Development of a TaqMan allelic discrimination assay for the distinction of two major subtypes of the grapevine yellows phytoplasma Bois noir

Abstract

Bois noir is a grapevine yellows disease that is gaining importance in many regions of Europe. The Bois noir phytoplasma (“Candidatus Phytoplasma solani”) was shown to be transmitted by the planthopper Hyalesthes obsoletus, which normally feeds on herbaceous weeds, and occasionally also on grapevines. Three subtypes of the Bois noir phytoplasma have been described and were shown to be associated with distinctive host plants. In this study, we developed a novel and rapid real-time PCR allelic discrimination assay for the distinction of the two major Bois noir phytoplasma subtypes, VK type I and II. Two TaqMan probes carrying different fluorescent dyes were designed to specifically bind to a polymorphism characteristic for the two Bois noir phytoplasma subtypes, thereby allowing discriminative amplification in a single-tube and single-step assay. A total of 259 bois noir-positive grapevine samples collected over 5 years were analysed using the conventional PCR-RFLP method and our newly developed TaqMan allelic discrimination assay. 257 out of 259 samples could be typed with the TaqMan method, compared to 200 out of 259 samples when using the conventional method. The overall concordance of the two methods was 100%. Our newly developed TaqMan assay represents a useful tool for fast and reliable determination of Bois noir phytoplasma subtypes in infected grapevine, insect vector, and host plant samples. The test is suitable for high-throughput analysis and will thereby facilitate further characterisation of Bois noir epidemiology.