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Development and evaluation of Taqman assays for the differentiation of Dickeya (sub)species

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Abstract

TaqMan assays were developed for the detection of seven Dickeya species, namely D. dianthicola, D. dadantii, D. paradisiaca, D. chrysanthemi, D. zeae, D. dieffenbachiae and D. solani. Sequences of the gene coding for dnaX were used for the design of primers and probes. In studies with axenic cultures of bacteria, the assays were highly specific and only reacted with strains of the target species, and not with non-target bacteria, including those belonging to other Dickeya species and other genera. The detection thresholds for DNA extracted from pure cultures of target strains ranged from 10 to 100 fg. The TaqMan assays for D. dianthicola and D. solani were more extensively evaluated as part of a method validation procedure. The threshold level for target bacteria added to a potato peel extract diluted ten-times in a semi-selective broth, was strain dependent and ranged from 1,000 to 100,000 cfu/ml. The coefficients of variation for repeatability and reproducibility were low and results were largely independent of the type of substrate, i.e. potato tuber or carnation leaf extracts. However, during routine testing of seed potatoes, false-positive reactions were found with the assay for D. solani. The use of the TaqMan assays for inspection of plant propagation material, ecological studies and studies on the effect of control strategies in disease management strategies is discussed.

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Acknowledgments

This work has been financially supported by the Dutch Ministry of Economic Affairs, (project BO-06-004-2.1)and FES project Strengthening the infrastructure of plant health. Thanks are indebted to Dr M.C.M. Pérombelon (Dundee, UK) for his editorial work.

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Author notes

  1. R. van Hoof

    Present address: RIKILT, Akkermaalsbos 2, 6708 WB, Wageningen, The Netherlands

Authors and Affiliations

  1. Plant Research International, P.O. Box 69, 6700 AB, Wageningen, The Netherlands

    J. M. van der Wolf, B. H. de Haas & R. van Hoof

  2. Dutch General Inspection Service for Agricultural Seed and Seed Potatoes (NAK), P.O. Box 1115, Randweg 14, 8300 BC, Emmeloord, The Netherlands

    E. G. de Haan & G. W. van den Bovenkamp

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  1. J. M. van der Wolf
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  2. B. H. de Haas
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  4. E. G. de Haan
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  5. G. W. van den Bovenkamp
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Correspondence to J. M. van der Wolf.

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van der Wolf, J.M., de Haas, B.H., van Hoof, R. et al. Development and evaluation of Taqman assays for the differentiation of Dickeya (sub)species. Eur J Plant Pathol 138, 695–709 (2014). https://doi.org/10.1007/s10658-013-0343-z

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  • DOI: https://doi.org/10.1007/s10658-013-0343-z

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