Abstract
AbstractXylella fastidiosa (Xf) is a gram‐negative bacterial plant pathogen that can infect over 500 plant species. While it is endemic in America, X. fastidiosa subsp. pauca was reported for the first time in Europe in 2013 on olive trees in southern Italy. The availability of fast, sensitive, and reliable diagnostic tools is indispensable for managing current and future outbreaks of Xf. In this paper, we use the OXford Nanopore Technologies (ONT) MinION platform for detecting and identifying Xf at species, subspecies, and sequence type (ST) level. Two workflows were developed: the first one provided a “shotgun” strategy, that is, exploring the possibility of detecting Xf within DNA extracted from plant samples. This allowed detection of Xf by direct DNA sequencing and identifying the subspecies only in samples with high bacterial levels. Nanopore amplicon sequencing was pursued as a second workflow. This consists of PCR amplification of a set of seven multilocus sequence typing (MLST) fragments, officially adopted for identifying Xf at type strain level, followed by Nanopore‐sequencing of the amplicons and an ad hoc pipeline to generate MLST consensus calls. This combined approach, which takes only a few hours, allowed the detection and identification of Xf at ST level in plant material with low bacterial infection.
Highlights
Xylella fastidiosa (Xf), a gram-negative phytopathogenic bacterium (Wells et al, 1987), has a very broad host range, causing different diseases in important crops (Hopkins, 1989) and in many urban shade trees (Sherald & Kostka, 1992)
Xf subspecies include different sequence types (STs) that are determined by multilocus sequence typing (MLST) analysis based on the sequencing of seven housekeeping genes (EPPO, 2019b; Nunney et al, 2012; Yuan et al, 2010)
Several diagnostic methods are available for the detection of Xf (EPPO, 2019b), the identification of subspecies and ST are currently laborious and time-consuming
Summary
Xylella fastidiosa (Xf), a gram-negative phytopathogenic bacterium (Wells et al, 1987), has a very broad host range, causing different diseases in important crops (Hopkins, 1989) and in many urban shade trees (Sherald & Kostka, 1992). MLST analysis is recommended for unambiguous identification of subspecies and ST in the case of a new outbreak or new plant hosts (EPPO, 2019b). A low concentration of the bacteria, and contaminants derived from the plant material, makes the MLST amplification, and subspecies and ST determination, extremely challenging (EPPO, 2019b). In our study we propose a strategy that exploits OXford Nanopore Technologies (ONT) to diagnose emerging and invasive pathogens such as Xf. For this, we investigated two strategies: one relies on a shotgun approach, that is, direct sequencing of plant extracts, and the other on amplicon sequencing. We investigated two strategies: one relies on a shotgun approach, that is, direct sequencing of plant extracts, and the other on amplicon sequencing The latter was the most reliable and robust strategy for subspecies and ST determination
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