First Report of Potato spindle tuber viroid in Cape Gooseberry (Physalis peruviana) from Turkey and Germany

Since the recent identification of Potato spindle tuber viroid (PSTVd) in vegetatively propagated ornamental plant species (4), many growers have asked to have their mother plants tested for this viroid. In December of 2007, a grower from Turkey submitted cuttings of cape gooseberry (Physalis peruviana) to be tested for PSTVd. Initial testing by real-time reverse transcription (RT)-PCR according to Boonham et al. (1) indicated the presence of either Mexican papita viroid, PSTVd, or Tomato chlorotic dwarf viroid in four samples. To identify the viroid(s) present, isolated RNA from these samples was used for RT-PCR (2), and products of the expected full genome size for the three viroids were amplified from each sample. One of the PCR products was sequenced (GenBank Accession No. EU862230) and analysis of the 357 nt sequence indicated it was most related to PSTVd sequences belonging to the so-called ‘Oceanian’ strain of the viroid (3), with 99.7% identity to GenBank Accession No. AY962324. Therefore, the viroid was identified as PSTVd. Pathogenicity of this PSTVd genotype was demonstrated when 4 weeks after mechanical inoculation with sap extracts seedlings of tomato cv. Money-maker showed the expected viroid symptoms of chlorosis and stunting, and the presence of the viroid in these plants was confirmed by RT-PCR (2). In March of 2008, by use of RT-PCR (2) and sequencing of the PCR product (GenBank Accession No. EU862231), PSTVd was identified in young seedlings of P. peruviana from a German grower. The German isolate differed at only three nucleotide positions from the Turkish isolate. The identification of PSTVd in young seedlings indicates that seeds had been source of infection, whereas in the case of the PSTVd infected cuttings from Turkey, the infection originated from infected mother plants. To our knowledge, these are the first reports of PSTVd in P. peruviana. Although infected P. peruviana plants did not show symptoms, they might act as sources of inoculum for crops like potato and tomato, which may suffer serious damage. References: (1) N. Boonham et al. J. Virol. Methods 116:139, 2004. (2) A. M. Shamloul et al. Can. J. Plant Pathol. 19:89, 1997. (3) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004. (4) J. Th. J. Verhoeven et al. Plant Pathol. 57:399, 2008.

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

Plant Disease ◽

10.1094/pdis-94-4-0479a ◽

2010 ◽

Vol 94 (4) ◽

pp. 479-479 ◽

Cited By ~ 8

Author(s):

L. I. Ward ◽

J. Tang ◽

S. Veerakone ◽

B. D. Quinn ◽

S. J. Harper ◽

...

Keyword(s):

New Zealand ◽

Real Time ◽

Consensus Sequence ◽

Potato Spindle Tuber Viroid ◽

Rt Pcr ◽

Indicator Plants ◽

First Report ◽

Physalis Peruviana ◽

Plant Pathol ◽

Cape Gooseberry

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. References: (1) N. Boonham et al. J. Virol. Methods 116:139, 2004. (2) B. S. M. Lebas et al. Australas. Plant Pathol. 34:129, 2005. (3) A. M. Shamoul et al. Can. J. Plant Pathol. 19:89, 1997. (4) J. T. H. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004. (5) J. T. H. Verhoeven et al. Plant Dis. 93:316, 2009.

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

Plant Disease ◽

10.1094/pdis-08-11-0711 ◽

2012 ◽

Vol 96 (1) ◽

pp. 150-150 ◽

Cited By ~ 3

Author(s):

M. Viršček Marn ◽

I. Mavrič Pleško

Keyword(s):

New Zealand ◽

Sequence Analysis ◽

Unknown Origin ◽

Potato Spindle Tuber Viroid ◽

Adult Plant ◽

Total Rna ◽

First Report ◽

Physalis Peruviana ◽

Plant Pathol ◽

Cape Gooseberry

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. References: (1) F. Di Serio. J. Plant Pathol. 89:297, 2007. (2) A. M. Shamloul et al. Can. J. Plant Pathol. 19:89, 1997. (3) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004. (4) J. Th. J. Verhoeven et al. Plant Dis. 93:316, 2009.

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First Report of Potato spindle tuber viroid in Tomato in Belgium

Plant Disease ◽

10.1094/pdis-91-8-1055a ◽

2007 ◽

Vol 91 (8) ◽

pp. 1055-1055 ◽

Cited By ~ 13

Author(s):

J. Th. J. Verhoeven ◽

C. C. C. Jansen ◽

J. W. Roenhorst ◽

S. Steyer ◽

D. Michelante

Keyword(s):

Potato Spindle Tuber Viroid ◽

Growth Reduction ◽

Rt Pcr ◽

Tomato Plants ◽

Final Destination ◽

The United Kingdom ◽

Pcr Product ◽

Viroid Infection ◽

Young Leaves ◽

Plant Pathol

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. References: (1) N. Boonham et al. J. Virol. Methods 116:139, 2004. (2) R. A. Mumford et al. Plant Pathol. 53:242, 2004. (3) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004.

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

Plant Disease ◽

10.1094/pdis-94-9-1171b ◽

2010 ◽

Vol 94 (9) ◽

pp. 1171-1171 ◽

Cited By ~ 6

Author(s):

M. Viršček Marn ◽

I. Mavrič Pleško

Keyword(s):

Real Time ◽

Coastal Region ◽

Leaf Tissue ◽

Positive Sample ◽

Rt Pcr ◽

Total Rna ◽

Production Site ◽

Pcr Products ◽

Tomato Chlorotic Dwarf Viroid ◽

Plant Pathol

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. References: (1) Anonymous. EPPO Bull. 34:257, 2004. (2) F. Di Serio. J. Plant Pathol. 89:297, 2007. (3) A. M. Shamloul et al. Can. J. Plant Pathol. 19:89, 1997. (4) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004.

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

Plant Disease ◽

10.1094/pdis-12-20-2730-re ◽

2021 ◽

Author(s):

Yi-Wen Tseng ◽

Chien-Fu Wu ◽

Chia-Hwa Lee ◽

Chung Jan Chang ◽

Yuh-Kun Chen ◽

...

Keyword(s):

Potato Spindle Tuber Viroid ◽

Detection Methods ◽

Universal Primers ◽

Rt Pcr ◽

Degenerate Primers ◽

Highly Sensitive ◽

One Step ◽

Tomato Chlorotic Dwarf Viroid ◽

Minimal Concentration ◽

Solanaceous Plants

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

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AtypicalCTSKTranscripts andARNTTranscription Read-Through intoCTSK

Comparative and Functional Genomics ◽

10.1002/cfg.483 ◽

2005 ◽

Vol 6 (5-6) ◽

pp. 268-276

Author(s):

Fabienne S. Giraudeau ◽

Jean-Philippe Walhin ◽

Paul R. Murdock ◽

Nigel K. Spurr ◽

Ian C. Gray

Keyword(s):

Negative Impact ◽

Cathepsin K ◽

Chromosome 1 ◽

Rt Pcr ◽

Transcription Start ◽

Aryl Hydrocarbon ◽

Pcr Product ◽

Close Proximity ◽

Pcr Products ◽

Human Chromosome 1

The aryl hydrocarbon receptor nuclear translocator (ARNT) and cathepsin K (CTSK) genes lie in a tandem head-to-tail arrangement on human chromosome 1. The two genes are in extremely close proximity; the usualCTSKtranscription start site is less than 1.4 kb downstream of the end of the longest reportedARNTtranscript. By generating an RT-PCR product that overlaps both the 3′ end ofARNTand the 5′ end ofCTSK, we show thatARNTtranscripts may extend through theARNT–CTSKintergenic region and progress into theCTSKgene. Furthermore, by using quantitative RT-PCR from several tissues to detect theARNTexpression signature inCTSKintrons, we show thatARNTtranscripts can read through intoCTSKas far asCTSKintron 3, extending approximately 3.7 kb downstream of the end of the longest previously describedARNTmRNA. Given thatARNTandCTSKare expressed in an overlapping range of tissues,ARNTread-through may have a negative impact onCTSKtranscript levels by interfering withCTSKexpression. We also present evidence for novelCTSKtranscripts following sequence analysis ofCTSK-derived ESTs and RT-PCR products. These transcripts show alternate 5′ splicing and or 5′ extension and are sometimes initiated from a cryptic alternative promoter which is upstream of the knownCTSKpromoter and possibly in the 3′ UTR ofARNT.

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An outbreak of viral gastroenteritis associated with consumption of sandwiches: implications for the control of transmission by food handlers

Epidemiology and Infection ◽

10.1017/s0950268898001150 ◽

1998 ◽

Vol 121 (3) ◽

pp. 615-621 ◽

Cited By ~ 107

Author(s):

U. D. PARASHAR ◽

L. DOW ◽

R. L. FANKHAUSER ◽

C. D. HUMPHREY ◽

J. MILLER ◽

...

Keyword(s):

Food Handler ◽

Common Source ◽

Viral Gastroenteritis ◽

Food Handlers ◽

Rt Pcr ◽

Source Of Infection ◽

Food Borne ◽

Pcr Product ◽

Stool Specimens ◽

Polymerase Chain

Although food handlers are often implicated as the source of infection in outbreaks of food-borne viral gastroenteritis, little is known about the timing of infectivity in relation to illness. We investigated a gastroenteritis outbreak among employees of a manufacturing company and found an association (RR=14·1, 95% CI=2·0–97·3) between disease and eating sandwiches prepared by 6 food handlers, 1 of whom reported gastroenteritis which had subsided 4 days earlier. Norwalk-like viruses were detected by electron microscopy or reverse transcriptase-polymerase chain reaction (RT-PCR) in stool specimens from several company employees, the sick food handler whose specimen was obtained 10 days after resolution of illness, and an asymptomatic food handler. All RT-PCR product sequences were identical, suggesting a common source of infection. These data support observations from recent volunteer studies that current recommendations to exclude food handlers from work for 48–72 h after recovery from illness may not always prevent transmission of Norwalk-like viruses because virus can be shed up to 10 days after illness or while exhibiting no symptoms.

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First Report of Grapevine Pinot gris virus (GPGV) in grapevine in France

Plant Disease ◽

10.1094/pdis-10-14-1008-pdn ◽

2015 ◽

Vol 99 (2) ◽

pp. 293-293 ◽

Cited By ~ 25

Author(s):

M. Beuve ◽

T. Candresse ◽

M. Tannières ◽

O. Lemaire

Keyword(s):

Large Scale ◽

De Novo ◽

Pinot Noir ◽

Rt Pcr ◽

Wine Quality ◽

First Report ◽

Italian Isolate ◽

Pcr Products ◽

Grapevine Pinot Gris Virus ◽

Plant Pathol

Grapevine Pinot gris virus (GPGV), belonging to the genus Trichovirus of the family Betaflexiviridae, was first identified by siRNA sequencing in northern Italy in 2012, in the grapevine varieties Pinot gris, Traminer, and Pinot Noir, which exhibited mottling and leaf deformation (1), and in asymptomatic vines, with a lower frequency. Since 2012, this virus has also been reported in South Korea, Slovenia, Greece (3), Czech Republic (2), Slovakia (2), and southern Italy (4). In 2014, GPGV was identified by Illumina sequencing of total RNAs extracted from leaves of the Merlot variety (Vitis vinifera) grafted onto Gravesac rootstock originated from a vineyard in the Bordeaux region of France. This Merlot plant exhibited fanleaf-like degeneration symptoms associated with Tomato black ring virus (TBRV) infection. Cuttings were collected in 2010 and maintained thereafter in a greenhouse. The full-length genome was assembled either de novo or by mapping of the Illumina reads on a reference GPGV genome (GenBank FR877530) using the CLC Genomics workbench software (CLC Bio, Qiagen, USA). The French GPGV isolate “Mer” (7,223 nucleotides, GenBank KM491305) is closely related to other European GPGV sequences; it exhibits 95.4% nucleotide identity with the reference Italian isolate (NC_015782) and 98 to 98.3% identity with Slovak isolates (KF134123 to KF134125). The higher divergence between French and Italian GPGV isolates was mainly due to differences in the 5′ extremity of the genome, as already shown with the Slovak GPGV isolates. RNA extracted from phloem scrapings of 19 cv. Merlot vines from the same plot collected in 2014 were analyzed by RT-PCR using the specific primer pair Pg-Mer-F1 (5′-GGAGTTGCCTTCGTTTACGA-3′) and Pg-Mer-R1 (5′-GTACTTGATTCGCCTC GCTCA-3′), designed on the basis of alignments of all available GPGV sequences from GenBank. The resulting amplicon of 770 bp corresponded to a fragment of the putative movement protein (MP) gene. Seven (35%) of the tested plants gave a strong positive amplification. Three RT-PCR products were directly sequenced and showed 99.3 to 99.5% identity within the MP gene of the GPGV-Mer isolate. Given the mixed viral infection status of the vines found infected by GPGV, it was not possible to associate a specific symptomatology with the presence of GPGV. Furthermore, similar RT-PCR tests were also performed on RNA extracts prepared from two plants of cv. Carignan that originated from a French grapevine collection, exhibiting fanleaf-like symptoms without any nepovirus detection. These samples similarly gave a strong positive amplification. The sequences obtained from the two Carignan vines showed 98.4 and 97.8% identity with the GPGV-Mer isolate. To our knowledge, this is the first report of GPGV in France. GPGV has been discovered in white and red berry cultivars, suggesting that its prevalence could be important in European vineyards (2). Further large-scale studies will be essential to determine the world prevalence of GPGV and to evaluate its potential effects on yield and on wine quality, as well as to shed light on GPGV epidemiology. Of particular concern is whether, like the other grapevine-infecting Trichovirus, Grapevine berry inner necrosis virus (GPGV) can be transmitted by the eryophid mite Colomerus vitis. References: (1) A. Giampetruzzi et al. Virus Res. 163: 262, 2012. (2) M. Glasa et al. Arch. Virol. 159: 2103, 2014. (3) G. P. Martelli, J. Plant Pathol. 96: S105, 2014. (4) M. Morelli et al. J. Plant Pathol. 96:431, 2014.

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

Plant Disease ◽

10.1094/pdis-93-10-1075b ◽

2009 ◽

Vol 93 (10) ◽

pp. 1075-1075 ◽

Cited By ~ 18

Author(s):

K.-S. Ling ◽

J. Th. J. Verhoeven ◽

R. P. Singh ◽

J. K. Brown

Keyword(s):

Direct Sequencing ◽

Leaf Tissue ◽

Natural Occurrence ◽

Rt Pcr ◽

Nucleotide Substitutions ◽

Tomato Production ◽

First Report ◽

Sequence Identity ◽

Tomato Chlorotic Dwarf Viroid ◽

Plant Pathol

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. References: (1) A. M. Shamloul et al. Can. J. Plant Pathol. 19:89, 1997. (2) R. P. Singh et al. J. Gen. Virol. 80:2823, 1999. (3) R. P. Singh and A. D. Dilworth. Eur. J. Plant Pathol. 123:111, 2009. (4) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004.

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Development and validation of a real-time RT-PCR test for screening pepper and tomato seed lots for the presence of pospiviroids

10.1101/2020.04.17.046508 ◽

2020 ◽

Author(s):

Marleen Botermans ◽

Johanna W. Roenhorst ◽

Marinus Hooftman ◽

Jacobus Th.J. Verhoeven ◽

Eveline Metz ◽

...

Keyword(s):

Real Time ◽

Seed Transmission ◽

Potato Spindle Tuber Viroid ◽

Rt Pcr ◽

Tomato Seed ◽

Stunt Viroid ◽

Testing Laboratories ◽

Tomato Chlorotic Dwarf Viroid ◽

Development And Validation ◽

Pcr Test

AbstractPotato 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.

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