Identification of indigenous and introduced symbiotic fungi in ectomycorrhizae by amplification of nuclear and mitochondrial ribosomal DNA

Abstract

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 a 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. proximo, 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. Key words: internal transcribed spacer, LrDNA gene, mycorrhizal ecology, polymerase chain reaction, rDNA.