Real-Time On-Site Diagnosis of Quarantine Pathogens in Plant Tissues by Nanopore-Based Sequencing

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.

Next‐generation methods for early disease detection in crops

“…It is able to sequence individual DNA molecules as they are driven through biological nanopores by an applied electrical field. The MinION is a promising fast‐detection technology for plant pathogen detection 137–139 because the instrument is smaller than a smartphone and provides output data within a few minutes. It has been explored for the sequencing of bacterial and virus pathogens, 132 detecting and genotype potato viruses 103 ; identifying Xylella fastidiosa subspecies and sequence in naturally infected olive samples 140 and in grapevine 141 and for the detection Fusarium oxysporum on Zinnia hybrida (zinnia) plants 142 …”

Nanopore Metagenomics Sequencing for Rapid Diagnosis and Characterization of Lily Viruses

“…The ONT platform was introduced as a portable MinION sequencer that meets the minimum requirements for technical skills and bioinformatics knowledge and can be performed under field conditions and in small laboratories with a minimal package of laboratory equipment ( Mehetre et al, 2021 ). The ease of point-of-care testing using MinION has been demonstrated and reported in several field studies ( Faria et al, 2017 ; Maestri et al, 2019 ; Marcolungo et al, 2022 ; Quick et al, 2016 ). Therefore, these technologies will provide new avenues for point-of-care diagnostics for plant viral diseases.…”

“…Similar to other HTS methods, benchtop sequencers are routinely used in laboratories because ONT-based studies such as MinION require sophisticated bioinformatics tools for data management and analysis ( Marcolungo et al, 2022 ). A single run can detect and diagnose multiple virus species in real time and has great potential for a variety of applications ( Ben Chehida et al, 2021 ).…”

Recombinase Polymerase Amplification Assay for the Field Detection of Mal Secco Disease byPlenodomus tracheiphilus

“…For this reason, nowadays, these emergencies must rely on technologically advanced means, such as molecular techniques able to satisfy the major requirements of the detection, i.e. specificity, sensitivity and rapidity (Aslam et al ., 2017; Boonham et al ., 2016; Donoso and Valenzuela, 2018; Hariharan and Prasannath, 2021; Marcolungo et al ., 2022; Patel et al ., 2022).…”