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

Abstract

SummaryXylella fastidiosa (Xf) is a polyphagous gram-negative bacterial plant pathogen that can infect more than 300 plant species. It is endemic in America while, in 2013, Xf subsp. pauca was for the first time reported in Europe on olive tree in the Southern Italy. The availability of fast and reliable diagnostic tools is indispensable for managing current and future outbreaks of Xf.In this work, we used the Oxford Nanopore Technologies (ONT) device MinION platform for detecting and identifying Xf at species, subspecies and Sequence Type (ST) level straight from infected plant material. The study showed the possibility to detect Xf by direct DNA sequencing and identify the subspecies in highly infected samples. In order to improve sensitivity, Nanopore amplicon sequencing was assessed. Using primers within the set of the seven MLST officially adopted for identifying Xf at type strain level, we developed a workflow consisting in a multiple PCR and an ad hoc pipeline to generate MLST consensus after Nanopore-sequencing of the amplicons. The here-developed combined approach achieved a sensitivity higher than real-time PCR allowing within few hours, the detection and identification of Xf at ST level in infected plant material, also at low level of contamination.Originality Significance StatementIn this work we developed a methodology that allows the detection and identification of Xylella fastidiosa in plant using the Nanopore technology portable device MinION. The approach that we develop resulted more sensitive than methods currently used for detecting X. fastidiosa, like real-time PCR. This approach can be extensively used for X. fastidiosa detection and it may pave the road for the detection of other tedious vascular pathogens.

From Nucleotides to Satellite Imagery: Approaches to Identify and Manage the Invasive Pathogen Xylella fastidiosa and Its Insect Vectors in Europe

“…To this aim, SNPs provides deeper resolution, but their discovery is heavily based on an appropriate reference genome(s) and results from different studies might not be comparable [68]. Developing faster, more reliable and efficient genetic tools that allow the simultaneous detection of Xf and its identification at the subspecies/strain level as well as the detection of mixed infections of multiple subspecies in one sample has been the focus of the most recent assays [91,[129][130][131]. Other efforts have aimed to portability, low-cost and ease of use with the development of miniaturized devices such as lab-on-chip, which shows intermediate performance between ELISA and RT-PCR methods [132].…”

“…Genome approaches are not yet time and cost effective for the rapid identification of pathogens and for field applications, but they allow for the quick discovery of multiple, genome-wide makers (e.g., SNPs) for strain/isolate classification and for tracing and monitoring projects [77,129]. Recently, metagenomic approaches allowed detecting and characterizing Xf without the need for pathogen cultivation and amplification of specific bacterial genes, but subspecies or strain classification was limited to material with high bacterial concentrations [129][130][131][132].…”

Investigating plant disease outbreaks with long-read metagenomics: sensitive detection and highly resolved phylogenetic reconstruction applied to Xylella fastidiosa

“…Metagenomic sequencing of plant samples with the MinION sequencer or Illumina sequencing platforms had been used previously to detect viral, fungal and bacterial plant pathogens [16], including X. fastidiosa [17,45,46]. However, when the MinION was used previously to detect X. fastidiosa, the obtained sequencing reads were simply aligned to reference genomes to find the genome to which the highest number of reads mapped without further phylogenetic analysis [17]; identification was limited to subspecies, or sequencing was performed only after PCR of MLST loci making whole-genome-based phylogenetic analysis impossible [45]. More recently, the Illumina iSeq and MiSeq were used for detection and identification of X. fastidiosa as well [18].…”

Trends in Molecular Diagnosis and Diversity Studies for Phytosanitary Regulated Xanthomonas

“…This RFLP approach was informed by the comparison of complete genome sequences of representatives of each sub-clade [ 302 ]. Another discriminatory approach is the sequencing of informative loci, i.e., an MLSA; that could allow high-resolution identification of the pathogen type directly from the DNA of infected plant material, as exemplified in Xylella by Faino et al [ 303 ].…”