Development of a TaqMan Assay for the Detection of 'Candidatus Phytoplasma brasiliense' and Assessment by...

'Candidatus Phytoplasma brasiliense' (CPB) is a phytoplasma originally discovered in South America and is known to infect a wide variety of economically important crops. It is most prevalent in Hibiscus spp., where it causes witches broom symptoms, and papaya, where it causes bunchy top. Recently, CPB was documented for the first time in North America in a new host, globe sedge. In this study, two quantitative PCR assays are developed: one using high-resolution melt curve analysis (HRMA) based on the secA gene and the other a TaqMan assay based on the dnaK gene. The secA/HRMA and dnaK/TaqMan assay successfully amplified two of the three isolates of CPB. Both assays were screened against available isolates of 16SrI, 16SrII, and 16SrIV phytoplasmas. The secA/HRMA assay failed to amplify 16SrI and 16SrIV phytoplasmas but successfully amplified 16SrII phytoplasmas. The resulting melting point (Tm) products of CPB and 16SrII phytoplasmas displayed a difference of 0.5°C, easily distinguishing them by melt curves. The dnaK/TaqMan assay failed to amplify all non-CPB phytoplasma isolates in the study. The development of these assays provides a valuable tool that will significantly improve monitoring programs in Florida and will aid in developing a better fundamental understanding of the epidemiology of this phytoplasma.