Tomato Chlorotic Dwarf Viroid Research Articles

First Report of Tomato Chlorotic Dwarf Viroid in Dahlia in China

First Report of Tomato Chlorotic Dwarf Viroid in Dahlia in China

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 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.

Tomato chlorotic dwarf viroid

This datasheet on Tomato chlorotic dwarf viroid covers Identity, Distribution, Hosts/Species Affected, Further Information.

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.

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.

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.

Detection, molecular characterisation and aspects involving the transmission of tomato chlorotic dwarf viroid in eggplant

AbstractTomato chlorotic dwarf viroid (TCDVd) is a pospiviroid that causes severe disease symptoms in tomato. TCDVd is also naturally found in other crops and plants, in most occasions being asymptomatic. Apart from the natural hosts reported up to now, artificial inoculations have revealed that TCDVd can infect other plants, including eggplant (Solanum melongena). In a screening of seedlings of eggplant from a breeding programme we detected a pospiviroid, which we identified as TCDVd, representing the first report of natural infection of eggplant by TCDVd. The new TCDVd isolate of eggplant was detected by reverse transcription polymerase chain reaction (RT‐PCR) using primers TG21/CT20, initially designed to detect potato spindle tuber viroid. The new isolate sequence is close to a Brugmansia sanguinea isolate of TCDVd from the Netherlands, and most of the nucleotidic changes with respect to this isolate and to the reference genome sequence of TCDVd are found in the TR region. Naturally infected plants of eggplant with this TCDVd isolate did not display any disease symptoms. We demonstrated that in eggplant TCDVd is mechanically transmitted with low to moderate efficiency with cultivation practices, but not by plant‐to‐plant contact. Tomato plants artificially inoculated with the eggplant isolate of TCDVd tested positive for the presence of the viroid at 50 days after inoculation, but did not display any disease symptoms. Seed transmission to germinated seedlings of eggplant was variable among progenies from infected plants, ranging from 7.7% to 100.0%. Disinfection of seeds with chemical treatments with sodium hypochlorite and trisodium phosphate solutions plus thermotherapy at 80°C for 24 hr or 90°C for 6 hr was ineffective in reducing the rate of transmission by seed. We did not find evidence of horizontal transmission of TCDVd by pollen, but vertical transmission was highly efficient when healthy eggplant plants were pollinated with infected pollen. Our results indicate that asymptomatic infection of eggplant by TCDVd and high seed and pollen transmission rates may contribute to the spread of this viroid. The information we obtained is useful in order to implement measures for the prevention, control and eradication of TCDVd in eggplant crops, as well as to avoid their transmission to other hosts.

First Report of Tomato Chlorotic Dwarf Viroid Infecting Greenhouse Tomato in Hawaii

First Report of Tomato Chlorotic Dwarf Viroid Infecting Greenhouse Tomato in Hawaii

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.

Transmission of Tomato chlorotic dwarf viroid by Myzus persicae assisted by Potato leafroll virus

In this study, we have shown Myzus persicae transmissibility of Tomato chlorotic dwarf viroid (TCDVd) by exposing Potato leafroll virus (PLRV) plus TCDVd carrying aphids to tomato plants. The efficiency of PLRV-assisted TCDVd transmission by M. persicae was higher at 26–28 °C than that at 20–22 °C, with a rate of infection of 25% compared to that of 5%, respectively. Moreover, serial passages of PLRV + TCDVd into host plants and non-host plants showed that TCDVd infections still could be detected in host plants (tomato) even after passage over non-host plants Brassica oleracea (20% compared to 33% only host plant passages). This finding indicates that PLRV-assisted TCDVd transmission occurs in a circulative manner. The foliar responses of singly TCDVd and doubly TCDVd + PLRV-infected tomatoes developed similarly for the first 5 weeks after inoculation. From the sixth week onward, however, doubly infected tomatoes began to produce new sprouts with small and stunted leaves, whereas the singly TCDVd-infected tomatoes remained stunted without forming new sprouts.

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.

Development of a rapid diagnostic assay for the detection of tomato chlorotic dwarf viroid based on isothermal reverse-transcription-recombinase polymerase amplification

A molecular diagnostic assay utilizing reverse transcription-recombinase polymerase amplification (RT-RPA) at an isothermal constant temperature of 39°C and target-specific primers and probe were developed for the rapid, sensitive, and specific detection of tomato chlorotic dwarf viroid (TCDVd) in infected leaf and seed tissues. The performance of the AmplifyRP® Acceler8™ RT-RPA diagnostic assay, utilizing a lateral flow strip contained within an amplicon detection chamber, was evaluated and the results were compared with a standard RT-PCR assay. The AmplifyRP® Acceler8™ assay was specific for TCDVd in leaf and seed tissues, its sensitivity was comparable to conventional RT-PCR in leaf tissues, and it does not require extensive sample purification, specialized equipment, or technical expertise. This is the first report utilizing an RT-RPA assay to detect viroids and the assay can be used both in the laboratory and in the field for TCDVd detection.

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.

λ-Carrageenan Suppresses Tomato Chlorotic Dwarf Viroid (TCDVd) Replication and Symptom Expression in Tomatoes.

The effect of carrageenans on tomato chlorotic dwarf viroid (TCDVd) replication and symptom expression was studied. Three-week-old tomato plants were spray-treated with iota(ɩ)-, lambda(λ)-, and kappa(κ)-carrageenan at 1 g·L−1 and inoculated with TCDVd after 48 h. The λ-carrageenan significantly suppressed viroid symptom expression after eight weeks of inoculation, only 28% plants showed distinctive bunchy-top symptoms as compared to the 82% in the control group. Viroid concentration was reduced in the infected shoot cuttings incubated in λ-carrageenan amended growth medium. Proteome analysis revealed that 16 tomato proteins were differentially expressed in the λ-carrageenan treated plants. Jasmonic acid related genes, allene oxide synthase (AOS) and lipoxygenase (LOX), were up-regulated in λ-carrageenan treatment during viroid infection. Taken together, our results suggest that λ-carrageenan induced tomato defense against TCDVd, which was partly jasmonic acid(JA) dependent, and that it could be explored in plant protection against viroid infection.

Detection of PSTVd and TCDVd in seeds of tomato using real‐time RT‐PCR

Worldwide outbreaks of pospiviroids in potato and tomato have increased the need for a reliable test for the detection of pospiviroids in seeds. This study describes the development and validation of a sensitive and fast test for the detection of Potato spindle tuber viroid (PSTVd) and Tomato chlorotic dwarf viroid (TCDVd) in tomato seeds. The test is based on RNA isolation using a commercial kit and is suitable for routine application. The test is able to detect one PSTVd or TCDVd contaminated seed in sub samples of 1000 seeds and results were both repeatable and reproducible.

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 Potato spindle tuber viroid Naturally Infecting Field Tomatoes in the Dominican Republic.

In recent years, viroid disease outbreaks have resulted in serious economic losses to a number of tomato growers in North America (1,2,3). At least three pospiviroids have been identified as the causal agents of tomato disease, including Potato spindle tuber viroid (PSTVd), Tomato chlorotic dwarf viroid (TCDVd), and Mexican papita viroid (MPVd). In the spring of 2013, a severe disease outbreak with virus-like symptoms (chlorosis and plant stunting) was observed in a tomato field located in the Dominican Republic, whose tomato production is generally exported to the United States in the winter months. The transplants were produced in house. The disease has reached an epidemic level with many diseased plants pulled and disposed of accordingly. Three samples collected in May of 2013 were screened by ELISA against 16 common tomato viruses (Alfalfa mosaic virus, Cucumber mosaic virus, Impatiens necrotic spot virus, Pepino mosaic virus, Potato virus X, Potato virus Y, Tobacco etch virus, Tobacco mosaic virus, Tobacco ringspot virus, Tomato aspermy virus, Tomato bushy stunt virus, Tomato mosaic virus, Tomato ringspot virus, Tomato spotted wilt virus, Groundnut ringspot virus, and Tomato chlorotic spot virus), a virus group (Potyvirus group), three bacteria (Clavibacter michiganensis subsp. michiganensis, Pectobacterium atrosepticum, and Xanthomonas spp.), and Phytophthora spp. No positive result was observed, despite the presence of symptoms typical of a viral-like disease. Further analysis by RT-PCR using Agdia's proprietary pospiviroid group-specific primer resulted in positive reactions in all three samples. To determine which species of pospiviroid was present in these tomato samples, full-genomic products of the expected size (~360 bp) were amplified by RT-PCR using specific primers for PSTVd (4) and cloned using TOPO-TA cloning kit (Invitrogen, CA). A total of 8 to 10 clones from each isolate were selected for sequencing. Sequences from each clone were nearly identical and the predominant sequence DR13-01 was deposited in GenBank (Accession No. KF683200). BLASTn searches into the NCBI database demonstrated that isolate DR13-01 shared 97% sequence identity to PSTVd isolates identified in wild Solanum (U51895), cape gooseberry (EU862231), or pepper (AY532803), and 96% identity to the tomato-infecting PSTVd isolate from the United States (JX280944). The relatively lower genome sequence identity (96%) to the tomato-infecting PSTVd isolate in the United States (JX280944) suggests that PSTVd from the Dominican Republic was likely introduced from a different source, although the exact source that resulted in the current disease outbreak remains unknown. It may be the result of an inadvertent introduction of contaminated tomato seed lots or simply from local wild plants. Further investigation is necessary to determine the likely source and route of introduction of PSTVd identified in the current epidemic. Thus, proper control measures could be recommended for disease management. The detection of this viroid disease outbreak in the Dominican Republic represents further geographic expansion of the viroid disease in tomatoes beyond North America.

First report of tomato chlorotic dwarf viroid isolated from symptomless petunia plants (Petunia spp.) in Japan

A viroid was detected for the first time in symptomless petunia plants (Petunia spp.) and identified as Tomato chlorotic dwarf viroid (TCDVd) based on an analysis of the complete genomic sequence. These petunia plants are a likely source of inoculum for tomato or potato plants because TCDVd induces severe symptoms on these plants. The genomic sequence of this petunia isolate from Japan shared 100 % identity with petunia isolates from the Netherlands and United Kingdom and a tomato isolate from Japan. Phylogenetic analysis showed that all petunia isolates and the tomato isolate from Japan formed a monophyletic clade.

First report of Tomato chlorotic dwarf viroid (TCDVd) in tomato in Norway and subsequent eradication.

In January 2012 a sample of tomato ( Solanum lycopersicon cv Juanita) was submitted to the Food and Environment Research Agency (Fera), via a horticultural consultant, from an established crop in the county Rogaland in Southwest Norway. The…