Identification of indigenous and introduced symbiotic fungi in
ectomycorrhizae by amplification of nuclear and mitochondrial
Gardes, M; White, T J; Fortin, J A; Bruns, T D; Taylor, J W
Department of Plant Pathology,
University of California, Berkeley CA 94720
Canadian Journal Of Botany. 1991 69(1):180-190.
We used the polymerase chain reaction (PCR) to amplify specific
regions of the nuclear and mitochondrial genomes of fungi using DNA
extracted from pure cultures as well as that directly from
ectomycorrhizal rootlets. The internal transcribed spacer (ITS)
of the nuclear ribosomal repeat unit and portion of the
mitochondrial large subunit ribosomal RNA gene were chosen as target
sequences because both exist in high copy number and amplification
primers for both discriminate between plant and fungal DNAs.
These features provided a sensitivity and specificity sufficient for
detection and analysis of a single mycorrhizal rootlet. We evaluated
the variations in the amplified products with regard to the length,
restriction endonuclease sites, and primary sequence for use in
identification of genera, species and strains of ectomycorrhizal
fungi, with particular attention to selected Laccaria species.
Accidental contamination of jack pine seedlings by Telephora
terrestris was easily recognized. Amplification and direct DNA
sequencing of a portion of the ITS were done for three strains of
L. bicolor, one of L. laccata, one of L. proxima, and one of T.
terrestris. The nucleotide sequence variation was 32% between
L. bicolor and T. terrestris, and it ranged from 3 to 5% among the
three Laccaria species examined and from 1 to 2% within L. bicolor.
The degree of variation observed is sufficient to allow the use of
specific oligonucleotides to characterize amplified ITS products.
To demonstrate the feasibility of this approach we designed and tested
a probe that enabled two isolates of L. bicolor to be distinguished
by a single base-pair difference in a filter-based hybridization
assay. In combination these methods now provide an important set
of tools for the study of mycorrhizal ecology.