Mycological Society of America
A PCR-Based Identification Method for Species of Armillaria
Author(s): T. C. Harrington and B. D. Wingfield
Source: Mycologia, Vol. 87, No. 2 (Mar. - Apr., 1995), pp. 280-288
Published by: Mycological Society of America
Stable URL: http://www.jstor.org/stable/3760915 .
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Mycologia, 87(2), 1995, pp. 280-288.
? 1995, by The New York Botanical Garden, Bronx, NY 10458-5126
A
identification
PCR-based
method
Department of Plant Pathology, Iowa State University,
Ames, Iowa 50011
ofthe
Department of Microbiology and Biochemistry, University
of the Orange Free State, P. O. Box 339, Bloemfontein,
9300 South Africa
major species. Until the 1970s, most
to all Northern
Hemireferred
in species identification
et al.,
(Guillaumin
et al., 1992; Guil?
1991; Watling et al., 1991; Harrington
laumin et al., 1993). Characteristics
of mycelial fans
and rhizomorphs
vary among the species only to a
ficulties
limited
are seasonal and rare
and basidiomes
in many regions. The fungus may be
difficult to isolate from some substrata, it grows slowly
in culture, there is little variation among Armillaria
and production
of
species in cultural characteristics,
A portion of the Intergenic
Spacer (IGS) of
RNA operon of 74 isolates of 11 Ar?
species from Europe and North America was
extent,
to uncommon
the ribosomal
chain reaction.
Amusing the polymerase
were
made
from
of
plifications
scrapes
living mycelium
Alu I digests ofthe amplified
without DNA extraction.
in agarose and stained
product were electrophoresed
basidiomes
in culture
each tax?
with ethidium bromide. With few exceptions,
on had a unique combination
of restriction fragments.
Most taxa had a single Alu I pattern, but two restric?
ing.
Critical
identifications
on
amplified
but each of these
taxa could
amplification
pattern
products
and dried
identical
to A. calvescens.
were obtained
prints
Spacer,
Words:
Armillaria,
IGS
from 8-year-old
as well as fresh
identification,
tester
of Armillaria
rely
(Korhonen,
are haploid and are
strains
connections
(Larsen
isolates (Siepmann,
1987; Shaw and
Rizzo
and
Harrington,
1992). These
1988;
Loopstra,
for
months
identification
to
tests
2
require up
pairing
cation
day.
Key
of cultures
may also be visible in compatible
et
al., 1992). A haploid tester can be
pairings
diploidized
upon pairing with a diploid isolate of the
from the pairing plates is
same species. Subculturing
et al., 1992), but still, the
recommended
(Harrington
for identifi?
results are often ambiguous,
particularly
clamp
basidiomes,
The technique
decay without DNA extraction.
from
allows for identification
wood, basi?
decayed
diomes or mycelia of these Armillaria species in a single
spore
wood
haploid
and time-consum-
rial mycelium,
and the mycelium often has a reddishstrains of
brown crust. Pairing of single basidiospore
the same species will usually result in diploidization,
tends to flatten. Transient
and the fluffy mycelium
be dis?
after restriction
by their polymorphisms
tinguished
with the enzymes Nde I, Bsm I, or Hind II. European
isolates of A. gallica had a distinct Alu I restriction
isolates of this species
pattern, but North American
had a restriction
pairings
with
is unreliable
stains
1978). Single basidiospore
on malt extract agar.
and
unpigmented
generally fluffy
are diploid
from
fans
or
Isolates
rhizomorphs
decay,
(Hintikka, 1973), generally produce relatively little ae?
tion patterns were seen among isolates of A. borealis,
A. cepistipes, A. gallica, A. tabescens, and A. mellea. Ar?
millaria ostoyae, A. gemina, one of the A. borealis types,
and one of the A. cepistipes types had identical sizes of
Alu I fragments,
Armillaria
plant pathologists
sphere species of Armillaria as A. mellea (Vahl: Fr.)
Kummer. Separating
the common
species and defining their biology has been seriously impeded by dif-
B. D. Wingfield
millaria
of
species
the biology
T. C. Harrington
Abstract:
for
of diploid
in pure culture.
for differ?
have been explored
techniques
of isoof
Armillaria.
Electrophoresis
entiating species
restriction
et
al., 1985),
fragments of
zymes (Morrison
et al.,
DNA (Anderson
ribosomal
or mitochondrial
after isolation
Intergenic
Other
rDNA
introduction
et al., 1989; Jahnke et al., 1987; Smith
1987; Anderson
and Anderson,
hybridization
1989), or DNA-DNA
some of these
et
al., 1987) can distinguish
(Jahnke
have been
species. However, none of these techniques
routine
for
or
to
be
feasible
tested
spe?
proven
widely
cies identifications.
Anderson and Stasovski (1992) published partial DNA
Species of Armillaria (Fr.: Fr.) Staude (Agaricales, Triroot
are among the most important
cholomataceae)
disease pathogens of trees, but the confused taxonomy
of
a clear understanding
of this genus has precluded
Accepted for publication November 30, 1994.
280
HARRINGTON AND WlNGFIELDI ARMILLARIA IDENTIFICATION
for the IGS (Intergenic
Spacer) region of
sequences
the ribosomal
RNA (rRNA) operon for most of the
species of Armillaria. Sequence
Hemisphere
number of isolates samthe
limited
among
that
restriction
enzyme digests of this
pled suggested
discriminate
region may
among the species. Using the
chain
reaction
(PCR) and the primers of
polymerase
Anderson
and Stasovski (1992), we amplified the IGS
region and screened the PCR products using a number
of restriction enzymes for unique restriction fragment
Northern
variation
(RFLP) among the European
A 1-day
species of Armillaria.
was developed
that can identify the 11 taxa
length polymorphisms
and North American
procedure
examined.
oration
ofthe
\00-u\
281
final reaction
volume.
The ther-
Massachu?
Inc, Watertown,
mocycler
(MJ Research,
at 95 C
were an initial denaturation
setts) conditions
of
C
for
60
40 see
35
for 95 see, followed
cycles
by
min
for
and
90
C for
C
72
2
(elongation)
(annealing),
was allowed
A final elongation
30 see (denaturation).
for 10 min at 72 C to ensure a double stranded am?
plification
DNA
product.
number
restriction.?A
for polymorphisms
of restriction
enzymes
among a select group
of Armillaria isolates. The enzyme Alu I gave the great?
some species had iden?
est polymorphisms,
although
of the
tical RFLP with this enzyme. DNA sequence
IGS region from the end of the LSU to the 5S gene
were tested
for all the
operon had been published
Armillaria species except A. mellea and A. taand Stasovski,
bescens (Scop.: Fr.) Emel. (Anderson
were
and
these
used
to
identify fur?
sequences
1992),
of the rRNA
tested
MATERIALS AND METHODS
least four haploid or diploid isolates of
Isolates.?At
each of the nine described and two nondescribed
spe?
cies of Armillaria in Europe and North America were
in the study (Table
included
I). These isolates were
various
identified
by
investigators
using pairing tests.
All the isolates were grown on MYEA plates (2% malt
extract, 0.2% yeast extract, 1.5% agar) at room tem?
perature prior to amplification.
Template DNA.?DNA
number of the cultures
was
isolated
from
the method
a limited
of Lee and
using
most ofthe results presented
Taylor (1990). However,
here were from amplifications
done directly from Ar?
on MYEA. Neither
millaria mycelium
the amplifica?
tions nor the results from the restriction
digests were
influenced
by the origins
For direct amplification
tip was scraped
of the DNA template.
from mycelium,
a pipette
1
cm
across
the
approximately
actively
growing mycelium at the edge of the colony. The tip
was then dipped in the PCR reaction vessel containing
the reaction mix and the mineral oil, and the mixture
was vigorously
fungal material
the reaction
stirred
attached
tip. In this manner
in
to the tip was suspended
with
the
mix.
Polymerase chain reaction (PCR). ?The
Intergenic
Spac?
er region (IGS) between the 3' end ofthe large subunit
ribosomal
(LSU) RNA (rRNA) gene and the 5' end of
the 5S rRNA gene was amplified using PCR. The prim?
ers used were
Stasovski
those
recommended
and
by Anderson
5'CTGAACGCCTCTAAG-
(1992): LR12R,
(Veldman et al., 1981) and O-l, 5'AGTCCTATGGCCGTGGAT3'
(Duchesne and Anderson,
1990).
The PCR reaction mixture included 2.5 units Taq poly?
merase (Promega,
Madison, Wisconsin)
per reaction,
TCAGAA3'
the buffer supplied
with the enzyme, 4 mM MgCl2,
200 uM dNTPs, and 0.5 a*M of each primer. DNA (10
were added as template
for
ng) or fungal mycelium
the reaction. Mineral oil was overlaid to prevent evap-
ther diagnostic
restriction
enzymes. The five enzymes
utilized were Alu I, Nde I or Hind II (Promega,
MadBsm
I
La
Califor?
ison, Wisconsin),
(Stratagene,
Jolla,
nia), and Tha I (Gibco BRL, Life Science Technologies, Gaithersburg,
Maryland).
The amplified DNA was not purified before
tion enzyme digestion.
Alu I, Nde I or Hind
restric?
II (2-4
to the PCR re?
units per reaction) was added directly
mix (20 u\) after amplification
and the digestion
allowed to proceed for l-16hat37C.
The Bsm I and
Tha I (2-4 units per reaction)
were per?
digestions
action
formed at 65 C for 1-16 h. NaCl was added to a final
concentration
of 50 mM for both the Nde I and Tha
I digestions
and to a final concentration
of 100 mM
for Bsm I digestions.
the amplified DNA and the re?
Electrophoresis.?Both
of these products
were
striction
enzyme fragments
in
a
in
TBE
mM
Tris,
[89
electrophoresed
agarose gels
89 mM boric acid, 2 mM EDTA (pH 8)] buffer system
to determine
the size of the amplification
and restric?
tion products.
For routine analysis, 2% agarose gels
were run at 100 V for 2 h, but more definite determinations of fragment sizes were based on larger gels
of 3% MetaPhor
Rockagarose (FMC BioProducts,
run at 200 V for 3 h at 10 C. The gels
with ethidium
bromide
and visualized
land,
were
Maine)
stained
using
UV light.
RESULTS
Good amplification
of the IGS region was obtained
from my?
with all isolates using direct amplification
celium. At least two amplifications
were made of each
ofthe isolates listed in Table I. The amplified product
from all isolates of A. mellea was 875 base pairs (bp),
and each isolate of A. tabescens yielded a product of
282
Mycologia
Table I. Alul restriction fragments of the amplified IGS region of rDNA and origins of the North American and European
isolates of Armillaria
Species (RFLP group)
A. ostoyae
A. gemina
A. borealis (A)
A. borealis (B)
A. sinapina
A. cepistipes (A)
A. cepistipes (B)
NABS IX
NABSX
A. gallica (European)
A. gallica (American)
Isolate
number
Determined by,
other isolate number3
State, province, or
country of origin
Fragment sizes (bp)b
Harrington
and
Wingfield:
Armillaria
Identification
283
Table I. Alul restriction fragments of the amplified IGS region of rDNA and origins of the North American and European
isolates of Armillaria (Cont.)
aJ. B. Anderson, H. H. Burdsall,
provided isolates identified through
b
Fragment sizes determined from
tabescens, which were determined by
845
bp. All other
isolates
S. Gregory, J. J. Guillaumin, K. Korhonen, K. I. Mallett, J. Rishbeth, andj. J. Worrall
pairing tests.
the reported sequence data (Anderson and Stasovski, 1992), except for A. mellea and A.
electrophoresis and staining with ethidium bromide.
yielded
a product
of 920
bP.
The product of at least two amplifications
of each
with Alu I and electrophoresed
isolate was digested
the sizes of the
separately with markers to determine
restriction
(Fig. 1). Only fragments
fragments
larger
100 bp were scored because fragments
smaller
than this were difficult to see clearly and tended to be
than
obscured
duced
by the prominent
"primer dimer" band pro?
The number and sizes of
during amplification.
from the two or more digestions
fragments
sistent for each isolate.
were con?
One or two Alu I digestion patterns were found in
each of the 11 taxa tested (Table
I, Fig. 1). Twelve
different patterns were found among the isolates test?
ed. Based on the published
of Anderson
sequences
and Stasovski (1992), it was possible to create a restric?
tion map (Fig. 2) for eight of these patterns,
which
includes all of the tested species of Armillaria except
A. mellea and A. tabescens. Two patterns were seen in
A. borealis Marxmiiller
8c Korhonen,
A. cepistipes Veand
A.
Marxmiiller
8c
The
lenovsky,
gallica
Romagnesi.
restriction
show
that
the
two
within
maps
patterns
each of these species is due to a difference
in one or
two restriction sites. Two restriction patterns were also
seen in A. mellea and A. tabescens, but the lack of pub?
lished sequence
data for these two species prevented
unequivocal
tion sites.
The IGS
determination
of the variation
in restric?
of isolates of A.
amplification
products
Herink, A. gemina Berube & Desostoyae (Romagnesi)
sureault, A. borealis type A, and A. cepistipes type B had
the same Alu I restriction sites. However, examination
of the IGS DNA sequences
revealed other diagnostic
restriction
enzymes for these species. Amplified prod?
ucts of all isolates listed in Table I were digested with
Nde I, but only the isolates of A. borealis and A. ostoyae
were cleaved, yielding products
of 550 and 370 bp
of
the
listed isolates of
(Fig. 3). Amplified
products
A. gemina and A. borealis did not digest with Bsm I, but
the products of all ofthe listed A. ostoyae isolates except
B177 restricted,
of 620 and 300
yielding fragments
bp (FiG. 3). The amplified product of another A. ostoyae
isolate, B747, gave inconsistent
digestion with Bsm I.
As predicted from the sequence data, Hind II digested
A. cepistipes isolates, giving fragment sizes of 580 and
340 bp, but did not cleave the IGS amplification
prod?
ucts of A. ostoyae, A. gemina or A. borealis.
North American and European isolates of A. gallica
differed in their Alu I restriction
I,
patterns (Table
Fig. 2). The North American
isolates had an Alu I
to that of A. calvescens Berube 8c
pattern identical
Dessureault
isolates.
Examination
of the IGS DNA
from North American A. gallica and A. calsequences
284
Mycologia
OST
BOR:GEI\
^W^tfHSWeSifTJfliPHi
'"
M||
wt dHmM
:*
?-^jit
-ii^?^^| .g^^jj^Jjjt
yigyH
lii
.; ,*? **
- .-??
a-w#._
S-^rllpB^
--iH:;i^:
.:5^(SP
,*&m
iNMfe
-ifiiP%MJ
j^^^^?^^^MltM:
-JMjfe
jj|fe:.$|fe/
Fig. 1. Ethidium bromide stained agarose gels (3%) of A/w I digestion products of the IGS region of representative isolates
of Armillaria. Markers (M) are 100-bp ladders, the lowest band being 100 bp in size. Lanes are designated with abbreviations
for the species listed in Table I. Two patterns within a species are denoted as "A" and "B," or "NA" (North American) and
"EUR" (European) above the appropriate lanes.
that
and Stasovski, 1992) indicated
vescens (Anderson
the two taxa differ only at a single nucleotide/base
should result in differential
pair, and this difference
we found
the
restriction
enzyme Tha I. However,
by
DNA from only some isolates of
that the amplified
each taxon were cleaved at this site with Tha I, and
were also found between two amplified
inconsistencies
we were
from
the same isolate. Therefore,
products
DNA from the
the amplified
not able to distinguish
North American isolates of A. gallica and A. calvescens
using this or any other enzyme.
from dried samples
We attempted
amplifications
of DNA by scraping the
and decay without extraction
for the ampli?
tissues with a pipette tip as described
fication from fresh mycelia. We successfully
amplified
the IGS region from small amounts of 6- to 8-yr-old
spore prints of A. gallica, gill tissue of a 7-yr-old dried
of A. mellea, two 8-yr-old dried basidiomes
basidiome
of A. ostoyae, and wood from two oak trees that were
small (barely visible)
decayed by A. gallica. Generally,
amounts of material worked better than larger amounts
Small pieces of wood were likely
for amplifications.
in
the
scrapings from decay. We also attempt?
present
from the core of dried rhizomorph
ed amplifications
tissue
tissue, but without success; fresh rhizomorph
The
to the technique.
might prove more amenable
the
other
tissues
were
the
from
amplified
products
Alu
l
The
correct length for the respective
species.
in
other
North
as
found
restriction
were
patterns
American isolates of A. gallica, A. mellea (type A), and
A. ostoyae (Table I).
DISCUSSION
of DNA from the IGS region with the re?
Digestions
striction enzyme Alu I proved reliable for unambigof most of the
of pure cultures
uous identification
Armillaria species known to Europe and North Amer?
ica. Because DNA extraction from pure cultures, dried
restric?
diagnostic
samples or decay was unnecessary,
in a single day. The
tion patterns could be obtained
IGS region was amplified from 8-yr-old spore prints
and basidiome
tissue, which should allow for nondestructive study of herbarium
samples critical to the
and nomenclature
of this difficult genus.
taxonomy
Further, the IGS region was amplified directly from
decayed wood, which will greatly facilitate rapid and
of fresh field materials for
identification
unambiguous
work.
ecological
Some care must be taken in interpreting
the restric-
HARRINGTON AND WlNGFlELDI ARMILLARIA IDENTIFICATION
A. ostoyae
I
285
Bsm\_
Nde\_
200
310_foMII
|ost|bor
|gem|m
|ost|bor
|gem
| m |
A. gemina
310
|
A. borealis
(type
310
|
A)
|89|135|
A. borealis
I
200
|89|135|H|
(type
||
200
II
200
||
200
||
200
||
200
B)
31?_IoqHcmII
>\. sinapina
|
399_|135|
A. cepistipes
I
(type
399_Il83
A. cepistipes
(type
310
|
NABS
A)
B)
18911351
IX
|
200
534
NABSX
I
399_|183
A. gallica
|
||
142|
||
240
(European)
399_|183
A. gallica
|
tion
for species
identification.
patterns
Secondary
smaller than the IGS product were somefragments
times found in amplifications,
and these bands may be
as
restriction
misinterpreted
fragments. Also, the con?
ditions for digestion
of the PCR product
were not
DNA
ideal, and incompletely
digested
fragments were
sometimes
with the restriction
en?
seen, especially
that
zymes Bsm I and Nde I. It should be reiterated
the digestions
were conducted
in the PCR reaction
mixture, not in the buffer recommended
by the manufacturer
of the restriction
and
enzyme. Purification
of the PCR product before digestion,
quantification
(N. American)
582_||
240
A. calvescens
|
A 2% agarose gel stained with ethidium bromide
the
Nde I and Bsm I digestion products of the IGS
showing
region of representative isolates of Armillaria ostoyae (OST),
A. gemina (GEM) and A. borealis (BOR). The occurrence of
digested and undigested DNA fragments in the same lane is
interpreted as incomplete digestion. Markers (M) are 100bp ladders with the lowest band being 100 bp in size.
Fig. 3.
582_||
240
Fig. 2. IGS restriction maps for the enzyme Alu I based
on the sequences published by Anderson and Stasovski (1992)
and restriction patterns seen in the isolates listed in Table
I and illustrated in Fig. 1. Numbers designate the approximate length (bp) of diagnostic fragments. Restriction maps
for A. tabescens and A. mellea were not developed.
286
use
Mycologia
of the
recommended
restriction
buffer, use of
concentrations
of
the
restriction
higher
enzyme, and
would
more
longer digestion
periods
give
complete
but these steps would add some expense
digestions,
and considerable
time to the technique.
the
restriction
Among
enzymes that we screened,
Alu I proved the most informative
for separating
the
but
A.
A.
and
of
A.
some
the
taxa,
ostoyae,
gemina
borealis isolates had the same restriction
The
pattern.
IGS sequences
and Stasovski,
for the three taxa are similar (Anderson
1992). The first two species are mor?
similar (Berube and Dessureault,
phologically
1989)
but much different in biology (Rizzo and Harrington,
North America, the amplified
1993). In northeastern
from A. ostoyae, but not A. gemina, should
products
be cleaved by Bsm I or Nde I. In Europe, where both
A. ostoyae and A. borealis are known, digestions
with
Bsm I could distinguish
the two species in most cases.
for A. cepistipes differs
substantially
species (Anderson
and Stasovski, 1992), the A. cepistipes type B restriction
pattern with Alu l was the same as the A. ostoyae pat?
the IGS product of A. cepistipes can
tern. However,
Although
the
IGS sequence
from the above
three
be distinguished
from the other
II restriction
of
a
Hind
presence
Both A. borealis isolates
species
site.
tested
by the
by Anderson and
A. borealis type B
sequenced
Stasovski (1992) had the predicted
Alu I digestion
pattern and not the type A pattern.
The type A A. borealis pattern produced
by Alu I di?
gestion is identical to that of A. ostoyae, but, unlike A.
ostoyae, all isolates of A. borealis lack a Bsm I restriction
site in this region. However, the amplified DNA from
our isolate B177 (Germany) of A. ostoyae failed to digest with Bsm I, and A. ostoyae isolate 337 (Germany)
of Anderson and Stasovski (1992) had an IGS sequence
lacking the Bsm I site. Thus, our A. ostoyae isolate Bl 77
and their A. ostoyae isolate 337 would be identified
as
A. borealis type A using our technique.
Comparisons
of the IGS sequences
and Stasovski, 1992)
(Anderson
show that the sequence of isolate 337 is similar to both
A. borealis and A. ostoyae. Thus, it could be speculated
that isolates B177 and 337, if accurately identified by
intermediates
be?
evolutionary
pairings,
represent
the closely related A. borealis and A. ostoyae.
The IGS region for A. cepistipes type A and type B
differed
at two Alu I restriction
sites. Our type A
isolates had a restriction
consistent
with pubpattern
lished sequences
of isolates 311 (Finland) and 316
and Stasovski (1992). Their iso?
(France) of Anderson
late 304 (Finland),
had an IGS sequence
however,
much different from that of the other two isolates of
A. cepistipes they studied and had a predicted
Alu I
restriction
from
different
that
we
tested.
pattern
any
None ofthe isolates studied by Anderson and Stasovski
that would result in the
(1992) had an IGS sequence
B
Alu I digestion.
A.
with
There
type
cepistipes pattern
tween
at least two, and possibly
are, therefore,
ent A. cepistipes IGS types.
The distinction between A. sinapina, A.
NABS XI remains clouded (Anderson et
rube and Dessureault,
1988; Guillaumin
three,
differ?
cepistipes and
al., 1987; Be?
et al., 1989).
Armillaria
North
sinapina is known from northern
and
Shaw
and
(Berube
Dessureault,
1988;
and
Worrall,
Loopstra,
1988; Mallett, 1990; Blodgett
and Rizzo, 1993). Armillaria cepis?
1992a; Harrington
tipes is reported from Europe (Guillaumin et al., 1993),
but NABS XI (group F sensu Morrison)
from northAmerica
western
North America is at least partially interfertile
in pairing studies with A. cepistipes and may be the
same species (Morrison
et al., 1985). We found only
one Alu I restriction
pattern among our A. sinapina
and this pattern was predicted
isolates,
by the se?
quence for A. sinapina isolate 48 (New York) of An?
derson and Stasovski (1992). The sequence
of their A.
sinapina isolate 205 (British Columbia), however, would
give an Alu I restriction pattern identical to A. cepistipes
we did not have isolates of
type A. Unfortunately,
NABS XI available for testing, nor did Anderson
and
Stasovski (1992) sequence
the IGS region of NABS XI
isolates.
The other two undescribed
North American taxa of
Armillaria (NABS IX and X) have unique IGS sequenc?
es (Anderson and Stasovski, 1992) and Alu I restriction
patterns. It should be pointed out, however, that only
a limited number of isolates of NABS IX and X were
used in these
In most
studies.
respective
cases where two Alu I restriction
were seen within an Armillaria
species,
patterns
a difference
at
a single restriction site would explain the discrepancy.
The geographic
origin of the isolates did not appear
to correspond
with the within-species
variation, except
in A. gallica. The North American A. gallica pattern
is consistent
with the sequence published for the North
434 (Michigan),
137 (Michigan) and
90 (Vermont) studied by Anderson and Stasovski (1992).
Their sequence for the European isolate 332 (France),
from the North Ameri?
however, differs substantially
can A. gallica isolates, and the predicted Alu I restric?
American
isolates
tion pattern for this isolate is consistent
with the pat?
tern obtained with our European A. gallica isolates.
The sequence ofthe IGS region ofthe North Amer?
ican A. gallica isolates is more similar to the North
American species A. calvescens than to the sequence in
the European A. gallica isolate (Anderson and Stasov?
found in the
ski, 1992). In fact, the only difference
of
these
two
North
American species is at a
sequence
I
Tha
but
we
found
of the IGS
site,
single
digestion
with
this
restriction
to
be
unreliable.
region
enzyme
Basidiomes
of the two species are morphologically
dis?
tinct (Berube and Dessureault,
but
these
species
1989),
are similar in other respects,
their weak
including
and large, monopodially
branched rhipathogenicity
Harrington
and
Wingheld:
(Berube and Dessureault,
zomorphs
1989; Blodgett
and Worrall, 1992a, b; Harrington
and Rizzo, 1993;
Rizzo and Harrington,
North
1993). In northeastern
where A. calvescens is known to occur, A.
America,
calvescens
may have recently evolved from A. gallica
and is better adapted to the more northern hardwood
forests dominated
by maple, whereas A. gallica re?
mains more prevalent in forests dominated
by oak and
at locations
south
of
the
known
distribution
typically
(Berube and Dessureault,
1989; Blod?
and
and
Worrall, 1992a, b; Harrington
Rizzo, 1993;
gett
Rizzo and Harrington,
1993).
Only the A. tabescens isolates had amplified products
Armillaria
287
Identification
Anderson is greatly appreciated, as is the technical assistance
of Joe Steimel. This material is based upon work supported
in part by a cooperative agreement with the U.S. Forest
Service, Forest Products Laboratory. We also thank the
Foundation for Research Development and Mondi Paper
Company, South Africa for financial assistance to BDW dur?
ing her sabbatical in Ames, Iowa. Journal Paper No. J-15969
of the Iowa Agriculture and Home Economics Experiment
Station, Ames, Iowa Project No. 0159.
of A. calvescens
of 845 bp. One isolate appeared to have an extra Alu
I restriction
the six isolates had
site, but otherwise,
the same restriction
pattern. Two isolates of A. tabes?
cens from Korea also had this pattern (data not shown).
The A. tabescens isolates from Europe and North Amer?
ica also had the same restriction
pattern with Mse I
in restriction
(data not shown). This near uniformity
patterns suggests that A. tabescens from Europe and
is a single species (Darmono
North America
et al.,
in
of
the
of
be?
spite
intersterility
1992),
suggestion
tween isolates from the two continents
et
(Guillaumin
al., 1993).
Each of the A. mellea isolates
tested had an amplified
of
one
of two unique Alu I
875
and
product
bp
gave
restriction
the
shown
patterns:
pattern
by five collec?
tions from northeastern
North America and the pat?
tern shown by the isolates from Britain and California.
from Japan had the Alu I pattern of the
latter group (data not shown). All A. mellea isolates
tested had the same pattern when digested with Mse
I (data not shown). When digested with Cfo I, the two
An isolate
British
had a pattern that differed from the
(data not shown). Thus, there
was some variability in A. mellea in the IGS restriction
other
isolates
A. mellea isolates
with the geographic
or?
patterns that may correspond
igin of the isolates, but the data are very limited.
More sampling would likely reveal new restriction
patterns among
North America,
the Armillaria species
but the most common
of Europe and
Alu I patterns
were
likely revealed
by this study. With our limited
of Alu I digests
data, it appears that a combination
and digestions
with other select enzymes can distinArmillaria species of these two
guish all the recognized
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