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

Abstract

Techniques to rapidly identify the basidiomycete fungal partner of ectomycorrhizal associations would be a major advantage for ecological, fungal population dynamics and life history studies of epigeous and hypogeous forms in plantations, forests, wild lands and other native or natural vegetation. PCR-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 discrimination of six different primer pairs, three targeting nuclear and three mitochondrial regions, for use in identification of Australian basidiomycete fungi 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 amplifying a fragment of mitochondrial large subunit ribosomal DNA met the requirements of high specificity and sensitivity, amplifying DNA from a broad range of larger basidiomycetes, with no amplification of plant, bacterial or 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 species level for 91 fungal species from 28 families. Hence these two DNA regions and the specific primers are a potential practical PCR-RFLP tool for identifying basidiomycetes associated with plants from field samples.

Polymorphism in the internal transcribed spacer (ITS) of the ribosomal DNA of 26 isolates of ectomycorrhizal fungi

“…However, in some cases, heterologous DNA from the plant material did not interfere with PCR amplification (Gardes et al, 1991;Henrion et al, 1992;Erland, 1995). Glen et al (2001b) tested six primers pairs (targeting three nuclear and three mitochondrial regions) for specificity, sensitivity and species discrimination on identified collections of fungi. Two sets of these primers, one newly designed and targeting the ITS region and the other amplifying a ribosomal DNA fragment of the large mitochondrial subunit met the requirements of high specificity and sensitivity, amplifying DNA from a broad range of the larger basidiomycetes, with no amplification of plant, bacterial or ascomycete DNA.…”

Interspecific and intraspecific variation of ectomycorrhizal fungi associated with Eucalyptus ecosystems as revealed by ribosomal DNA PCR-RFLP

“…n et al 1997 and this is critical if the target DNA is in complex mixtures of plant, fungal (including soil ascomycete), bacterial and possibly insect and other DNA. The new primers amplified DNA from a larger number of species than previous basidiomycete-specific primers, and from diverse orders of basidiomycetes (Glen et al 2001).…”

“…To distinguish a wide array of Australian fungi and to discriminate them from plants Glen et al (2001) developed sensitive larger basidiomycete specific primers for rDNA ITS. Importantly, they did not also amplify plant, ascomycete or bacterial DNA as had previous commonly-used primers (Gardes & Bruns 1993, Ka/ re!…”

“…A list of the collections of 109 species of larger basidiomycetes from the study site is given in Table 1. PCR-RFLP profiles for 41 of these species (Table 1) are given in Glen et al (2001).…”

“…DNA extraction, primers, PCR amplification, restriction digestion and PAGE DNA was extracted, amplified, digested, electrophoresed and the fragments sized as described in Glen et al (2001). Primers ITSF and ITSR (Glen et al 2001) for the rDNA ITS and ML3 and ML4 (White et al 1990) for the mitochondrial LSU rDNA were used with the conditions specified in Glen et al (2001) to selectively amplify basidiomycete DNA.…”