Use of PCR and DNA hybridization for identification of pear powdery mildew caused by Podosphaera leucotricha

Abstract

Polymerase chain reaction (PCR) amplification of the ribosomal gene internal transcribed spacer (ITS) region has been used to classify Podosphaera leucotricha from apple but not pear. Molecular information is needed to show the relationship between apple and pear powdery mildew. DNA was successfully extracted from cleistothecia, conidia and mycelial fragments from leaves and shoots of apple, pear and six unrelated hosts with symptoms of powdery mildew. PCR amplification of the ITS regions of P. leucotricha with primers that amplify non-specific fungal DNA resulted in a 506 bp PCR product. Sequencing of the PCR products from apple and pear from different orchard blocks produced identical sequences for the ITS region that did not differ from P. leucotricha sequences in the GenBank database. The sequence data was also used to select species-specific oligonucleotides for P. leucotricha that could be used in a confirmatory hybridization assay. The hybridization reactions were positive for eight out of nine apple and pear powdery mildew samples. Six other hosts with powdery mildew symptoms were negative when tested with the assay and showed that this DNA test was specific for P. leucotricha. In conclusion the PCR and hybridization assays should prove to be useful tools in monitoring both apple and pear powdery mildew for epidemiological studies.

Keywords:

• ITS region
• phylogenetic relationship
• reverse dot blot
• apple
• Pyrus
• Malus