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Fast and sensitive on-site isothermal assay (LAMP) for diagnosis and detection of three fruit tree phytoplasmas

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Abstract

Over the years, real-time PCR outflanked endpoint PCR in phytopathogen diagnostics, mainly because of the increase in sensitivity and timesaving aspects of the technique. However, a time consuming 16S rRNA-based nested PCR method is still the gold standard for phytoplasma diagnosis. This is also the case for phytoplasma detection in Malus, Pyrus and Prunus, the three main host plants of apple proliferation (AP), pear decline (PD) and European stone fruit yellows (ESFY) phytoplasma, respectively. The last decade, loop-mediated isothermal amplification (LAMP) (Notomi et al. 2000) is gaining a lot in significance and is also for phytoplasmas expected to become a widely used reliable diagnostic tool. High specificity and sensitivity which also requires a less stringent need for DNA purification, and the short analysis time and the limited equipment requirements makes the LAMP method a fast and affordable alternative with great point-of-care diagnostic potential. In this paper, we present a LAMP primer set for the ribosomal group 16SrX, containing the important fruit tree phytoplasmas AP, PD and ESFY. The primers were developed and validated for fast and sensitive detection and general use for diagnosis. We foresee that the LAMP technique will also have its application in on-site diagnosis of the fruit tree phytoplasmas during inspections and surveys.

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Acknowledgments

We wish to thank Vision4Care, Zottegem, Belgium for the use of the Genie®III instrument during the validation experiments. The authors are also grateful to Bernd Schneider (JKI, Germany), Santiago Schaerer (ACW, Switzerland) and Helga Reisenzein (AGES, Austria) of the Euphresco ERAnet APOPHYT project group for the provision of the ESFY isolates included in this study. This study was supported by the Belgian Plant Protection Service (FASFC and FAVV). We wish to thank Barbara Van Steendam for her help with the ESFY validation experiments.

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  1. Institute for Agricultural and Fisheries Research (ILVO), Burgemeester Van Gansberghelaan 96, 9820, Merelbeke, Belgium

    K. De Jonghe, I. De Roo & M. Maes

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  1. K. De Jonghe
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  2. I. De Roo
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  3. M. Maes
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Correspondence to K. De Jonghe.

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De Jonghe, K., De Roo, I. & Maes, M. Fast and sensitive on-site isothermal assay (LAMP) for diagnosis and detection of three fruit tree phytoplasmas. Eur J Plant Pathol 147, 749–759 (2017). https://doi.org/10.1007/s10658-016-1039-y

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