Specificity, sensitivity and discrimination of primers for PCR-RFLP of larger basidiomycetes and their applicability to identification of ectomycorrhizal fungi in Eucalyptus forests and plantations

Techniques to rapidly identify the basidiomycete fungal partner of ectomyco rrhizal associations would be a major advantage for ecological. fungal popu lation dynamics and life history studies of epigeous and hypogeous forms in plantations, forests, wild lands and other native or natural vegetation. P CR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism ) identification of DNA regions is an available technique; however, primers which have a high probability of amplifying only the basidiomycete DNA are needed. Here we have assessed the specificity, sensitivity and discriminat ion of six different primer pairs, three targeting nuclear anti three mitoc hondrial regions, for use in identification of Australian basidiomycete fun gi from Eucalyptus forests by matching PCR-RFLP patterns to morphologically defined species. Two sets of primers, one newly designed and targeting the nuclear ribosomal DNA internal transcribed spacers (ITS) and the other amp lifying a fragment of mitochondrial large subunit ribosomal DNA met the req uirements of high specificity and sensitivity, amplifying DNA from a broad range of larger basidiomycetes. with no amplification of plant. bacterial o r ascomycete DNA. The specificity of the ITS primer pair was compared with that of ITS1-F/ITS4-B. PCR-RFLP of the two regions discriminated fungi to s pecies level for 91 fungal species from 28 families. Hence these two DNA re gions and the specific primers are a potential practical PCR-RFLP tool for identifying basidiomycetes associated with plants from field samples.