A New TaqMan Real-Time PCR Assay for Detecting the Blueberry Pathogen Monilinia vaccinii-corymbosi

Abstract

Monilinia vaccinii-corymbosi (Mvc) is an important fungal pathogen of blueberry, causing mummy berry disease. While the symptoms of the advanced stages of the disease can be obvious, diagnosing its early stages can be challenging. To facilitate fast and sensitive screening of asymptomatic or latently infected plant material for Mvc, we developed a specific TaqMan real-time PCR assay targeting the internal transcribed spacer (ITS) region. The assay was shown to be specific to Mvc and did not cross react with any of the other tested Monilinia species or other blueberry pathogens. Using the multicopy ITS region ensured high analytical sensitivity, enabling very low concentrations of Mvc DNA (0.1 pg) to be detected both in water and host DNA matrix. Comparable results were obtained in interlaboratory testing, showing that the assay is robust, and can be effectively used in other laboratories. Assay sensitivity was also confirmed on infected plant tissue, showing that it is effective in detecting the pathogen in infected asymptomatic stem tissue, as well as infected tissue that was mixed with healthy tissue at a ratio of 1:10 by weight. The assay was duplexed with a plant internal control (cytochrome oxidase gene) for simultaneous amplification of the pathogen and plant internal control in a single reaction. This new diagnostic tool can be used for sensitive and rapid screening of blueberry plants for the presence of Mvc in many different settings, e.g., for breeding programs, research, or biosecurity diagnostics.