LAMP for in-field quantitative assessments of airborne grapevine downy mildew inoculum

Abstract

Aims: Cheap, rapid tools for measuring emissions of Plasmopara viticola sporangia directly in the field are required to protect grapevines efficiently and sustainably against downy mildew. To this end, we adapted an existing loop-mediated isothermal amplification (LAMP) protocol based on ITS2 sequences, coupled with a rotating-arm sampler and simple cell lysis, for the in-field measurement of airborne sporangia of P. viticola. Methods and Results: We estimated the sensitivity and specificity of the molecular reaction with an unpurified DNA template in controlled conditions, using the droplet digital PCR (ddPCR) as a reference. We show that the LAMP lower limit of quantification is 3.3 sporangia.m−3 air sampled. Cell lysis in KOH solution was less efficient than CTAB for DNA extraction, but the repeatability of the method was good. We tested this protocol directly in a plot at Chateau Dillon (Blanquefort, France) in which we monitored P. viticola sporangia concentrations from March to October 2020 (88 samples which revealed concentrations ranging from 0 to 243 sporangia.m−3). There was a significant quantitative correlation (R2 = 0.52) between ddPCR and LAMP results. Conclusion: LAMP analysis of an unpurified DNA matrix is a simple and reliable method for in-field estimations of the concentration of airborne P. viticola sporangia. Significance and Impact of the Study: This study constitutes a first step towards the development of a regional grapevine downy mildew monitoring network in the vineyards of Bordeaux.