General phytoplasma detection by a q-PCR method using mycoplasma primers

Phytoplasmas and mycoplasmas are bacteria belonging to the class Mollicutes. In this study, a fine tuning of quantitative polymerase chain reaction (qPCR) with a universal mycoplasma primer pair (GPO3F/MGSO) targeting the 16S rRNA gene was carried out on phytoplasmas. The dissociation curves of DNAs from Catharanthus roseus phytoplasma-infected micropropagated shoots and from phytoplasma field infected plant samples showed a single peak at 82.5 degrees C (+/- 0.5) specifically detecting phytoplasmas belonging to several ribosomal groups. Assay specificity was determined with DNA of selected bacteria: 'Candidatus Liberibacter solanacearum', Xylella fastidiosa, Ralstonia solanacearum and Clavibacter michiganensis. No amplification curves were observed with any of these tested bacteria except 'Ca. L. solanacearum' that was amplified with a melting temperature at 85 degrees C. Absolute quantification of phytoplasma titer was calculated using standard, curves prepared from serial dilutions of plasmids containing the cloned fragment GPO3F/MGSO from European stone fruit yellows phytoplasma. Phytoplasma copy number ranged from 10(6) to 10(3) according with the sample. The sensitivity evaluated comparing plasmid serial dilutions resulted 10(-6) for conventional PCR and 10(-7) for qPCR. The latter method resulted therefore able to detect very low concentrations of phytoplasma in plant material. (C) 2017 Elsevier Ltd. All rights reserved