Dahlia latent viroid: a recombinant new species of the family Pospiviroidae posing intriguing questions about its origin and classification

[EN] A viroid-like RNA has been detected in two asymptomatic dahlia accessions by return and double PAGE. It appeared smaller than Chrysanthemum stunt viroid and Potato spindle tuber viroid, the two members of the genus Pospiviroid, family Pospiviroidae, reported in this ornamental previously. RT-PCR with primers designed for amplifying all pospiviroids produced no amplicons, but RT-PCR with random primers revealed a 342 nt RNA. The sequence of this RNA was confirmed with specific primers, which additionally revealed its presence in many dahlia cultivars. The RNA was named Dahlia latent viroid (DLVd) because it replicates autonomously, but symptomlessly, in dahlia and shares maximum sequence identity with other viroids of less than 56 %. Furthermore, DLVd displays characteristic features of the family Pospiviroidae: a predicted rod-like secondary structure of minimum free energy with a central conserved region (CCR), and the ability to form the metastable structures hairpins I and II. Its CCR is identical to that of Hop stunt viroid (HSVd, genus Hostuviroid). However, DLVd: (i) has the terminal conserved region present in members of the genus Pospiviroid, but absent in HSVd, and (ii) lacks the terminal conserved hairpin present in HSVd. Phylogenetic reconstructions indicate that HSVd and Pepper chat fruit viroid (genus Pospiviroid) are the closest relatives of DLVd, but DLVd differs from these viroids in its host range, restricted to dahlia so far. Therefore, while DLVd fulfils the criteria to be a novel species of the family Pospiviroidae, its recombinant origin makes assignment to the genera Pospiviroid or Hostuviroid problematicResearch in R. F.'s laboratory is presently supported from the Ministerio de Educacion y Ciencia (MEC) of Spain by grant BFU2011-28443. During this work P. S. has been supported by postdoctoral contracts from the Generalitat Valenciana (APOSTD/2010, program VALi + d) and the MEC (Program Juan de la Cierva).Verhoeven, JTJ.; Meekes, ETM.; Roenhorst, JW.; Flores Pedauye, R.; Serra Alfonso, P. (2013). Dahlia latent viroid: a recombinant new species of the family Pospiviroidae posing intriguing questions about its origin and classification. Journal of General Virology. 94(4):711-719. https://doi.org/10.1099/vir.0.048751-0S71171994

Development and validation of a real-time RT-PCR test for screening pepper and tomato seed lots for the presence of pospiviroids.

Potato spindle tuber viroid and other pospiviroids can cause serious diseases in potato and tomato crops. Consequently, pospiviroids are regulated in several countries. Since seed transmission is considered as a pathway for the introduction and spread of pospiviroids, some countries demand for the testing of seed lots of solanaceous crops for the presence of pospiviroids. A real-time RT-PCR test, named PospiSense, was developed for testing pepper (Capsicum annuum) and tomato (Solanum lycopersicum) seeds for seven pospiviroid species known to occur naturally in these crops. The test consists of two multiplex reactions running in parallel, PospiSense 1 and PospiSense 2, that target Citrus exocortis viroid (CEVd), Columnea latent viroid (CLVd), pepper chat fruit viroid (PCFVd), potato spindle tuber viroid (PSTVd), tomato apical stunt viroid (TASVd), tomato chlorotic dwarf viroid (TCDVd) and tomato planta macho viroid (TPMVd, including the former Mexican papita viroid). Dahlia latent viroid (DLVd) is used as an internal isolation control. Validation of the test showed that for both pepper and tomato seeds the current requirements of a routine screening test are fulfilled, i.e. the ability to detect one infested seed in a sample of c.1000 seeds for each of these seven pospiviroids. Additionally, the PospiSense test performed well in an inter-laboratory comparison, which included two routine seed-testing laboratories, and as such provides a relatively easy alternative to the currently used tests

Tomato Brown Rugose Fruit Virus Nextstrain Build Version 3: Rise of a Novel Clade

In the Netherlands, tomato brown rugose fruit virus (ToBRFV; genus Tobamovirus) was first identified in tomato crops in 2019. Since then, the National Plant Protection Organization (NPPO-NL) has performed surveys to track and trace this regulated virus aiming for its eradication. To gain more insight in the epidemiology of ToBRFV, genomes were assembled from Illumina sequence data. Whole-genome phylogenetics was integrated with epidemiological metadata in a Nextstrain build. Two new clades were defined, one of which displayed a rapid increase in comparison to the previous version of the Nextstrain build. This rapid increase could be attributed to the unauthorized application of an isolate of ToBRFV as a cross-protection product. Further analysis of the test results of positive samples from tomato production sites suggests that both deliberate application and accidental introduction had occurred. This report introduces the inclusion of 61 new (near) complete ToBRFV genomes in version three of the Nextstrain build, available from https://nextstrain.nrcnvwa.nl/ToBRFV/20220412. [Graphic: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license

Facilitating the adoption of high‐throughput sequencing technologies as a plant pest diagnostic test in laboratories: A step‐by‐step description

International audienceHigh-throughput sequencing (HTS) is a powerful tool that enables the simultaneous detection and potential identification of any organisms present in a sample. The growing interest in the application of HTS technologies for routine diagnostics in plant health laboratories is triggering the development of guidelines on how to prepare laboratories for performing HTS testing. This paper describes general and technical recommendations to guide laboratories through the complex process of preparing a laboratory for HTS tests within existing quality assurance systems. From nucleic acid extractions to data analysis and interpretation, all of the steps are covered to ensure reliable and reproducible results. These guidelines are relevant for the detection and identification of any plant pest (e.g. arthropods, bacteria, fungi, nematodes, invasive plants or weeds, protozoa, viroids, viruses), and from any type of matrix (e.g. pure microbial culture, plant tissue, soil, water), regardless of the HTS technology (e.g. amplicon sequencing, shotgun sequencing) and of the application (e.g. surveillance programme, phytosanitary certification, quarantine, import control). These guidelines are written in general terms to facilitate the adoption of HTS technologies in plant pest routine diagnostics and enable broader application in all plant health fields, including research. A glossary of relevant terms is provided among the Supplementary Material