Real‐time Quantitative PCR: DNA Determination in Isolated Spores of the Mycorrhizal Fungus Glomus mosseae and Monitoring of Phytophthora infestans and Phytophthora citricola in their Respective Host Plants

Abstract

AbstractSpecific primers and dual‐labelled fluorogenic probes were designed for polymerase chain reaction (PCR)‐based detection of both, mycorrhizal and pathogen DNA. Based on the on‐line connection with an automated ABI Prism 7700 sequence detector, amplicon quantification was directly performed during the PCR. The starting copy numbers of target sequences present in each reaction were calculated by comparing the Ct‐values of unknown samples to the Ct‐values of standards with known amounts of DNA. The Ct‐value depends on the input of starting copies and is defined as that cycle number at which a statistically significant increase in the reporter fluorescence can first be detected. DNA was extracted from 11 as well as 100 spores of the mycorrhizal fungus Glomus mosseae and quantified by using the fluorescent PCR technology. Furthermore, DNA of Phytophthora infestans, causal agent of late blight of potatoes, was quantified after extraction from artificially infected potato tubers and naturally infected field plants. Phytophthora citricola DNA, causing root‐rot diseases, was quantified after isolation from artificially inoculated seedling roots of beech and oak. The results demonstrate that novel real‐time PCR techniques are a powerful universal tool in modern phytopathological research.