Guidelines for the reliable use of high throughput sequencing technologies to detect plant pathogens and pests

High-throughput sequencing (HTS) technologies have the potential to become one of the most significant advances in molecular diagnostics. Their use by researchers to detect and characterize plant pathogens and pests has been growing steadily for more than a decade and they are now envisioned as a routine diagnostic test to be deployed by plant pest diagnostics laboratories. Nevertheless, HTS technologies and downstream bioinformatics analysis of the generated datasets represent a complex process including many steps whose reliability must be ensured. The aim of the present guidelines is to provide recommendations for researchers and diagnosticians aiming to reliably use HTS technologies to detect plant pathogens and pests. These guidelines are generic and do not depend on the sequencing technology or platform. They cover all the adoption processes of HTS technologies from test selection to test validation as well as their routine implementation. A special emphasis is given to key elements to be considered: undertaking a risk analysis, designing sample panels for validation, using proper controls, evaluating performance criteria, confirming and interpreting results. These guidelines cover any HTS test used for the detection and identification of any plant pest (viroid, virus, bacteria, phytoplasma, fungi and fungus-like protists, nematodes, arthropods, plants) from any type of matrix. Overall, their adoption by diagnosticians and researchers should greatly improve the reliability of pathogens and pest diagnostics and foster the use of HTS technologies in plant health

A pospiviroid from symptomless portulaca plants closely related to iresine viroid 1

[EN] In symptomless plants of portulaca a potential new pospiviroid was characterized. Analysis by both double and return PAGE showed the presence of a circular RNA. RT-PCR and sequencing revealed a genome of 351 nt with properties characteristic of members of the genus Pospiviroid and with highest sequence identity (circa 80%) with iresine viroid 1 (IrVd-1). The circular RNA from portulaca was shown to replicate independently in its original host, thus showing that it is indeed a viroid. Based on its molecular characteristics, it should be considered a new species. However, since no biological differences have yet been found with its closest relative IrVd-1, the viroid from portulaca does not fulfil all criteria for species demarcation of the ICTV. © 2015 Elsevier B.V. All rights reserved.Work in R.F. laboratory has been funded by grants BFU2011-28443 and BFU2014-56812-P from Ministerio de Economia y Competititvidad (MINECO, Spain) and ACOMP/2014/A/103 from Generalitat Valenciana (GV). P.S. has been supported by post-doctoral contracts from GV (APOSTD/2010, program VALi+d) and MINECO (program Juan de la Cierva).Verhoeven, JJ.; Roenhorst, J.; Hooftman, M.; Meekes, E.; Flores Pedauye, R.; Serra Alfonso, P. (2015). A pospiviroid from symptomless portulaca plants closely related to iresine viroid 1. Virus Research. 205:22-26. doi:10.1016/j.virusres.2015.05.005S222620

Development of a multiplexed bead-based suspension array for the detection and discrimination of Pospiviroid plant pathogens

Efficient and reliable diagnostic tools for the routine indexing and certification of clean propagating material are essential for the management of pospiviroid diseases in horticultural crops. This study describes the development of a true multiplexed diagnostic method for the detection and identification of all nine currently recognized pospiviroid species in one assay using Luminex bead-based suspension array technology. In addition, a new data-driven, statistical method is presented for establishing thresholds for positivity for individual assays within multiplexed arrays. When applied to the multiplexed array data generated in this study, the new method was shown to have better control of false positives and false negative results than two other commonly used approaches for setting thresholds. The 11-plex Luminex MagPlex-TAG pospiviroid array described here has a unique hierarchical assay design, incorporating a near-universal assay in addition to nine species-specific assays, and a co-amplified plant internal control assay for quality assurance purposes. All assays of the multiplexed array were shown to be 100% specific, sensitive and reproducible. The multiplexed array described herein is robust, easy to use, displays unambiguous results and has strong potential for use in routine pospiviroid indexing to improve disease management strategies