Abstract
Rapid and sensitive assays for the identification of plant pathogens are necessary for the effective management of crop diseases. The main limitation of current diagnostic testing is the inability to combine broad and sensitive pathogen detection with the identification of key strains, pathovars, and subspecies. Such discrimination is necessary for quarantine pathogens, whose management is strictly dependent on genotype identification. To address these needs, we have established and evaluated a novel all-in-one diagnostic assay based on nanopore sequencing for the detection and simultaneous characterization of quarantine pathogens, using Xylella fastidiosa as a case study. The assay proved to be at least as sensitive as standard diagnostic tests and the quantitative results agreed closely with qPCR-based analysis. The same sequencing results also allowed discrimination between subspecies when present either individually or in combination. Pathogen detection and typing were achieved within 13 min of sequencing owing to the use of an internal control that allowed to stop sequencing when sufficient data had accumulated. These advantages, combined with the use of portable equipment, will facilitate the development of next-generation diagnostic assays for the efficient monitoring of other plant pathogens.
Highlights
Plant pathogens and the diseases they cause are severe threats to global food security, resulting in yield losses of up to 30% in major staple crops and requiring the use of large quantities of pesticides for pathogen and/or vector control [1,2,3]
A900-bp region of the Xylella gene encoding protein HL was amplified in triplicate from each sample with primers annealing to sequences conserved in each subspecies, and the resulting amplicons were used to generate a multiplex Oxford Nanopore Technologies (ONT) sequencing library
The effective management of quarantine pathogens according to international regulations requires fast, sensitive, and cost-effective diagnostic tests that detect the pathogen and distinguish specific genotypes in a quantitative manner [27,37,38]
Summary
Plant pathogens and the diseases they cause are severe threats to global food security, resulting in yield losses of up to 30% in major staple crops and requiring the use of large quantities of pesticides for pathogen and/or vector control [1,2,3]. Gold standard methods include the enzyme-linked immunosorbent assay (ELISA) to detect pathogen proteins, and the polymerase chain reaction (PCR), quantitative real-time PCR (qPCR), or loop-mediated isothermal amplification (LAMP) to detect pathogen nucleic acids. These are quicker and more reliable than traditional methods requiring pathogen cultures, but sample preparation and analysis can take a long time, and the results are not quantitative unless standards of known concentration are available. The effective management of quarantine pathogens, those subject to international legislation [9], requires diagnostic procedures that are, at the same time, both broad and specific, as well as fast, sensitive, reliable, quantitative, and inexpensive
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