Optimisation of techniques for quantification of Botrytis cinerea in grape berries and receptacles by quantitative polymerase chain reaction

Authors

  • S. Saito,

    1. Department of Plant Pathology and Plant-Microbe Biology, New York State Agricultural Experiment Station, Cornell University, Geneva, NY, USA
    Search for more papers by this author
  • K.J. Dunne,

    1. Perennial Horticulture Centre, Tasmanian Institute of Agriculture, University of Tasmania, New Town, Tas., Australia
    Search for more papers by this author
  • K.J. Evans,

    1. Perennial Horticulture Centre, Tasmanian Institute of Agriculture, University of Tasmania, New Town, Tas., Australia
    Search for more papers by this author
  • K. Barry,

    1. Perennial Horticulture Centre, Tasmanian Institute of Agriculture, University of Tasmania, New Town, Tas., Australia
    Search for more papers by this author
  • L. Cadle-Davidson,

    Corresponding author
    1. USDA-ARS, Grape Genetics Research Unit, Cornell University, Geneva, NY, USA
    • Department of Plant Pathology and Plant-Microbe Biology, New York State Agricultural Experiment Station, Cornell University, Geneva, NY, USA
    Search for more papers by this author
  • W.F. Wilcox

    1. Department of Plant Pathology and Plant-Microbe Biology, New York State Agricultural Experiment Station, Cornell University, Geneva, NY, USA
    Search for more papers by this author

Correspondence author: Dr Lance Cadle-Davidson, email lance.cadledavidson@ars.usda.gov

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.

Ancillary