Tomato Apical Stunt Viroid Research Articles

Reverse-transcription recombinase-aided amplification and CRISPR/Cas12a-based universal detection for fast screening and accurate identification of six pospiviroids infecting Solanaceae crops.

Pospiviroids, members of the genus Pospiviroid, can cause severe diseases in tomato and other Solanaceae crops, causing considerable economic losses worldwide. Six pospiviroids including potato spindle tuber viroid (PSTVd), tomato chlorotic dwarf viroid (TCDVd), tomato planta macho viroid (TPMVd), Columnea latent viroid (CLVd), pepper chat fruit viroid (PCFVd), and tomato apical stunt viroid (TASVd) are regulated in many countries and organizations. Rapid, accurate detection is thus crucial for controlling the spread of these pospiviroids. For simultaneous detection of these six pospiviroids, we developed a rapid, visual method that uses a reverse transcription recombinase-aided amplification (RT-RAA) assay coupled with a clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 12a (CRISPR/Cas12a) system. In particular, this technique could achieve both universal detection and specific identification of the six target pospiviroids within 40 min. The universal detection could diagnose the six target pospiviroids in a single reaction, and the specific identification could identify each target pospiviroid without cross-reactivity of other pospiviroids. The sensitivity limits for the target pospiviroids detection with the proposed detection method were higher than those of the conventional reverse transcription-polymerase chain reaction (RT-PCR) method. We designed an RT-RAA-CRISPR/Cas12a-based universal detection method for both large-scale screening and accurate identification of the six target pospiviroids, which is appropriate for on-site detection. Our study results can aid in performing rapid, large-scale screening of multiple pests simultaneously. © 2024 Society of Chemical Industry.

A universal probe for simultaneous detection of six pospiviroids and natural infection of potato spindle tuber viroid (PSTVd) in tomato in China

Several viroid species in the genus Pospiviroid can infect tomato (Solanum lycopersicum) and cause severe diseases, posing a serious threat to tomato production. For simultaneous detection of six tomato-infecting pospiviroids–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)–we developed a universal probe based on a highly conserved 61 nt long sequence shared among them. Compared with their specific probes, the universal probe had a similar, though slightly reduced, detection sensitivity and has the advantages of simple and cost-effective preparation and simultaneous detection of the six pospiviroids. In addition, the universal probe was used in dot-blot hybridization assays for a large-scale survey of viroid(s) in tomato plantings in China. Only PSTVd was detected in a few greenhouse-planted tomato plants. Sequence analysis revealed that these tomato PSTVd isolates may have been introduced from tomato seeds imported from abroad.

Rapid diagnostic detection of tomato apical stunt viroid based on isothermal reverse transcription-recombinase polymerase amplification

Tomato apical stunt viroid (TASVd) is a serious threat to tomato plants that can cause a considerable yield loss. In the present study, two isothermal molecular diagnostic assays based on reverse transcription-recombinase polymerase amplification (RT-RPA) utilizing the AmplifyRP® platform for plant pathogen detection were developed. The results of this research demonstrated distinct specificity of both developed assays, AmplifyRP® Acceler8™ and AmplifyRP® XRT, expressed in the absence of any cross-reaction activity to all total RNA extracts obtained from plants infected with other pospiviroids. The RT-RPA assays detected viroid RNA in 81- and 27-fold dilutions of the original TASVd-infected crude extract for AmplifyRP® Acceler8™ and AmplifyRP® XRT, respectively. The sensitivity tests in serial water dilutions showed the ability of AmplifyRP® Acceler8™ and AmplifyRP® XRT to detect 8 and 80 fg of pure TASVd RNA transcript, respectively. The influence of crude extract on viroid RNA transcript detection was also examined and a decrease of sensitivity of approximately 100-fold for both RT-RPA assays was revealed. To our knowledge, this is the first report describing development of RT-RPA assays to detect TASVd in plants using the AmplifyRP® platform that can be further employed both in laboratory conditions and in the field for on-site diagnosis.

Universal Primers for Rapid Detection of Six Pospiviroids in Solanaceae Plants Using One-Step Reverse-Transcription PCR and Reverse-Transcription Loop-Mediated Isothermal Amplification.

A number of viruses and viroids infect solanaceous plants causing severe yield losses. Several seed-borne viroids are listed as quarantine pathogens in many countries. Among them, columnea latent viroid, pepper chat fruit viroid, potato spindle tuber viroid, tomato apical stunt viroid, tomato chlorotic dwarf viroid, and tomato planta macho viroid 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 reverse transcription PCR (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 1 fg to 10 ng, 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 in use for these viroids require at least two different sets of primers. The assays developed in this research could facilitate the ability 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.

Pospiviroid infections in Capsicum annuum: disease symptoms and lack of seed transmission

Four pospiviroids, columnea latent viroid (CLVd), pepper chat fruit viroid (PCFVd), potato spindle tuber viroid (PSTVd) and tomato apical stunt viroid (TASVd), were mechanically inoculated on Capsicum annuum (pepper) cv. Yolo Wonder. Symptoms were different, though most pronounced for PCFVd, i.e. delay in flower and fruit onset, increase of plant height, and reduction of fruit size. For studying seed transmission, seeds with high loads of infectious viroid were sown at different time intervals after harvesting, i.e. 0, 2 and 77 days. No transmission from seed to seedlings was recorded in a total of 765 seedlings, irrespective viroid species and time interval. For PCFVd, the experiment was repeated in another pepper cultivar with the similar outcome for 179 seedlings. The results of this study are discussed in view of the role of infested seed lots in causing outbreaks in practice.

Rasta Disease of Tomato in Ghana is Caused by the Pospiviroids Potato spindle tuber viroid and Tomato apical stunt viroid.

Rasta is a virus-like disease of unknown etiology affecting tomato (Solanum lycopersicum) plants in Ghana. Symptoms include stunting; epinasty, crumpling, and chlorosis of leaves; and necrosis of leaf veins, petioles, and stems. Leaf samples with rasta symptoms were collected from commercial tomato fields in Ghana in October 2012 and applied to FTA cards, and RNA extracts were prepared. Reverse-transcription polymerase chain reaction (RT-PCR) tests with primers for Columnea latent viroid, which causes rasta-like symptoms in tomato plants in Mali, were negative, whereas tests with degenerate viroid primer pairs were inconclusive. However, tomato seedlings (Early Pak 7) mechanically inoculated with RNA extracts of 10 of 13 samples developed rasta-like symptoms. In RT-PCR tests with RNA from leaves of the 10 symptomatic seedlings and primers for Potato spindle tuber viroid (PSTVd) or Tomato apical stunt viroid (TASVd), the expected size (approximately 360 bp) of DNA fragment was amplified from eight and two seedlings, respectively. Sequence analyses confirmed that these fragments were from PSTVd and TASVd isolates, and revealed a single PSTVd haplotype and two TASVd haplotypes. The PSTVd and TASVd isolates from Ghana had high nucleotide identities (>94%) with isolates from other geographic regions. In a host range study, PSTVd and TASVd isolates from Ghana induced rasta symptoms in the highly susceptible tomato cultivar Early Pak 7 and mild or no symptoms in Glamour, and symptomless infections in a number of other solanaceous species. PSTVd and TASVd isolates were seed associated and possibly seed transmitted.

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.

Characterization of tomato apical stunt viroid isolated from a 24-year old seed lot of Capsicum annuum.

Tomato apical stunt viroid (TASVd) has been identified in a 24-year old seed lot of Capsicum annuum produced in Taiwan. It is the first finding of TASVd in this plant species. The isolate could be discriminated from all reported isolates of TASVd based on its nucleotide sequence, which showed only 94.8% identity with the most related genotype of TASVd. This discrimination was substantiated by phylogenetic analysis. Inoculation of a RNA extract of contaminated seeds to healthy pepper plants showed that the infectivity of the viroid had remained over time. Nevertheless, no transmission to seedlings was observed.

Phylogeny of five predominant pospiviroid species in Belgium

In Belgium pospiviroids are routinely detected in various hosts. The most frequently found pospiviroids are: Citrus exocortis viroid (CEVd), Chrysanthemum stunt viroid (CSVd), Potato spindle tuber viroid (PSTVd), Tomato apical stunt viroid (TASVd) and Tomato chlorotic dwarf viroid (TCDVd). Apart from the high incidence of pospiviroids in latently-infected ornamentals, viroids have also been found in plants where they cause disease: PSTVd and TCDVd in tomatoes and CSVd in chrysanthemum. In order to gain more epidemiological data on these infections, this study has conducted phylogenetic analyses of Belgian isolates for each of these five pospiviroid species. PSTVd and CEVd-isolates show a clustering depending on host plant identity. This was not observed for TCDVd and TASVd. A very high degree of sequence similarity was noticeable for CSVd-isolates from various hosts. During the past decade, PSTVd and CSVd-infected mother plants have been systematically eradicated in Belgium after positive detection results, also when found in symptomless plants, leading to a decreased trend of these quarantine pests in the past few years. However, other non-quarantine pospiviroid species are still ubiquitously present in many ornamentals. Since these pospiviroids can be equally harmful to crops as the two quarantine pests PSTVd and CSVd, there is still a risk that transmission occurs from symptomless-infected ornamental plants to economically important crops in Belgium such as tomato, pepper and chrysanthemum.

RNA-directed DNA methylation efficiency depends on trigger and target sequence identity.

RNA-directed DNA methylation (RdDM) in plants has been extensively studied, but the RNA molecules guiding the RdDM machinery to their targets are still to be characterized. It is unclear whether these molecules require full complementarity with their target. In this study, we have generated Nicotiana tabacum (Nt) plants carrying an infectious tomato apical stunt viroid (TASVd) transgene (Nt-TASVd) and a non-infectious potato spindle tuber viroid (PSTVd) transgene (Nt-SB2). The two viroid sequences exhibit 81% sequence identity. Nt-TASVd and Nt-SB2 plants were genetically crossed. In the progeny plants (Nt-SB2/TASVd), deep sequencing of small RNAs (sRNAs) showed that TASVd infection was associated with the accumulation of abundant small interfering RNAs (siRNAs) that mapped along the entire TASVd but only partially matched the SB2 transgene. TASVd siRNAs efficiently targeted SB2 RNA for degradation, but no transitivity was detectable. Bisulfite sequencing in the Nt-SB2/TASVd plants revealed that the TASVd transgene was targeted for dense cis-RdDM along its entire sequence. In the same plants, the SB2 transgene was targeted for trans-RdDM. The SB2 methylation pattern, however, was weak and heterogeneous, pointing to a positive correlation between trigger-target sequence identity and RdDM efficiency. Importantly, trans-RdDM on SB2 was also detected at sites where no homologous siRNAs were detected. Our data indicate that RdDM efficiency depends on the trigger-target sequence identity, and is not restricted to siRNA occupancy. These findings support recent data suggesting that RNAs with sizes longer than 24nt (>24-nt RNAs) trigger RdDM.

Callose Deposition in Plasmodesmata and Viroid Invasion of the Shoot Apical Meristem.

Callose Deposition in Plasmodesmata and Viroid Invasion of the Shoot Apical Meristem.

Assessment of pospiviroid transmission by myzus persicae, macrolophus pygmaeus and bombus terrestris

During the past decade, pospiviroid outbreaks in tomato and sweet pepper crops have been regularly reported worldwide. Although seed transmission was often suspected, transmission from asymptomatic ornamentals and weeds through pruning tools, or via insect vectors could not be excluded. To evaluate the risk of pospiviroid transmission via insects, three insect spp. commonly observed in these crops were used in multiple transmission experiments with Potato spindle tuber viroid (PSTVd), Tomato apical stunt viroid (TASVd), Pepper chat fruit viroid (PCFVd) and Tomato chlorotic dwarf viroid (TCDVd)-infected source plants, such as tomatoes, petunias and peppers. The insect species belong to three different functional groups: a phloem-feeding pest (Myzus persicae Sulzer), a pollinator (Bombus terrestris L.) that feeds on pollen and nectar, and a generalist predator (Macrolophus pygmaeus Rambur), that consumes several species of insect pests and feeds on plant saps and pollen. For each insect, four transmission experiments were conducted in which different receptor host plants were challenged. During the experiments, insects that were seen probing or feeding on were captured and tested for viroids using PCR. The percentage of each insect species that was pospiviroid positive was 100 % for M. persicae, 4 % B. terrestris, and 0 % for M. pygmaeus. Although these results indicate that M. persicae may be a vector, none of the receiving host plants in four separate experiments tested positive. However, transmission by B. terrestris is possible since one of 18 receiving tomato plants tested positive for TCDVd in one of four transmission experiments, resulting in an overall transmission rate of 2.6 %. Based on these results, we believe that the use of pollinating insects and biological control agents in greenhouses does not imply a major phytosanitary risk for viroid dispersal.

The role of weeds in the epidemiology of pospiviroids

SummaryMany weeds that are closely associated with horticultural activities are known as natural reservoirs of plant viruses. However, whether these weeds can also serve as hosts of pospiviroids is not well known. Pospiviroids are naked, non‐coding RNA pathogens that cause severe economic damage in many solanaceous crops. In this study, we examined the overall risk of pospiviroid spreading from weeds to economically important crops, by combining the results from previous inoculation studies with new results coming from a survey, a contact experiment and an inoculation experiment. A survey of commercial ornamental glasshouses revealed that ornamental plants mainly belonging to the Solanaceae harbour pospiviroids, in contrast to weed species sampled in the same places. No new weed hosts could be identified after testing weeds that grew in contact with Tomato apical stunt viroid (TASVd)‐infected plants of tomato (Solanum lycopersicum) and jasmine nightshade (Solanum jasminoides) in an experimental glasshouse. Finally, in mechanical inoculation experiments with TASVd, none of the six tested weed species were determined to be a host at 6 weeks after inoculation. Commonly occurring weed species therefore do not appear to play a significant role as reservoir hosts for pospiviroids. This does not rule out other potential weed hosts that have not yet been tested. Inoculation studies should include rigorous experimental protocols with a sufficient number of replicated as well as adequate positive controls. The information gained through this study may prove useful in future risk assessments for the pospiviroid group.

An assessment of the transmission rate of four pospiviroid species through tomato seeds

Greenhouse experiments were conducted to investigate the importance of tomato seeds as a route of pospiviroid dissemination. More than 7000 seeds were collected from tomato plants mechanically inoculated with Tomato apical stunt viroid, Citrus exocortis viroid, Columnea latent viroid and Potato spindle tuber viroid. Fruits and part of the seeds were found to be 100 % infected by the inoculated pospiviroid by means of RT-PCR testing. Much of the rest of the seeds were sown and produced about 4700 seedlings. None of the seedlings were found positive for the respective pospiviroid. This shows that pospiviroid seed transmission in tomato is very rare.

DETECTION OF POSPIVIROIDS IN THE CZECH REPUBLIC AND THEIR DISCRIMINATION BY RESTRICTION ANALYSIS

Potato spindle tuber viroid (PSTVd), a member of the genus Pospiviroid, is a quarantine pest in the European Union and its presence in ornamental solanaceous crops represents a serious threat to susceptible vegetable and potato crops. Since the year 2007 the Czech Plant Protection Service (PPS) carries out a survey for PSTVd on ornamental solanaceous species. Within the first year of the survey, several new hosts of PSTVd were identified. Also, a new species of Pospiviroid was detected in the Czech Republic. At the beginning of the survey, a method for detection of PSTVd recommended in the EPPO diagnostic protocol was adopted. Reverse transcription-polymerase chain reaction with a semi-specific primer pair was carried out in the diagnostic laboratory of PPS while the sequencing of PCR products was performed in an external laboratory. Due to this time consuming process, which proved inefficient for the needs of rapid application of phytosanitary measures, the laboratory developed a faster procedure. The entire genomic sequences of all viroids which contained primer identical segments were downloaded from the GenBank database and aligned to search for viroid-specific restriction sites. A combination of four enzymes was chosen to distinguish the detected viroids. Restriction analysis was successfully tested and applied for the PSTVd identification. Later on, after confirmation of other pospiviroids detected for the first time in the Czech Republic (Tomato apical stunt viroid and Chrysanthemum stunt viroid on Solanum jasminoides and Citrus exocortis viroid on Solanum jasminoides and Verbena A— hybrid), the diagnostic method was improved to detect a wide range of Pospiviroid species with the introduction of primers detecting the five most common pospiviroids in the ornamental solanaceous plants in Europe. Similarly, for identification of the amplified PCR products, a new set of restriction enzymes was identified.

Tomato apical stunt viroid. [Distribution map

Abstract A new distribution map is provided for Tomato apical stunt viroid. Pospiviroidae: Pospiviroid. Hosts: tomato ( Solanum lycopersicum ). Information is given on the geographical distribution in Europe (Austria, Belgium, Croatia, Finland, France, Germany, Italy, Netherlands, Poland, Slovenia), Asia (Indonesia, Israel), Africa (Cote d'Ivoire, Senegal, Tunisia).

Host ranges of Potato spindle tuber viroid, Tomato chlorotic dwarf viroid, Tomato apical stunt viroid, and Columnea latent viroid in horticultural plants

Host ranges of Potato spindle tuber viroid (PSTVd), Tomato chlorotic dwarf viroid (TCDVd), Tomato apical stunt viroid (TASVd), and Colmunea latent viroid (CLVd) were investigated in 30 species from 12 genera of horticultural plants that are frequently traded internationally. They were mechanically inoculated, and viroid infection was checked using both ways of reverse-transcription polymerase chain reaction and back-inoculation to tomato. Among 30 species examined, eight to 10 were susceptible to PSTVd, TCDVd, TASVd, and CLVd. Most of these species belong to the Compositae and Solanaceae families, and symptoms were only observed in Calendula officinalis, Petunia × hybrida, and Solanum melongena. These data indicate a risk of unexpected viroid expansion by international trading of vegetatively propagated asymptomatic plant materials.

First Report of Tomato apical stunt viroid in Tomato in Italy.

During a survey conducted in October 2013 in tomato greenhouses in Diano Marina (Imola Province, northwest Italy), in a single greenhouse, unusual disease symptoms were observed in four out 1,400 (~0.3%) of tomato plants cv. Ingrid, grafted on 'Beaufort' rootstock. Symptoms including shortened apical internodes associated with tiny, deformed, and brittle chlorotic leaves, while ripe fruits appeared reduced in size and pale red. Samples of leaves from the four plants were collected and examined using commercial antisera (Bioreba AG, Reinach, Switzerland) by double antibody sandwich (DAS)-ELISA against Tomato spotted wilt virus, Cucumber mosaic virus, Alfalfa mosaic virus, Tomato/Tobacco mosaic viruses, and by indirect plate trapped antigen (PTA)-ELISA against potyviruses (potygroup test). None of the tested viruses were detected in the four leaf samples. In addition, PCR tests for begomoviruses and phytoplasmas were also negative. In a host range study, the original symptoms, consisting mainly of stunting and chlorosis, were reproduced within ~10 days in tomato seedlings (Momor line), mechanically inoculated at two true leaves stage with sap extract obtained from the four symptomatic tomato plants, whereas no symptoms were observed in Chenopodium amaranticolor, C. quinoa, Nicotiana tabacum (cv. Xanthi nc), N. glutinosa, or Phaseolus vulgaris (cv. Borlotto rosso). Total RNAs extracted with the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) from symptomatic samples were tested in RT-PCR using pospiviroids generic primers PSTVd-32/33 (1), designed to amplify the whole genome of Potato spindle tuber viroid (PSTVd), Tomato apical stunt viroid (TASVd), and Columnea latent viroid (CLVd), and CEVd-FW/RE primers, designed to amplify the whole genome of Citrus exocortis viroid (CEVd) and TASVd (3). Each of the four samples yielded amplicons of the same size (364 bp) with both primer combinations. The identity of the viroid was then determined by sequencing, on both strands, amplicons obtained from the four symptomatic plants at MWG (Ebersberg, Germany). Sequences obtained were identical, showing the highest nucleotide identity (99.7%) with the TASVd isolate Sj1 (AM777161), identified in Germany on Solanum jasminoides. The sequence was deposited in GenBank (Accession No. HG916812) and the field isolate named To1-IT. Two other cases of pospiviroid infection in tomato in Italy have been reported so far and the viroid species detected were PSTVd (2) and CLVd (4), respectively. To our knowledge, this is the first report of TASVd infecting tomato in Italy. The origin of this infection is still unclear, although based on the biological properties and sequence similarity, the To1-IT isolate probably originated from an ornamental species, most likely S. jasminoides, as recently reported for other tomato TASVd isolates, according to their biological and genetic features (5).

Occurrence and molecular variability of Potato spindle tuber viroid and Tomato apical stunt viroid in ornamental plants in Croatia

Viroids of the genus Pospiviroid are able to induce diseases in a wide range of host plants including important crop species. Although occasional disease outbreaks of Potato spindle tuber viroid (PSTVd) and closely related pospiviroids have been reported in potato and tomato, recent studies found an increase in number of latent infections in ornamental solanaceous species. In order to verify the presence of PSTVd and other pospiviroids in Croatia, a survey was conducted between 2009 and 2012. A total of 182 samples belonging to five ornamental species and two solanaceous crops were analyzed. Eight plants belonging to two different species (Solanum jasminodes and Lycianthes rantonnetii) were found infected by PSTVd and, in addition, one S. jasminoides plant infected by Tomato apical stunt viroid (TASVd). Viroid infection was confirmed by mechanical inoculation on tomato plants to observe symptom expression. Molecular characterization of the isolates was done and complete viroid sequences were submitted to the GenBank. This is the first evidence of the presence of PSTVd and TASVd and their variability in Croatia.