Variation within the internal transcribed spacer (ITS) and the
intergenic spacer (IGS) of the ribosomal RNA gene of isolates
representing seven European species of Armillaria was examined
coupled with RFLP analysis and partial sequencing of the ITS region.
The amplified-ITS region was about 840 bp long and uniform in length
the European isolates, while the amplified-IGS
region showed four different lengths which corresponded to
A. mellea, A. tabescens, A. ectypa and a group
including the four other species. A. borealis, A. ostoyae,
A. cepistipes and A. gallica.
Restriction digestions of the ITS and IGS regions by Alu I
Rsa I, respectively, gave rise to the same four polymorphic groups.
However, A. borealis and A. ostoyae were easily separated
from A. cepistipes and A. gallica by digestion of the
two rDNA spacers
with Hinf I and Taq I. Nde II digests of the
ITS could distinguish A. borealis from A. ostoyae and
each of the seven species
were separated by Alu I digestion of the IGS region. A North
American A. mellea isolate, partly examined in this work, was
to be different from the European A. mellea isolates, while there
was a close similarity between the European A. ostoyae and an
isolate of the same species isolated from the U.S.A.
Cluster analysis based on the presence or absence of comigrating restriction
fragments indicated more than 80% similarity
between A. borealis and A. ostoyae and between
A. cepistipes and A. gallica, while A. mellea,
A. tabescens and A. ectypa were found in
separate clusters exhibiting, respectively, about 40, 38 and 32% average
similarity with the other species.
Although little intraspecific variation was observed in many species,
A. gallica and A. cepistipes were found to be heterogeneous.
view of recent results suggesting several groups in A. borealis
A. cepistipes, the Alu I restriction patterns obtained
in this work
would identify the former species as type B, characterized by an Alu
pattern different from that of A. ostoyae, and the latter species
composed of one isolate (C4) of type B and three isolates (C1, C2 and C3)
of type A.
Nucleotide sequences of the rDNA internally transcribed spacer 1 (ITS1)
one isolate of each species showed that A. mellea and
A. ectypa differed from the other species by several insertions
point mutations giving rise to a level of similarity ranging from
66 to 79%. This DNA region was highly conserved within the other species
which revealed a similarity of 97 to 100%.
These results demonstrate that analysis of rDNA spacers provides appropriate
data to circumscribe taxonomic entities within the
Armillaria complex and that the phylogenic relationships among
deduced from the present study are consistent with
previous analyses based on pairing tests as well as morphological and physiological