Development and evaluation of a triplex TaqMan assay and Next Generation Sequence Analysis for improved detection of Xylella in plant material

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Abstract

Xylella fastidiosa is a heterogenous gram-negative bacterial plant pathogen with a wide host range covering over 300 plant species. Since 2013, in Europe, the presence of the pathogen is increasing in a part of the Mediterranean area, but causes in particular severe disease problems in olive orchards in the Southern part of Italy. Various subspecies of the pathogen were also diagnosed in natural outbreaks and intercepted ornamental plants in Europe, among them Olea europaea, Coffea arabica and Nerium oleander. The host range of the pathogen can vary, depending on the subspecies and even the strain. The availability of fast and reliable diagnostic tools are indispensable in management strategies to control diseases caused by X. fastidiosa. To improve the reliability of the TaqMan assay, currently widely used in surveys, a triplex TaqMan assay was developed in which two specific and sensitive TaqMan assays, previously designed for X. fastidiosa, were combined with an internal control. The triplex assay exhibited the same diagnostic sensitivity as the simplex assays. In addition, the usefulness of a metagenomic approach using next generation sequencing (NGS) was demonstrated, in which total DNA extracted from plant material was sequenced. DNA extracts from plant material free of X. fastidiosa, from artificially inoculated hosts plants or from naturally infected plants sampled in France, Spain and Italy, or intercepted in Austria and The Netherlands, were analysed for the presence of X. fastidiosa using the metagenomic approach. In all samples, even in samples with a low infection level, but not in the pathogen-free samples, DNA reads were detected specific for X. fastidiosa. In most cases, the pathogen could be identified up to the subspecies level and for one sample even the whole genome could be assembled and the sequence type could be determined. All results of NGS analysed samples were confirmed with the triplex TaqMan PCR and LAMP.
LanguageEnglish
Number of pages36
JournalPlant Disease
Volume103
Issue number4
Early online date18 Feb 2019
DOIs
Publication statusPublished - Apr 2019

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Xylella
Xylella fastidiosa
sequence analysis
assays
pathogens
host range
DNA
sampling
Italy
Nerium oleander
diagnostic sensitivity
Olea europaea
Coffea arabica
ornamental plants
Austria
plant pathogens
Netherlands
disease control
orchards
host plants

Cite this

@article{20efa6bbf39b4b12b7666d68e3b3235d,
title = "Development and evaluation of a triplex TaqMan assay and Next Generation Sequence Analysis for improved detection of Xylella in plant material",
abstract = "Xylella fastidiosa is a heterogenous gram-negative bacterial plant pathogen with a wide host range covering over 300 plant species. Since 2013, in Europe, the presence of the pathogen is increasing in a part of the Mediterranean area, but causes in particular severe disease problems in olive orchards in the Southern part of Italy. Various subspecies of the pathogen were also diagnosed in natural outbreaks and intercepted ornamental plants in Europe, among them Olea europaea, Coffea arabica and Nerium oleander. The host range of the pathogen can vary, depending on the subspecies and even the strain. The availability of fast and reliable diagnostic tools are indispensable in management strategies to control diseases caused by X. fastidiosa. To improve the reliability of the TaqMan assay, currently widely used in surveys, a triplex TaqMan assay was developed in which two specific and sensitive TaqMan assays, previously designed for X. fastidiosa, were combined with an internal control. The triplex assay exhibited the same diagnostic sensitivity as the simplex assays. In addition, the usefulness of a metagenomic approach using next generation sequencing (NGS) was demonstrated, in which total DNA extracted from plant material was sequenced. DNA extracts from plant material free of X. fastidiosa, from artificially inoculated hosts plants or from naturally infected plants sampled in France, Spain and Italy, or intercepted in Austria and The Netherlands, were analysed for the presence of X. fastidiosa using the metagenomic approach. In all samples, even in samples with a low infection level, but not in the pathogen-free samples, DNA reads were detected specific for X. fastidiosa. In most cases, the pathogen could be identified up to the subspecies level and for one sample even the whole genome could be assembled and the sequence type could be determined. All results of NGS analysed samples were confirmed with the triplex TaqMan PCR and LAMP.",
author = "P.J.M. Bonants and Y. Griekspoor and I.M. Houwers and M.C. Krijger and {van der Zouwen}, P.S. and {van der Lee}, T.A.J. and {van der Wolf}, J.M.",
year = "2019",
month = "4",
doi = "10.1094/PDIS-08-18-1433-RE",
language = "English",
volume = "103",
journal = "Plant Disease",
issn = "0191-2917",
publisher = "American Phytopathological Society",
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}

TY - JOUR

T1 - Development and evaluation of a triplex TaqMan assay and Next Generation Sequence Analysis for improved detection of Xylella in plant material

AU - Bonants, P.J.M.

AU - Griekspoor, Y.

AU - Houwers, I.M.

AU - Krijger, M.C.

AU - van der Zouwen, P.S.

AU - van der Lee, T.A.J.

AU - van der Wolf, J.M.

PY - 2019/4

Y1 - 2019/4

N2 - Xylella fastidiosa is a heterogenous gram-negative bacterial plant pathogen with a wide host range covering over 300 plant species. Since 2013, in Europe, the presence of the pathogen is increasing in a part of the Mediterranean area, but causes in particular severe disease problems in olive orchards in the Southern part of Italy. Various subspecies of the pathogen were also diagnosed in natural outbreaks and intercepted ornamental plants in Europe, among them Olea europaea, Coffea arabica and Nerium oleander. The host range of the pathogen can vary, depending on the subspecies and even the strain. The availability of fast and reliable diagnostic tools are indispensable in management strategies to control diseases caused by X. fastidiosa. To improve the reliability of the TaqMan assay, currently widely used in surveys, a triplex TaqMan assay was developed in which two specific and sensitive TaqMan assays, previously designed for X. fastidiosa, were combined with an internal control. The triplex assay exhibited the same diagnostic sensitivity as the simplex assays. In addition, the usefulness of a metagenomic approach using next generation sequencing (NGS) was demonstrated, in which total DNA extracted from plant material was sequenced. DNA extracts from plant material free of X. fastidiosa, from artificially inoculated hosts plants or from naturally infected plants sampled in France, Spain and Italy, or intercepted in Austria and The Netherlands, were analysed for the presence of X. fastidiosa using the metagenomic approach. In all samples, even in samples with a low infection level, but not in the pathogen-free samples, DNA reads were detected specific for X. fastidiosa. In most cases, the pathogen could be identified up to the subspecies level and for one sample even the whole genome could be assembled and the sequence type could be determined. All results of NGS analysed samples were confirmed with the triplex TaqMan PCR and LAMP.

AB - Xylella fastidiosa is a heterogenous gram-negative bacterial plant pathogen with a wide host range covering over 300 plant species. Since 2013, in Europe, the presence of the pathogen is increasing in a part of the Mediterranean area, but causes in particular severe disease problems in olive orchards in the Southern part of Italy. Various subspecies of the pathogen were also diagnosed in natural outbreaks and intercepted ornamental plants in Europe, among them Olea europaea, Coffea arabica and Nerium oleander. The host range of the pathogen can vary, depending on the subspecies and even the strain. The availability of fast and reliable diagnostic tools are indispensable in management strategies to control diseases caused by X. fastidiosa. To improve the reliability of the TaqMan assay, currently widely used in surveys, a triplex TaqMan assay was developed in which two specific and sensitive TaqMan assays, previously designed for X. fastidiosa, were combined with an internal control. The triplex assay exhibited the same diagnostic sensitivity as the simplex assays. In addition, the usefulness of a metagenomic approach using next generation sequencing (NGS) was demonstrated, in which total DNA extracted from plant material was sequenced. DNA extracts from plant material free of X. fastidiosa, from artificially inoculated hosts plants or from naturally infected plants sampled in France, Spain and Italy, or intercepted in Austria and The Netherlands, were analysed for the presence of X. fastidiosa using the metagenomic approach. In all samples, even in samples with a low infection level, but not in the pathogen-free samples, DNA reads were detected specific for X. fastidiosa. In most cases, the pathogen could be identified up to the subspecies level and for one sample even the whole genome could be assembled and the sequence type could be determined. All results of NGS analysed samples were confirmed with the triplex TaqMan PCR and LAMP.

U2 - 10.1094/PDIS-08-18-1433-RE

DO - 10.1094/PDIS-08-18-1433-RE

M3 - Article

VL - 103

JO - Plant Disease

T2 - Plant Disease

JF - Plant Disease

SN - 0191-2917

IS - 4

ER -