Quantitative PCR assay for detection of Bois noir phytoplasmas in grape and insect tissue

In Europe's vineyards "Bois noir" (BN) is an expanding yellows disease on Vitis vinifera. It is associated with phytoplasmas of the stolbur group (16SrXII-A). Two subtypes are important, one is associated with Urtica dioica and one with Convolvulus arvensis. Both phytoplasma types are transmitted by the insect Hyalesthes obsoletus. A nucleic acid extraction method for V. vinifera and H. obsoletus was developed together with a real time PCR (qPCR) assay based on a polymorphic sequence with homology to a putative dimethyladenosine transferase. The comparison of the conventional detection method with the qPCR assay of 40 insect and 40 V. vinifera samples showed a 10 % higher sensitivity of qPCR in plant samples. The titer of phytoplasmas in H. obsoletus was 2643-fold increased in the strongest infected samples compared to the lowest ones. The results suggest this real-time PCR as a valid and fast alternative procedure for the detection and quantification of BN phytoplasmas. The assay allows to discriminate the two phytoplasma types and to quantify phytoplasmas in H. obsoletus.