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Research article - Peer-reviewed, 2016

Real-time PCR for detection and quantification, and histological characterization of Neonectria ditissima in apple trees

Ghasemkhani, Marjan; Holefors, Anna; Marttila, Salla; Dalman, Kerstin; Zborowska, Anna; Rur, Mira; Rees-George, Jonathan; Nybom, Hilde; Everett, Kerry R.; Scheper, Reiny W. A.; Garkava-Gustavsson, Larisa


Neonectria ditissima, the causal pathogen of fruit tree canker, is a sordariomycete fungus that affects apple orchards, especially in north-western Europe. To prevent serious disease epidemics, an accurate, rapid, and sensitive method for detection of N. ditissima is needed for pathogen identification. A quantitative real-time PCR (qPCR) assay was developed for both detection and quantification of this pathogen in infected apple cultivars. Several primer sets were designed from regions of the β-tubulin gene. One primer set passed several validation tests, and the melting curve confirmed species-specific amplification of the correct product. In addition, the N. ditissima biomass could be detected at variable amounts in samples from the infection sites of six different cultivars, with ‘Aroma' having the lowest amount of N. ditissima biomass and ‘Elise' the highest. To complement the qPCR results, tissue from detached shoots and 1-year-old trees of ‘Cox's Orange Pippin' (susceptible) and ‘Santana' (partially resistant) was used in a histopathology study. In both detached shoots and trees, fungal hyphae were found in cells of all tissues. No qualitative differences in the anatomy of the infected samples were observed between the cultivars. In the detached shoot experiment, both cultivars were affected but differences in the rate of disease progression suggest that the partially resistant cultivar could resist the fungus longer. The qPCR assay developed in our study produced reproducible results and can be used for detection of N. ditissima in infected trees.


Apple canker; Anatomy; B-tubulin gene; Neonectria galligena; Nectriaceae; qPCR

Published in

Trees - Structure and Function
2016, Volume: 30, number: 4, pages: 1111-1125