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 it 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 is 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 analyzed 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 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-analyzed samples were confirmed with the triplex TaqMan polymerase chain reaction and loop-mediated isothermal amplification.

Nanopore sequencing for the detection and the identification of Xylella fastidiosa subspecies and sequence types from naturally infected plant material

“…Nanopore direct DNA sequencing of these samples provides a very low number of reads which reflect an even lower number of reads mapping to Xf genomes making the analysis doubtable. A similar result was also obtained by Bonants et al (2019) using Illumina, who reported the ability of NGS to determine the ST of Xf only in highly infected samples. The reason of the low sensitivity of the direct Nanopore sequencing should be addressed to the low quality of the DNA for the tested samples.…”

“…In the last fifteen years, NGS transformed genomic research with an inevitable impact also on diagnostics, taking us to a new scenario. These technologies have recently been applied for the diagnosis of plant pathogens (Bronzato Badial et al , 2018; Chalupowicz et al , 2019) and for the detection and identification of Xf (Bonants et al , 2019). Although several diagnostic methods are available for the detection of Xf (EPPO, 2016b), the identification of subspecies and ST are currently laborious and time-consuming.…”

PM 7/24 (4) Xylella fastidiosa

“…Appendix 9 -Triplex real-time PCR (Bonants et al, 2019) The test below is described as it was carried out to generate the validation data provided in section 4. Other equipment, kits or reagents may be used provided that verification (see PM 7/98) is carried out.…”

“…naturally infected host tissue or host tissue spiked with the target organism). For a series of analyses including samples from different plant species, whenever possible one PIC should be included per plant species to be analysed, or per botanical genus • Negative amplification control (NAC) to rule out false positives due to contamination during the preparation of the reaction mix: the molecular-grade water that was used to prepare the reaction mix The gBlock containing the Acidovorax cattleyae (Acat) internal control amplicon sequence as described by Bonants et al (2019) can be used to detect the Acat internal control.…”

Novel Tetraplex Quantitative PCR Assays for Simultaneous Detection and Identification of Xylella fastidiosa Subspecies in Plant Tissues

“…For plant pathogens, these methods have been tested on samples of naturally infected plants, spiked samples (Li et al, 2009;Willsey et al, 2018), and on mixtures of plant and pathogen DNAs (Abraham et al, 2018). Xf-specific multiplexed qPCR assays have already been developed based on the combination of primers designed by Harper et al (2010) and Ouyang et al (2013) (Bonants et al, 2018). Other tests were proposed to differentiate Xf from phytoplasmas sharing common host plants (Ito and Suzaki, 2017) and to differentiate the subspecies fastidiosa from the subspecies multiplex (Burbank and Ortega, 2018).…”