Development of a new real-time TaqMan PCR assay for the detection of the Prunus pathogen Monilinia kusanoi

Abstract

Monilinia kusanoi causes shoot blight, leaf blight and fruit rot of several Prunus species, and is currently known to be present only in Japan and Korea. Despite the restricted distribution range, the risk remains that the pathogen could spread with the transportation of plant material, especially since no DNA based diagnostic methods are available to detect this fungus. In this study, a new TaqMan probe-based real-time assay was developed for specific and rapid detection of M. kusanoi. The assay targets the single copy elongation factor gene and enables the detection down to 1 pg of pathogen DNA in a plant sample, which corresponds to approximately 25 hyphal cells. When testing a panel of fungal cultures, all M. kusanoi isolates were detected and there was no cross-reaction with other Monilinia species or related fungi. The assay was duplexed with a plant internal control assay to allow simultaneous detection of the pathogen and cytochrome oxidase gene from host plants. This newly developed real-time PCR assay will facilitate rapid and sensitive pathogen screening of imported plant material, as M. kusanoi is a pathogen of biosecurity concern in New Zealand and Australia and would also be useful for research and screening purposes in areas where the pathogen is already established.