Development of real-time PCR using Minor Groove Binding probe to monitor the biological control agent Candida oleophila (strain O)

A real-time PCR assay using a 3'-Minor Groove Binding (MGB) probe was developed for specific detection and monitoring of Candida oleophila (strain O), a biocontrol agent against Botrytis cinerea and Penicillium expansum, on harvested apples. The application of the RAPD technique on C. oleophila strains followed by reproducible sequence characterized amplified region (SCAR) amplifications allowed the identification of a semi-specific fragment of 244 bp, observed in the profiles of strain O and three other C. oleophila strains. After sequencing, polymorphisms (3%) were observed between the strain O sequence and the three other sequences. A 3'-Minor Groove Binding probe was designed to specifically match a region of the strain O sequence and was able to discriminate a single base mutation or a two-base difference in the corresponding sequences of the non-target strains. This specific detection method was applied to monitor strain 0 population, recovered by a washing buffer, from harvested apples. Population densities were calculated using an external standard curve consisting in a serial dilution of strain 0 cells in the washing buffer from untreated apples. Linearity in the standard curve was kept between 1.64x10(2) and 1.64x10(5) cfu cm(-2) of apple surface. During a first practical experiment, the calculated population densities were similar to those obtained by plating on semi-selective media. This new real-time PCR method is a promising tool to monitor quickly and specifically strain O population on apple surface in middle- or large-scale experiments. (C) 2004 Elsevier B.V. All rights reserved.