Detection of Bois Noir phytoplasma in grapevine roots by reverse transcription-Real Time TaqMan assays

Bois noir (BN) is a phytoplasma disease of grapevine spread in the Mediterranean basin, causing relevant economic losses. BN phytoplasma diagnosis is currently carried out by detecting the pathogen DNA sequences in the leaf. Even if reliable and usable in routine analyses, molecular diagnosis is nevertheless restricted to the late summer, when BN symptoms become evident. This is because the above methods are not enough sensitive to carry out diagnosis in other periods of the year, when symptoms are not evident, or on other plant organs such as roots. This consideration suggests that more sensitive and focused detection methods for BN would be needed. Because of the lack of diagnostic methods for root tissues, it is not known where the pathogen overwinters inside the host. We aim at expanding diagnosis effectiveness to root tissues in BN-infected grapevine, to better understand phytoplasma/ grapevine interactions and to give new insights to BN epidemiology. Different root samples from healthy, BN-infected and recovered grapevines, have been collected and RNA was extracted. As the low concentration of template in the host, we performed a nested Real-Time PCR using a BN specific TaqMan probe, following the method already described by Margaria et al. (Plant Pathol. 58:838-845, 2009). Preliminary analyses showed positive signal in roots of symptomatic and recovered plants, whereas no amplification was observed in healthy samples. This result suggests that BN phytoplasma persists in the root phloem tissues of recovered individuals. The epidemiological significance of this finding will be discussed.