ITS primers with enhanced specificity for basidiomycetes ‐ application to the identification of mycorrhizae and rusts

We have designed two taxon‐selective primers for the internal transcribed spacer (ITS) region in the nuclear ribosomal repeat unit. These primers, ITS1‐F and ITS4‐B, were intended to be specific to fungi and basidiomycetes, respectively. We have tested the specificity of these primers against 13 species of ascomycetes, 14 of basidiomycetes, and 15 of plants. Our results showed that ITS4‐B, when paired with either a ‘universal’ primer ITS1 or the fungal‐specific primer ITS1‐F, efficiently amplified DNA from all basidiomycetes and discriminated against ascomycete DNAs. The results with plants were not as clearcut. The ITS1‐F/ITS4‐B primer pair produced a small amount of PCR product for certain plant species, but the quantity was in most cases less than that produced by the ‘universal’ ITS primers. However, under conditions where both plant and fungal DNAs were present, the fungal DNA was amplified to the apparent exclusion of plant DNA. ITS1‐F/ITS4‐B preferential amplification was shown to be particularly useful for detection and analysis of the basidiomycete component in ectomycorrhizae and in rust‐infected tissues. These primers can be used to study the structure of ectomycorrhizal communities or the distribution of rusts on alternate hosts.

[1]  C H Porter,et al.  Species-diagnostic differences in a ribosomal DNA internal transcribed spacer from the sibling species Anopheles freeborni and Anopheles hermsi (Diptera:Culicidae). , 1991, The American journal of tropical medicine and hygiene.

[2]  P Simonet,et al.  Frankia genus-specific characterization by polymerase chain reaction , 1991, Applied and environmental microbiology.

[3]  T. White,et al.  Identification of indigenous and introduced symbiotic fungi in ectomycorrhizae by amplification of nuclear and mitochondrial ribosomal DNA , 1991 .

[4]  M. Bobola,et al.  Five major nuclear ribosomal repeats represent a large and variable fraction of the genomic DNA of Picea rubens and P. mariana. , 1992, Molecular biology and evolution.

[5]  G. Baura,et al.  Gastrosuillus laricinus is a recent derivative of Suillus grevillei : molecular evidence , 1992 .

[6]  J. Deacon,et al.  Experimental study of establishment of ectomycorrhizas in different regions of birch root systems , 1988 .

[7]  R. Nazar,et al.  Potential use of PCR-amplified ribosomal intergenic sequences in the detection and differentiation of verticillium wilt pathogens , 1991 .

[8]  J. W. Taylor,et al.  Phylogeny of five fungus-like protoctistan Phytophthora species, inferred from the internal transcribed spacers of ribosomal DNA. , 1992, Molecular biology and evolution.

[9]  F. Gannon,et al.  The 16s/23s ribosomal spacer region as a target for DNA probes to identify eubacteria. , 1991, PCR methods and applications.

[10]  K. Mullis,et al.  Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. , 1987, Methods in enzymology.

[11]  James B. Anderson,et al.  Molecular phylogeny of northern hemisphere species of Armillaria , 1992 .

[12]  B. Kropp,et al.  Ectomycorrhizae in reforestation. , 1990 .

[13]  C. Levenson,et al.  Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies. , 1990, Nucleic acids research.

[14]  T. White.  Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics , 1990 .

[15]  R. Sommer,et al.  Minimal homology requirements for PCR primers. , 1989, Nucleic acids research.

[16]  R. Schneider,et al.  Species-specific polymorphisms in transcribed ribosomal DNA of fivePythium species , 1992 .

[17]  R. Danielson,et al.  The ectomycorrhizal status of urban spruce , 1989 .