Pospiviroid Primers Research Articles

First report of tomato chlorotic dwarf viroid infecting litchi tomato (Solanum sisymbriifolium).

Litchi tomato (LT) (Solanum sisymbriifolium) is a solanaceous weed that is considered a biological control tool to manage potato cyst nematode (PCN) in Europe and is being explored for use in Idaho. Two Several LT lines were clonally maintained as stocks in the university greenhouse since 2013 and were also established in tissue culture at the same time. In 2018, tomato (Solanum lycopersicum cv. Alisa Craig) scions were grafted onto two LT rootstocks originating either from healthy-looking greenhouse stocks or from tissue culture-maintained plants. Unexpectedly, tomatoes grafted onto the greenhouse-maintained rootstocks of LT displayed severe symptoms of stunting, foliar deformation, and chlorosis, while grafts onto the same LT lines from tissue culture produced healthy-looking tomato plants. Tests for the presence of several viruses known to infect solanaceous plants were conducted on symptomatic tomato scion tissues using ImmunoStrips (Agdia, Elkhard, IN) and RT-PCR (Elwan et al. 2017) but yielded negative results. High throughput sequencing (HTS) was then used to identify possible pathogens that could have been responsible for the symptoms observed in tomato scions. Samples from two symptomatic tomato scions, two asymptomatic scions grafted onto the tissue culture-derived plants, and two greenhouse-maintained rootstocks were subjected to HTS. Total RNA from the four tomato and two LT samples was depleted of ribosomal RNA and subjected to HTS on an Illumina MiSeq platform producing 300-bp paired-end reads and raw reads were adapter and quality cleaned. For the tomato samples, the clean reads were mapped against the S. lycopersicum L. reference genome, and unmapped paired reads were assembled producing between 4,368 and 8,645 contigs. For the LT samples, all clean reads were directly assembled, producing 13,982 and 18,595 contigs. In the symptomatic tomato scions and the two LT rootstock samples, a 487-nt contig was found, comprising an ~1.35 tomato chlorotic dwarf viroid (TCDVd) genome and exhibiting 99.7% identity with it (GenBank accession AF162131; Singh et al. 1999). No other virus-related or viroid contigs were identified. RT-PCR analysis using a pospiviroid primer set Pospi1-FW/RE (Verhoeven et al. 2004), and a TCDVd-specific primer set TCDVd-Fw/TCDVd-Rev (Olmedo-Velarde et al. 2019) produced 198-nt and 218-nt bands, respectively, thus confirming the presence of TCDVd in tomato and LT samples. These PCR products were Sanger sequenced and confirmed to be TCDVd-specific; the complete sequence of the Idaho isolate of TCDVd was deposited in GenBank under the accession number OQ679776. Presence of TCDVd in LT plant tissue was confirmed by the APHIS PPQ Laboratory in Laurel, MD. Asymptomatic tomatoes and LT plants from tissue culture were found negative for TCDVd. Previously, TCDVd was reported to affect greenhouse tomatoes in Arizona and Hawaii (Ling et al. et al. 2009; Olmedo-Velarde et al. 2019), however, this is the first report of TCDVd infecting litchi tomato (S. sisymbriifolium). Five additional greenhouse-maintained LT lines were found TCDVd-positive using RT-PCR and Sanger sequencing. Given the very mild or asymptomatic infection of TCDVd in this host, molecular diagnostic methods should be used to screen LT lines for the presence of this viroid to avoid inadvertent spread of TCDVd. Another viroid, potato spindle tuber viroid, was reported to be transmitted through LT seed (Fowkes et al. 2021), and transmission of TCDVd through LT seed may also be responsible for this TCDVd outbreak in the university greenhouse, although no direct evidence was collected. To the best of our knowledge, this is the first report of TCDVd infection in S. sisymbriifolium and also the first report of the TCDVd occurrence in Idaho.

A DIAGNOSTIC METHOD FOR THE SIMULTANEOUS DETECTION AND IDENTIFICATION OF POSPIVIROIDS

Potato spindle tuber viroid (PSTVd) was the first species of the genus Pospiviroid detected in naturally infected symptomless ornamental plants (Solanum jasminoides) in 2006. Since then, other pospiviroid species were found in several naturally infected solanaceous ornamentals. The combination of latent infection, the widespread occurrence and the possibility of transmission to edible solanaceous crops (tomato and potato) has made the development of accurate and reliable molecular diagnostic detection methods a high priority. The aim of this study was the development of a genus-specific RT-PCR protocol, using universal pospiviroid primers, which would allow also a species-specific identification through restriction fragment length polymorphism (RFLP) analysis. The primers were designed to amplify the majority of the pospiviroid genome (ca. 80%) and the identification of each species using appropriate restriction endonuclease enzymes (AluI and Sau96I). The method was evaluated for specificity using healthy and/or non-target viroid-infected plants and for sensitivity using serial dilutions of total RNA extracted from plants infected by different pospiviroid species. The reliability of the method was successfully ascertained during a survey when numerous plants of the families Solanaceae and Asteraceae were assayed for single and mixed infection of pospiviroid species.

First Report of Pepper chat fruit viroid in Traded Tomato Seed, an Interception by Australian Biosecurity.

Pepper chat fruit viroid (PCFVd), a species of Pospiviroid, was first discovered in a capsicum crop in the Netherlands in 2006 (4) and was then reported only in Thailand (2) and Canada. The mechanism of international spread was not known, but movement with traded seed was suspected. PCFVd is transmissible through capsicum seed (4) and very probably through tomato seed, like other pospiviroids. The viroid causes disease in capsicum and tomato and experiments by others indicate a capacity to cause disease in potato. It poses a biosecurity threat to crops internationally. PCFVd was intercepted by the Australian Government Department of Agriculture, Fisheries, and Forestry (DAFF) in five shipments of tomato seed (Solanum lycopersicum) exported from Israel and Thailand in September and October 2012. Batches of up to 20,000 seeds were sampled from each seed lot in a shipment and total nucleic acids were extracted from sub-samples, each of about 400 seeds, following a method similar to Hoshino et al. (1). PCFVd was initially detected when reverse transcription PCR using the generic pospiviroid primers Pospi1-FW and Pospi1-RE (3) produced amplicons of 189 bp, which were then sequenced. The PCFVd specific primers AP FW1 and AP RE2 (4) were used to amplify the remainder of the viroid genome, which was directly sequenced. Overlapping sequences were aligned to produce complete sequences of 349 bases, one from seed from Thailand and two from seed from Israel (GenBank: KC762952, KC762953, KC762954). Searches of the GenBank nucleotide non-redundant database indicated close matches with sequences from PCFVd isolates from tomato in Thailand (2); alignments generated by BLAST showed the sequences differed from those from Thailand at only 2 to 18 nucleotide positions, equating to 95 to 99% identity. PCFVd sequences from seed from Thailand were almost identical (>99%) to the sequences from seed from Israel. Many sub-samples were negative, indicating that the number of contaminated seeds was very small in some shipments. The positive sub-samples as a proportion of the total number of sub-samples tested from the five shipments was 1/1, 1/5, 1/1, 12/50, and 7/50. Tomato and capsicum seed are produced in many countries and often traded through second countries. The infected tomato seed shipments intercepted by DAFF were destroyed or re-exported following Australian regulations. Other countries were informed through the International Plant Protection Convention. This pest viroid has not been intercepted by Australian authorities before and has not been detected in recent Australian survey work (data not shown).

First Report of Columnea latent viroid (CLVd) in Tomato in Mali.

During surveys of tomato (Solanum lycopersicum) fields in Niono, Mali, conducted in March 2011, unusual disease symptoms, including stunted growth, epinasty, and chlorosis of leaves and necrosis of leaf veins and stems were observed in multiple fields. The incidence of these symptoms was low (~1 to 5%), but they were distinct from those associated with known diseases in the region. A representative leaf sample with these symptoms was applied to filter paper (FTA cards, Whatman), and DNA and RNA extracts were prepared according to manufacturer instructions. RT-PCR tests for Tomato spotted wilt virus, Tobacco streak virus, Tomato necrotic spot virus, Tobacco/tomato mosaic viruses, Cucumber mosaic virus, Alfalfa mosaic virus, torradoviruses, and potyviruses, and PCR tests for begomoviruses, phytoplasmas, and 'Candidatus Liberibacter' infection were also negative. However, virus-like symptoms developed in all 16 tomato seedlings (cv. Early Pak 7) 7 to 10 days after mechanical (sap) inoculation with inoculum prepared from the FTA sample. No symptoms developed in mock-inoculated control plants (n = 3). Symptoms induced included stunted growth and severe epinasty of leaves, followed by necrosis of leaf veins, petioles, and stems. These symptoms were similar to those observed in plants in Mali. When RNA extracts prepared from leaves of these symptomatic plants were mechanically inoculated onto 24 tomato seedlings, similar symptoms developed in all plants, suggesting the causal agent might be a viroid. RT-PCR tests with RNA from symptomatic tomato leaves and universal (3) and various specific Pospiviroid primer pairs were negative. However, equivalent RT-PCR tests conducted with the pCLV4/pCLVR4 primer pair specific for Columnea latent viroid (CLVd) (2) generated a DNA fragment of the expected size (~370 bp). The sequence of this DNA fragment (GenBank Accession No. JQ362419) was 99% identical with those of CLVd isolates from the Netherlands (AY373446 and AY372396). In host range studies, the CLVd isolate from Mali induced symptoms in all 48 mechanically-inoculated tomato plants, whereas no symptoms developed (up to 90 days after inoculation) in inoculated Chenopodium quinoa, C. amaranticolor, Nicotiana benthamiana, N. tabacum (cvs. Havana, Glurk and Turkish), N. glutinosa, Datura stramonium, common bean (cvs. Topcrop and Pinto bean), pumpkin (cv. Small Sugar), pepper (Capsicum annuum, cv. Yolo Wonder) and cucumber (cvs. Emparator and Poinsett 76) plants (results of three independent experiments with six plants per experiment). Symptomless infections were detected in pepper (24 of 30), N. benthamiana (25 of 25), and N. tabacum cv. Turkish (11 of 24) plants by RT-PCR with the pCLV4/pCLVR4 primer pair. To our knowledge, this is the first report of CLVd infecting tomato in Mali. RT-PCR tests of seeds collected from CLVd-infected tomato, pepper, and N. benthamiana plants also detected CLVd (1). Thus, it is possible that CLVd was introduced into Mali in association with seed.

First report of Iresine viroid 1 in Portulaca sp. in Slovenia.

In October 2011, during a survey for pospiviroids in ornamental plants, leaf samples were collected from two symptomless Portulaca sp. plants of two different varieties grown in a flower bed. Total RNA of each sample was extracted twice from 15 mg of leaf tissue using the MagMaxTM-96 total RNA isolation kit (Ambion, USA). The samples were tested by RT-PCR using semiuniversal pospiviroid primers [Pospi1-RE/FW and Vid-RE/FW (Verhoeven et al., 2004)]. A product was amplified with primer pair Pospi1-RE/FW but not with primer pair Vid-RE/FW. The amplicon from each total RNA extract was custom sequenced (Macrogen, The Netherlands). Blast sequence analysis showed high identity with Iresine viroid 1 (IrVd-1). Primer pair IrVdFW1/IrVd-RE1 (Verhoeven et al., 2010) was used to amplify the full-length viroidal genomes. Amplicons were successfully cloned into pGEM-T easy vector (Promega, USA) and transformed into E. coli JM109 competent cells (Promega, USA). At least eight clones per sample were sequenced. Master sequences of each sample were deposited in GenBank under accession Nos. JQ889689 and JQ889690. They showed 99.7% identity between each other and 96.7-98.6% identity with other IrVd-1 sequences (GU911350, DQ094294, NC_003613 and DQ94293). This is the first report of IrVd-1 in Slovenia and the second report of IrVd-1 in Portulaca sp. worldwide. Portulaca sp. was first reported as a host of IrVd-1 in 2010 (Verhoeven, 2010). Unlike other pospiviroids IrVd-1 does not seem to present a threat to potato and tomato (Verhoeven et al., 2010).

First report of Tomato apical stunt viroid in Solanum jasminoides in Slovenia

A survey of Potato spindle tuber viroid (PSTVd) in ornamental host plants was initiated in Slovenia in December 2006 after a PSTVd infection was confirmed in an ornamental plant Brugmansia cordata imported from the Netherlands. By the end of 2010, PSTVd had also been detected in Solanum jasminoides, S. rantonnetii, S. muricatum and Petunia spp. Furthermore, after the detection of Tomato chlorotic dwarf viroid (TCDVd) in Petunia sp. (Viršček Marn & Mavrič Pleško, 2010) and Citrus exocortis viroid (CEVd) in S. jasminoides (Viršček Marn & Mavrič Pleško, 2011) in 2010, an assessment of the risk of pospiviroids for Slovenia was performed at the beginning of 2011. The results of pest risk analysis showed that pospiviroids pose a serious threat to important agricultural plants. Symptomless pospiviroid-infected ornamental plants were proposed to be a probable source of infection for agricultural crops. Sampling of all pospiviroid hosts and destruction of all pospiviroid-infected lots was therefore implemented in Slovenia in 2011. In 2011, 14 samples were taken from symptomless S. jasminoides plants in greenhouses and retail trade and were tested by one-step RT-PCR using semi-universal pospiviroid primers Pospi1-RE/FW and Vid-RE/FW (cited in Verhoeven et al., 2008). With three samples, PCR products were obtained with both primer pairs. They were all directly sequenced (Macrogen, The Netherlands). Sequence analysis of PCR products obtained with both primer pairs from two samples confirmed infection with PSTVd. Sequences of amplification products obtained with the Vid-RE/FW primer pair from the third sample showed the highest similarity with PSTVd whereas the sequences of the amplicon obtained with Pospi1-RE/FW primers suggested the presence of Tomato apical stunt viroid (TASVd). A second total RNA extraction was made; both extracted RNAs were further used in RT-PCRs using primer pairs of Shamloul et al. (1997) and of Di Serio (2007) for detection of PSTVd, and primer pairs of Elleuch et al. (2003) and Verhoeven et al. (2008) for detection of TASVd and CEVd. Sequence analysis confirmed the presence of PSTVd and TASVd. PSTVd and TASVd sequences of this doubly infected sample were deposited in the GenBank (Accession Nos. JN559763 and JN543957, respectively). Successful amplification with only Pospi1-RE/FW was obtained with 10 samples. These amplification products were also directly sequenced and sequence analysis revealed the presence of CEVd in three samples and the presence of TASVd in seven samples. To obtain full viroid sequences further analyses were made using primer pairs of Elleuch et al. (2003) and Verhoeven et al. (2008). These sequences have also been deposited in GenBank (JQ083645 to JQ083647 for CEVd and JN872141 to JN872144 and JQ904911 for TASVd). This is the first record of TASVd in Slovenia. All Slovene TASVd sequences had 370 nt and were identical to each other at 366 positions. They shared 98.9 to 100% identity among themselves and 97.5 to 100% identity with other TASVd sequences from S. jasminoides. The variability of sequences detected in S. jasminoides in Slovenia and worldwide is rather small. This data suggest a possible common origin of TASVd infection of S. jasminoides. Higher variability was observed between TASVd sequences from S. jasminoides and from tomato. The most distant sequences shared only 92.0% identity.

First Report of Citrus exocortis viroid in a Verbena sp. in Montenegro.

Numerous ornamental plants have been found to be symptomless hosts of various pospiviroids including Citrus exocortis viroid (CEVd), and hence, may serve as potential inoculum reservoirs for susceptible vegetable plants. Production of tomato, potato, and pepper, on which viroids from the genus Pospiviroid can cause severe damage, represents two-thirds of the vegetable production in Montenegro. We tested vegetable, ornamental, and weed host plants for the presence of pospiviroids in September 2011. Altogether 80 samples were taken. Samples of ornamental plants (15 of Petunia spp., 7 of Impatiens spp., 4 of Verbena spp., 3 of Dahlia spp., 3 of Pittosporum tobira, 3 of Vinca spp., 2 of Brugmansia spp., 2 of avocado, 2 of Portulaca spp., and 1 of Datura sp.) were taken from three places of production. One sample per species was collected from symptomless eggplants, tomatoes, sweet peppers, and avocados in the vicinity of one glasshouse with ornamental plants. Twenty-two samples from sweet pepper and seven samples from tomato, all grown under cover and all showing potential virus-like symptoms, were collected from three places of vegetable production. Two samples of Solanum nigrum and three samples of unidentified weed species belonging to genus Solanum were taken from two glasshouses. With the exception of weed plants, samples consisted of fully developed leaves collected from five plants. All sampled ornamental and weed plants were symptomless. RNA was extracted from approximately 15 mg of leaf tissue with the MagMAX-96 Total RNA Isolation Kit (Life Technologies, Foster City, CA) in accordance with the manufacturer's instructions for the MagMAX Express Magnetic Particle Processor. Samples were tested by reverse transcription (RT)-PCR using semi-universal pospiviroid primers (Pospi1-RE/FW and Vid-RE/FW [3]). None of the samples reacted with the Vid-RE/FW primer pair. An amplicon of an expected size (approximately 196 nt) was produced with the Pospi1-RE/FW primer pair from one Verbena sp. sample. Direct sequencing was performed by Macrogen (Amsterdam, The Netherlands). Sequence analysis indicated the presence of CEVd. This finding was confirmed by sequence analysis of the DNA product obtained by RT-PCR using Pospi1-FW/RE from a new extraction. Further analyses using primer pairs CEVd-AS/S (1) and CEVd-FW2/RE2 (4) were performed to obtain the full viroid sequence. The sequence of 372 nt was deposited in GenBank (Accession No. JN872140) and had 99% identity with two CEVd sequences from Verbena spp. (Accession Nos. EF192396 and DQ094297). To our knowledge, this is the first report of CEVd in a Verbena sp. in Montenegro and the second report in Europe (4). CEVd has been detected in Verbena spp. also in India and Canada and can be transmitted by seed (2). The infected Verbena sp. plants were not destroyed, since CEVd is not listed as a quarantine organism in Montenegro. The spread of CEVd infection to tomato could devastate the production of this crop in Montenegro.

First Report of Potato spindle tuber viroid in Cape Gooseberry in Slovenia.

Cape gooseberry (Physalis peruviana) was first reported as a host of Potato spindle tuber viroid (PSTVd) in 2009 (4). In Slovenia, 10 young plants of cape gooseberry that were grown in a glasshouse were inspected in April 2011. Plants were multiplied from an adult plant of unknown origin. During sampling, the inspected plants showed no disease symptoms. Total RNA was extracted twice from leaves of five plants with an RNeasy Plant Mini Kit (QIAGEN, Hilden, Germany). Reverse transcription (RT)-PCR assay employing two pairs of semi-universal pospiviroid primers (Pospi1-RE/FW and Vid-RE/FW [3]) yielded amplicons of the expected size (approximately 196 and 360 bp) from each total RNA preparation. All four DNA products were sequenced directly (Macrogen, Amsterdam, the Netherlands). Sequence analysis confirmed the identity of a viroid as PSTVd. Further RT-PCRs using primer pairs of Shamloul et al. (2) and Di Serio (1) were made to obtain a full viroid sequence. The sequence was deposited in the NCBI GenBank under Accession No. JN543964. Sequence analysis confirmed the identity of the viroid as PSTVd. The Slovenian isolate had 358 nucleotides and was 100% identical to the cape gooseberry isolate from Germany (GenBank Accession No. EU862231) and the tomato isolate from New Zealand (GenBank Accession No. AF369530). The analyzed sample was the only sample of cape gooseberry taken from the start of the survey for PSTVd in 2006 because P. peruviana is rarely grown in Slovenia. To our knowledge, this is the first report of PSTVd infection of P. peruviana in Slovenia and the fourth reported case after the detection of PSTVd infection in Germany, Turkey, and New Zealand. This emerging host for PSTVd could potentially serve as a source of infection for tomato and potato, where the viroid can cause severe losses.

First Report of Chrysanthemum stunt viroid in Various Cultivars of Argyranthemum frutescens in France.

Described for the first time in Chrysanthemum indicum in the United States, Chrysanthemum stunt viroid (CSVd) was reported to naturally infect species in the Asteraceae family (1,3), as well a few hosts in other families. In May 2010 in a nursery in southwest France, the occurrence of stunted A. frutescens plantlets of cv. Butterfly showing yellow deformed leaves with terminal necrosis, which resembled the growth reduction, flower distortion or leaf necrosis symptoms reported for CSVd in Argyranthemum spp. (3), was reported. Mother plants from which the plantlets originated were asymptomatic. Reverse transcription (RT)-PCR with universal pospiviroid primers Pospi1-FW/RE (4) was performed on five symptomatic plants. A fragment of expected size (197 bp) was obtained in all cases. Viroid infection was confirmed by RT-PCR with two sets of primers: Vid-FW/RE using a 59°C annealing temperature instead of the recommended 62°C (4) and Vir-plus/minus that allows the amplification of the full-length viroid genome (2). Sequences of the three different uncloned amplicons were determined and a 355-nt contig was assembled (GenBank No. JF938538). A BLAST analysis of this full-length sequence revealed 99% identity with CSVd isolates from Chrysanthemum from Korea and Germany (GenBank Accession Nos. AF394452 and X16408). The Argyranthemum CSVd sequence differed from the Chrysanthemum ones by an A insertion at position 289 and substitutions (A to T) at positions 65 and 299. The insertion at position 289 is currently unique among CSVd sequences in GenBank. Thirty-five symptomless mother plants of A. frutescens cv. Butterfly were tested by PCR and all were shown to be infected. The difference in symptomatology observed between the mother plants and the commercial potting plants cannot be explained at this stage, but may reflect the different physiologies or growing conditions of the two kinds of plants, since these are known to affect CSVd symptoms in other hosts (1). To estimate the extent of CSVd contamination in A. frutescens, samples of 11 other cultivars originating from different nurseries were similarly analyzed. In addition to Butterfly, cvs. Sonnenstral, Maya Bofinger, Lili, Blanc Double, and Daisy Solenio were found to be infected by CSVd in the absence of clear symptomatology. The CSVd-free cultivars were Angelic Bordeaux, Dark Pink, Pink Delight, Angelic White, Dana, and Summer. The Pospi1-FW/RE amplicons from Blanc Double, Lili, and Daisy Solenio were identical to the Butterfly isolate sequence while the Maya Bofinger sequence showed one substitution (C to T) at position 256 and Sonnenstral had one substitution (T to A) at position 254. Although CSVd infection of Butterfly had been reported from Germany (3), to our knowledge, the results reported here represent the first report of CSVd in Argyranthemum for France and implicate a range of cultivars. CSVd being classified as a quarantine pest in Chrysanthemum spp. in the European Union, the finding of its significant prevalence in A. frutescens cultivars, frequently in the absence of clear symptomatology, raises the possibility that contaminated Argyranthemum may constitute a reservoir for future Chrysanthemum contamination.

First Report of a Brugmansia sp. Infected by Tomato apical stunt viroid in Belgium.

During a routine screening of ornamentals by the Federal Agency for the Safety of the Food Chain (FASFC) in Belgium, a pospiviroid was detected in a symptomless Brugmansia sp. (angel's trumpets) coming from the Netherlands. Detection was performed on a leaf sample by reverse transcription (RT)-PCR, first using universal pospiviroid primer pair (2) VIR1/VIR2 and subsequently with semispecific primer pair (3) CEVd-FW/RE, to amplify the whole viroid genome. Amplicons of expected sizes (260 and 360 bp, respectively) were detected and the sequencing of the CEVd-FW/RE amplicon (GenBank Accession No. FN994891) revealed that the viroid from a Brugmansia sp. was a Tomato apical stunt viroid (TASVd) showing 99.7% similarity with the Sj1 isolate found in Solanum jasminoides (GenBank Accession No. AM777161). Diluted-sap inoculation attempts on tomato from lyophilized material were unsuccessful in agreement with previous results obtained with a Brugmansia isolate of Potato spindle tuber viroid (4). TASVd is mentioned on the alert list of EPPO (European and Mediterranean Plant Protection Organization) because of the heavy yield losses it can induce in tomato, its asymptomatic presence on different ornamental plants (S. jasminoides, Lycianthes rantonnetii, and Streptosolen jamesonii), and its capacity to be transmitted by contact to different Solanaceae as well as by bumble bees to tomato (1). The contaminated plants found in the survey by FASFC have been destroyed to avoid accidental transmission to potato and tomato. To our knowledge, this is the first report of TASVd in a Brugmansia sp.

Chrysanthemum stunt viroid newly reported in Slovenia

In August 2009, samples of chrysanthemum (Dendranthema × grandiflorum) were received for disease diagnosis from glasshouse crops growing in West Slovenia. Twelve out of 200 plants of cv. Miral, originating from Germany, showed viroid-like symptoms including stunting and yellow leaf spots. Plants showing symptoms were distributed randomly in the glasshouse. ELISA tests of leaves with symptoms for Tomato spotted wilt virus (TSWV), Impatiens necrotic spot virus (INSV), Chrysanthemum stem necrosis virus (CSNV), Chrysanthemum B virus (CVB) and Tomato aspermy virus (TAV) were negative and examination by electron microscopy revealed no virus particles. Leaf samples were also tested using reverse transcription-polymerase chain reaction (RT-PCR) with two sets of semi-universal pospiviroid primers: Pospi1-RE/FW and Vid-RE/FW (Verhoeven et al., 2004). With Pospi1-RE/FW primers, symptom-bearing chrysanthemum leaves yielded an amplicon of approximately 196 bp, whereas no product was obtained with Vid-RE/FW primers. PCR products were sequenced and a BLAST analysis of the sequenced amplicons revealed 100% identity with Chrysanthemum stunt viroid (CSVd) (GenBank Accession No. AB279769). Infection by CSVd was confirmed by RT-PCR using CSVd specific primers (Hooftman et al., 1996) and a product of the expected size (354 bp) was obtained. The PCR product was sequenced and subject to BLAST analysis. This sequence also shared 100% identity with the same isolate of CSVd (AB279769). Infectivity of the CSVd isolate from chrysanthemum was confirmed by mechanical inoculation on to tomato plants cv. Moneymaker in a glasshouse under quarantine conditions. Six weeks after inoculation the tomato plants were found to be infected with CSVd using RT-PCR testing. To our knowledge, this is the first report of CSVd in Slovenia. Thanks to Slovenian PARS and M.Tusek Znidaric for their help.

First Report of Tomato chlorotic dwarf viroid in Petunia spp. in Slovenia.

In early April 2010, 30 samples of Petunia spp. were taken by phytosanitary inspectors from 22 production sites in Slovenia in the frame of surveying host plants for the presence of Potato spindle tuber viroid (PSTVd). Samples were taken in accordance with the plan of the survey for the year 2010 and were tested for the presence of PSTVd by real-time RT-PCR according to the EPPO protocol (1). At the time of sampling, there were no disease symptoms on the plants. Samples consisted of fully developed leaves collected from as many as five plants. Total RNA was isolated from 50 ± 5 mg of leaf tissue with an RNeasy Plant Mini Kit (Qiagen, Chatsworth, CA). One sample of cv. Surfinia Purple from a production site from the coastal region and another of cv. Surfinia Hot Pink 05 from a production site near Ljubljana, both multiplied through cuttings, were positive by real-time RT-PCR, confirming the presence of PSTVd or Tomato chlorotic dwarf viroid (TCDVd). To identify the viroid, RT-PCR with primer pairs of Shamloul et al. (3) and Di Serio (2) were performed with isolated total RNA of each positive sample. RT-PCR products were obtained only with primer pairs of Shamloul et al. (3). To obtain the full sequence, additional RT-PCR was done for each sample with semi-universal pospiviroid primers Vid-RE/FW (4). RT-PCR products obtained with primer pair of Shamloul et al. (3) and primer pair Vid RE/FW were sequenced (Macrogen, Seoul, Korea). Sequence analysis confirmed the identity of a viroid as TCDVd. Both isolates consisted of 360 nucleotides and were 100% identical to an isolate from tomato deposited in NCBI GenBank under Accession No. AF162131. They showed 98% identity with sequences from petunias (GQ396664, EF582392, EF582393, and DQ859013). The infected Petunia spp. stocks were destroyed. Although the infection of Petunia spp. with TCDVd is symptomless, the infected plants could be a source of infection for tomato and potato. TCDVd infection can cause severe damage on potato and tomato, similar to that caused by infection with PSTVd, to which it is closely related. To our knowledge this is the first finding of TCDVd in Petunia spp. in Slovenia.

First Report of Potato spindle tuber viroid in Cape Gooseberry (Physalis peruviana) in New Zealand.

In February 2009, 10 cape gooseberry plants (Physalis peruviana) grown from seed on a domestic property in Christchurch, New Zealand, showed severe leaf distortion, fasciation and etiolation of growing tips, and weak flowering. Symptoms were first observed in the emerging seedlings. No virus particles were observed in sap from infected plants with the electron microscope. Total RNA was isolated from leaves of the 10 plants with a Qiagen RNeasy Plant Mini Kit (Valencia, CA). All 10 plants tested positive for Potato spindle tuber viroid (PSTVd) by real-time reverse transcription (RT)-PCR (1) and by RT-PCR with PSTVd-specific primers (3) and generic pospiviroid primers (4). For both conventional PCRs, the expected 359-bp amplicons were sequenced directly and sequences were aligned together to create a consensus sequence (GenBank Accession No. FJ797614). BLASTn analysis showed 98% nucleotide identity to PSTVd (EU862231, DQ308556, X17268, and AY532801-AY532804). Sap from one of the infected plants was mechanically inoculated onto healthy P. peruviana, Solanum lycopersicum 'Rutgers', Chenopodium amaranticolor, C. quinoa, Cucumis sativum 'Crystal Apple', Gomphrena globosa, Nicotiana benthamiana, N. clevelandii, N. occidentalis '37B', N. tabacum 'WB', N. sylvestris, and Phaseolus vulgaris 'Prince'. After 4 weeks, the leaves of the 'Rutgers' tomato plants were showing severe distortion, purpling, and necrosis of mid-veins and P. peruviana plants were showing distortion of newly emerging apical leaves. Healthy control P. peruviana were asymptomatic. Symptoms appeared milder than that observed in the original P. peruviana plants, but this may be related to different environmental conditions or age or growth stage of the plants when inoculated. All other indicator plants were symptomless, but along with P. peruviana, tested positive for PSTVd by real-time RT-PCR (1). The presence of PSTVd was further confirmed in one original symptomatic and the mechanically inoculated P. peruviana plants and in the indicator plants by dot-blot hybridization with a digoxygenin-labeled synthetic ssRNA probe specific to the full-length PSTVd genome. PSTVd has been reported in New Zealand previously in commercial glasshouse crops of tomatoes and peppers (2), but was eradicated and so remains a regulated pest. The plants were grown from seeds imported from Germany and it is possible that the infection was seedborne. PSTVd was reported in young cape gooseberry seedlings in Germany and Turkey but the infection was asymptomatic (5). Symptoms were associated with the PSTVd-infected cape gooseberry in New Zealand. To our knowledge, this is the first report of the viroid in domestically grown plants in New Zealand, and only the second report of PSTVd in cape gooseberry worldwide. Our findings suggest that this species is an emerging host for PSTVd and that dissemination of seed may provide a pathway for international movement of the viroid.

First Report of Tomato chlorotic dwarf viroid in Greenhouse Tomatoes in Arizona

Tomato chlorotic dwarf viroid (TCDVd), a member of the genus Pospivroid, family Pospiviroidae, was first identified on greenhouse tomato (Solanum lycopersicum) in Canada (2). Since then, it has also been reported elsewhere, e.g., on tomato in Colorado (4). During 2006 in Arizona, tomato plants in a large greenhouse facility with continuous tomato production exhibited viroid-like symptoms of plant stunting and chlorosis of the young leaves. Symptomatic plants were often located along the edge of the row, indicating the presence of a mechanical transmissible agent. Approximately 4% of the plants in this greenhouse were symptomatic in 2008. Symptoms were distinctly different from those caused by Pepino mosaic virus (PepMV), a virus that was generally present in this greenhouse and also in our test samples. Other commonly occurring tomato viruses were ruled out by serological, PCR, or reverse transcription (RT)-PCR tests in multiple laboratories. RT-PCR with two sets of universal pospiviroid primers, PospiI-FW/RE and Vid-FW/RE (4), yielded amplicons of the expected sizes of 196 and 360 bp in three samples collected from symptomatic plants. Direct sequencing of the amplicons revealed that the genome was 360 nt and 100% identical to the type TCDVd from Canada (GenBank Accession No. AF162131) (2). Mechanical inoculation with leaf tissue extract from four samples to plants of the tomato 'Money-Maker' resulted in the same viroid-like symptoms and TCDVd was confirmed in these plants by RT-PCR and sequencing. In both 2007 and 2008, 18 samples were tested using primers PSTVd-F and PSTVd-R (1), which are capable of amplifying the full TCDVd genome. Analysis of the sequences from the amplicons revealed two genotypes of TCDVd. The first genotype (GenBank Accession No. FJ822877) was identical to the type TCDVd and found in 11 samples from 2007 and one from 2008. The second genotype (GenBank Accession No. FJ822878) was 361 nt, differing from the first by nine nucleotide substitutions, 2 insertions, and 1 deletion. This second genotype was found in 7 and 17 samples from 2007 and 2008, respectively, and showed the highest sequence identity (97%) to a Japanese tomato isolate (AB329668) and a much lower sequence identity (92%) to a U.S. isolate previously identified in Colorado (AY372399) (4). The origin of TCDVd in this outbreak is not clear. The genotype identified first could have been introduced from a neighboring greenhouse where the disease was observed before 2006 and where this genotype also was identified in 2007. The second genotype may have been introduced from infected seed since TCDVd has recently been shown to be seed transmitted in tomato (3). To our knowledge, this is the first report of natural occurrence of TCDVd in Arizona.

First Report of Potato spindle tuber viroid in Tomato in Belgium.

During August of 2006, a sample of a tomato plant (Solanum lycopersicum, formerly Lycopersicum esculentum) from a greenhouse in Belgium was received for diagnosis. The plant showed severe growth reduction and the young leaves were chlorotic and distorted. In the greenhouse, the disease had been spreading slowly along the row. These observations suggested the presence of a viroid infection, and reverse transcriptase (RT)-PCR with two sets of universal pospiviroid primers (Pospi1-RE/FW and Vid-FW/RE; 3) yielded amplicons of the expected size (approximately 196 and 360 bp). Sequence analysis of the larger PCR product revealed that the genome was 358 nt and 100% identical to two isolates of Potato spindle tuber viroid (PSTVd) previously submitted to the NCBI GenBank (Accession Nos. AJ583449 from the United Kingdom and AY962324 from Australia). A pathogen associated with the symptomatic tomato plants was therefore identified as PSTVd. Tracing the origin of the infection revealed the following information: during November of 2005, 8-day-old tomato seedlings raised from seed by a Dutch nursery were transferred to a small part of the greenhouse of the Belgian grower; 7 to 8 weeks later, the plants were transplanted to their final destination; during May of 2006, the grower first observed growth reduction in a single plant; several weeks later, similar symptoms were observed in two more plants in the same row close to the first symptomatic plant; and by September, there were approximately 20 symptomatic tomato plants, all located in two adjacent rows. The viroid outbreak was fully eradicated by destroying all tomato plants in the affected rows as well as in two adjacent rows at both sides. The absence of further infections was confirmed by testing approximately 1,200 tomato plants in pooled samples for PSTVd by RT-PCR (2) and real-time RT-PCR (1). The origin and the method of introduction and spread of the viroid remain unclear.

First Report of Tomato chlorotic dwarf viroid in Petunia hybrida from the United States of America.

In November 2005, 13 accessions of Petunia hybrida from the United States of America entered the post-entry quarantine station of the Plant Protection Service in the Netherlands. The plants were inspected and tested for quarantine organisms according to Directives 95/44 and 97/46 of the European Union. No virus and viroid symptoms were observed in the imported plants or in mechanically inoculated plants of Chenopodium quinoa, Nicotiana benthamiana, and N. occidentalis-P1 (3). Testing for pospiviroids by return-polyacrylamide gel electrophoresis (1) and reverse transcriptase-PCR with universal pospiviroid primers Pospi1-RE/FW (2) indicated the presence of pospiviroids in 3 and 11 P. hybrida accessions, respectively. The 196-bp amplicons of six accessions were sequenced. Sequence analysis showed the highest identity for all amplicons to both isolates of Tomato chlorotic dwarf viroid (TCDVd) in NCBI GenBank, Accession Nos. AF162131and AY372399, from Canada and the United States, respectively. Additional RT-PCRs with the Pospi1-RE/FW primers in opposite order and the semi-universal pospiviroid primers Vid-RE/FW (2) for one isolate, followed by sequence analysis, confirmed the identity as TCDVd. The isolate consisted of 359 nucleotides (GenBank Accession No. DQ859013) and showed sequence identities of 98.6 and 96.1% to the Canadian and American tomato isolates of this viroid, respectively. The next highest sequence identity was 90.0% to two accessions of Potato spindle tuber viroid (GenBank Accession Nos. AJ593449 and AY962324). On the basis of these results, the viroid from P. hybrida was identified as TCDVd. To our knowledge, this is the first report of TCDVd in this plant species. Reference: (1) J. W. Roenhorst et al. EPPO Bull. 30:453, 2000. (2) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004. (3) J. Th. J. Verhoeven and J. W. Roenhorst. EPPO Bull. 33:305, 2003.

First Report of Potato virus M and Chrysanthemum stunt viroid in Solanum jasminoides.

In 2005, a plant of the ornamental crop Solanum jasminoides from the Netherlands was submitted for testing on viruses and viroids because of its intended use for propagation. Sap from this plant was mechanically inoculated to the test plant species Chenopodium quinoa, Nicotiana benthamiana, N. hesperis-67A, and N. occidentalis-P1 (3). N. hesperis-67A showed chlorotic local lesions and rugosity followed by vein necrosis, N. occidentalis-P1 showed necrotic local lesions and systemic leaf distortion, and the two other test plant species remained symptomless. Potato virus M (PVM) was identified by double antibody sandwich enzyme-linked immunosorbent assay using leaves from S. jasminoides and N. hesperis-67A. The plant of S. jasminoides was also tested for the presence of viroids by reverse transcriptase-polymerase chain reaction (RT-PCR) with universal pospiviroid primers Pospi1-RE/FW (2). This reaction yielded an amplicon of the expected size of 198 bp. The sequence showed 100% identity to an isolate of Chrysanthemum stunt viroid (CSVd; NCBI GenBank Accession No. AF394453). Subsequently, the complete sequence of our viroid isolate (GenBank Accession No. DQ406591) was determined from the amplicon obtained after RT-PCR using specific primers for the detection of CSVd (1). The viroid isolate from S. jasminoides consisted of 354 nucleotides and showed the highest identity (98.6%) to a chrysanthemum isolate of CSVd (GenBank Accession No. AB055974). Therefore, the viroid was identified as CSVd. To our knowledge, this is the first report of PVM and CSVd in S. jasminoides. Reference: (1) R. Hooftman et al. Acta Hortic. 432:120, 1996. (2) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004. (3) J. Th. J. Verhoeven and J. W. Roenhorst, EPPO Bull. 33:305, 2003.

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

In May 2005, the Plant Protection Service in the Netherlands received two tomato (Lycopersicon esculentum) plant specimens for diagnosis from a protected crop production facility of 2.5 ha near Kebili in Tunisia. Growth of the plants was reduced and leaves were chlorotic and brittle. Ripening of the fruits was delayed and their storage life was reduced from 3 weeks to 1 week. The grower reported that initially only 5% of plants showed symptoms; however, the number of symptomatic plants increased quickly to 100% as a result of increasing temperatures in the production facility. Test plant species Chenopodium quinoa, Datura stramonium, Nicotiana glutinosa, N. hesperis-67A, N. occidentalis-P1, and L. esculentum 'Money-maker' were mechanically inoculated with sap from the affected plants. Symptoms including chlorosis and stunting were observed only on L. esculentum. Reverse transcriptase-polymerase chain reaction (RT-PCR) with universal pospiviroid primers Pospi1-RE/FW (2) yielded amplicons of the expected size (196 bp) for each of the two samples. One of these amplicons was sequenced and showed the highest identity to the four isolates of Tomato apical stunt viroid (TASVd) in the NCBI Gen-Bank. Subsequently, the complete sequence of the Tunisian isolate (Gen-Bank Accession No. DQ144506) was determined by sequencing the am-plicon obtained after RT-PCR using primers developed for the detection of Citrus exocortis viroid (CEVd) (1). The isolate consisted of 363 nucleotides and showed the highest sequence identity (96.7%) to tomato isolates of TASVd from Indonesia and Israel (GenBank Accession Nos. X06390 and AY062121, respectively), 92.6% to a tomato isolate from the Ivory Coast (GenBank Accession No. K00818), and 87.7% to an isolate from Solanum pseudocapsicum (GenBank Accession No. X95293). The next highest sequence identity was 81.5% to an isolate of CEVd (GenBank Accession No. X53716). On the basis of these results, the viroid was identified as TASVd. To our knowledge, this is the first report of TASVd in Tunisia. Reference: (1) N. Önelge. Turkish J. Agric. For. 21:419, 1997. (2) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004.

An RT-PCR primer pair for the detection of Pospiviroid and its application in surveying ornamental plants for viroids

A primer pair for reverse transcription-polymerase chain reaction (RT-PCR), based on the conserved sequences of the members of genus Pospiviroid was designed to yield a fragment of about 200 base pairs (bp). Since pospiviroids infect a large number of plants species and a few members of the genus Pospiviroid have been already detected in some ornamental plants, the primer pair was evaluated for its efficacy using ornamental plants. The method of return-polyacrylamide gel electrophoresis (R-PAGE) was used to determine the general presence of viroids in the test samples. Efficacy of the primer pair for members of genus Pospiviroid was demonstrated by the detection of Potato spindle tuber viroid (PSTVd) and Tomato chlorotic dwarf viroid (TCDVd) in potato, Chrysanthemum stunt viroid and Iresine viroid in Verbena and Vinca species, and Citrus exocortis viroid in Impatiens species. Specificity of the primer pair became evident, where additional viroids were detected by R-PAGE in Coleus and Magilla species, but they were not amplified by the Pospiviroid primer. This primer pair would be of benefit in indexing ornamental plants in quarantine samples or in viroid-free certification schemes, irrespective of their actual identity.