the
event
that
an
insufficient
number
of
virulent
infections
are
present
at
the
onset
of
the
study,
six
virus­
free,
virulent
C.
parasitica
strains
(
see
page
13),
will
be
used
APHIS
PERMIT­
West
Virginia
University,
Dr.
William
MacDonald
16
to
initiate
cankers.
These
strains
were
among
sixty
collected
in
December
2003
from
the
Pendleton.
These
strains
were
assayed
for
vegetative
compatibility
and
mating
type.
Two
transgenic
isolates
(
Ep146/
pXHE7
and
Ep155/
pXHE7)
will
be
used
to
spermatize
virulent
cankers.
Isolates
Ep
146
and
Ep
155
(
ATCC
38755)
were
used
as
recipients
for
transformation
with
plasmid
pXHE7,
which
contains
a
full­
length
cDNA
copy
of
hypovirus
CHV1­
Euro
7
RNA,
to
produce
transgenic
hypovirulent
strains.
In
addition
to
the
CHV1­
Euro
7
full­
length
cDNA,
plasmid
pXHE7
also
contains
the
Escherichia
coli
hygromycin
B
phosphotransferase
gene
as
a
selectable
marker
flanked
by
the
Aspergillus
nidulans
trpC
promoter
and
terminator
domains;
this
confers
resistance
to
hygromycin
(
HygR).
Strains
Ep146
and
Ep155
transfected
with
infectious
CHV1­
Euro7
synthetic
transcripts
and
desingated
Ep146/
tpTE7
and
Ep155/
tpTE7
will
be
in
a
second
set
of
replicate
plots.
Virus­
free
isolates
Ep
146
and
Ep
155
will
be
deployed
in
a
third
set
of
replicate
plots.

DEPLOYMENT
PROCEDURES:
A
series
of
nine
plots
will
be
established
in
the
cut­
over
area.
Each
of
three
treatments
will
be
evaluated
in
three
replicate
plots:
1)
the
transgenic
inoculum
[
Ep146/
pXHE7
and
Ep155/
pXHE7];
2)
non­
transgenic
cytoplasmic
inoculum
[
Ep146/
tpTE7
and
Ep155/
tpTE7];
and,
3)
virus­
free
virulent
strains
(
Ep146
and
Ep155).
All
living
trees
in
each
plot
that
are
4
cm
in
diameter
or
larger
will
be
numbered
and
mapped
for
future
reference.
Their
diameters
and
disease
status
also
will
be
recorded.
A
plot
will
contain
20­
30
stems
that
meet
the
above
criteria
and
will
be
physically
separated
from
an
adjacent
plot
by
at
least
15m.
a)
Treatment
1
(
transgenic
inoculum).
Three
of
the
replicate
plots
will
receive
transgenic
inoculum
that
will
be
deployed
via
two
application
techniques.
The
first
is
to
apply
a
slurry
mixture
of
the
two
transgenic
isolates
to
virulent
cankers
that
exist
at
the
time
of
the
initial
release
and
to
new
cankers
that
develop
during
June,
July
and
August,
a
period
of
high
sexual
receptivity.
If
any
artificially
inoculated
virulent
infections
are
established,
they
will
be
treated
with
the
slurry
inoculum.
The
application
of
the
strain
mixture
is
intended
to
spermatize
developing
wild
type
cankers,
regardless
of
mating
type
(
MAT­
1
or
MAT­
2),
to
promote
outcrossing
of
the
canker­
inciting
strains,
thereby
resulting
in
the
production
of
sexual
spores
(
ascospores)
that
are
hypovirus
laden.
The
hypovirus
mixture
will
be
prepared
by
growing
each
transgenic
isolate
on
PDA
under
high
light
conditions
for
10­
14
days.
Conidia
will
be
liberated
from
each
plate
using
0.1%
peptone,
collected
and
adjusted
to
107
conidia/
ml.
Conidia
from
each
transgenic
strain
will
be
mixed
and
then
thickened
with
4%
water
agar
to
the
consistency
of
applesauce.
The
transgenic
slurry
will
be
transported
to
the
field
in
sterile,
1.8L
plastic
containers.
Two­
thirds
of
the
trees
in
each
plot
will
be
selected
for
the
spermatizing
straininoculation
procedure.
One­
third
of
the
trees
will
not
receive
any
inoculum
and
serve
as
`
trap'
trees
to
detect
dissemination
of
the
introduced
strains.
A
second
introduction
method
will
be
employed
that
is
intended
to
initiate
hypovirulent
cankers.
To
accomplish
this,
areas
of
healthy
bark
(
5
X
7
cm)
will
be
scratch­
wounded
and
inoculated
with
a
slurry
of
inoculum
containing
either
the
Ep146/
pXHE7
or
Ep155/
pXHE7
inoculum
source.
Slurry
preparation
will
proceed
as
above
but
the
two
isolates
will
not
be
mixed.
The
scratch
wounds
will
be
covered
with
an
absorbent
underpad
(
Fisher
Scientific)
for
a
3­
week
period
to
promote
the
establishment
of
the
treatment
inoculum.
The
artificially
established
cankers
will
be
inititated
(
2/
stem)
on
the
same
set
of
trees
that
receive
the
spermatization
treatment
(
described
above).
The
intent
of
the
scratch
wound
inoculations
is
to
create
artificial
hypovirulent
cankers
that
will
produce
asexual
hypovirulent
inoculum
(
conidia
and
mycelial
fragments)
that
can
serve
to
spermatize
cankers
or
initiate
hypovirulent
infections.
APHIS
PERMIT­
West
Virginia
University,
Dr.
William
MacDonald
17
b)
Treatment
2
(
cytoplasmic
inoculum).
A
second
series
of
three
replicate
plots
will
be
established
and
the
trees
treated
and
the
inoculum
introduced
identically
to
procedures
described
in
Treatment
1.
Isolates
Ep146/
tpTE7
and
Ep155/
tpTE7
will
carry
only
the
cytoplasmically
replicating
CHV1­
Euro7
RNA
intoduced
by
transfection.
These
plots
will
serve
to
compare
the
biological
control
efficacy
of
the
transgenic
strains
to
their
transfected
hypovirulent
counterparts.
The
treatment
scheme
used
in
Treatment
1
(
two­
thirds
inoculuated
and
one­
third
noninoculated)
also
will
be
used
in
these
plots.
c)
Treatment
3
(
virulent
inoculum).
The
third
set
of
replicate
plots
will
be
established
identically
to
Treatments
1
and
2.
The
introduced
inoculum
source,
however,
will
consist
of
Ep146
and
Ep155
virulent
strains
that
are
hypovirus
free.
The
treatment
scheme
used
in
Treatment
1
(
two­
thirds
inoculuated
and
one­
third
noninoculated)
also
will
be
used
in
these
plots.

EVALUATION
AND
SAMPLING
a)
Canker
and
tree
health
evaluation.
All
trees
will
be
scored
as
healthy
or
infected
at
the
initiation
of
the
experiment.
Cankers
will
be
rated
on
a
scale
of
1­
4
to
represent
the
range
of
infection
states,
from
lethal/
girdling
infections
with
abundant
stromata
to
those
that
possess
heavy
callus
with
little
evidence
of
sporulation.
Each
November,
subsequent
evaluations
will
be
made
noting
whether
trees
are
alive
or
dead
and
the
status
(
rating)
of
each
canker.
A
number
of
photos
from
representative
trees
and
cankers
will
be
taken
to
establish
a
photo
log
of
each
treatment
type.
b)
Pre­
treatment
canker
sampling.
At
the
initiation
of
the
experiment,
all
pre­
existing
cankers
will
be
sampled.
This
will
be
accomplished
by
removing
2­
4
two­
mm
bark
plugs
from
each
canker.
The
plugs
will
be
surface
sterilized
and
cultured;
the
resulting
isolates
will
be
transferred
to
agar
slants
to
establish
a
reference
collection
of
virulent
strains
representative
of
those
at
the
site
at
the
time
the
experiment
is
initiated.
c)
Post­
treatment
sampling.
After
the
experiment
is
initiated,
new
cankers
that
arise
on
numbered
stems
will
be
sampled
at
4­
6
week
intervals
when
the
plots
are
visited
from
June­
November.
Sampling
will
be
conducted
by
removing
4­
6
two­
mm
bark
plugs
from
each
infection.
The
resulting
C.
parasitica
colonies
obtained
from
culturing
the
bark
plugs
will
be
evaluated
for
their
hypovirus
infection
status
(
see
culture
analysis
of
bark
samples,
next
page).
Additionally,
a
representative
number
of
scratch­
wounded
areas
will
be
sampled
to
verify
if
the
treatment
inoculum
became
established.
.
d)
Determining
inoculum
production.
Each
fall
for
three
consecutive
years,
7­
mm
bark
disks
will
be
removed
from
representative
cankers
in
each
treatment
plot.
To
assess
transgenic
inoculum
production,
pycnidia
and
perithecia
will
be
removed
individually
from
the
stromata
embedded
in
the
bark
disks;
liberated
conidia
and
ascospores
will
be
serially
diluted
to
obtain
individual
spores.
The
colonies
resulting
from
the
asexual/
sexual
spores
will
be
evaluated
for
virulent
or
hypovirulent
phenotypes.
All
ascospore
colonies
will
be
grown
on
PDA
amended
with
hygromycin
for
screening,
as
described
above.

DATA
ANALYSIS
a)
Canker
and
tree
health
analysis.
Tree
health
data,
collected
initially
and
each
fall,
will
be
compared
to
establish
the
rate
of
disease
increase
in
each
set
of
replicate
plots.
Of
particular
interest
is
whether
tree
survival
and
the
morphology
of
cankers
improve
over
the
threeyear
duration
of
the
experiment
and
if
the
data
differs
significantly
among
the
transgenic,
cytoplasmic
and
virulent
release
plots.
APHIS
PERMIT­
West
Virginia
University,
Dr.
William
MacDonald
18
b)
Cultural
analysis
of
bark
samples.
The
collection
and
culture
of
bark
samples
from
cankers
on
treated
and
nontreated
trees
will
provide
evidence
of
hypovirus
establishment
in
the
thallus
of
either
canker
type.
Transgenic
CHV1­
Euro7
input
strains
are
lightly
pigmented
due
to
the
suppression
of
orange
pigment
production
by
the
CHV1­
Euro7
hypovirus.
They
also
are
hygromycin
resistant
(
HygR)
due
to
the
presence
of
the
hygromycin
resistance
gene
encoded
on
the
transformation
vector
pXHE7
adjacent
to
the
CHV1­
Euro7
cDNA.
Thus,
highly
pigmented
isolates
recovered
from
bark
plugs
are
expected
to
behypovirus
free.
In
contrast,
white
isolates
are
likely
to
be
infected
with
CHV1­
Euro7.
These
white
CHV1­
Euro7
 
containing
isolates
can
be
further
distinguished
by
incubation
on
PDA
amended
with
hygromycin
(
40
µ
g/
ml).
Resistance
to
hygromycin
(
HygR)
indicates
the
presence
of
the
integrated
pXHE7
vector
and
CHV1­
Euro7cDNA.
Thus,
any
(
HygR)
isolates
are
either
derived
directly
from
input
transgenic
conidia
or
from
transgenic
ascospore
progeny
that
result
from
spermizitation
by
input
transgenic
conidia.
The
latter
will
be
of
the
same
vegetative
compatibility
group
as
the
input
transgenic
strains
while
the
former
will
be
of
a
different
vegetative
compatibility
group
due
to
allelic
rearrangement
at
the
vegetative
compatibility
loci
following
mating.
White
isolates
that
acquire
CHV1­
Euro7
hypovirus
by
anastomosis
from
transgenic
input
strains
or
germinated
transgenic
ascospore
progeny
will
be
sensitivity
to
hygromycin
(
HygS).
c)
Analysis
of
inoculum
production.
Evaluation
of
conidia
and
ascospore
inoculum
production
will
be
possible
each
fall
by
determining
the
phenotype
of
the
single
spore
colonies
that
arise
from
spores
collected
from
pycnidia
and
perithecia
obtained
from
bark
disks
(
see
section
b
above).
Comparisons
of
inoculum
generated
by
treated
cankers
to
that
produced
on
nontreated
trap
trees
is
of
paramount
interest,
as
these
data
can
be
used
to
provide
a
measure
of
treatment
efficacy
and
to
ascertain
whether
tree­
to­
tree
spread
has
contributed
to
the
production
of
hypovirulent
inoculum.
APHIS
PERMIT­
West
Virginia
University,
Dr.
William
MacDonald
19
ENCLOSURE
13f.
DETAILED
DESCRIPTION
OF
PROCESSES,
PROCEDURES
AND
SAFEGUARDS
Manipulation
of
fungal
isolates
in
the
laboratory
will
be
conducted
in
a
Labconco
Class
II
biosafety
cabinet.
Transgenic
isolates
will
be
grown
in
temperature­
controlled
growth
chamber,
secured
from
the
general
public.
Slurry
inoculum
for
field
deployment
will
be
prepared
in
the
laboratory
using
"
good
microbiological
practices".
Our
Institutional
Biosafety
Committee
has
approved
all
practices
described
in
this
proposal.

All
fungi
will
be
transported
from
West
Virginia
University
to
the
field
test
sites
in
an
automobile
under
the
supervision
of
West
Virginia
University
personnel
who
are
directly
responsible
for
supervising
the
field
trial.
Fungi
will
be
transported
in
containers
that
meet
the
requirements
of
§
340.7
(
container
requirements
for
movement
of
regulated
articles).

Bark
samples
from
cankers
will
be
collected
in
plastic
microtiter
or
tissue
culture
plates
(
13
x
8.5
cm),
secured
with
tape
to
prevent
mixing
or
escape,
and
transported
in
a
cooler
to
the
laboratory
at
West
Virginia
University.
Bark
samples
will
be
stored
in
a
low
temperature
freezer
until
processed.
Isolates
will
be
maintained
in
incubators
and
temperature­
controlled
growth
chambers,
secured
from
the
general
public.
Following
cultural
assessment
and
molecular
analyses,
all
fungal
cultures
will
be
autoclaved
prior
to
disposal.

In
preparation
for
APHIS
permit
#
94­
189­
02M,
issued
in
1994,
a
full­
length
copy
of
CHV1­
EP713
dsRNA
was
integrated
into
a
virulent
strain
of
Cryphonectria
parasitica.
The
CHV1­
EP713
transgenic
isolate
was
inoculated
into
the
following
woody
plants
in
a
greenhouse
pursuant
to
of
the
1994
permit:
sweet
birch,
white
pine,
American
chestnut,
red
maple,
sugar
maple,
black
walnut,
blueberry,
American
beech,
white
oak,
red
oak
and
pin
oak.
Isolates
recovered
from
inoculated
tissues
retained
the
hypovirulence
phenotype
and
integrated
transformation
plasmid
DNA.
A
virulent
strain
used
as
a
control
was
pathogenic
on
American
chestnut
and
slightly
pathogenic
on
red
maple.
Findings
from
this
greenhouse
study
revealed
no
indication
of
altered
host
range
for
the
transgenic
strain.
CHV1­
EP713
and
CHV1­
Euro7,
the
hypovirus
proposed
for
the
2004
release,
show
extensive
sequence
identities:
87
to
93%
and
90
to
98%
at
the
nucleotide
and
amino
acid
levels,
respectively.
APHIS
PERMIT­
West
Virginia
University,
Dr.
William
MacDonald
20
ENCLOSURE
13g.
DETAILED
DESCRIPTION
OF
INTENDED
DESTINATION
The
intended
destination
of
the
genetically
engineered
(
transgenic)
organisms
is
the
field
test
site,
located
in
Pendleton
County,
WV,
described
in
Figures
5
and
6.
This
experiment
is
unique
in
that
the
focus
is
hypovirus
dissemination,
not
containment.
To
achieve
biological
control
of
the
invasive
pathogen
(
Cryphonectria
parasitica),
the
hypovirus
must
be
disseminated
effectively.
In
most
stands
that
have
moderate
numbers
of
Castanea
dentata,
the
fungal
pathogen
reaches
log
phase
5­
7
years
following
release
(
logging,
storm
damage,
etc).
The
virulent
population
of
C.
parasitica
is
both
formidable
and
diverse
(
in
terms
of
vegetative
compatibility
types).
Therefore,
hypovirus
dissemination
also
must
be
aggressive;
otherwise,
biological
control
will
not
be
effected.
It
is
the
intent
of
this
experiment
to
follow
hypovirus
dissemination
through
the
assessment
of
cultural
morphology,
and
dsRNA
content
of
fungal
isolates
made
over
the
course
of
three
years.
It
is
possible
that
the
diversity
of
the
native,
virusfree
pathogen
will
prohibit
any
extensive
acquisition
of
hypovirus.
The
end
result
will
be
dead
trees.
Conversely,
successful
dissemination
and
acquisition
of
hypovirus
may
allow
the
chestnut
trees
to
survive.
In
this
instance,
living
trees
will
remain.
This,
in
turn,
will
allow
the
hypovirus
to
perpetuate
and
become
established
in
this
WV
field
site.
Given
either
success
or
failure
of
the
experiment,
removal
of
the
entire
stand
of
chestnut
at
this
field
site
is
not
practical.
APHIS
PERMIT­
West
Virginia
University,
Dr.
William
MacDonald
21
ENCLOSURE
13h.
DETAILED
DESCRIPTION
OF
PROPOSED
PROCEDURES
AND
SAFEGUARDS
At
West
Virginia
University:
Genetically
engineered
(
transgenic)
strains
will
be
incubated
or
stored
in
incubators
or
temperature­
controlled
growth
chambers
secured
from
the
general
public.
All
Petri
plates
will
be
autoclaved
prior
to
disposal.
The
university
building
and
laboratories
are
locked
after
normal
work
hours
and
on
weekends;
university
buildings
are
staffed
by
night
custodians
and
patrolled
periodically
by
the
WVU
Campus
Police.

At
the
field
site
in
the
Monongahela
National
Forest:
The
goal
of
this
experiment
is
to
evaluate
hypovirus
dissemination
with
the
intended
purpose
of
achieving
biological
control
of
Cryphonectria
parasitica,
an
invasive
pest.
To
be
an
effective
control,
hypovirus
dissemination
and
establishment
are
desired.
The
field
test
site
is
located
in
a
sparsely
populated
area
of
Pendleton
County,
WV
in
the
Monongahela
National
Forest.
The
field
plots
are
located
beyond
a
locked
US­
Forest
Service
gate
and
not
visible
from
USFS
access
road
#
48.

While
C.
parasitica
is
capable
of
infecting
other
tree
species
(
particularly
members
of
the
genus,
Castanea),
the
only
other
potential
host
(
Castanea
pumila)
is
not
detectable
in
the
mixed
hardwood
forest
of
eastern
WV.
A
survey
of
the
proposed
area
did
not
identify
C.
pumila,
thus
spread
of
the
hypovirus
should
accompany
C.
parasitica
to
only
C.
dentata.
Since
the
fungal
pathogen
is
limited
to
C.
dentata,
the
accompanying
hypovirus
should,
likewise,
be
limited
to
strains
of
C.
parasitica
infecting
C.
dentata.

In
1996,
Anagnostakis
conducted
a
pathogenicty
study
in
the
laboratory
using
woody
stems
of
many
eastern
U.
S.
plants,
comparing
the
lesion
size
of
a
virulent
and
hypovirulent
isolate
of
C.
parasitica
(
data
unpublished).
The
results
of
that
study
are
listed
in
Table
7.
Results
indicate
that
the
hypovirulent
C.
parasitica
isolate
is
non­
pathogenic
on
all
woody
stems
used
in
the
test.
APHIS
PERMIT­
West
Virginia
University,
Dr.
William
MacDonald
22
Table
7.
Lesion
length
(
mm)
following
49­
day
inoculation
of
Cryphonectria
parasitica
virulent
strain
Ep155
and
hypovirulent
strain
CN2
(
CHV1­
Ep713/
pXH9)
into
various
woody
stems.
Plant
Lesion
Length
(
mm)*
of
Virulent
Ep155*
Lesion
Length
(
mm)*
of
Hypovirulent
CN2**
Blueberry
0,
0
0,
0,
0,
0
Apple
3,
3
1,
1,
3,
3
Red
Maple
28,
14
4,
3,
6,
11
Sugar
Maple
2,
8
6,
1,
2,
1
Sweet
Birch
9,
1
8,
1,
4,
3
American
Beech
6
1,
1,
1
American
Chestnut
59,
34
4,
1,
3,
4
Pin
Oak
6,
1
1,
1,
3,
1
White
Oak
1
3,
3,
1
Red
Oak
6,
9
3,
3,
6,
4
Black
Walnut
6,
9
3,
3,
3,
3
White
Pine
3,
3
3,
1,
1,
0
*
Lesion
length
includes
only
visible
growth
beyond
the
4­
mm­
diamater
inoculation
hole.
*
Two
lesions
were
made
to
each
woody
stem
with
the
virulent
isolate.
**
Four
lesions
were
made
to
each
woody
stem
with
the
hypovirulent
isolate.
ENCLOSURE
13i.
DETAILED
DESCRIPTION
OF
PROPOSED
METHOD
OF
FINAL
DISPOSITION
All
fungal
material
will
be
autoclaved
prior
to
disposal.
APHIS
PERMIT­
West
Virginia
University,
Dr.
William
MacDonald
24
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25
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William
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26
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