Abstract
A fluorescence-based real-time loop-mediated isothermal amplification (LAMP) assay for ‘Candidatus Phytoplasama solani’ (Bois noir phytoplasma; BNp) detection was developed and optimised for rapid laboratory and on-site BNp detection. This assay is highly specific, rapid and as sensitive as qPCR. It was validated according to European and Mediterranean Plant Protection Organisation recommendations. In addition, 286 grapevine leaf samples from the 2015 growing season were tested with this new real-time LAMP assay and an assay previously developed for detection of Flavescence dorée phytoplasma (FDp). These LAMP assays for detection of both BNp and FDp used without any DNA extraction step, which is a required step for qPCR analysis, were comparably effective to qPCR, and positive results were obtained in less than 35 min.
Highlights
In grapevine (Vitis vinifera L.), phytoplasmas are associated with grapevine yellows (GYs) diseases, that occur in the majority of grapevine growing countries worldwide (Constable et al 2003)
We evaluated the analytical sensitivity of direct testing of leaf-vein homogenates prepared with Ultra Turrax Tube Drive device (UTTD, IKA)
The performance of the crude leaf-vein homogenate testing with the loop-mediated isothermal amplification (LAMP) assay validated here for BNp and the earlier developed LAMP assay for Flavescence dorée phytoplasma (FDp) (Kogovšek et al 2015) was compared to routine qPCR testing of the extracted DNA
Summary
In grapevine (Vitis vinifera L.), phytoplasmas are associated with grapevine yellows (GYs) diseases, that occur in the majority of grapevine growing countries worldwide (Constable et al 2003). ‘Candidatus Phytoplasma solani’ is from the stolbur group/ 16SrXII-A of phytoplasmas (Quaglino et al 2013), and is associated with Bois noir disease (i.e., Bois noir phytoplasma; BNp). Flavescence dorée is associated with phytoplasmas belonging to the 16SrV group (i.e., Flavescence dorée phytoplasma; FDp) and this is the most severe of the GYs diseases. Typical symptoms of all GYs include leaf curling and discoloration of leaf veins and lamina, inter-vein yellowing or reddening (according to the variety), uneven or total lack of lignification of canes, flower abortion, and berry withering. Symptoms caused by BNp and FDp are not distinguishable by visual inspection and as the distribution of phytoplasma is uneven within a host that has a very low titre (Prezelj et al 2013), only specific molecular approaches are suitable for the accurate and reliable detection of BNp and FDp
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