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PCR‐basierte Identifizierung von Erysiphe pulchra und Phyllactinia guttata von Cornus florida mit Hilfe von ITS‐spezifischen Primern
Author(s) -
Mmbaga M. T.,
Klopfenstein N. B.,
Kim M.S.,
Mmbaga N. C.
Publication year - 2004
Publication title -
forest pathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.535
H-Index - 49
eISSN - 1439-0329
pISSN - 1437-4781
DOI - 10.1111/j.1439-0329.2004.00374.x
Subject(s) - biology , genbank , primer (cosmetics) , internal transcribed spacer , botany , powdery mildew , agarose gel electrophoresis , genetics , ribosomal rna , gene , chemistry , organic chemistry
Summary 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, ITS 1 and ITS 4 . 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 from E. pulchra ‐specific primer pair was 568 bp while the P. guttata band was 597 bp; the two primer pairs were highly specific to E. pulchra and P. guttata . Comparison of ITS sequences with information in the GenBank showed a very close similarity between sequences of E. pulchra isolates from Cornus florida in the USA and isolates collected on Cornus kousa in Japan. BLAST analysis 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 .