Direct amplification by PCR of DNA from ungerminated teliospores of Tilletia species

Abstract

Abstract A simple technique of conducting polymerase chain reaction (PCR) on single ungerminated teliospores of Telleria species was developed. Teliospores were manually cracked under a stereo microscope prior to adding to the PCR reaction mixture. Amplification product was obtained using primers for either a portion of the nuclear ribosomal intergenic spacer region or a portion of the mitochondria) DNA. Collections of teliospores from Tilletia indica, Tilletia barclayana, Tilletia controversa, Tilletia tritici, Tilletia laevis and an unidentified Tilletia sp, from Lolium varied in the proportion of spores from which amplification product could be detected, with the success rate ranging from 100 to 10%. This technique avoids the difficulty and time delay in having to germinate teliospores prior to extracting DNA from a mycelial matte and thus will be of great value in the application of PCR methods for regulatory testing and phylogenetic studies of Tilletia species.