PCR-based identification of Erysiphe pulchra and Phyllactinia guttata from Cornus florida using ITS-specific primers

The internal transcribed spacer (ITS) regions of rDNA and the intervening 5.8S rRNA gene for the powdery mildew fungi Erysiphe (sect. Microsphaera) pulchra and Phyllactinia guttata were amplified using standard polymerase chain reaction (PCR) protocols and the universal primer pairs, ITS1 and ITS4. PCR products for ITS were analysed by electrophoresis in a 1.5% agarose gel and sequenced. The size of the amplified ITS products (approximately 650 bp) were not sufficiently different to allow reliable differentiation of E. pulchra and P. guttata; however, their sequences were distinct. Specific primers for E. pulchra and P. guttata were developed and evaluated for use as diagnostic tools. The diagnostic band size fromE. pulchra-specific primer pairwas 568 bpwhile theP. guttatabandwas 597 bp; the twoprimer pairs were highly specific toE. pulchra and P. guttata. Comparison of ITS sequences with information in theGenBank showed a very close similarity between sequences ofE. pulchra isolates fromCornus florida in theUSAand isolates collectedonCornus kousa in Japan.BLASTanalysis of the sequence of the 650-bp band from P. guttata revealed a close alignment with sequences of P. moricola (92%), P. kakicola (94%), and P. fraxini (92%). The sequence of P. guttata in C. florida also had a 98% identity with P. guttata in Calycanthus occidentalis and 94% identity with P. guttata in Corylus cornuta.