Development of  a qPCR assay for specific quantification of Botrytis cinerea on grapes

Abstract

The aim of this study was to develop a system for rapid and accurate real-time quantitative PCR (qPCR) identification and quantification of Botrytis cinerea, one of the major pathogens present on grapes. The intergenic spacer (IGS) region of the nuclear ribosomal DNA was used to specifically detect and quantify B. cinerea. A standard curve was established to quantify this fungus. The qPCR reaction was based on the simultaneous detection of a specific IGS sequence and also contained an internal amplification control to compensate for variations in DNA extraction and the various compounds from grapes that inhibit PCR. In these conditions, the assay had high efficiency (97%), and the limit of detection was estimated to be 6.3 pg DNA (corresponding to 540 spores). Our method was applied to assess the effects of various treatment strategies against Botrytis in the vineyard. Our qPCR assay proved to be rapid, selective and sensitive and may be used to monitor Botrytis infection in vineyards.

Development of a Nested Quantitative Real-Time PCR for DetectingPhytophthora cinnamomiinPersea americanaRootstocks

“…This assay was highly sensitive, with detection limits as low as 20 fg of P. cinnamomi DNA. This is an improvement in sensitivity vs other DNA based detection methods for P. cinnamomi where 100 fg could be detected (15 as a normalization parameter as has been shown in various studies (3,4).…”

A quantitative PCR assay for accurate in planta quantification of the necrotrophic pathogen Phytophthora cinnamomi

“…In a recent study, Diguta et al (2010) also noted the potential for inaccuracy of pathogen quantification using normalization to host DNA or sample fresh weight and they added intact yeast, Yarowia lipolitica, as an internal control prior to DNA extraction for detection of the fungal pathogen Botrytis cinerea on grapes. However, variation in the efficiency and reproducibility of a given DNA extraction method for extracting the yeast DNA may still influence the accuracy of pathogen quantification.…”

A real-time PCR assay for detection and quantification of Botrytis cinerea in Pelargonium x hortorum plants, and its use for evaluation of plant resistance

“…Accurate quantification of a pathogen in diseased plant tissues is an important means for the evaluation of the degree of host susceptibility and it provides essential information for breeding programs for plant resistance. Several PCR and real-time PCR assays have been developed for detection and quantification of B. cinerea in many types of samples (Rigotti et al 2002;Suarez et al 2005;Cadle-Davidson 2008;Diguta et al, 2010;Tomlinson et al 2010;Sanzani et al 2012). …”