Optimisation of a one-step PCR assay for the diagnosis of Flavescence doreerelated phytoplasmas in field-grown grapevines and vector populations

Abstract

Field-infected grapevines and natural populations of Scaphoideus titanus have been analysed to detect group V phytoplasmas associated with flavescence doree in northwestern Italy using nested PCR. A first amplification driven by universal ribosomal primers R16SF2/R2 was followed by a second round assisted by R16(V)F1/R1 primers and subsequent RFLP analysis. To optimize the test, nested PCRs were compared with direct amplification assisted by the group V-specific fAY/rEY primer pair, directed towards other ribosomal sequences. In nested and direct PCRs, respectively, DNAs from 71 and 57 out of 96 grapevines (i.e. 73.9 and 59.3 %) and 51 and 50 out of 108 insects (i.e. 47.2 and 46.3 %) reacted positively. Although it was not possible to determine the subgroup of the phytoplasmas after fAY/rEY amplification, these primers could be used successfully in mass screening of plant material and insect populations. They could detect, in single-step amplification, the phytoplasmas in 80 and 98 % of the plant and insect samples, respectively, that were already indexed as positive using nested PCR. This strongly reduced the number of samples requiring the nested approach, with beneficial effects on costs, labour and risks of the analysis.