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Comparative analysis of techniques for detection of quiescent Botrytis cinerea in grapes by quantitative PCR.
S. SAITO (1), L. Cadle-Davidson (2), W. F. Wilcox (1). (1) Cornell University NYSAES, Geneva, NY, U.S.A.; (2) USDA-ARS, Grape Genetics Research Unit, Geneva, NY, U.S.A.

Quantitative PCR (qPCR) can be used to detect and monitor pathogen colonization, but early attempts to apply the technology to quiescent <i>Botrytis cinerea</i> infections of grape berries identified some specific limitations. In this study, four DNA extraction methods, two tissue-grinding methods, two grape (<i>Vitis vinifera</i>) organs, two probe sets and two enzymes were compared in order to improve the sensitivity of <i>B. cinerea</i> detection in grapevine. Furthermore, duplex qPCR for concurrent detection of <i>B. cinerea</i> and <i>V. vinifera</i> DNA was developed as a check against false negative assays, and to establish a Pathogen Coefficient (PC) that addresses variability among samples caused by DNA quality, PCR efficiency and pipetting error. The resulting optimized qPCR technique was highly sensitive, providing a Ct value < 33 for as little as 0.001 ng of <i>B. cinerea</i> DNA, and could be applied as a tool to monitor quiescent <i>B. cinerea</i> infections in vineyards.<p><p>Keywords: Fungus, Fruits-Nuts, Grape

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