Mycological Society of America
Isozyme Variation and Species Delimitation in the Ceratocystis coerulescens Complex
Author(s): T. C. Harrington, J. P. Steimel, M. J. Wingfield, G. A. Kile
Source: Mycologia, Vol. 88, No. 1 (Jan. - Feb., 1996), pp. 104-113
Published by: Mycological Society of America
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Mycologia, 88(1), 1996, pp. 104-113.
? 1996, by The New York Botanical Garden, Bronx, NY 10458-5126
variation
Isozyme
and
Ceratocystis
coerulescens
T. C. Harrington
J. P. Steimel
cause
of blue-stain in the wood of spruce (Picea) and
in Europe (Miinch, 1907). The anamorph
(Pinus)
pine
state may be Chalara ungeri Sacc. (Nag Raj and Ken?
drick, 1975).
of C. coerulescens isolates suggest
Our examinations
that there are up to five morphological
variants of this
on
conifers.
Two
of
these
from west?
variants
species
ern North America were recognized by Davidson (1953,
Department of Microbiology and Biochemistry,
University ofthe Orange Free State, P.O. Box 339,
Bloemfontein, 9300 South Africa
G. A. Kile
CSIRO, Division of Forestry, P.O. Box 4008,
Queen Victoria Terrace, ACT 2600, Australia
1955), one as C. c. f. douglasii on wood of Douglasfir [Pseudotsuga menziesii (Mirb.) Franco] and the other
as an associate of the spruce beetle, Dendroctonus ru-
Nineteen
electrophoretic
phenotypes
of electromorphs)
were found
among 98 isolates of Ceratocystis coerulescens and mor?
similar species using 10 isozymes. Analysis
phologically
of the isozyme data and morphological
comparisons
suggested that there are five variants of C. coerulescens
found
combinations
on conifers:
three are associated
fipennis (Kirby) (Coleoptera:
Scolytidae).
Two other conifer taxa related to C. coerulescens are
also bark beetle
maszko)
of Picea or Pinus, one (C. coerulescens f. douglasii) with
of Pseudotsuga, and one associated with the
bark beetle Dendroctonus rufipennis on Picea. Ceratocystis
and C. laricicola, associated
with bark beetle
in
the
on
Picea
and
Larix, respec?
species
genus Ips
inare morphologically
tively, had similar isozymes,
from
each
and
should
other,
distinguishable
probably
be synonymized.
Ceratocystis virescens, cause of stain of
polonica
distinct
disease
southeastern
United States, and Davidson (1944) later
the hardwood
fungus as new: C. virescens
Moreau. Others (Hunt, 1956; Upadhyay,
(Davidson)
this hardwood species as a syn?
1981) have considered
described
similarity to those of
an undescribed
species of Cer?
Eucalyptus and Chalara australis. The
species,
atocystis from
Chalara states of these three hardwood
Chalara neocaledoniae are morphologically
Key Words: Acer, blue-stain,
Eucalyptus, Larix, Picea, Pinus,
C. laricicola
from
zyme electromorphs
zymes of C. virescens show some
two Australian
associates.
Ceratocystis polonica (Siewas described from spruce attacked
is difficult to distinguish
C.
morphologically
polonica.
Davidson (1935) and Verrall (1939) reported C. coe?
rulescens as a cause of stain in hardwood lumber in the
this recognition,
of Acer saccharum, is
of
species
Ceratocystis in iso?
and anamorph morphology.
Iso?
and sapstreak
from the conifer
Moreau
by Ips typographus L. (Siemaszko, 1939) and C. laricicola
Redfern 8c Minter from larch (Larix) attacked by Ips
cembrae Heer (Redfern et al., 1987). The Ips typogra?
phus associate had been known as Ophiostoma polonicum
Siemaszko but recent studies (Visser et al., 1994) have
in Ceratocystis. With
shown that the species belongs
with blue-stain
blue-stain
hardwoods
complex
importance,
species limits within the genus are
in the group of taxa re?
defined,
poorly
particularly
ferred to here as the C. coerulescens complex.
Cerato?
coerulescens
Bakshi
was
described
as a
(Miinch)
cystis
M. J. Wingfield
(unique
the
nomic
Department of Plant Pathology, Iowa State University,
Ames, Iowa 50011
Abstract:
in
delimitation
species
of C. coerulescens, although
this was not sup?
ported by Kile and Walker (1987). The hardwood spe?
cies is recognized
as a cause of sapstreak of maple
United States, and similar
(Acer) in the northeastern
diseases on angiosperms
in New Caledonia
and Aus?
tralia (Kile, 1993) have been associated
with Chalara
species; Chalara neocaledoniae Kiffer 8c Delon was re?
ported on coffee and guava and Ch. australis Walker
8c Kile on Nothofagus. The anamorph of C. virescens is
similar to the two Chalara species and also to the ana?
on Eucalyptus, an unde?
morph of a weak pathogen
onym
species and
similar.
Chalara, Dendroctonus,
Pseudotsuga
INTRODUCTION
Ceratocystis sensu stricto (excluding
Ophiostoma) is a rel?
atively small genus of often insect-vectored
ascomy?
and
cetes, comprised
primarily of plant pathogens
wood-staining
fungi (Kile, 1993). In spite of their eco-
scribed species of Ceratocystis. Ascospores
of the Eu?
calyptus fungus are much larger than those of C. vi?
rescens or C. coerulescens (Kile et al., 1994).
Isozymes have proved useful in delimiting
species
Accepted for publication August 24, 1995.
104
105
HARRINGTON ET AL.: CERATOCYSTIS COERULESCENS COMPLEX
taxa in other fungal groups (e.g.,
and infra-specific
1989; 1992).
Leptographium, Zambino and Harrington,
new taxa or synonymizing
Before describing
species
we chose to apply isozyme anal?
based on morphology,
Besides the five
ysis to the C. coerulescens complex.
we
studied
C. polonica, C.
variants of C. coerulescens,
the
undescribed
laricicola, C. virescens,
Ceratocystis spe?
similar
cies from Eucalyptus, and two morphologically
no
Chalara
australis
with
known
teleomorphs,
species
FU?S
DEFFED
ACDDCA
and Ch. neocaledoniae.
MATERIALS AND METHODS
isolates of Ceratocystis and Chalara from
Ninety-eight
Australia, New Caledonia, Japan, North America and
I).
Europe were tested for isozyme variation (Table
were
obtained
from
the
isolates
of
recognized
Many
collections
(ATCC and CBS), and others were supplied
Forest
H. Roll-Hansen
and H. Solheim,
Norwegian
Norway; D. Redfern, Forestry Commission,
Research Station, Edinburgh, U.K.; J. Gibbs,
Alice Holt
Research
Commission
Station,
Forestry
of
Y.
Yamaoka,
Department
Lodge, Surrey, England;
of Tsukuba,
Plant Pathology,
Ibaraki, Ja?
University
Forest Service, Hamden,
U.S.D.A.
pan; D. Houston,
Forest Service,
and T. Hinds, U.S.D.A.
Connecticut;
Institute,
Northern
Ft. Collins, Colorado.
was grown
for enzyme extraction
Fresh mycelium
in 30 ml of liquid medium (20 g Difco malt extract
and 2 g yeast extract per liter of water) in 125 ml
flasks and incubated at room temperature
Erlenmeyer
two weeks. Buffers and protocols
for approximately
for extraction ofthe enzymes from mycelia onto paper
wicks were as previously described (Zambino and Har?
rington, 1989; 1992). Wicks were frozen at-70 C until
as
Starch gels (12 %) were prepared
electrophoresis.
described by Marty et al. (1984) and poured into gel
forms such as those described
by Cardy et al. (1983).
are shown in
conditions
Buffers and electrophoresis
Table
II. Staining for FUM and G6PD activity fol?
of Marty et al. (1984), but stain?
lowed the procedures
the procedures
of
followed
ing for other isozymes
extracCardy et al. (1983). At least two independent
tions from each isolate were tested for isozyme activity.
Among the isozymes tested, only 10 gave consistent
results for all 98 isolates. For each isolate, only one
but
was evident for most of the 10 isozymes,
seen in some isolates
bands were inconsistently
tested for DIA, G6PD, and PGD activity. With
latter three isozymes, only the consistently
pro?
of represenband was scored. Electromorphs
isozymes are shown in FiG. 1. For each isozyme,
were designated
by letters in order of
electromorphs
band
second
when
these
duced
tative
anodal migration.
decreasing
phenotypes
Electrophoretic
of electromorphs
combinations
were defined as unique
for the 10 isozymes.
AB
ABEGHGEBA
DED
BA
Fig. 1. Representative electromorphs, designated by letter in order of decreasing anodal migration, for fumerase,
and
isocitrate dehydrogenase, aspartate aminotransferase,
phosphogluconate
dehydrogenase.
were used to cluster the ETs into
us?
units. A matrix was developed
distance
(NTSYS-PC
program,
ing Rogers'
genetic
(Felsenstein,
Rohlf, 1990) or Nei's genetic distance
was generated
1993) and a phenogram
using neigh?
The electromorphs
putative taxonomic
bor-joining
(Felsenstein,
1993).
RESULTS
Nineteen electrophoretic
(ETs) were iden?
phenotypes
tified among the 98 isolates of Ceratocystis and Chalara
tested (Table III). One to four unique ETs were seen
in each of 11 putative taxonomic
units. The ETs within
unit generally varied for only one or two
a taxonomic
but the 10 tested isolates from Eucalyptus
isozymes,
for four of the ten
showed variation in electromorphs
isozymes. The isozymes AAT, DIA and GDH were the
with eight electro?
of the enzymes,
most variable
morphs for each found among the 98 isolates (Table
were seen for
II). In contrast, only two electromorphs
G6PD.
Genetic
distances
phe?
among the electrophoretic
by both Nei's and Rogers'
notypes were determined
and neighbor-joining
methods,
analyses of these maThe single isolate of C.
trixes gave similar topologies.
a phenotype
c. f. douglasii represented
quite distinct
from the others, and principal
analyses
component
had little
(Rohlf, 1990) indicated that this phenotype
in
affinity to the others. It was used as an outgroup
The
other
the tree in Fig. 2.
phenotypes
generating
fell into two clusters, one primarily ofthe isolates from
hardwoods
and the other of conifer isolates only.
similar ETs
Analysis of the isozyme data grouped
units or
taxonomic
into what appear to be natural
Table
Species
I.
Isolate numbers,
Isolate
No.a
substrate,
origin and electrophoretic
Other No./Collectorb
phenotype
of isolates of Ceratocystis and Chalara examined
Substrate
fo
C
o
Table
I.
Continued.
Table
I.
Continued.
Substrate
Nothofagus cunninghamii
Nothofagus cunninghamii
Nothofagus cunninghamii
Nothofagus cunninghamii
Nothofagus cunninghamii
Nothofagus cunninghamii
Nothofagus cunninghamii
Nothofagus cunninghamii
Nothofagus cunninghamii
Nothofagus cunninghamii
Nothofagus cunninghamii
Nothofagus cunninghamii
Nothofagus cunninghamii
Nothofagus cunninghamii
Nothofagus cunninghamii
Nothofagus cunninghamii
Nothofagus cunninghamii
Nothofagus cunninghamii
Nothofagus cunninghamii
Nothofagus cunninghamii
Nothofagus cunninghamii
Nothofagus cunninghamii
Nothofagus cunninghamii
Nothofagus cunninghamii
Nothofagus cunninghamii
Nothofagus cunninghamii
Coffea robusta
a Isolate numbers from the collection of the senior
author.
b Collectors and their institutions are listed in the
Materials and Methods.
Harrington
et al.:
Ceratocystis
coerulescens
109
found for 10 enzymes tested for variation in the
Abbreviations, buffer systems and number of electromorphs
coerulescens
complex
Ceratocystis
Table
Complex
II.
a Nomenclature Committee of the International Union of Biochemistry.
b Buffer B was a continuous histine citrate
system, adjusted to pH 5.7, run at 9 watts constant wattage for 4 hours. Buffer
E was a continuous morpholine citrate system, adjusted to pH 8.1, run at 15 watts constant wattage for 6 hours, except for
diaphorase, which was run at 9 watts for 6 hours.
species (Fig. 2). The single ET of C. polonica clustered
with the three ETs of C. laricicola. Two ETs of C.
virescens differed by only a single isozyme and were
units. The four ETs
distinct from all other taxonomic
of the undescribed
species from Eucalyptus clustered
was found among the 30 isovariation
No
together.
Table III.
Isozyme electromorphs
for 19 electrophoretic
ElectroSpecies
phoretic
phenotype
Num?
ber
of
iso- -__
AAT
lates
1 Letters
represent isozyme electromorphs
b No
activity for DIA.
of Ch. australis,
neocalidoniae.
lates
which
grouped
nearest
to Ch.
five clusters of ETs could be
seen: sp. A, sp. B, sp. C, sp. D and f. douglasii. The
two ETs of sp. A differed by only one enzyme, as did
the two ETs of sp. C (Table III). Two of the variants
Within
C. coerulescens,
phenotypes among 98 isolates of the Ceratocystis coerulescens complex
Isozymes
DIA
FUM
G6PD
GPI
for each isozyme in order of decreasing
GDH
IDH
MDH
anodal migration.
PGM
PGD
Mycologia
110
0
L.
4
6
_J_
8
IV
V
\c. coerulescens
VI C. coerulescens
XII
XIII
C
D
XV
sp.
Ceratocystis
from Eucalyptus
XVII
XVIII Chalara
australis
XIX Ch. neocaledonlae
j-
VIII C. polonica
X
primary dispersal propagule, an isolate would be forced
less variation
to cross to be dispersed.
Substantially
in
C. virescens.
17
was
found
ETs
isolates)
(two
among
on
the
the
from
Aside
Ceratocystis
species
Eucalyptus,
C. virescens, are ho?
species studied here, including
of mating type
perhaps due to a mechanism
and McNew,
unpublished).
switching
(Harrington
can be produced
Thus, ascospores
by selfing in C.
level of variation might
virescens, and an intermediate
mothallic,
C. laricicola
IX
III C. coerulescens
B
C. coerulescens
VII C.c.t.
the Eucalyptus fungus had the
species studied,
in
variation
(four ETs
isozyme electromorphs
greatest
is
heterothallic
This
10
(Kile
isolates).
species
among
are the
that ascospores
et al., 1994), and assuming
the
C. virescens
XIV
XVI
in pathogenesis
(Kile, 1993), but the species on Eu?
is
(Kile, unpublished).
only weakly pathogenic
calyptus
variation in the hardThe degree of within-species
of the sexual com?
wood group may be a reflection
taxa. Among all
of
these
respective
patibility systems
Chalara australis showed no variation in
and has no known teleomorph;
isozyme electromorphs
most
it is apparently dispersed as asexual propagules,
be expected.
A
douglasii
Phenogram (Neighbor-joining analysis) based on
of 19 electrophoretic
phenotypes
isozyme electromorphs
(Roman numerals) in the Ceratocystis coerulescens complex.
Fig. 2.
probably in the frass of ambrosia beetles
This low level of isozyme polymorphism
fungus (Zambino and Har?
The
data would suggest that
1989; 1992).
rington,
since spethere has been little sexual recombination
of Ch. australis to Aus?
ciation or since introduction
pected
in such an asexual
(sp. A and sp. B) grouped most closely to C. polonica
and C. laricicola, and sp. C and D grouped most closely
to C. virescens (Fig. 2). C. c. f. douglasii was represented
tralia, but the species
ern Australia.
by only one isolate, which had unique electromorphs
for five of the ten isozymes tested and did not cluster
with the other phenotypes.
Conifer species.?Isozyme
guishing five morphological
as distinct taxa. Differences
DISCUSSION
Analysis of the isozyme data clustered morphologically
of many spe?
similar isolates and supported delineation
the
cies within the C. coerulescens complex.
Although
of isozymes tested was too low to confidently
among all the tested
compare the relative relatedness
one
taxa, two broadly defined groups were suggested:
number
around the hardwood species C. virescens and
centered
around C. coerulescens sensu stricto
(sp. A). The isolate of C. c. f. douglasii did not cluster
closely with other isolates.
centered
another
Hardwood species. ?Four
taxa on hardwoods
(C. vires?
from
Ch.
australis
and
cens, Ceratocystis sp.
Eucalyptus,
Ch. neocalidoniae) share many isozyme electromorphs
and have similar conidiophore
states: tapering
and
as
to
the
proliferating
phialides
opposed
non-tapering
phialides typically found in the conifer species of the
C. coerulescens complex. Three ofthe hardwood species
are primary pathogens
and are believed to be similar
(Kile, 1993).
might be ex?
is not known
variation
variants
outside
southeast-
distinsupports
of C. coerulescens
in electromorphs
between
any two of these variants was substantial,
greater than
that found within the other Ceratocystis species studied.
Two of the variants of C. coerulescens showed similarity
to C. polonica and C. laricicola, two were closer to the
hardwood
species C virescens, and the single isolate
of C. c. f. douglasii differed markedly from all of the
other isolates in isozyme electromorphs.
An isolate of
C. coerulescens examined by Wingfield et al. (1994) for
rRNA homology
had greater similarity to C. virescens
than to C. laricicola. It is not clear to which of the five
of C. coerulescens their isolate belongs, but the
data
isozyme
presented herein would suggest that their
isolate was C. coerulescens sp. C.
Among the variants of C. coerulescens, the isolates of
closest to Miinch's (1907)
sp. A are morphologically
variants
of the species. The four isolates we studied
from stained wood of Pinus spp. in Enoriginated
Perithecia
gland.
produced
by pairing isolate C490
with any of the three other isolates (C487, C488, or
C489) were large and robust with long necks, fitting
the dimensions
given by Munch (1907) and as rec?
ognized by Lagerberg et al. (1927), Siemaszko (1939),
and Bakshi (1951).
concept
Harrington
et al.:
Ceratocystis
Only one isolate (C301) representing
sp. B produces
in culture, and these perithecia
are smaller
perithecia
than those of sp. A. However,
Campbell (1957), who
of per?
originally isolated C301, reported dimensions
ithecia
for C. coerulescens
similar
to those
(1907), and crossing C301
in perithecia with necks comparable
Miinch
pine and
species
America.
in
and
North
Similarity in
Europe
spruce
material
for mor?
of
and
biology
paucity
perithecial
a
clear
separation of
preclude
phological
comparisons
A. Our
B isolates
were
reported by
with C693 results
to those of species
from
both
sp. A from sp. B.
Perithecia of sp. C produced in culture have shorter
of sp. A. Isolates were ob?
necks than the perithecia
tained from Europe and North America. At least two
of the isolates representing
sp. C were from wounds
of living trees, this
in spruce. As a wound colonizer
which is also true
species may be weakly pathogenic,
similar variant, sp. D.
for the most electrophoretically
by
Species D appears to be the variant recognized
in
Col?
Davidson (1955) from beetle-attacked
spruce
of sp. D grow slowly at room tem?
and
only at 20 C or less.
produce perithecia
perature
are
this
variant
of
larger than those of the
Ascospores
other conifer species. Our isolates of sp. D all origi?
nated from Dendroctonus rufipennis or from Picea engelmannii attacked by this bark beetle in Canada. It
orado.
Our isolates
to spruce (Solheim,
1994)
appears to be pathogenic
an
and is likely to be
symbiont of this bark
important
would tend to coincide with
beetle. Its pathogenicity
of the other species with which it
the pathogenicity
shared isozyme electromorphs
(i.e., sp. C and the hard?
of the complex).
of isolates of C. coe?
(1953) recognition
as a separate form is well
rulescens from Douglas-fir
by the isozyme data, although C. c. f. doug?
supported
in this study by only one isolate.
lasii was represented
and the two types
Its sensitivity to warm temperatures
wood
species
Davidson's
described
of conidiophores
(1953), parby Davidson
distin?
further
the
long, tapering phialides,
ticularly
a
as
treated
this
synonym of
fungus. Although
guish
C. coerulescens in the past (Hunt, 1956; Upadhyay, 1981)
we believe that C. c. f. douglasii should be recognized
at the rank of species.
The other examined
C. po?
conifers,
species
for their as?
are noteworthy
from
lonica and C. laricicola,
which are relatively
cospores,
small, more broadly elsheaths (outer walls),
lipsoid with broadly-thickened
at each end. The as?
and have two distinct guttules
of the other Ceratocystis species studied are
cospores
considerably
longer and have sheaths that are most
are
ends. Conidiophores
at their terminal
obvious
found
often
and
scarce in C. polonica and C. laricicola
on the base of peri?
only as hairs or ornamentation
et
described
thecia. When C. laricicola was
(Redfern
which
to
C.
al., 1987), it was not compared
polonica,
coerulescens
Complex
111
was then believed to be an Ophiostoma species with a
Now that C. polonica is rec?
Leptographium anamorph.
to
have
a
Chalara
(Visser et al.,
ognized
anamorph
distinction
between
C. lar?
1994), the morphological
icicola and C. polonica is difficult to discern. Isolates
of C. polonica from Poland (an isolate from the holo?
Norway and Japan had identical isozyme elec?
and these differed only slightly from the
tromorphs,
ETs of C. laricicola, which originated
from Japan and
type),
Scotland.
These
related
closely
their insect
two taxa are strongly associated
species of Ips, perhaps co-evolving
with
with
symbionts.
Bark beetle associations.?Several
and C. laricicola
characteristics
are also found
of C.
in C. coerules?
polonica
cens sp. D, the other bark beetle associate in this study.
These three taxa produce few conidiophores
and gen?
to
erally lack strong, fruity odors, features important
of mats with conidia carried by fungalspermatization
of these
feeding insects. Because the protoperithecia
to the galleries of bark
species are mostly confined
would
for cross-fertilization
beetles, the opportunity
be less than in the other Ceratocystis species, which
on exposed
tend to produce mats with protoperithecia
Because mats would not be exposed for acqui?
and dispersal of spermatizing
conidia, the bark
to suppress co?
be
beetle associates
expected
might
of attracand
the
production
production
nidiophore
tive volatiles (such as isobutyl acetate). They may also
wood.
sition
of the mycelium (feed?
strongly to wounding
to
a
cue
as
bark
beetles)
many per?
producing
ing by
and cultures of these species
ithecia with ascospores,
in abun?
on malt agar do tend to produce perithecia
respond
Because the /^5-associated
dance after wounding.
spe?
cies (C. polonica and C. laricicola) and Dendroctonusassociated
species (sp. D) showed dissimilar isozyme
of conidi?
it may be that suppression
electromorphs,
the low level of odors and the
production,
response evolved separately in the Ips and the
Dendroctonus-associated
Ceratocystis species.
of Ophiostoma species with
the
association
Although
bark beetles has been much discussed
(most recently
of Ceratocystis
association
the
1993),
by Harrington,
ophore
wound
bark beetles has only
species with conifer-infesting
on whether C.
been recognized
recently. Depending
laricicola and C. polonica turn out to represent distinct
taxa or not, we are now aware of two or perhaps three
species of Ceratocystis carried by conifer infesting bark
tests with C. polonica and
beetles. From pathogenicity
and Solheim, 1990; Redfern
C. laricicola (Christiansen
et al., 1987; Solheim,
1993) and also with C. coerules?
are that the
cens sp. D (Solheim,
1994), indications
than
virulent
more
are
considerably
Ceratocystis species
im?
of
more
and
hosts
to
the
tree
Ophiostoma species
trees.
to the beetle in killing mass-attacked
portance
In contrast, the Ophiostoma species might be relatively
Mycologia
112
to the beetles, as
As
tree pathogens,
by Harrington
proposed
these few Ceratocystis species may have evolved a mu?
tualistic symbiosis with bark beetles, where both part-
unimportant
and even
a hindrance
(1993).
from the relationship.
of
the
Many
fungi studied here are believed to be
dispersed by insects, which has apparently led to sub?
in morphological
stantial convergence
characteristics
et
Hausner
et
1994;
al, 1993; Wingfield
(Blackwell,
and limited variation has
al., 1994). This convergence
ners benefit
made species delimitation
based on morphology
alone
difficult. However, variation in isozyme patterns with?
in C. coerulescens and among related species generally
well with the limited
corresponded
morphological
variation in the group. The technique
proved useful
in helping to delineate
taxa of common biology, and
this information
will be applied to revise the taxonomy
of this important
complex.
ACKNOWLEDGMENTS
We are grateful to all those who contributed isolates for this
study. Portia Hsiau and Jonathan Wendel provided invaluable assistance with analysis of the data. Doug McNew has
assisted with morphological comparisons of the species stud?
ied. The support of the Foundation for Research Devel?
opment, Mondi Paper Company and the Ernest Oppenheimer Trust to MJW during sabbatical leave is acknowledged.
Journal Paper No. J?16188 of the Iowa Agriculture and
Home Economics Experiment Station, Ames, Iowa Project
0159.
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