Rapid identification of genetic variation of ectomycorrhizal fungi by amplification of ribosomal RNA genes.

Abstract

There is a clear requirement to develop sensitive methods for detecting denned isolates of ectomycorrhizal fungi within the complex microbial communities of natural ecosystems and reforestation sites. We present a method that permits the rapid identification of an ectomycorrhizal isolate using enzymatic amplification (polymerase chain reaction) of DNA extracted either from pure cultures or ectomycorrhizas. A set of oligonucleotide primers capable of amplifying full-length nuclear 17S and 25S ribosomal RNA genes, together with the ribosomal internal transcribed spacer and intergenic spacer, have been designed and could be used for amplifying target sequences from a wide range of ectomycorrhizal genera. Length polymorphism in the amplified rDNA and restriction endonuclease analysis of nearly 6-0 kbp of amplified rDNA provided useful criteria for the rapid typing of isolates from different genera and species. Restriction endonuclease analysis of amplified DNA from 26 isolates representing four species of Laccaria (L. bicolor, L. laccata, L. proxima, L. tortilis) yielded up to 20 scored RFLPs and revealed interspecific and intraspecific polymorphism. Most of the polymorphisms were located within the regions corresponding to the internal transcribed spacer and intergenic spacer. The degree of variation observed was sufficient to discriminate several isolates from the same species. Genetic variation was correlated to some extent with geographical origin of the isolates. However, RFLPs of the rRNA genes cannot unambiguously discriminate all selected isolates within Laccaria species, requiring the development of additional DNA probes. Alone, or in combination with other DNA probes, the amplified rDNA genes may serve in the determination of pure fungal cultures and in the characterization of genetic variation of field ectomycorrhizal populations.