Development of a Real-Time, Quantitative PCR for Detection of the Columbia Basin Potato Purple Top Phytoplasma in Plants and Beet Leafhoppers.

Abstract

A quantitative, real-time "TaqMan" polymerase chain reaction assay (real-time PCR) was developed which was capable of detecting and quantifying a group 16SrVI phytoplasma in DNA extracts prepared from infected tomatoes, potatoes, and beet leafhoppers (Circulifer tenellus). Primers and probe were designed from the 16S rRNA gene of the Columbia Basin potato purple top phytoplasma, which is closely related to the beet leafhopper transmitted virescence agent. The detection limit in phytoplasma-infected tomato DNA was approximately 50 pg. The concentration of phytoplasma varied considerably among potato plants showing symptoms of purple top. The pathogen was readily detected in extracts from single or groups of five beet leafhoppers. As with infected potatoes, the concentration of phytoplasma in individual leafhoppers was variable. The assay also detected aster yellows (group 16SrI) and pigeon pea witches'-broom (group 16SrIX) phytoplasmas in infected periwinkle plants. The real-time PCR was at least as sensitive as the commonly used and more labor-intensive nested PCR for detection of the pathogen.