The early development of indigenous and introduced Scots pine
(Pinus sylvestris L.) ectomycorrhiza in natural
forest humus was examined using molecular fingerprinting techniques. Non-mycorrhizal
or mycorrhizal (Suillus
bovinus (L. ex Fr.) O. Kuntze or Paxillus involutus (Batsch
ex Fr.)
Fr.) seedlings were placed in transparent two-dimensional microcosms on
a thin
layer of sieved humus to allow observation of ectomycorrhizal development
and
fungal growth. Twelve ectomycorrhizal morphotypes, mainly based on colour,
gross
morphology and outer mantle
structure, were identified over a 3-month period. No successional trends
in
mycorrhiza formation were observed.
For further characterization, individual ectomycorrhiza representing the
different
morphotypes were subjected to
analyses of esterase (EST) isozyme profiles and RFLPs of the internal transcribed
spacer (ITS) from fungal rRNA
genes following amplification using PCR. All 10 morphotypes analysed displayed
different esterase isozyme
profiles, and the characteristic S. bovinus species diagnostic
band
(S. b. EST8) was detected in two white
morphotypes. Ten Pink and Black ectomycorrhiza were all separated into
one and
three groups, respectively. A
fast-running plant-specific polymorphic locus (Z) was also confirmed in
most
ectomycorrhizal morphotypes.
Diagnostic species-specific (S. bovinus and Pink) EST bands were
detected in intact external mycelium colonizing
soil. Successful amplification of the ITS from individual ectomycorrhiza,
of
eight different morphotypes, was
found to be mainly influenced by the DNA template concentration. Different
RFLPs
(Hinf I, Mbo I and Hha I)
of the ITS placed the white morphotypes into two groups, one corresponding
to
S. bovinus, in agreement with the
esterase fingerprinting. By contrast, Pink and Beige morphotypes displayed
different EST profiles but nearly
identical RFLP fingerprints. The ITS amplification success rate of standardized
DNA template concentrations
from 10 individual Pink and Black ectomycorrhiza was 90 and 50%, respectively.
In all successful amplifications,
the black morphotype yielded the smallest ITS fragment, similar to earlier
reports for Cenococcum geophilum, that
gave identical RFLPs. The ecological significance of the observed mycorrhizal
diversity and combined application of these two identification methods
is discussed.