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

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 problematic.

Possible Overestimation of Seed Transmission in the Spread of Pospiviroids in Commercial Pepper and Tomato Crops Based on Large-Scale Grow-Out Trials and Systematic Literature Review.

Several outbreaks of pospiviroids have been reported in pepper and tomato crops worldwide. Tracing back the origin of the infections has led to different sources. In some cases, the infections were considered to result from seed transmission. Other outbreaks were related to transmission from ornamental crops and weeds. Pospiviroids, in particular potato spindle tuber viroid, are regulated by many countries because they can be harmful to potatoes and tomatoes. Seed transmission has been considered an important pathway of introduction and spread. However, the importance of this pathway can be questioned. This paper presents data on seed transmission from large-scale grow-out trials of infested pepper and tomato seed lots produced under standard seed-industry conditions. In addition, it presents the results of a systematic review of published data on seed transmission and outbreaks in commercial pepper and tomato crops. Based on the results of the grow-out trials and review of the literature, it was concluded that the role of seed transmission in the spread of pospiviroids in practice is possibly overestimated.

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.