Plant Pathology (2021) 70, 1860-1870

Luigi Faino, Valeria Scala, Alessio Albanese, Vanessa Modesti, Alessandro Grottoli, Nicoletta Pucci, Andrea Doddi, Alessia L'Aurora, Giuseppe Tatulli, Massimo Reverberi and Stefania Loreti (2021)
Nanopore sequencing for the detection and identification of Xylella fastidiosa subspecies and sequence types from naturally infected plant material
Plant Pathology 70 (8), 1860-1870
Abstract: Xylella 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.
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Database assignments for author(s): Massimo Reverberi, Stefania Loreti

Research topic(s) for pests/diseases/weeds:
identification/taxonomy

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Pest and/or beneficial records:

Beneficial	Pest/Disease/Weed	Crop/Product	Country	Quarant.
	Xylella fastidiosa	Almond (Prunus dulcis)	Italy
	Xylella fastidiosa	Olive (Olea europaea)	Italy
	Xylella fastidiosa	Lavender (Lavandula)	Italy
	Xylella fastidiosa	Rosemary (Salvia rosmarinus)	Italy
	Xylella fastidiosa	Polygala (crop)	Italy
	Xylella fastidiosa	Rhamnus (crop)	Italy