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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
Trees 2016 v.30 no.4 pp. 1111-1125
DNA primers, Malus domestica, Neonectria ditissima, apples, biomass, cultivars, disease course, disease outbreaks, fruit trees, fungi, genes, histopathology, hyphae, light microscopy, melting, odors, orchards, pathogen identification, pathogens, quantitative polymerase chain reaction, shoots, tissues, tubulin, wood, Europe
KEY MESSAGE : We designed a pair of primers from a region of the β-tubulin gene to detect and quantify Neonectria ditissima in wood of some infected apple cultivars, and optimized light microscopy to study fungal-plant interactions. Neone ctria 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.