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

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Abstract

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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.

Abstract

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.

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Acknowledgments

Financial assistance was received from Swedish Farmer’s organization and Partnership Alnarp to Larisa Garkava-Gustavsson, from the Nordic Ministries of Food and Agriculture through the Nordic collaboration on Public–Private Partnership for pre-breeding, administered by NordGen, to Hilde Nybom and the New Zealand Institute for Plant & Food Research Ltd. The authors gratefully acknowledge the help of Kerstin Brismar with microscopy samples, Prof. Erland Liljeroth for suggestions with regard to the isolation of Neonectria ditissima and useful discussions, Marlene Jaspers and Margaret Dick for providing LUPP and NZFS isolates and Helle Turesson for help with qPCR analyses.

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Authors and Affiliations

  1. Department of Plant Breeding, Swedish University of Agricultural Sciences, Box 101, 230 53, Alnarp, Sweden

    Marjan Ghasemkhani, Anna Zborowska & Larisa Garkava-Gustavsson

  2. In vitro Plant-tech AB, Geijersgatan 4, 216 18, Limhamn, Sweden

    Anna Holefors

  3. Department of Plant Protection Biology, Swedish University of Agricultural Sciences, Box 102, 230 53, Alnarp, Sweden

    Salla Marttila & Mira Rur

  4. Department of Chemistry and Biotechnology, Uppsala Biocenter, Swedish University of Agricultural Sciences, Box 7026, 750 07, Uppsala, Sweden

    Kerstin Dalman

  5. Department of Plant Breeding, Swedish University of Agricultural Sciences, Balsgård, Fjälkestadsvägen 459, 29 194, Kristianstad, Sweden

    Hilde Nybom

  6. The New Zealand Institute for Plant and Food Research Ltd, PB 92169, Mt Albert, Auckland, New Zealand

    Jonathan Rees-George & Kerry R. Everett

  7. The New Zealand Institute for Plant and Food Research Ltd, PB 1401, Havelock North, New Zealand

    Reiny W. A. Scheper

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  1. Marjan Ghasemkhani
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  3. Salla Marttila
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  11. Larisa Garkava-Gustavsson
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Correspondence to Marjan Ghasemkhani.

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Communicated by W. Osswald.

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Ghasemkhani, M., Holefors, A., Marttila, S. et al. Real-time PCR for detection and quantification, and histological characterization of Neonectria ditissima in apple trees. Trees 30, 1111–1125 (2016). https://doi.org/10.1007/s00468-015-1350-9

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  • DOI: https://doi.org/10.1007/s00468-015-1350-9

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