IDENTIFICATION OF SOME ECTOMYCORRHIZAL BASIDIOMYCETES BY PCR AMPLIFICATION OF THEIR GPD (GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE) GENES

Degenerated oligonucleotide primers designed to Bank an approximately 1.2-kb fragment of the gene encoding glyceraldehyde-3-phosphate dehydr ogenase (gpd) from ascomycetes and basidiomycetes were used to amplify the corresponding gpd fragments from several species of the ectomycor rhizal fungal taxa Boletus, Amanita, and Lactarius. Those from B, edul is, A, muscaria, and L, deterrimus were cloned and sequenced, The resp ective nucleotide sequences of these gene fragments showed a moderate degree of similarity (72 to 76%) in the protein-encoding regions and o nly a low degree of similarity in the introns (56 to 66%). Introns, wh ere present, occurred at conserved positions, but the respective posit ions and numbers of introns in a given taxon varied, The amplified fra gment from a given taxon could be distinguished from that of others by both restriction nuclease cleavage analysis and Southern hybridizatio n. A procedure for labeling DNA probes with fluorescein-12-dUTP by PCR was developed, These probes were used in a nonradioactive hybridizati on assay, with which the gene could be detected in 2 ng of chromosomal DNA of L. deterrimus on slot blots, Taxon-specific amplification was achieved by the design of specific oligonucleotide primers, The applic ation of the gpd gene for the identification of mycorrhizal fungi unde r field conditions was demonstrated, with Picea abies (spruce) mycorrh izal roots harvested from a northern alpine forest area as well as fro m a plant-breeding nursery. The interference by inhibitory substances, which sometimes occurred in the DNA extracted from the root-fungus mi xture, could be overcome by using very diluted concentrations of templ ate DNA for a first round of PCR amplification followed by a second ro und with nested oligonucleotide primers, We conclude that gpd can be u sed to detect ectomycorrhizal fungi during symbiotic interaction.