Detection of a wide range of phytoplasmas using a qPCR methodology developed for mycoplasmas

The available methodologies for quantitative PCR (qPCR) amplification of phytoplasmas are very often group specific and not very sensitive, therefore having a generic qPCR assay to verify the presence of these prokaryotes when they are at very low concentrations, such as in propagation materials or under the presence of inhibition factors (cultivation broths), is of relevance to the researches in this field. Total DNAs extracted from the micropropagated phytoplasma-infected periwinkle collection maintained at the University of Bologna were used. They were preliminary tested in conventional PCR using 16S ribosomal primers. DNA samples (100 ng/μl) used belonged to groups 16SrI-B, 16SrII-C and –D, 16SrIII-A, 16SrV-A, 16SrVI-C, 16SrVII-A, 16SrIX-C, 16SrX-A, -B and C, 16SrXI-C, 16SrXII-A and 16SrXV. Botes (Botes et al., 2005) and van Kuppeveld (van Kuppeveld et al., 1992) primers developed for mycoplasmas were used in qPCR. The results were consistent for all the phytoplasma groups tested with both primers, they showed a very high sensitivity also for phytoplasmas as it is known already for other Mollicutes, both in qPCR and in conventional PCR assays. Repetition for validation was then carried out only with primers Botes that confirmed the phytoplasma specific melting temperature at 84.5°C and Ct values enclosed between 21 and 24. System sensitivity was tested on European stone fruit yellows (ESFY) phytoplasma DNA template at the concentrations of 20, 10, 5, 2, 0.2 and 0.02 ng/μl, only the latter concentration gave negative results, while other concentrations gave Ct values ranging from 24 to 30. Moreover, the qPCR assay with Botes primers showed positive result also for DNA extracted from leaves of corn deriving from seeds produced by phytoplasma infected plants, and from leaf material from field collected symptomatic apricot with Ct values of 31 and 27 respectively.