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J W Roenhorst - One of the best experts on this subject based on the ideXlab platform.
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Pospiviroid infections in Capsicum annuum: disease symptoms and lack of seed transmission
European Journal of Plant Pathology, 2020Co-Authors: J.th.j. Verhoeven, Harrie Koenraadt, A. Jodlowska, L. Hüner, J W RoenhorstAbstract:Four Pospiviroids, columnea latent viroid (CLVd), pepper chat fruit viroid (PCFVd), potato spindle tuber viroid (PSTVd) and tomato apical stunt viroid (TASVd), were mechanically inoculated on Capsicum annuum (pepper) cv. Yolo Wonder. Symptoms were different, though most pronounced for PCFVd, i.e. delay in flower and fruit onset, increase of plant height, and reduction of fruit size. For studying seed transmission, seeds with high loads of infectious viroid were sown at different time intervals after harvesting, i.e. 0, 2 and 77 days. No transmission from seed to seedlings was recorded in a total of 765 seedlings, irrespective viroid species and time interval. For PCFVd, the experiment was repeated in another pepper cultivar with the similar outcome for 179 seedlings. The results of this study are discussed in view of the role of infested seed lots in causing outbreaks in practice.
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a Pospiviroid from symptomless portulaca plants closely related to iresine viroid 1
Virus Research, 2015Co-Authors: Th J J Verhoeven, J W Roenhorst, E. T. M. Meekes, Ricardo Flores, M Hooftman, P SerraAbstract:In symptomless plants of portulaca a potential new Pospiviroid was characterized. Analysis by both double and return PAGE showed the presence of a circular RNA. RT-PCR and sequencing revealed a genome of 351 nt with properties characteristic of members of the genus Pospiviroid and with highest sequence identity (circa 80%) with iresine viroid 1 (IrVd-1). The circular RNA from portulaca was shown to replicate independently in its original host, thus showing that it is indeed a viroid. Based on its molecular characteristics, it should be considered a new species. However, since no biological differences have yet been found with its closest relative IrVd-1, the viroid from portulaca does not fulfil all criteria for species demarcation of the ICTV.
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dahlia latent viroid a recombinant new species of the family Pospiviroidae posing intriguing questions about its origin and classification
Journal of General Virology, 2013Co-Authors: Jacobus Th J Verhoeven, J W Roenhorst, E. T. M. Meekes, Ricardo Flores, P SerraAbstract:A viroid-like RNA has been detected in two asymptomatic dahlia accessions by return and double PAGE. It appeared smaller than Chrysanthemum stunt viroid and Potato spindle tuber viroid, the two members of the genus Pospiviroid, family Pospiviroidae, reported in this ornamental previously. RT-PCR with primers designed for amplifying all Pospiviroids produced no amplicons, but RT-PCR with random primers revealed a 342 nt RNA. The sequence of this RNA was confirmed with specific primers, which additionally revealed its presence in many dahlia cultivars. The RNA was named Dahlia latent viroid (DLVd) because it replicates autonomously, but symptomlessly, in dahlia and shares maximum sequence identity with other viroids of less than 56 %. Furthermore, DLVd displays characteristic features of the family Pospiviroidae: a predicted rod-like secondary structure of minimum free energy with a central conserved region (CCR), and the ability to form the metastable structures hairpins I and II. Its CCR is identical to that of Hop stunt viroid (HSVd, genus Hostuviroid). However, DLVd: (i) has the terminal conserved region present in members of the genus Pospiviroid, but absent in HSVd, and (ii) lacks the terminal conserved hairpin present in HSVd. Phylogenetic reconstructions indicate that HSVd and Pepper chat fruit viroid (genus Pospiviroid) are the closest relatives of DLVd, but DLVd differs from these viroids in its host range, restricted to dahlia so far. Therefore, while DLVd fulfils the criteria to be a novel species of the family Pospiviroidae, its recombinant origin makes assignment to the genera Pospiviroid or Hostuviroid problematic.
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Mexican papita viroid and tomato planta macho viroid belong to a single species in the genus Pospiviroid
Archives of Virology, 2011Co-Authors: J.th.j. Verhoeven, J W Roenhorst, Robert A OwensAbstract:Tomato planta macho viroid (TPMVd) and Mexican papita viroid (MPVd) are two closely related (>90% sequence identity) members of the genus Pospiviroid. Their current status as members of separate species is based upon the reported ability of TPMVd to replicate in Gomphrena globosa and the inability of this viroid to evoke flower break in N. glutinosa. Characterization of a viroid recently isolated from diseased tomato plants grown in Mexico (identical to GenBank accession GQ131573) casts doubt on this earlier report and indicates that these viroids should be classified as members of a single species. Giving priority to the older name, we propose including both of these viroids in the current species Tomato planta macho viroid.
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High stability of original predominant Pospiviroid genotypes upon mechanical inoculation from ornamentals to potato and tomato
Archives of Virology, 2010Co-Authors: J.th.j. Verhoeven, J W RoenhorstAbstract:Eleven Pospiviroid isolates from ornamental plants and one from pepper were successfully transmitted to potato and tomato by mechanical inoculation. All isolates with characteristic predominant genotypes were inoculated to a series of potato and tomato plants and propagated for up to four passages. In total, 385 nucleotide sequences were determined, in which 17 new predominant genotypes were identified with minimal differences to the original predominant genotype. In addition, in the original ornamental hosts, only one of eleven predominant Pospiviroid genotypes had changed during the experiments over a period of 2 years. These results confirm the high stability of predominant Pospiviroid genotypes.
Th J J Verhoeven - One of the best experts on this subject based on the ideXlab platform.
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a Pospiviroid from symptomless portulaca plants closely related to iresine viroid 1
Virus Research, 2015Co-Authors: Th J J Verhoeven, J W Roenhorst, E. T. M. Meekes, Ricardo Flores, M Hooftman, P SerraAbstract:In symptomless plants of portulaca a potential new Pospiviroid was characterized. Analysis by both double and return PAGE showed the presence of a circular RNA. RT-PCR and sequencing revealed a genome of 351 nt with properties characteristic of members of the genus Pospiviroid and with highest sequence identity (circa 80%) with iresine viroid 1 (IrVd-1). The circular RNA from portulaca was shown to replicate independently in its original host, thus showing that it is indeed a viroid. Based on its molecular characteristics, it should be considered a new species. However, since no biological differences have yet been found with its closest relative IrVd-1, the viroid from portulaca does not fulfil all criteria for species demarcation of the ICTV.
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pepper chat fruit viroid biological and molecular properties of a proposed new species of the genus Pospiviroid
Virus Research, 2009Co-Authors: Th J J Verhoeven, C C C Jansen, J W Roenhorst, Ricardo Flores, M PenaAbstract:In autumn 2006, a new disease was observed in a glasshouse-grown crop of sweet pepper (Capsicum annuum L.) in the Netherlands. Fruit size of the infected plants was reduced up to 50%, and plant growth was also slightly reduced. Here we show that the disease is caused by a previously non-described viroid. The pepper viroid is transmitted by both mechanical inoculation and pepper seeds and, when inoculated experimentally, it infects several solanaceous plant species inducing vein necrosis and reduced fruit and tuber size in tomato and potato, respectively. The viroid RNA genome consists of 348 nucleotides and, with minor modifications, it has the central conserved and the terminal conserved regions characteristic of members of the genus Pospiviroid. Classification of the pepper viroid within the genus Pospiviroid is further supported by the presence and structure of hairpins I and II, the presence of internal and external RY motifs, and phylogenetic analyses. The primary structure of the pepper viroid only showed a maximum of 66% nucleotide sequence identity with other viroids, which is far below the main species demarcation limit of 90%. According to its biological and molecular properties, we propose to assign the pepper viroid to a new species within the genus Pospiviroid, and to name this new species Pepper chat fruit viroid.
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first report of solanum jasminoides infected by citrus exocortis viroid in germany and the netherlands and tomato apical stunt viroid in belgium and germany
Plant Disease, 2008Co-Authors: Th J J Verhoeven, C C C Jansen, J W Roenhorst, S Steyer, N Schwind, M WasseneggerAbstract:Recent identifications of Chrysanthemum stunt viroid (CSVd) and Potato spindle tuber viroid (PSTVd) in Solanum jasminoides (3,4) prompted the testing of this plant species for infections with other Pospiviroids. From autumn of 2006 to spring of 2007, samples from symptomless plants of S. jasminoides were collected in Belgium (3 samples ranging from 75 to 150 plants), Germany (3 samples ranging from 1 to 200 plants), and the Netherlands (3 samples ranging from 2 to 200 plants). Samples were tested for Pospiviroids by reverse transcription (RT)-PCR assays using the Pospi1-FW/RE and Vid-FW/RE (2) and PSTV-Nb-FW (5′-ggatccccggggaaacctgga-3′)/RE (5′-ggatccctgaagcgctcctcc-3′) primer sets. Each set amplifies several but not all Pospiviroids. The first and last primer sets amplified PCR products from six samples. The full-length genomes of all six isolates were amplified using primer pairs CEVd-FW1/RE1 (1) and CEVd-FW2 (5′-gtgctcacctgaccctgcagg-3′)/RE2 (5′-accacaggaacctcaagaaag-3′), which are fully complementary ...
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first report of tomato chlorotic dwarf viroid in petunia hybrida from the united states of america
Plant Disease, 2007Co-Authors: Th J J Verhoeven, C C C Jansen, A W Werkman, J W RoenhorstAbstract: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-P...
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natural infections of tomato by citrus exocortis viroid columnea latent viroid potato spindle tuber viroid and tomato chlorotic dwarf viroid
European Journal of Plant Pathology, 2004Co-Authors: Th J J Verhoeven, C C C Jansen, T M Willemen, Robert A Owens, J W RoenhorstAbstract:Since 1988, viroids have been occasionally detected in samples of tomato (Lycopersicon esculentum) originating both in the Netherlands and other countries. Infected plants showed chlorosis, bronzing, leaf distortion and growth reduction. Initial diagnosis of these viroids was by return-polyacrylamide gel electrophoresis, which did not allow a further identification. This paper reports the identification of these viroids by reverse transcription-polymerase chain reaction and sequence analysis. Three known viroids of tomato, i.e. Citrus exocortis viroid, Potato spindle tuber viroid and Tomato chlorotic dwarf viroid were identified. In addition, six isolates were identified as Columnea latent viroid, a viroid so far only detected in some ornamental species. Like the isolates previously isolated from ornamental species, the isolates from tomato share genetic characteristics of both the genera Hostuviroid and Pospiviroid. The biological characteristics of all four viroids, especially their potential effects on both potato (Solanum tuberosum) and tomato, stress the need for reconsideration of their phytosanitary risks.
Ricardo Flores - One of the best experts on this subject based on the ideXlab platform.
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ICTV Virus Taxonomy Profile: Pospiviroidae.
The Journal of general virology, 2021Co-Authors: Francesco Di Serio, Georgios Vidalakis, Jacobus Th J Verhoeven, Jaroslav Matousek, Robert A Owens, Teruo Sano, Vicente Pallás, John W. Randles, Ricardo FloresAbstract:Members of the family Pospiviroidae have single-stranded circular RNA genomes that adopt a rod-like or a quasi-rod-like conformation. These genomes contain a central conserved region that is involved in replication in the nucleus through an asymmetric RNA-RNA rolling-circle mechanism. Members of the family Pospiviroidae lack the hammerhead ribozymes that are typical of viroids classified in the family Avsunviroidae. The family Pospiviroidae includes the genera Apscaviroid, Cocadviroid, Coleviroid, Hostuviroid and Pospiviroid, with >25 species. This is a summary of the ICTV Report on the family Pospiviroidae, which is available at ictv.global/report/Pospiviroidae.
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Iresine Viroid 1 and a Potential New Pospiviroid From Portulaca
Viroids and Satellites, 2017Co-Authors: Jacobus Th J Verhoeven, Ricardo Flores, P SerraAbstract:Abstract Iresine viroid 1 (IrVd-1) and a potential new Pospiviroid from portulaca (portulaca latent viroid, PoLVd) cause symptomless infections in a small number of ornamental species. Both viroids display the main characteristics of members of the family Pospiviroidae; i.e., a predicted rod-like secondary structure of minimum free energy with a central conserved region (CCR). Further features allow classification of both viroids in the genus Pospiviroid. The CCR is similar to that of potato spindle tuber viroid (genus Pospiviroid), and the conformations of both IrVd-1 and PoLVd contain or can adopt relevant structural elements such as the terminal conserved region, Hairpins I and II, and an external RY motif. Both viroids are more closely related to each other than to any other viroid according to sequence alignment, phylogenetic analysis, and biological features (only these two Pospiviroids cannot infect tomato). Based on the currently available biological data, however, PoLVd does not fulfil all criteria for species demarcation of the ICTV.
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a Pospiviroid from symptomless portulaca plants closely related to iresine viroid 1
Virus Research, 2015Co-Authors: Th J J Verhoeven, J W Roenhorst, E. T. M. Meekes, Ricardo Flores, M Hooftman, P SerraAbstract:In symptomless plants of portulaca a potential new Pospiviroid was characterized. Analysis by both double and return PAGE showed the presence of a circular RNA. RT-PCR and sequencing revealed a genome of 351 nt with properties characteristic of members of the genus Pospiviroid and with highest sequence identity (circa 80%) with iresine viroid 1 (IrVd-1). The circular RNA from portulaca was shown to replicate independently in its original host, thus showing that it is indeed a viroid. Based on its molecular characteristics, it should be considered a new species. However, since no biological differences have yet been found with its closest relative IrVd-1, the viroid from portulaca does not fulfil all criteria for species demarcation of the ICTV.
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dahlia latent viroid a recombinant new species of the family Pospiviroidae posing intriguing questions about its origin and classification
Journal of General Virology, 2013Co-Authors: Jacobus Th J Verhoeven, J W Roenhorst, E. T. M. Meekes, Ricardo Flores, P SerraAbstract:A viroid-like RNA has been detected in two asymptomatic dahlia accessions by return and double PAGE. It appeared smaller than Chrysanthemum stunt viroid and Potato spindle tuber viroid, the two members of the genus Pospiviroid, family Pospiviroidae, reported in this ornamental previously. RT-PCR with primers designed for amplifying all Pospiviroids produced no amplicons, but RT-PCR with random primers revealed a 342 nt RNA. The sequence of this RNA was confirmed with specific primers, which additionally revealed its presence in many dahlia cultivars. The RNA was named Dahlia latent viroid (DLVd) because it replicates autonomously, but symptomlessly, in dahlia and shares maximum sequence identity with other viroids of less than 56 %. Furthermore, DLVd displays characteristic features of the family Pospiviroidae: a predicted rod-like secondary structure of minimum free energy with a central conserved region (CCR), and the ability to form the metastable structures hairpins I and II. Its CCR is identical to that of Hop stunt viroid (HSVd, genus Hostuviroid). However, DLVd: (i) has the terminal conserved region present in members of the genus Pospiviroid, but absent in HSVd, and (ii) lacks the terminal conserved hairpin present in HSVd. Phylogenetic reconstructions indicate that HSVd and Pepper chat fruit viroid (genus Pospiviroid) are the closest relatives of DLVd, but DLVd differs from these viroids in its host range, restricted to dahlia so far. Therefore, while DLVd fulfils the criteria to be a novel species of the family Pospiviroidae, its recombinant origin makes assignment to the genera Pospiviroid or Hostuviroid problematic.
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pepper chat fruit viroid biological and molecular properties of a proposed new species of the genus Pospiviroid
Virus Research, 2009Co-Authors: Th J J Verhoeven, C C C Jansen, J W Roenhorst, Ricardo Flores, M PenaAbstract:In autumn 2006, a new disease was observed in a glasshouse-grown crop of sweet pepper (Capsicum annuum L.) in the Netherlands. Fruit size of the infected plants was reduced up to 50%, and plant growth was also slightly reduced. Here we show that the disease is caused by a previously non-described viroid. The pepper viroid is transmitted by both mechanical inoculation and pepper seeds and, when inoculated experimentally, it infects several solanaceous plant species inducing vein necrosis and reduced fruit and tuber size in tomato and potato, respectively. The viroid RNA genome consists of 348 nucleotides and, with minor modifications, it has the central conserved and the terminal conserved regions characteristic of members of the genus Pospiviroid. Classification of the pepper viroid within the genus Pospiviroid is further supported by the presence and structure of hairpins I and II, the presence of internal and external RY motifs, and phylogenetic analyses. The primary structure of the pepper viroid only showed a maximum of 66% nucleotide sequence identity with other viroids, which is far below the main species demarcation limit of 90%. According to its biological and molecular properties, we propose to assign the pepper viroid to a new species within the genus Pospiviroid, and to name this new species Pepper chat fruit viroid.
P Serra - One of the best experts on this subject based on the ideXlab platform.
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Iresine Viroid 1 and a Potential New Pospiviroid From Portulaca
Viroids and Satellites, 2017Co-Authors: Jacobus Th J Verhoeven, Ricardo Flores, P SerraAbstract:Abstract Iresine viroid 1 (IrVd-1) and a potential new Pospiviroid from portulaca (portulaca latent viroid, PoLVd) cause symptomless infections in a small number of ornamental species. Both viroids display the main characteristics of members of the family Pospiviroidae; i.e., a predicted rod-like secondary structure of minimum free energy with a central conserved region (CCR). Further features allow classification of both viroids in the genus Pospiviroid. The CCR is similar to that of potato spindle tuber viroid (genus Pospiviroid), and the conformations of both IrVd-1 and PoLVd contain or can adopt relevant structural elements such as the terminal conserved region, Hairpins I and II, and an external RY motif. Both viroids are more closely related to each other than to any other viroid according to sequence alignment, phylogenetic analysis, and biological features (only these two Pospiviroids cannot infect tomato). Based on the currently available biological data, however, PoLVd does not fulfil all criteria for species demarcation of the ICTV.
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a Pospiviroid from symptomless portulaca plants closely related to iresine viroid 1
Virus Research, 2015Co-Authors: Th J J Verhoeven, J W Roenhorst, E. T. M. Meekes, Ricardo Flores, M Hooftman, P SerraAbstract:In symptomless plants of portulaca a potential new Pospiviroid was characterized. Analysis by both double and return PAGE showed the presence of a circular RNA. RT-PCR and sequencing revealed a genome of 351 nt with properties characteristic of members of the genus Pospiviroid and with highest sequence identity (circa 80%) with iresine viroid 1 (IrVd-1). The circular RNA from portulaca was shown to replicate independently in its original host, thus showing that it is indeed a viroid. Based on its molecular characteristics, it should be considered a new species. However, since no biological differences have yet been found with its closest relative IrVd-1, the viroid from portulaca does not fulfil all criteria for species demarcation of the ICTV.
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dahlia latent viroid a recombinant new species of the family Pospiviroidae posing intriguing questions about its origin and classification
Journal of General Virology, 2013Co-Authors: Jacobus Th J Verhoeven, J W Roenhorst, E. T. M. Meekes, Ricardo Flores, P SerraAbstract:A viroid-like RNA has been detected in two asymptomatic dahlia accessions by return and double PAGE. It appeared smaller than Chrysanthemum stunt viroid and Potato spindle tuber viroid, the two members of the genus Pospiviroid, family Pospiviroidae, reported in this ornamental previously. RT-PCR with primers designed for amplifying all Pospiviroids produced no amplicons, but RT-PCR with random primers revealed a 342 nt RNA. The sequence of this RNA was confirmed with specific primers, which additionally revealed its presence in many dahlia cultivars. The RNA was named Dahlia latent viroid (DLVd) because it replicates autonomously, but symptomlessly, in dahlia and shares maximum sequence identity with other viroids of less than 56 %. Furthermore, DLVd displays characteristic features of the family Pospiviroidae: a predicted rod-like secondary structure of minimum free energy with a central conserved region (CCR), and the ability to form the metastable structures hairpins I and II. Its CCR is identical to that of Hop stunt viroid (HSVd, genus Hostuviroid). However, DLVd: (i) has the terminal conserved region present in members of the genus Pospiviroid, but absent in HSVd, and (ii) lacks the terminal conserved hairpin present in HSVd. Phylogenetic reconstructions indicate that HSVd and Pepper chat fruit viroid (genus Pospiviroid) are the closest relatives of DLVd, but DLVd differs from these viroids in its host range, restricted to dahlia so far. Therefore, while DLVd fulfils the criteria to be a novel species of the family Pospiviroidae, its recombinant origin makes assignment to the genera Pospiviroid or Hostuviroid problematic.
C C C Jansen - One of the best experts on this subject based on the ideXlab platform.
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pepper chat fruit viroid biological and molecular properties of a proposed new species of the genus Pospiviroid
Virus Research, 2009Co-Authors: Th J J Verhoeven, C C C Jansen, J W Roenhorst, Ricardo Flores, M PenaAbstract:In autumn 2006, a new disease was observed in a glasshouse-grown crop of sweet pepper (Capsicum annuum L.) in the Netherlands. Fruit size of the infected plants was reduced up to 50%, and plant growth was also slightly reduced. Here we show that the disease is caused by a previously non-described viroid. The pepper viroid is transmitted by both mechanical inoculation and pepper seeds and, when inoculated experimentally, it infects several solanaceous plant species inducing vein necrosis and reduced fruit and tuber size in tomato and potato, respectively. The viroid RNA genome consists of 348 nucleotides and, with minor modifications, it has the central conserved and the terminal conserved regions characteristic of members of the genus Pospiviroid. Classification of the pepper viroid within the genus Pospiviroid is further supported by the presence and structure of hairpins I and II, the presence of internal and external RY motifs, and phylogenetic analyses. The primary structure of the pepper viroid only showed a maximum of 66% nucleotide sequence identity with other viroids, which is far below the main species demarcation limit of 90%. According to its biological and molecular properties, we propose to assign the pepper viroid to a new species within the genus Pospiviroid, and to name this new species Pepper chat fruit viroid.
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first report of solanum jasminoides infected by citrus exocortis viroid in germany and the netherlands and tomato apical stunt viroid in belgium and germany
Plant Disease, 2008Co-Authors: Th J J Verhoeven, C C C Jansen, J W Roenhorst, S Steyer, N Schwind, M WasseneggerAbstract:Recent identifications of Chrysanthemum stunt viroid (CSVd) and Potato spindle tuber viroid (PSTVd) in Solanum jasminoides (3,4) prompted the testing of this plant species for infections with other Pospiviroids. From autumn of 2006 to spring of 2007, samples from symptomless plants of S. jasminoides were collected in Belgium (3 samples ranging from 75 to 150 plants), Germany (3 samples ranging from 1 to 200 plants), and the Netherlands (3 samples ranging from 2 to 200 plants). Samples were tested for Pospiviroids by reverse transcription (RT)-PCR assays using the Pospi1-FW/RE and Vid-FW/RE (2) and PSTV-Nb-FW (5′-ggatccccggggaaacctgga-3′)/RE (5′-ggatccctgaagcgctcctcc-3′) primer sets. Each set amplifies several but not all Pospiviroids. The first and last primer sets amplified PCR products from six samples. The full-length genomes of all six isolates were amplified using primer pairs CEVd-FW1/RE1 (1) and CEVd-FW2 (5′-gtgctcacctgaccctgcagg-3′)/RE2 (5′-accacaggaacctcaagaaag-3′), which are fully complementary ...
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first report of tomato chlorotic dwarf viroid in petunia hybrida from the united states of america
Plant Disease, 2007Co-Authors: Th J J Verhoeven, C C C Jansen, A W Werkman, J W RoenhorstAbstract: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-P...
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application of real time rt pcr for large scale testing of potato for potato spindle tuber Pospiviroid
Eppo Bulletin, 2005Co-Authors: J W Roenhorst, C C C Jansen, L F F Kox, E G De Haan, G Van Den Bovenkamp, N Boonham, T Fisher, R A MumfordAbstract:The real-time RT-PCR protocol of Boonham and coworkers performed extremely well in a recent ring test, comparing different methods for detection of Potato spindle tuber Pospiviroid (PSTVd) in several laboratories. Since, in addition, real-time PCR technology has proved suitable for high-throughput testing, this method was chosen as the starting point for the development of a protocol for the large-scale testing of potato. The initial experiments focused on the specificity of the primers and probes with regard to different isolates of PSTVd and other (pospi-) viroids. Further experiments were performed with leaf material from both primarily and secondarily infected plants. The parameters studied were sampling position, growing-on temperature and bulking rate. In addition, different grinding, nucleic-acid extraction and disinfection methods were compared. To monitor false negatives and positives, different controls were included and tested in duplex and triplex formats. The final protocol was tested using a hundred samples from the Dutch potato-monitoring programme. The results of this pilot experiment were promising. Future plans include the development of a protocol for direct tuber testing and inter-laboratory ring testing of the protocols.
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reliability of nucleic acid amplification techniques modified target rna as exogenous internal standard for a real time rt pcr for potato spindle tuber Pospiviroid
Eppo Bulletin, 2005Co-Authors: L F F Kox, C C C Jansen, I L A Boxman, J W RoenhorstAbstract:Techniques based on nucleic acid amplification techniques, like PCR, are quick, sensitive and specific, and therefore very suitable for the development of diagnostic tests. These techniques enable the detection of very small amounts of target organisms by specific amplification of part of its genome. This exquisite sensitivity puts a high demand on measures to prevent false-positive reactions due to contamination of the laboratory with nucleic acid, hence the need for inclusion of negative controls. In addition, to exclude false negatives, the performance of the reactions must be measured by the inclusion of several positive controls, e.g. cytochrome oxidase (COX) primers (and probes) to monitor efficiency of the nucleic acid extraction and an internal control to monitor inhibition of the target PCR. For the RT-PCR assay for Potato spindle tuber Pospiviroid (PSTVd) recently described by Boonham and coworkers, we have developed an exogenous internal standard, i.e. in vitro RNA transcribed from a plasmid containing a modified PSTVd sequence (a 17-bp sequence of cloned PSTVd (isolate Howell) was substituted for a 118-bp sequence of Escherichia coli). To this exogenous sequence, a specific probe was designed with a fluorescent label different from that of the PSTVd-specific probe. By making use of the same primers as the target organism PSTVd, this internal standard provides a tool to measure the performance of the specific reaction. Moreover, by using another fluorescent probe, this standard can easily be discriminated from the target organism. The approach used for the construction of the internal control for PSTVd offers a tool for the construction of internal standards for other pathogens.