Detection of Forest Ectomycorrhizal Fungi- Pisolithus tinctorius by Nested PCR

Abstract

For investigating a suitable molecular approach to detect ectomycorrhizal fungi(EMF),the internal transcribed spacer(ITS) region of the EMF Pisolithus tinctorius was amplified with universal primers ITS1-F/ITS4-B and sequenced.Based on the conserved sequence determined by alignment using Clustal W,a pair of specific primers named PtF/PtR were designed by Primer premier 5.0.The specificity of the primer pair was then analyzed in 10 P.tinctorius strains together with 6 reference strains,and a 347 bp product was amplified from all P.tinctorius strains but there was no product from the 6 reference strains.A nested PCR procedure using ITS1-F/ITS4-B as the first-round primers followed by PtF/PtR increased detection sensitivity 1 000-fold to 10 fg DNA template.The target EMF from the mycorrhiza of Pinus massoniana potted seedlings inoculated with P.tinctorius was detected by this technique.The results indicated that the nested PCR assay could be used as a sensitive and accurate way to detect forest EMF P.tinctorius.