Optimisation of techniques for quantification ofBotrytis cinereain grape berries and receptacles by quantitative polymerase chain reaction

Abstract

Background and Aims Bunch rot symptoms can appear weeks after the infection of grape flowers by Botrytis cinerea. Quantitative polymerase chain reaction (qPCR) detects changes in the DNA mass of a target organism and is a potential tool for studying quiescent infections. The aim was to optimise a duplex qPCR to quantify B. cinerea DNA in the background of endogenous Vitis vinifera DNA. Methods and Results Three DNA extraction techniques and three probe sets were compared. The optimised qPCR using the Bc3 probe set was 1000-fold more sensitive than other probe sets, with a threshold cycle value of <33 for as little as 1 picogram of B. cinerea DNA. The duplex assay successfully detected an increasing amount of B. cinerea DNA when mixed with V. vinifera DNA or of B. cinerea conidia when added to grape receptacles. Conclusions Duplex assays quantifying B. cinerea DNA in the background of endogenous grape DNA were efficient and sensitive, with calculation of a pathogen coefficient allowing comparison of results among assays. Significance of the Study The results demonstrate the potential to monitor symptomless, quiescent infections and to investigate the consequence of an intervention (e.g. a fungicide treatment) before disease symptoms are visible.