194

Forest pathology

DNA polymorphism and molecular diagnosis in

Inonotus spp.
Hugo Germain, Gaston Laflamme, Louis Bernier, Bruno Boulet, and
Richard C. Hamelin

Abstract: Specific polymerase chain reaction (PCR) primers were developed for the internal transcribed spacer (ITS)
region of the rDNA gene of Inonotus tomentosus, the causal agent of tomentosus root rot of conifers. The primers
were designed to specifically amplify DNA from I. tomentosus and allow its differentiation from Inonotus leporinus
and from Phellinus pini s.l., which are morphologically very similar to I. tomentosus in culture. The PCR amplification
was carried out successfully from DNA extracted from fruiting bodies and cultures and can potentially be used to
detect the pathogen from environmental samples for survey and management purposes. The PCR assay was validated
with 42 samples from seven coniferous hosts originating from eight provinces or states across the North American
continent. No cross reaction was observed with DNA of several other species of the same genus, with Phellinus pini or
with white spruce (Picea glauca), a conifer host of I. tomentosus. Phylogenetic analysis of the ITS region for six
species of Inonotus suggests that these resulted from the adaptation of a generalist ancestor to different ecological
niches. It also appears that divergent evolution of an ancestor occupying different ecological niches has driven the
speciation process, which subsequently conferred specificity to either coniferous or deciduous trees.
Key words: Inonotus, root rot, molecular diagnosis, ribosomal DNA.
Résumé : Des amorces spécifiques de la réaction en chaîne de la polymérase ont été développées pour la région de
l’espaceur transcrit interne de l’ADN ribosomique de l’Inonotus tomentosus, l’agent causal de la carie rouge alvéolaire
du pied chez les conifères, pour le distinguer de l’Inonotus leporinus, qui est morphologiquement identique en culture,
et du Phellinus pini s.l., qui est très similaire à l’I. tomentosus. L’amplification par réaction en chaîne de la polymérase
a été réalisée à partir d’ADN extrait de cultures ou de carpophores. Aucune réaction croisée n’a été observée contre
d’autres espèces du même genre, contre le Phellinus pini ou contre l’épinette blanche (Picea glauca), l’hôte de l’I.
tomentosus. L’analyse phylogénétique de la région des espaceurs transcrits internes effectuée pour six espèces de
l’Inonotus suggère que ceux-ci proviennent de l’adaptation d’un ancêtre commun à des niches écologiques différentes.
Cette adaptation à une niche écologique semble avoir mené à un processus de spéciation et par la suite à une
spécialisation pour les conifères ou pour les feuillus.

Mots clés : Inonotus, pourriture racinaire, diagnostique moléculaire, ADN ribosomique.

Germain et al.: Inonotus

tomentosus / molecular identification 199
Accepted 14 November 2001.
H. Germain. Natural Resources Canada, Canadian Forest
Service, Laurentian Forestry Centre, 1055 du PEPS, P.O.
Box 3800, Sainte-Foy, QC G1V 4C7, and Centre de
recherche en biologie forestière, Université Laval, Cité
universitaire, QC G1K 7P4, Canada.
G. Laflamme and R.C. Hamelin.1 Natural Resources
Canada, Canadian Forest Service, Laurentian Forestry Centre,
1055 du PEPS, P.O. Box 3800, Sainte-Foy, QC G1V 4C7,
Canada.
L. Bernier. Centre de recherche en biologie forestière,
Université Laval, Cité universitaire, QC G1K 7P4, Canada.
B. Boulet. Ministère des Ressources naturelles du Québec,
Département de la conservation forestière, 880, chemin
Sainte-Foy, Québec, QC G1S 4X4, Canada.
1
Corresponding author (e-mail: rhamelin@cfl.forestry.ca).
Introduction
The pathogenic fungus Inonotus tomentosus (Fr.) Teng
infects several North American coniferous species (Whitney
1977). The root and butt rot caused by this fungus is re-
sponsible for major economic losses, particularly in white
spruce (Picea glauca (Moench) Voss) stands. The symp-
toms resulting from an infection of I. tomentosus are very
similar to those caused by Inonotus leporinus (Fr.)
Gilbertson (I. leporinus = I. circinatus = Onnia leporina;
Wagner and Fischer 2001) and Phellinus pini (Brot.:Fr.) A.
Ames, which are other pathogenic, but less virulent, fungi
that cause root disease in white spruce. Growing the fungi
in pure culture does not facilitate identification, since
I. tomentosus and I. leporinus are indistinguishable from
each other in culture (Whitney and Bohaychuk 1977).
Phellinus pini rarely produces chlamydospore-like struc-

Can. J. Plant Pathol. 24: 194–199 (2002)

Germain et al.: Inonotus tomentosus / molecular identification
tures in culture, while the two others regularly do, and it
has thicker setae-like hyphae (Nobles 1958). Other tech-
niques, such as differential culture media (Hunt 1997),
polyclonal antibodies (Hunt et al. 1999), and intramycelial
protein electrophoresis (Hunt and Ekramoddoullah 1996),
may allow differentiation of I. tomentosus and I. leporinus.
However, these assays must currently be conducted with
material derived from cultures.
In addition to overcoming these problems, there are other
reasons why a rapid and accurate diagnostic assay for
I. tomentosus is desirable. Symptoms of the disease can
take several years to develop, and the presence of the patho-
gen (such as indicated by fruiting bodies) is often only visi-
ble when the fungus is well established. Management
options are often reduced at the time when the pathogen is
well established. If the pathogen can be identified from soil,
roots, or stems prior to appearance of the symptoms, the
disease may be easier to manage. In addition, a diagnostic
assay might help us better understand the epidemiology of
the pathogen and the etiology of the disease by providing
reliable data on spread and incidence. The extensive dam-
age caused by I. tomentosus justifies the development of a
reliable, expedient, and user-friendly diagnostic test.
The polymerase chain reaction (PCR) (Saiki et al. 1985)
has been used extensively in DNA identification and offers
the opportunity to selectively amplify DNA from an organ-
ism. The internal transcribed spacer (ITS) region is part of
the ribosomal DNA gene cluster. It is located on each side
of the highly conserved 5.8S region, between the small and
large nuclear ribosomal subunits. The ITS region shows a
high level of interspecific polymorphism, and multiple cop-
ies are present in the cells making it amplifiable from min-
ute amounts of DNA (Gardes and Bruns 1993). These
features make the ITS region a good candidate for design-
ing species-specific primer pairs. The ITS regions have
been used extensively in identification of forest fungi (Kim
et al. 1999; Hamelin et al. 1996, 2000), pathogens of crops
(Bounou et al. 1999), edible mushrooms (Amicucci et al.
1998), and for phylogenetic analysis (Roux et al. 1999). A
review of the subject was also made by Martin et al. (2000).
The objectives of the present study were to (1) study the
phylogenetic relationships among several species of the ge-
nus Inonotus to identify regions of the ITS that are poly-
morphic and discriminant among taxa and (2) develop a set
of PCR primers specific to the ITS region of I. tomentosus.
Materials and methods
Biological material
The following species were used: I. tomentosus, I. lepo-
rinus, Inonotus cuticularis (Bull.:Fr.) P. Karst., Inonotus
glomeratus (P.K.) Murr., Inonotus radiatus (Sowerby:Fries)
Karsten, Inonotus rheades (Pers.) Bond & Singer, and
Phellinus pini. Two isolates of I. tomentosus were used: one
collected in Quebec, and the other, in British Columbia. All
material used was diploid and originated from fruiting bod-
ies. The fungal material used, the host on which the
basidiocarp was found, and the GenBank accession num-
bers are listed in Table 1 for each species used. Forty iso-
lates previously identified as I. tomentosus were used to test
the specific primers (Table 2).
195
DNA isolation
DNA was extracted directly from fruiting body tissue
(stored at –20°C) or mycelium of I. tomentosus isolates us-
ing a modified version of the method described by Zolan
and Pukkila (1986). Approximately 5 mg of tissue were
taken from the sporocarp using a scalpel and placed in a
1.5-mL Eppendorf vial. For mycelium, 3-week-old cultures
(2% malt extract) were filtered on a double layer of
Whatman No. 2 filter paper, which had previously been
rinsed twice with distilled water, and placed in a 1.5-mL
Eppendorf vial. Approximately the same quantity of
diatomaceous earth (Sigma Chemical Co., St. Louis, Mo.)
and 100 µL of extraction buffer (100 mM Tris–HCl
(pH 9.5), 2% cetyltrimethylammonium bromide (CTAB),
1.4 M NaCl, 1% polyethylene glycol 6000, 20 mM EDTA,
and 0.25% β-2-mercaptoethanol) were added and the mix-
ture was ground for 3 min using disposable Kontes pestles
(VWR-Canlab, Toronto, Ont.). Then, 300 µL of extraction
buffer were added, and the tubes were incubated at 65°C for
1 h. Samples were extracted using 400 µL phenol – chloro-
form – isoamyl alcohol (25:24:1), vortexed, and centrifuged
at 10 000 × g for 10 min. The upper phase was transferred
to a 1.5-mL Eppendorf vial, and DNA was precipitated us-
ing 7.5 M ammonium acetate and 600 µL cold isopropanol
and placed at –20°C for 1 h. DNA was pelleted by
centrifugation at 10 000 × g for 10 min and was washed
with 800 µL 70% ethanol. The pellet was dried in a Speed-
vac for 10 min and resuspended in 20 µL Tris–EDTA
(10 mM Tris–HCl (pH 8), 1 mM EDTA). DNA extractions
were verified through migration in 0.8% agarose gel. DNA
extractions were diluted 1:50 and stored at –20°C.
Amplification with universal primers ITS-1F and ITS-
4 and with specific primers
Amplifications were performed using universal primers
ITS-1F (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and ITS-
4 (5′-TCCTCCGCTTATTGATATGC3′) under the following
conditions: 36 cycles of 30 s at 92°C, 30 s at 55°C, and
1 min at 72°C. Reactions were preceded by a 3-min dena-
turation step at 94°C and terminated with a 10-min elonga-
tion step at 72°C. Sequence analysis yielded two new
I. tomentosus specific primers it-ITS-209-f (GCTAAATCC-
ACTCTTAACAC) and it-ITS-700-rc (AGGAGCCGACCA-
CAAAAGAT) with the following parameters: 35 cycles of
30 s at 94°C, 30 s at 50°C, and 30 s at 72°C preceded by
3 min at 94°C and ending with a 5-min elongation step at
72°C. Amplification products were separated on an agarose
gel (1.5%) in 1× TAE (Tris–acetate–EDTA), stained in
ethidium bromide and visualized under an ultraviolet lamp.
All amplifications were carried out under the following con-
ditions: DNA was amplified in 25 µL volume containing
10 mM Tris–HCl, 50 mM KCl, 1.5 mM MgCl2 (pH 8.3);
50 µM of each deoxyribonucleotide triphosphate (dATP,
dCTP, dTTP, dGTP); 1 µM of each primer; and 1 unit of
Taq polymerase (Boehringer Gmbh, Mannheim, Germany).
Primers it-ITS-209-f and it-ITS-700-rc, designed to be
specific for I. tomentosus, were used in a PCR experiment
on seven different species of fungi (Fig. 1). Parallel amplifi-
cations were also performed using universal primers ITS-1F
and ITS-4 to exclude amplification failure due to poor DNA
quality.
196 Can. J. Plant Pathol. Vol. 24, 2002

Table 1. Isolate identification, GenBank accession number, and host for each species analyzed.
Host on which GenBank
Species Isolate it was found accession No.
Inonotus tomentosus 05-10-07, B.C.* Picea glauca AF237729
Inonotus leporinus QFB-813 Picea glauca AF237728
Inonotus cuticularis QFB-888 Unknown AF237730
Inonotus glomeratus QFB-893 Acer sp. AF247968
Inonotus radiatus QFB-322 Unknown AF237732
Inonotus rheades QFB-571 Unknown AF237731
Phellinus pini QFB-815 Picea glauca AF420589
*British Columbia.

Table 2. Results of the amplification of the Inonotus tomentosus isolates tested with it-ITS-209-f and it-ITS-700-rc.
Amplification with
Collection it-ITS-209-f and
Reference No. Origin year Host it-ITS-700-rc
Bud-1556 Ontario 1948 Picea mariana +
Bud-1703-A Ontario 1948 Picea glauca +
Bud-1848 Ontario 1948 Abies balsamea +
Colo-51-112 Colorado 1951 Picea engelmannii +
Colo-57-68-R Colorado 1957 Picea engelmannii +
FP-100229-Sp Colorado 1952 Picea engelmannii +
FP-100585-Sp Saskatchewan 1968 Picea glauca +
FP-100926-Sp Ontario 1971 Picea glauca +
FP-100927-Sp Ontario 1971 Picea glauca +
FP-100928-T Ontario 1971 Picea glauca +
FP-100929-Sp Ontario 1971 Picea glauca +
FP-100930-T Ontario 1971 Picea glauca +
FP-100931-Sp Ontario 1971 Picea glauca +
FP-100931-T Ontario 1971 Picea glauca +
FP-102419 New York 1990 Pinus strobus +
Loo-13789-Ov Pennsylvania 1931 Pinus rigida +
Mad-549-T Minnesota 1923 Picea mariana +
P2-2-R Ontario 1968 Picea glauca +
P3-2-R Ontario 1968 Picea glauca +
SSMF-695-4974-01 Ontario 1969 Pinus strobus +
FP90083-sp Colorado 1945 Picea engelmannii +
NoFC-41 Alberta 1947 Picea engelmannii +
NoFC-299 Alberta 1962 Larix lyalii +
NoFC-311 Alberta 1952 Picea engelmannii +
NoFC-312 Alberta 1952 Picea glauca +
NoFC-313 Alberta 1952 Picea glauca +
NoFC-540 Saskatchewan 1956 Picea glauca +
NoFC-543 Saskatchewan 1956 Picea glauca +
CFL-115 Quebec 1957 Picea mariana +
CFL-415 Quebec 1961 Picea glauca +
CFL-479 Quebec 1962 Picea glauca +
CFL-503 Quebec 1962 Picea glauca +
CFL-505 Quebec 1979 Picea mariana +
CFL-506 Quebec 1962 Picea glauca +
Marsboro940915 Quebec 1999 Picea glauca +
it-9905-a Quebec 1999 Picea glauca +
it-9901 Quebec 1999 Picea glauca +
it-Quebec-99 Quebec 1999 Picea glauca +
M1–99 Quebec 1999 Picea glauca +
M2–99 Quebec 1999 Picea glauca +
M3–99 Quebec 1999 Picea glauca +
M4–99 Quebec 1999 Picea glauca +
Germain et al.: Inonotus tomentosus / molecular identification 197

Fig. 1. Amplification product obtained with the specific primers it-ITS-209-f and it-ITS-700-rc and with the universal primers ITS-1F
and ITS-4. Lanes 1 and 11 are Gibco BRL 100-bp DNA ladders, lanes 2–10 show amplification with specific primers, and lanes 12–20
show amplification with universal primers. The lanes contained the following DNA: lanes 2 and 12, Inonotus tomentosus (culture, 05-
10-07, British Columbia); lanes 3 and 13, Inonotus tomentosus (fruit body, Quebec); lanes 4 and 14, Inonotus leporinus; lanes 5 and
15, Inonotus cuticularis; lanes 6 and 16, Inonotus glomeratus; lanes 7 and 17, Inonotus radiatus; lanes 8 and 18, Inonotus rheades;
lanes 9 and 19, Phellinus pini; and lanes 10 and 20, negative control.

Cloning and sequencing
The PCR products obtained with primers ITS-1F and
ITS-4 were cloned using the vector pCR 2.1 according to
the manufacturer’s instructions (Invitrogen, Carlsbad, Ca-
lif.). The clones were sequenced using primers ITS-1F and
ITS-4 (Gardes and Bruns 1993) on an ABI 373 (Applied
Biosystems, Foster City, Calif.) automatic sequencer. All
sequences were deposited in GenBank (Table 1).
Data analysis
Phylogenetic analysis of fragment sequences was used to
determine the relationship among the members of the
Inonotus genus and to test whether molecular data would
support the morphological information indicating that
I. tomentosus and I. leporinus are closely related. A pheno-
gram was inferred with PAUP (Wisconsin Package version
10.0; Genetic Computer Group, Madison, Wis., U.S.A.) us-
ing maximum parsimony analysis with branch support be-
ing calculated using a bootstrap (500 iterations) method.
Phellinus pini was used as an outgroup to provide rooting
of the tree. Sequences were manually edited and aligned us-
ing CLUSTALW version 1.8. This alignment was then used
to design primers specific to I. tomentosus and to draw the
phenogram. The program PRIMER3 (available from http://
www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi)
was used to design the specific primers for I. tomentosus.
Results
The DNA extraction protocol, described by Zolan and
Pukkila (1986), yielded amplifiable DNA for all fungal spe-
cies and tissues used in this study.
The DNA extraction protocol, described by Zolan and
Pukkila (1986), yielded amplifiable DNA for all fungal spe-
cies and tissues used in this study.
DNA amplifications using the universal primers (ITS-1F,
ITS-4) resulted in PCR products of various lengths for the
isolates tested. Inonotus tomentosus, I. leporinus, and
I. glomeratus showed fragment length of approximately 800
base pairs (bp), whereas I. radiatus, I. rheades, and Phellinus
pini showed a fragment of 750 bp and I. cuticularis showed
a fragment of approximately 750 bp in length. Length poly-
morphism between the different species of Inonotus ampli-
fied with the universal primers is shown in Fig. 1.
Electrophoresis of the PCR products generated with spe-
cific primers (it-ITS-209-f, it-ITS-700-rc) revealed a frag-
ment of approximately 500 bp; 491 bp was expected from
the sequence data used to design the primers. An
amplification product was observed for the two isolates of
I. tomentosus used in this study, and no amplification prod-
uct was generated for any of the other species (Fig. 1).
These primers were tested against 42 isolates (Table 2) of
I. tomentosus from different locations in Canada and the
United States. All specimens, cultures, or fruiting bodies
previously identified as I. tomentosus tested positive (data
not shown), while false negatives were not observed.
The phenogram inferred from the alignment suggested
that I. tomentosus and I. leporinus were evolutionarily
closely related to one another, with a divergence of 0.8%
over 491 nucleotides, compared with an average of 8.6%
between I. tomentosus and the other Inonotus species used
in this study. The two fungal species pathogenic on conifers
were in the same clade, while all three species from decidu-
198
Fig. 2. Phylogenetic relationships among the genus Inonotus using Phellinus pini as an outgroup. Rooted tree using maximum
parsimony analysis of ITS-1, 5.8S, and ITS-2 DNA sequences revealed that 105 characters were parsimony informative. Branch lengths
were drawn proportional to the number of changes. Bootstrap values are indicated on branches. The homoplasy index is 0.1542.
ous hosts clustered in a different clade. The conifer clade
was highly supported (bootstrap value of 100%), while the
deciduous clade was supported at a lower value (59%).
Discussion
Inonotus tomentosus causes one of the most important
root rots affecting coniferous species of the northern hemi-
sphere. The symptoms caused by the fungus are not specific
to I. tomentosus and, therefore, generally cannot be used to
diagnose an infection by this fungus. Fruiting bodies of the
fungus can provide material for identification of the fungus;
however, they are very ephemeral and do not necessarily
occur at the same time from one year to another, if they oc-
cur at all. In vitro cultures of the fungus are extremely simi-
lar to other root rot pathogenic cultures, such as other
Inonotus species and Phellinus pini, making identification
from cultures difficult. For these reasons, a molecular tech-
nique was developed to help with the identification of this
fungus.
Polymerase chain reaction amplifications revealed that
the set of primers designed for I. tomentosus was specific to
this species. In addition, since amplification was positive
for I. tomentosus from the provinces of British Columbia
and Quebec, we can assume that DNA extracted from both
cultured tissues and fruiting bodies from different origins
can be amplified using these primers. Furthermore, no cross
reactions were observed using this PCR assay with any of
the other species of Inonotus, Phellinus pini, or their host,
Picea glauca wood samples.
These results suggest that the test could provide a spe-
cific assay to detect this pathogen, not only from fruiting
bodies or culture, but also directly from roots or stems of
infected trees. A similar assay for detecting root rot infec-
tions caused by Cylindrocladium floridanum Sobers & C.P.
Seymour and Cylindrocarpon destructans (Zinssmeister)
Scholten from conifer seedlings has been reported to detect
all infected seedlings from which cultures of the pathogen
had been obtained (Hamelin et al. 1996). Positive control
Can. J. Plant Pathol. Vol. 24, 2002
tests with universal ITS primers allowed the exclusion of
PCR failure caused by low-quality DNA. These results indi-
cate that this protocol can be used to identify this pathogen
from different material without the time-consuming step of
culturing the fungus. However, proper validation that would
correlate the molecular assay with isolation of the fungus as
well as symptom development would be required before re-
liability of this assay using field material can be assessed.
The phylogenetic tree obtained is in agreement with eco-
logical and morphological data. Phylogenetic analysis indi-
cated the presence of two different groups within the genus
Inonotus. Inonotus tomentosus and I. leporinus constitute
one clade, and the other clade is composed of I. radiatus,
I. glomeratus, I. rheades, and I. cuticularis. All six species
have the ability to cause white rot decay, which is charac-
terized by a degradation of the lignin of the tree.
However, ecological and morphological data support the
fact that I. tomentosus and I. leporinus are separated from
all other taxa within this genus. Both are pathogenic to co-
niferous species, whereas the other four species are associ-
ated with deciduous tree species (Gilbertson and Ryvarden
1986). Even though they share a common ancestor with the
other species, I. tomentosus and I. leporinus probably
evolved separately, enabling them to occupy a different eco-
logical niche.
Inonotus radiatus is also ecologically different from all
other taxa included within this analysis. It is the only truly
saprophytic fungus of deciduous trees. It will attack trees
only when the tissue is dead and will often colonize wood
when degradation is already well underway (Gilbertson and
Ryvarden 1986). Its main role is to recycle woody material
of deciduous trees like birch and alder. This ecological ob-
servation is consistent with the phylogenetic tree where we
find I. radiatus in the clade of fungi that infect deciduous
species but with a fairly weakly supported branch (59%)
(Fig. 2).
The other three species within this clade, I. glomeratus,
I. rheades, and I. cuticularis, cause heartwood decay of de-
ciduous trees and cause progressive elimination of old trees
Germain et al.: Inonotus tomentosus / molecular identification
(Boulet 1995). The branch containing these taxa in the
phylogenetic analysis is supported by a bootstrap value of
86%. Of these three decay-causing fungi, I. glomeratus is
the most aggressive, particularly on maple and beech.
Inonotus cuticularis is an opportunist decay fungus of liv-
ing hardwood (mainly elms), whereas I. rheades is particu-
larly aggressive on aspens. The sporocarps of I. rheades
have a granular core with highly branched hyphae
(sclerids), a characteristic which is not encountered in other
species of this genus. This, however, is not paralleled by dif-
ferences in the molecular sequences analyzed in our study.
Based on morphological evidence, I. rheades is sometimes
classified in a different subgenus (Dai et al. 1997). The
grouping of I. cuticularis and I. rheades could be the only
ambiguity between ecological and molecular data of this
phylogenetic tree. The analysis of another locus or the anal-
ysis of a different character will help in deciding if this
morphological feature is significant. Also, a larger set of
taxa would be desirable to resolve the type of questions
raised above.
Ecological niches appear to have played a major role in
the speciation and evolution processes of these fungi. Evo-
lution of an ancestor infecting hosts from different ecologi-
cal niches appears to have led to a speciation process,
which conferred specificity to either coniferous or decidu-
ous species.
The specific primers developed in this study will allow reli-
able, sensitive, and expedient identification of I. tomentosus.
This molecular identification process (DNA extraction, am-
plification, and revelation) can be completed within 2 days,
leading to unambiguous results and providing a valuable
identification test for phytopathologists. The use of this as-
say directly from roots, soil samples, or stems would be ex-
tremely useful to predict and monitor the presence and
spread of this pathogen in forest plantations and natural
stands. This information would provide a valuable tool for
forest managers.
Acknowledgements
Special thanks are extended to Rita Rentmeester (USDA
Forest Service, Madison, Wis.) and Rich Hunt (Natural Re-
sources Canada, Canadian Forest Service, Pacific Forestry
Centre, Victoria, B.C.) for providing fungal material, to
Jean Bérubé (Natural Resources Canada, Canadian Forest
Service, Laurentian Forestry Centre, Sainte-Foy, Que.) for
reviewing the manuscript, and to Nicole Lecours (Natural
Resources Canada, Canadian Forest Service, Laurentian
Forestry Centre), whose technical expertise was very help-
ful throughout this study.
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