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United States Department of Agriculture

Agricultural Research Service

Research Project: IMPROVING GRAPE ROOTSTOCK AND SCION PEST AND DISEASE RESISTANCE Title: Optimisation of techniques for quantification of Botrytis cinerea in grape berries and receptacles by quantitative polymerase chain reaction

Authors
item Saito, Seiya -
item Dunne, Katie -
item Evans, Kathy -
item Barry, Karen -
item Wilcox, Wayne -
item Cadle-Davidson, Lance

Submitted to: Australian Journal of Grape and Wine Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 4, 2012
Publication Date: February 14, 2013
Citation: Saito, S., Dunne, K., Evans, K., Barry, K., Wilcox, W., Cadle Davidson, L.E. 2013. Optimisation of techniques for quantification of Botrytis cinerea in grape berries and receptacles by quantitative polymerase chain reaction. Australian Journal of Grape and Wine Research. 19:68-73.

Interpretive Summary: Quantitative PCR (qPCR) is a molecular biology technique that requires specialized equipment for quantification of DNA. qPCR can be used to detect and monitor pathogen colonization, but early attempts to apply the technology to Botrytis cinerea infection of grape berries have identified limitations to current techniques. In this study, we tested several limiting factors in the process to improve the sensitivity for B. cinerea detection in grapevine. Furthermore, we simultaneously quantified B. cinerea and Vitis vinifera DNA by qPCR to account for sources of sample-to-sample variation. The resulting qPCR method proved to be sensitive - detecting as little as one-trillionth of a gram of B. cinerea DNA. This method could be used to monitor B. cinerea infection in vineyards and is particularly suited to the detection and quantification of the pathogen prior to the development of symptoms.

Technical Abstract: Quantitative PCR (qPCR) can be used to detect and monitor pathogen colonization, but early attempts to apply the technology to Botrytis cinerea infection of grape berries have identified limitations to current techniques. In this study, four DNA extraction methods, two grinding methods, two grape organs, three probe sets and two enzymes were compared in order to improve the sensitivity for B. cinerea detection in grapevine. Furthermore, duplex qPCR including detection of Vitis vinifera DNA as reference gene was developed to establish Pathogen Coefficient (PC), allowing normalization for sample-to-sample variation caused by DNA quality, PCR efficiencies and pipetting error. The described qPCR method proved to be sensitive (Ct value <33 for as little as 0.001 ng of the B. cinerea DNA), and with the concept of PC this method could be used to monitor B. cinerea infection in vineyards and particularly suited to the detection and quantification of the pathogen prior to the development of symptoms.

Last Modified: 11/23/2014
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