Evaluation of Primer Pairs for the Reliable Diagnosisof Paulownia Witches'-Broom Disease Using a Polymerase Chain Reaction

Abstract

To establish a reliable method for detection of paulownia witches'-broom (PWB) phytoplasma by a polymerase chain reaction (PCR), several oligonucleotide primers that amplify ribosomal protein (rp) or 16S rRNA (rD) genes of PWB-phytoplasma were compared for their specificity and sensitivity in the amplification of PWB-specific fragments by PCR. Use of rp primer pairs for PCR resulted in amplified DNA fragments of expected sizes in samples from diseased leaves. A fragment was amplified from healthy samples using one primer pair (rp3/rp4), but the fragment size was different from disease-specific fragments. In contrast, all four rD primer pairs designed for the 16S rRNA gene amplified both PWB-specific DNA fragments from infected leaf samples and nonspecific fragments of the same size as PWB-specific fragments from healthy samples. Using the rp3/rp4 primer pair, we amplified a PWB-specific DNA fragment from 150 pg of nucleic acid samples. This method allowed detection at the 95% confidence level of PWB-phytoplasma from paulownia leaves from the ten asymptomatic infected trees used in this study when at least 3 leaves randomly collected in September were used.