West
Virginia
University
College
of
Agriculture,
Forestry
and
Consumer
Sciences
April
16,
2004
Dr.
William
R.
Schneider
Biopesticides
and
Pollution
Prevention
Division
Office
of
Pesticide
Programs
US
Environmental
Protection
Agency
1200
Pennsylvania
Avenue
Washington,
DC
20460
Dear
Dr.
Schneider:

This
letter
is
in
response
to
your
email
of
15
April
2004
regarding
the
submission
of
a
Biotechnology
Notification
letter
about
our
proposed
release
of
genetically
modified
(
transgenic)
Cryphonectria
parasitica
strains.
The
materials
and
methods
of
the
proposed
research
are
detailed
in
the
attached
APHIS
permit
(
04­
077­
02r,
granted
13
April
2004).
We
propose
to
compare
the
dissemination
of
two
hypovirulent
(
less
virulent)
treatments
(
genetically
modified
[
transgenic]
and
non­
genetically
modified
[
cytoplasmic])
with
a
treatment
of
virulent
C.
parasitica.
Treatment
isolates
will
be
grown
in
the
laboratory
at
West
Virginia
University,
mixed
with
water
agar
to
the
consistency
of
applesauce
and
applied
to
cankers
on
American
chestnut
with
a
paintbrush.
The
experiment
will
be
conducted
in
the
Monongahela
National
Forest,
near
Circleville,
WV
(
Pendleton
County)
on
nine
0.1
acre
plots
(
0.9
acres
or
0.36
hectares).
The
transgenic
strains
will
be
released
in
only
three
plots;
the
remaining
six
plots
will
test
the
cytoplasmic
hypovirulent
and
virulent
strains.
In
the
transgenic
plots,
only
a
percentage
of
trees
will
be
treated
with
transgenic
inoculum;
the
remaining
trees
will
be
untreated
and
serve
as
trap
trees.
Therefore,
the
total
area
proposed
for
treatment
with
trangenic
strains
is
less
than
0.3
acres
or
0.13
hectares.
The
research
is
proposed
to
be
conducted
from
May
2004
 
August
2006.

Referencing
40
CFR
172.4
(
b)
Name
and
address
of
applicant:
Dr.
William
L.
MacDonald,
401
Brooks
Hall,
West
Virginia
University,
Morgantown,
WV
26506­
6058
telephone
304­
293­
3911
ext
2236.
(
b)
(
iii)
Purpose
of
objective
of
proposed
testing:
The
goal
of
this
project
is
to
examine
the
dissemination
and
subsequent
establishment
of
transgenic
hypovirulent
strains
of
Cryphonectria
parasitica.
The
objectives
are
three­
fold:
(
1)
comparison
of
growth
and
sporulation
of
artificially
established
cankers
initiated
with
either
transgenic
or
cytoplasmic
hypovirulent
strains
or
virulent
strains
of
C.
parasitica;
(
2)
the
spermatizing
potential
of
transgenic
hypovirulent
conidia;
and,
(
3)
the
ability
of
transgenic
strains
to
disseminate.
(
b)
(
iv)
Name
and
address
of
all
participants:
William
Rittneour,
401
Brooks
Hall,
West
Virginia
University,
Morganwtown,
WV
26506­
6058,
304­
293­
3911
ext
2150;
Mark
Double,
401
Brooks
Hall,
West
Virginia
University,
Morgantown,
WV
26506­
6058,
304­
29303911
ext
2239.
(
b)
(
v)
Name
and
address
of
all
cooperators:
Dr.
Donald
L.
Nuss,
Center
for
Biosystems
Research,
University
of
Maryland
Biotechnology
Institute,
5115
Plant
Sciences
Building,
College
Park,
MD
20742,
phone
301­
405­
0334.
(
b)
(
vi)
Description
of
specific
results
of
prior
testing:
A
greenhouse
test,
utilizing
a
similar
transgenic
strain
(
CHV1­
EP713/
pXH9)
was
conducted
on
twelve
woody
stems.
The
results
are
listed
below.
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.
Lesion
length
(
mm)
following
49­
day
inoculation
of
Cryphonectria
parasitica
virulent
strain
Ep155
and
hypovirulent
strain
CN2
(
CHV1­
Ep713/
pXH9)
into
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.
(
b)
(
vii)
Proposed
method
of
storage
and
disposal:
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".
The
West
Virginia
University
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
(
2­
mm)
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.

I
trust
this
brief
narrative
is
sufficient
to
address
the
notification
issues.
If
further
information
is
required,
please
feel
free
to
contact
me.

Sincerely,

William
L.
MacDonald
Professor,
Plant
Pathology
Division
of
Plant
and
Soil
Sciences
401
Brooks
Hall
P.
O.
Box
6058
Morgantown,
WV
26506­
6058
Phone:
304­
293­
3911
Fax:
304­
293­
2872
