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

Abstract

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 .