Quantification of <i>Cadophora luteo-olivacea</i> From Grapevine Nursery Stock and Vineyard Soil Using Droplet Digital PCR

Abstract

<i>Cadophora luteo-olivacea</i> is the most prevalent <i>Cadophora</i> species associated with Petri disease and esca of grapevine. Accurate, early, and specific detection and quantification of <i>C. luteo-olivacea</i> are essential to alert growers and nurseries to the presence of the pathogens in soil and to prevent the spread of this pathogen through grapevine planting material. The aim of this study was to develop molecular tools to detect and quantify <i>C. luteo-olivacea</i> inoculum from environmental samples. Species specific primers based on the β-tubulin gene and a TaqMan probe for droplet digital PCR (ddPCR) and quantitative PCR (qPCR) were first developed to detect and quantify purified DNA of the target fungus. Specificity tests showed that the primers were able to amplify the <i>C. luteo-olivacea</i> DNA (20 isolates) while none of the 29 nontarget fungal species (58 isolates) tested were amplified. The ddPCR was shown to be more sensitive compared with qPCR in the detection and quantification of <i>C. luteo-olivacea</i> at very low concentrations and was further selected to accurately detect and quantify the fungus from environmental samples. Twenty-five of the 94 grafting plants (26.6%) analyzed by ddPCR tested positive to <i>C. luteo-olivacea</i> DNA (>3 copies/µl). <i>C. luteo-olivacea</i> was barely detected from vineyard soils. The procedure employed in this study revealed the presence of the pathogen in symptomless vines, which makes implementation of this technique suitable for certification schemes of <i>C. luteo-olivacea</i>-free grapevine planting material.