Symptom Severity, Infection Progression and Plant Responses in Solanum Plants Caused by Three Pospiviroids Vary with the Inoculation Procedure

Infectious viroid clones consist of dimeric cDNAs used to generate transcripts which mimic the longer-than-unit replication intermediates. These transcripts can be either generated in vitro or produced in vivo by agro-inoculation. We have designed a new plasmid, which allows both inoculation methods, and we have compared them by infecting Solanum lycopersicum and Solanum melongena with clones of Citrus exocortis virod (CEVd), Tomato chlorotic dwarf viroid (TCDVd), and Potato spindle tuber viroid (PSTVd). Our results showed more uniform and severe symptoms in agro-inoculated plants. Viroid accumulation and the proportion of circular and linear forms were different depending on the host and the inoculation method and did not correlate with the symptoms, which correlated with an increase in PR1 induction, accumulation of the defensive signal molecules salicylic (SA) and gentisic (GA) acids, and ribosomal stress in tomato plants. The alteration in ribosome biogenesis was evidenced by both the upregulation of the tomato ribosomal stress marker SlNAC082 and the impairment in 18S rRNA processing, pointing out ribosomal stress as a novel signature of the pathogenesis of nuclear-replicating viroids. In conclusion, this updated binary vector has turned out to be an efficient and reproducible method that will facilitate the studies of viroid–host interactions.

Universal primers for rapid detection of six pospiviroids in Solanaceae plants using one-step RT-PCR and RT-LAMP

A number of viruses and viroids infect solanaceous plants causing severe yield losses. Several seed-borne viroids are currently listed as quarantine pathogens in many countries. Among them, 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) are of major concerns. The objective of this study was to design and test universal primers that could be used to detect six viroids in solanaceous plants using one-step RT-PCR and reverse transcription loop-mediated isothermal amplification (RT-LAMP). Results revealed that a pair of degenerate primers could be used in a one-step RT-PCR to amplify six pospiviroids from Solanaceae seeds and plants. Moreover, five primers were designed and used in RT-LAMP to amplify six pospiviroids. The minimal concentration of viroid RNA required for a successful detection varied, ranging from one femtogram to 10 nanograms, depending on the species of viroid and detection method. In general, RT-LAMP was more sensitive than RT-PCR but both assays were rapid and highly sensitive tools to detect six pospiviroids. Detection methods currently in use for these viroids require at least two different sets of primers. The assays developed in this research could facilitate to screen a large number of solanaceous plants and seeds intended for import and export.

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.

Viroid-infected Tomato and Capsicum Seed Shipments to Australia

Pospiviroid species are transmitted through capsicum and tomato seeds. Trade in these seeds represents a route for the viroids to invade new regions, but the magnitude of this hazard has not been adequately investigated. Since 2012, tomato seed lots sent to Australia have been tested for pospiviroids before they are released from border quarantine, and capsicum seed lots have been similarly tested in quarantine since 2013. Altogether, more than 2000 seed lots have been tested. Pospiviroids were detected in more than 10% of the seed lots in the first years of mandatory testing, but the proportion of lots that were infected declined in subsequent years to less than 5%. Six pospiviroid species were detected: Citrus exocortis viroid, Columnea latent viroid, Pepper chat fruit viroid, Potato spindle tuber viroid, Tomato chlorotic dwarf viroid and Tomato apical stunt viroid. They were detected in seed lots exported from 18 countries from every production region. In many seed lots, the detectable fraction (prevalence) of infected seeds was estimated to be very small, as low as 6 × 10−5 (~1 in 16,000; CI 5 × 10−6 to 2.5 × 10−4) for some lots. These findings raise questions about seed production practices, and the study indicates the geographic distributions of these pathogens are uncertain, and there is a continuing threat of invasion.