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Real-time PCR as a promising tool to monitor growth of Venturia spp. in scab-susceptible and -resistant apple leaves

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Abstract

Apple scab, the most important disease of apple worldwide, is caused by Venturia inaequalis. Currently, evaluation of fungal pathogenicity and host resistance is based on a symptomatic disease rating. However, this method does not provide an accurate measurement of the degree of infection and cannot detect early fungal development in symptomless leaves. In this study, a Venturia-specific real-time PCR assay was developed using primers designed around the specific internal transcribed spacer 2 (ITS2) region of the 5.8S rRNA gene. Using SYBR® Green I technology, the assay can accurately quantify Venturia DNA over a concentration range of at least five orders of magnitude. Detection sensitivities were in the order of 100 fg. The method was used to quantify Venturia genomic DNA levels in leaves of three apple cultivars with different levels and types of scab resistance and artificially infected with V. inaequalis. The assay clearly discriminated between Venturia levels in monogenic resistant (‘Topaz’), polygenic resistant (‘Discovery’), and susceptible (‘Golden Delicious’) cultivars, and proved especially useful to quantify pathogen levels during the initial latent stage of infection. The real-time PCR data of ‘Golden Delicious’ were consistent with the observed evolution of the degree of sporulation during a time-course experiment. Although measurements were influenced by the presence of co-extracted PCR-inhibitors, the impact of these compounds was independent of the apple cultivar or the initial amount of fungal DNA present. In conclusion, real-time PCR amplification of the ITS2-5.8S rDNA of Venturia spp. is a faster, more objective and more sensitive method to monitor fungal growth and to evaluate host resistance than phenotypic disease rating scores.

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Acknowledgments

This research was funded by a PhD grant of the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen). The authors thank Valérie Caffier from INRA (Angers, France) for kindly providing V. inaequalis isolates on PDA. Our gratitude also goes to Jane Debode from the Flemish Institute for Agriculture and Fishery Research (ILVO) in Merelbeke (Belgium), and to Bart Lievens from the Scientia Terrae Research Institute (Sint-Katelijne-Waver, Belgium) for their advice and support on protocols for extraction of gDNA from plant tissue.

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Authors and Affiliations

  1. Laboratory for Fruit Breeding and Biotechnology, Division of Crop Biotechnics, KULeuven, De Croylaan 42, 3001, Heverlee, Leuven, Belgium

    Bruno Daniëls, Mark W. Davey & Johan Keulemans

  2. Bayer CropScience SA-NV, Bayer Environmental Science, J.E. Mommaertslaan 14, 1831, Diegem, Machelen, Belgium

    Anke De landtsheer

  3. Bayer CropScience SA-NV, BioScience - Regulatory Science, Technologiepark 38, 9052, Gent, Belgium

    Rozemarijn Dreesen

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  1. Bruno Daniëls
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  2. Anke De landtsheer
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  5. Johan Keulemans
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Correspondence to Johan Keulemans.

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Daniëls, B., De landtsheer, A., Dreesen, R. et al. Real-time PCR as a promising tool to monitor growth of Venturia spp. in scab-susceptible and -resistant apple leaves. Eur J Plant Pathol 134, 821–833 (2012). https://doi.org/10.1007/s10658-012-0058-6

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