scholarly journals First Report of Ranunculus white mottle ophiovirus in Slovenia in Pepper with yellow leaf curling symptom and in Tomato

Plant Disease ◽  
2021 ◽  
Author(s):  
Mark Paul Selda Rivarez ◽  
Zala Kogej ◽  
Nejc Jakos ◽  
Anja Pecman ◽  
Gabrijel Seljak ◽  
...  
Keyword(s):  
Coat Protein ◽  
Plant Protection ◽  
Rt Pcr ◽  
Disease Symptoms ◽  
Tomato Plants ◽  
Horizon 2020 ◽  
Interveinal Chlorosis ◽  
Leaf Curling ◽  
Italian Isolate ◽  
Pcr Targeting

Pepper (Capsicum annuum) and Tomato (Solanum lycopersicum) plants showing virus-like disease symptoms were collected in 2017, 2019, and 2020, in different parts of Slovenia (Supplementary Figure 1). Total RNA was extracted from leaf tissue of individual samples using RNeasy Plant Mini kit (Qiagen) and pooled in four composite samples as follows: 2 pepper plants from 2017 (D2017), 5 pepper and 4 tomato plants from 2019 (D2019_P1), 7 tomato plants (D2020_P1), and 2 pepper and 4 tomato plants (D2020_P3) from 2020. The pooled RNA samples were sequenced using Illumina platforms, details of the sequencing experiments are in Supplementary Table 1. Reads were analyzed using CLC Genomics Workbench (v. 20.0, Qiagen) following the pipelines for plant virus discovery (Pecman et al., 2017). Reads and contigs mapping to Ranunculus white mottle ophiovirus (RWMV, GenBank accession no. AY542957 or NC_043389) were detected in all pools. The longest contig (1,255 bp) was obtained from the 2019 composite sample, mapping to the coat protein-coding RNA 3 segment of the RWMV genome (accession no. AY542957). Details of mapping, genome coverage, and other viruses detected in the pools are summarized in the Supplementary Table 1. To identify individual RWMV-infected plants from the pools, primers were designed for detection by reverse transcription-polymerase chain reaction (RT-PCR) targeting the coat protein gene (see Supplementary Table 2). Two pepper samples from two different farms, collected in 2017 and 2019 in southwest Slovenia, and four tomato samples from two different farms, collected in 2020 in central Slovenia tested positive for RWMV in RT-PCR assays. To assess the diversity of RWMV isolates, amplicons were purified using QIAquick PCR purification kit (Qiagen) and sent for Sanger sequencing. Based on maximum likelihood phylogenetic analysis, RWMV Italian and Slovenian isolates form a monophyletic clade within the genus (see Supplementary Figure 2). Pairwise nucleotide identities of the Slovenian isolates (accession no. MZ507604-MZ507609), relative to the original Italian isolate coat protein (accession no. AY542957) range from 92-97%, indicating a moderate level of diversity among isolates (see Supplementary Figure 2). Since only RWMV, bell pepper alphaendornavirus (BPEV), and pepper cryptic virus 2 (PepCV2), were present in a pepper sample from 2017, and BPEV and PepCV2 infection in pepper are not known to be associated with any of the disease symptoms (Okada et al., 2011; Saritha et al., 2016), the symptoms observed on this plant might be associated with RWMV infection. We observed mottling with interveinal chlorosis or yellowing, slight to full curling of leaves from lamina inward, as well as necrotic and aborted flowers on this plant (see Supplementary Figure 1). We cannot easily associate observed symptoms with RWMV in RWMV-positive tomatoes, since several viruses were detected in the pools containing these samples. Nevertheless, the prominent symptoms in tomato were mottling with interveinal chlorosis and leaf curling, similar to those observed in pepper. RWMV was discovered and characterized in buttercup (Ranunculus asiaticus), and detected in anemone (Anemone coronaria), from Italy (Vaira et al., 1996, 1997, 2000, 2003). It was recently detected in pepper from Australia showing veinal yellowing (Gambley et al., 2019). Here, we detected RWMV for the first time in Slovenia, and reported its first detection in tomato and pepper from Europe. These findings call for further studies on the effects of RWMV infection on tomato and pepper production, and its monitoring in neighboring European countries. Acknowledgment This study received funding from the Administration of the Republic of Slovenia for Food Safety, Veterinary Sector and Plant Protection, Slovenian Research Agency (ARRS) core financing (P4-0165), and the Horizon 2020 Marie Skłodowska-Curie Actions Innovative Training Network (H2020 MSCA-ITN) project “INEXTVIR” (GA 813542), under the management of the European Commission-Research Executive Agency. References Gambley, C., et al. 2019. New Dis. Rep. 40:13. doi:10.5197/j.2044-0588.2019.040.013. Okada, R., et al. 2011. J. Gen. Virol. 92:2664-2673. doi:10.1099/vir.0.034686-0. Pecman, A., et al. 2017. Front. Microbiol. 8:1-10. doi:10.3389/fmicb.2017.01998. Saritha, R. K., et al. 2016. VirusDisease 27:327-328. doi:10.1007/s13337-016-0327-7. Vaira, A. M., et al. 2003. Arch. Virol. 148:1037-1050. doi:10.1007/s00705-003-0016-x. Vaira, A. M., et al. 1996. Acta Hortic., 432:36-43. doi:10.17660/ActaHortic.1996.432.3. Vaira, A. M., et al. 1997. Arch. Virol. 142:2131-2146. doi:10.1007/s007050050231. Vaira, A. M., et al. 2000. Plant Dis. 84:1046-1046. doi:10.1094/PDIS.2000.84.9.1046B.

scholarly journals First Report of Sharka Disease Caused by Plum Pox Virus in Lithuania

Plant Disease ◽  
1998 ◽  
Vol 82 (12) ◽  
pp. 1405-1405 ◽  
Author(s):  
J. Staniulis ◽  
J. Stankiene ◽  
K. Sasnauskas ◽  
A. Dargeviciute
Keyword(s):  
Coat Protein ◽  
Plant Protection ◽  
Virus Disease ◽  
Pcr Amplification ◽  
Enzyme Linked Immunosorbent Assay ◽  
Plum Pox Virus ◽  
Rt Pcr ◽  
First Report ◽  
Das Elisa ◽  
Sharka Disease

Plum pox (sharka) disease caused by plum pox potyvirus (PPV) is considered the most important virus disease of stone fruit trees in Europe and the Mediterranean region. Nearly all those countries that produce stone fruits are affected (3). The causal virus of the disease is a European Plant Protection Organization A2 quarantine pathogen. Symptoms of leaf mottling, diffuse chlorotic spots, rings, and vein banding of varied intensity characteristic for plum pox virus infection were observed in the plum (Prunus domestica) orchard tree collection of the Lithuanian Institute of Horticulture in Babtai in 1996. Presence of this virus in the diseased trees was confirmed by double antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA) with kits from BIOREBA (Reinach, Switzerland) and by polyclonal antibodies raised against a Moldavian isolate of PPV courtesy of T. D. Verderevskaya (Institute of Horticulture, Kishinev, Moldova). ELISAs with both sources of antiserum were positive for presence of PPV. Electron microscopy revealed the presence of potyvirus-like particles averaging 770 nm in extracts of mechanically inoculated plants of Chenopodium foetidum (chlorotic LL [local lesions]) and Pisum sativum cvs. Rainiai and Citron (mottling). For molecular diagnosis and characterization of this isolate, PPV-971, reverse transcription-polymerase chain reaction (RT-PCR) was employed. Total RNA from the leaves of infected pea was isolated as described (2). High molecular weight RNA selectively precipitated with 2 M lithium chloride was used for RT-PCR amplification of the coat protein encoding sequence by use of specific primers complementary to 5′ and 3′ parts of PPV coat protein L1 (GenBank accession no. X81081). Amino acid sequence comparison with GenBank data indicated 98.2% similarity with coat protein of PPV potyvirus isolated by E. Mais et al. (accession no. X81083) and 97.3% with PPV strain Rankovic (1).The specific DNA fragment, corresponding to predicted coat protein sequence size, was cloned into Escherichia coli pUC57 for DNA sequencing. Expression of the cloned sequence in bacteria and yeast expression systems is under investigation. The presence of PPV in plum trees in the 9-year-old collection at Babtai was confirmed by DAS-ELISA in 1997 and again in 1998. PPV was then detected in 20% of symptomatic trees of three cultivars. The Lithuanian PPV isolate reacted positively with “universal” Mab.5b and with a Mab (Mab.4DG5) specific for PPV-D. No reaction was observed with Mabs specific for PPV-M (Mab.AL), PPV-C (Mab.AC and Mab.TUV), and PPV-El Amar (Mab.EA24). PPV-971 seems to be a typical member of the less aggressive Dideron strain cluster of PPV (D. Boscia, personal communication). This is the first report of PPV in Lithuania and confirms the necessity for continuing the precautionary measures established in this country for indexing of nursery plum trees used for graft propagation. References: (1) S. Lain et al. Virus Res. 13:157, 1989. (2) J. Logemann et al. Anal. Biochem. 163:16, 1987. (3) M. Nemeth. OEPP/EPPO Bull. 24:525, 1994.

scholarly journals Tomato Brown Rugose Fruit Virus Contributes to Enhanced Pepino Mosaic Virus Titers in Tomato Plants

Viruses ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 879 ◽  
Author(s):  
Chen Klap ◽  
Neta Luria ◽  
Elisheva Smith ◽  
Lior Hadad ◽  
Elena Bakelman ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
High Throughput Sequencing ◽  
Amino Acid Position ◽  
Viral Disease ◽  
Mixed Infections ◽  
Rt Pcr ◽  
Tomato Production ◽  
Pepino Mosaic Virus ◽  
Disease Symptoms ◽  
Tomato Plants

The tobamovirus tomato brown rugose fruit virus (ToBRFV), a major threat to tomato production worldwide, has recently been documented in mixed infections with the potexvirus pepino mosaic virus (PepMV) CH2 strain in traded tomatoes in Israel. A study of greenhouse tomato plants in Israel revealed severe new viral disease symptoms including open unripe fruits and yellow patched leaves. PepMV was only detected in mixed infections with ToBRFV in all 104 tested sites, using serological and molecular analyses. Six PepMV isolates were identified, all had predicted amino acids characteristic of CH2 mild strains excluding an isoleucine at amino acid position 995 of the replicase. High-throughput sequencing of viral RNA extracted from four selected symptomatic plants showed solely the ToBRFV and PepMV, with total aligned read ratios of 40.61% and 11.73%, respectively, indicating prevalence of the viruses. Analyses of interactions between the co-infecting viruses by sequential and mixed viral inoculations of tomato plants, at various temperatures, showed a prominent increase in PepMV titers in ToBRFV pre-inoculated plants and in mixed-infected plants at 18–25 °C, compared to PepMV-single inoculations, as analyzed by Western blot and quantitative RT-PCR tests. These results suggest that Israeli mild PepMV isolate infections, preceded by ToBRFV, could induce symptoms characteristic of PepMV aggressive strains.

scholarly journals Chino del tomate virus:Relationships to Other Begomoviruses and Identification of A-Component Variants that Affect Symptom Expression

2000 ◽  
Vol 90 (5) ◽  
pp. 546-552 ◽  
Author(s):  
J. K. Brown ◽  
Kristin M. Ostrow ◽  
Ali M. Idris ◽  
Drake C. Stenger
Keyword(s):  
Amino Acid ◽  
Genotypic Variation ◽  
Symptom Expression ◽  
Wild Type ◽  
Disease Symptoms ◽  
Phenotypic Differences ◽  
Interveinal Chlorosis ◽  
Leaf Curling ◽  
Symptom Phenotype ◽  
Severe Leaf

Phylogenetic and distance analyses place Chino del tomate virus (CdTV) in the New World clade of begomoviruses and indicate that CdTV and Tomato leaf crumple virus (TLCrV) are closely related strains of the same virus. One cloned CdTV A component (pCdTV-H6), when inoculated to tomato with the B component (pCdTV-B52), produced mild symptoms and low DNA titers. Another cloned CdTV A component (pCdTV-H8), when coinoculated to tomato with the B component, produced moderate leaf curling and veinal chlorosis similar to that of TLCrV. Coinoculation of both CdTV A components and the B component to tomato produced wild-type chino del tomate (CdT) disease symptoms consisting of severe leaf curling, veinal and interveinal chlorosis, and stunting. The two CdTV A components were nearly identical, except at nucleotide positions 1,722 and 2,324. The polymorphism at nucleotide 1,722 resulted in a change at Rep amino acid 261. The second polymorphism at nucleotide 2,324 resulted in changes at Rep amino acid 60 and AC4 amino acid 10. Two chimeric A components constructed by reciprocal exchange of a fragment bearing the polymorphic site at nucleotide 1,722 were evaluated for symptom phenotype. One chimeric A component (pCdTV-H86) produced wild-type CdT symptoms when coinoculated to tomato with the B component. The reciprocal chimeric A component (pCdTV-H68), when coin-oculated to tomato with the B component, also produced severe leaf curling, veinal chlorosis, and stunting. However, pCdTV-H68 induced less obvious interveinal chlorosis than wild-type or pCdTV-H86. Examination of A component genotypes recovered from tomato coinoculated with pCdTV-H6 and pCdTV-H8 indicated that recombination occurred to produce a genotype identical to pCdTV-H86. These results indicate that subtle genotypic variation has significant effects on symptom expression and may explain phenotypic differences observed among isolates and cloned DNAs of CdTV and TLCrV.

scholarly journals Detection and Confirmation of Potato mop-top virus in Potatoes Produced in the United States and Canada

Plant Disease ◽  
2004 ◽  
Vol 88 (4) ◽  
pp. 363-367 ◽  
Author(s):  
H. Xu ◽  
T.-L. DeHaan ◽  
S. H. De Boer
Keyword(s):  
United States ◽  
Coat Protein ◽  
Coat Protein Gene ◽  
Protein Gene ◽  
The United States ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
The North ◽  
Potato Mop Top Virus ◽  
Pcr Targeting

Potato mop-top virus (PMTV) was detected in potatoes grown in the United States and Canada during surveillance testing by a reverse transcription-polymerase chain reaction (RT-PCR) targeting the coat protein gene in RNA3. Out of 3,221 lots of seed and ware potatoes that were tested, 4.3% were positive for PMTV. The reliability of the survey results was confirmed by reextraction of selected samples and additional RT-PCR tests using two primer sets targeting gene segments in RNA2 and RNA3. Amplicons generated from RNA2 and RNA3 were identified by analysis of fragment length polymorphisms after digestion with BamHI and HindIII, respectively. PMTV was further identified by enzyme-linked immunosorbent assay, bioassay on Nicotiana debneyi, and transmission electron microscopy. Sequencing of a portion of the coat protein gene revealed near 100% identity among isolates from the United States and Canada and >97% homology of the North American isolates with European isolates.

scholarly journals RNA silencing-related genes contribute to tolerance of infection with potato virus X and Y in a susceptible tomato plant

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Joon Kwon ◽  
Atsushi Kasai ◽  
Tetsuo Maoka ◽  
Chikara Masuta ◽  
Teruo Sano ◽  
...  
Keyword(s):  
Coat Protein ◽  
Reverse Transcription ◽  
Rna Silencing ◽  
Potato Virus X ◽  
Transgenic Tomato ◽  
Genomic Rna ◽  
Rt Pcr ◽  
Tomato Plants ◽  
Transgenic Tomato Plants ◽  
Polymerase Chain

Abstract Background In plants, the RNA silencing system functions as an antiviral defense mechanism following its induction with virus-derived double-stranded RNAs. This occurs through the action of RNA silencing components, including Dicer-like (DCL) nucleases, Argonaute (AGO) proteins, and RNA-dependent RNA polymerases (RDR). Plants encode multiple AGOs, DCLs, and RDRs. The functions of these components have been mainly examined in Arabidopsis thaliana and Nicotiana benthamiana. In this study, we investigated the roles of DCL2, DCL4, AGO2, AGO3 and RDR6 in tomato responses to viral infection. For this purpose, we used transgenic tomato plants (Solanum lycopersicum cv. Moneymaker), in which the expression of these genes were suppressed by double-stranded RNA-mediated RNA silencing. Methods We previously created multiple DCL (i.e., DCL2 and DCL4) (hpDCL2.4) and RDR6 (hpRDR6) knockdown transgenic tomato plants and here additionally did multiple AGO (i.e., AGO2 and AGO3) knockdown plants (hpAGO2.3), in which double-stranded RNAs cognate to these genes were expressed to induce RNA silencing to them. Potato virus X (PVX) and Y (PVY) were inoculated onto these transgenic tomato plants, and the reactions of these plants to the viruses were investigated. In addition to observation of symptoms, viral coat protein and genomic RNA were detected by western and northern blotting and reverse transcription-polymerase chain reaction (RT-PCR). Host mRNA levels were investigated by quantitative RT-PCR. Results Following inoculation with PVX, hpDCL2.4 plants developed a more severe systemic mosaic with leaf curling compared with the other inoculated plants. Systemic necrosis was also observed in hpAGO2.3 plants. Despite the difference in the severity of symptoms, the accumulation of PVX coat protein (CP) and genomic RNA in the uninoculated upper leaves was not obviously different among hpDCL2.4, hpRDR6, and hpAGO2.3 plants and the empty vector-transformed plants. Moneymaker tomato plants were asymptomatic after infection with PVY. However, hpDCL2.4 plants inoculated with PVY developed symptoms, including leaf curling. Consistently, PVY CP was detected in the uninoculated symptomatic upper leaves of hpDCL2.4 plants through western blotting. Of note, PVY CP was rarely detected in other asymptomatic transgenic or wild-type plants. However, PVY was detected in the uninoculated upper leaves of all the inoculated plants using reverse transcription-polymerase chain reactions. These findings indicated that PVY systemically infected asymptomatic Moneymaker tomato plants at a low level (i.e., no detection of CP via western blotting). Conclusion Our results indicate that the tomato cultivar Moneymaker is susceptible to PVX and shows mild mosaic symptoms, whereas it is tolerant and asymptomatic to systemic PVY infection with a low virus titer. In contrast, in hpDCL2.4 plants, PVX-induced symptoms became more severe and PVY infection caused symptoms. These results indicate that DCL2, DCL4, or both contribute to tolerance to infection with PVX and PVY. PVY CP and genomic RNA accumulated to a greater extent in DCL2.4-knockdown plants. Hence, the contribution of these DCLs to tolerance to infection with PVY is at least partly attributed to their roles in anti-viral RNA silencing, which controls the multiplication of PVY in tomato plants. The necrotic symptoms observed in the PVX-infected hpAGO2.3 plants suggest that AGO2, AGO3 or both are also distinctly involved in tolerance to infection with PVX.

scholarly journals First Report of Tomato torrado virus Infecting Tomato in Italy

Plant Disease ◽  
2010 ◽  
Vol 94 (9) ◽  
pp. 1172-1172 ◽  
Author(s):  
S. Davino ◽  
L. Bivona ◽  
G. Iacono ◽  
M. Davino
Keyword(s):  
Mosaic Virus ◽  
Sandwich Elisa ◽  
Fruit Production ◽  
Tomato Mosaic Virus ◽  
Serological Testing ◽  
Rt Pcr ◽  
Disease Symptoms ◽  
Tomato Plants ◽  
First Report ◽  
Pcr Products

In 2009 and 2010, approximately 2% of plants had disease symptoms, including initial leaflet chlorosis that later developed into necrotic spots and general necroses along the leaflet. Fruit production on affected plants was substantially reduced and necroses were also present. Total RNA was extracted from five symptomatic plant samples using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and analyzed by reverse transcription (RT)-PCR with specific primer pair: TR2F (5′ GAAGGACGAAGAGCGACTG 3′), and TR2R (5′ AAGGTAGGTATGCGTTTGC 3′) (1). The primers amplified a 575-bp fragment within the coat protein Vp23 of Tomato torrado virus (ToTV). No RT-PCR products were observed when water or asymptomatic tomato plants were used as controls. The RT-PCR products were purified and directly sequenced in both directions. Pair-wise similarity analysis confirmed the presence of ToTV with 99% similarity to isolate PRI-ToTV0301 (GenBank Accession No. DQ388880) and 98% similarity to isolate Kra (Accession No. EU652402). A representative sequence was deposited with GenBank (Accession No. GU903899). To further confirm the presence of ToTV, dsRNA analysis was conducted on all five symptomatic plants and one healthy tomato plant (2). Electrophoresis of dsRNA showed two bands of approximately 5,400 and 7,800 nucleotides long, typical of ToTV in all samples, while a third band between the other two (approximately 6,400 nt) was detected. Serological testing using double-antibody sandwich-ELISA was also conducted on the five symptomatic and 25 additional plants from the same greenhouse that displayed typical Pepino mosaic virus (PepMV) symptoms only. Antibodies used for serological testing screened for the presence of PepMV, Tomato spotted wilt virus, Cucumber mosaic virus, and Tomato mosaic virus (Loewe Biochemica, Sauerlach, Germany). These tests detected PepMV in all samples with disease symptoms typical of PepMV, and in three of the five samples with the newly described symptoms. To our knowledge, this is the first report of ToTV in Italy, and in some plants, co-infection with PepMV was likely. All ToTV-infected tomato plants in the greenhouse were destroyed. References: (1) H. Pospieszny et al. Plant Dis. 91:1364, 2007. (2) J. Sambrook et al. Molecular Cloning. A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory Press, Woodbury, NY, 1989.

scholarly journals Evaluation of an Automated High-Throughput Liquid-Based RNA Extraction Platform on Pooled Nasopharyngeal or Saliva Specimens for SARS-CoV-2 RT-PCR

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 615
Author(s):  
Allen Wing-Ho Chu ◽  
Cyril Chik-Yan Yip ◽  
Wan-Mui Chan ◽  
Anthony Chin-Ki Ng ◽  
Dream Lok-Sze Chan ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
High Throughput ◽  
Rna Extraction ◽  
Nasopharyngeal Swab ◽  
Acid Extraction ◽  
Rt Pcr ◽  
Nucleic Acid Extraction ◽  
Suitable Alternative ◽  
Pcr Inhibitors ◽  
Pcr Targeting

SARS-CoV-2 RT-PCR with pooled specimens has been implemented during the COVID-19 pandemic as a cost- and manpower-saving strategy for large-scale testing. However, there is a paucity of data on the efficiency of different nucleic acid extraction platforms on pooled specimens. This study compared a novel automated high-throughput liquid-based RNA extraction (LRE) platform (PHASIFYTM) with a widely used magnetic bead-based total nucleic acid extraction (MBTE) platform (NucliSENS® easyMAG®). A total of 60 pools of nasopharyngeal swab and 60 pools of posterior oropharyngeal saliva specimens, each consisting of 1 SARS-CoV-2 positive and 9 SARS-CoV-2 negative specimens, were included for the comparison. Real-time RT-PCR targeting the SARS-CoV-2 RdRp/Hel gene was performed, and GAPDH RT-PCR was used to detect RT-PCR inhibitors. No significant differences were observed in the Ct values and overall RT-PCR positive rates between LRE and MBTE platforms (92.5% (111/120] vs 90% (108/120]), but there was a slightly higher positive rate for LRE (88.3% (53/60]) than MBTE (81.7% (49/60]) among pooled saliva. The automated LRE method is comparable to a standard MBTE method for the detection of SAR-CoV-2 in pooled specimens, providing a suitable alternative automated extraction platform. Furthermore, LRE may be better suited for pooled saliva specimens due to more efficient removal of RT-PCR inhibitors.

scholarly journals First Report of Mosaic Disease Caused by Colombian datura virus on Solanum lycopersicum Plants Commercially Cultivated in Japan

Plant Disease ◽  
2014 ◽  
Vol 98 (5) ◽  
pp. 698-698 ◽  
Author(s):  
Y. Tomitaka ◽  
T. Usugi ◽  
R. Kozuka ◽  
S. Tsuda
Keyword(s):  
Nucleotide Sequence ◽  
Solanum Lycopersicum ◽  
Mosaic Disease ◽  
Causal Agent ◽  
Cultivated Plants ◽  
Rt Pcr ◽  
Degenerate Primers ◽  
Specific Primers ◽  
Tomato Plants ◽  
First Report

In 2009, some commercially grown tomato (Solanum lycopersicum) plants in Chiba Prefecture, Japan, exhibited mosaic symptoms. Ten plants from a total of about 72,000 cultivated plants in the greenhouses showed such symptoms. To identify the causal agent, sap from leaves of the diseased plants was inoculated into Chenopodium quinoa and Nicotiana benthamiana plants. Local necrotic lesions appeared on inoculated leaves of C. quinoa, but no systemic infection was observed. Systemic mosaic symptoms were observed on the N. benthamiana plants inoculated. Single local lesion isolation was performed three times using C. quinoa to obtain a reference isolate for further characterization. N. benthamiana was used for propagation of the isolate. Sap from infected leaves of N. benthamiana was mechanically inoculated into three individual S. lycopersicum cv. Momotaro. Symptoms appearing on inoculated tomatoes were indistinguishable from those of diseased tomato plants found initially in the greenhouse. Flexuous, filamentous particles, ~750 nm long, were observed by electron microscopy in the sap of the tomato plants inoculated with the isolate, indicating that the infecting virus may belong to the family Potyviridae. To determine genomic sequence of the virus, RT-PCR was performed. Total RNA was extracted from the tomato leaves experimentally infected with the isolate using an RNeasy Plant Mini kit (QIAGEN, Hilden, Germany). RT-PCR was performed by using a set of universal, degenerate primers for Potyviruses as previously reported (2). Amplicons (~1,500 bp) generated by RT-PCR were extracted from the gels using the QIAquick Gel Extraction kit (QIAGEN) and cloned into pCR-BluntII TOPO (Invitrogen, San Diego, CA). DNA sequences of three individual clones were determined using a combination of plasmid and virus-specific primers, showing that identity among three clones was 99.8%. A consensus nucleotide sequence of the isolate was deposited in GenBank (AB823816). BLASTn analysis of the nucleotide sequence determined showed 99% identity with a partial sequence in the NIb/coat protein (CP) region of Colombian datura virus (CDV) tobacco isolate (JQ801448). Comparison of the amino acid sequence predicted for the CP with previously reported sequences for CDV (AY621656, AJ237923, EU571230, AM113759, AM113754, and AM113761) showed 97 to 100% identity range. Subsequently, CDV infection in both the original and experimentally inoculated plants was confirmed by RT-PCR using CDV-specific primers (CDVv and CDVvc; [1]), and, hence, the causal agent of the tomato disease observed in greenhouse tomatoes was proved to be CDV. The first case of CDV on tomato was reported in Netherlands (3), indicating that CDV was transmitted by aphids from CDV-infected Brugmansia plants cultivated in the same greenhouse. We carefully investigated whether Brugmansia plants naturally grew around the greenhouses, but we could not find them inside or in proximity to the greenhouses. Therefore, sources of CDV inoculum in Japan are still unclear. This is the first report of a mosaic disease caused by CDV on commercially cultivated S. lycopersicum in Japan. References: (1) D. O. Chellemi et al. Plant Dis. 95:755, 2011. (2) J. Chen et al. Arch. Virol. 146:757, 2001. (3) J. Th. J. Verhoeven et al. Eur. J. Plant. Pathol. 102:895, 1996.

Clinical diagnosis of early dengue infection by novel one-step multiplex real-time RT-PCR targeting NS1 gene

2015 ◽  
Vol 65 ◽  
pp. 11-19 ◽  
Author(s):  
Je-Hyoung Kim ◽  
Chom-Kyu Chong ◽  
Mangalam Sinniah ◽  
Jeyaindran Sinnadurai ◽  
Hyun-Ok Song ◽  
...  
Keyword(s):  
Real Time ◽  
Clinical Diagnosis ◽  
Dengue Infection ◽  
Rt Pcr ◽  
One Step ◽  
Ns1 Gene ◽  
Pcr Targeting

Prevalence and Molecular Characterization Coat Protein Gene of Tobacco streak virus Causing Peanut Stem Necrosis Disease in Coastal and Rayalaseema Districts of Andhra Pradesh, South-India

2021 ◽  
Author(s):  
K. Saratbabu ◽  
K. Vemana ◽  
A.K. Patibanda ◽  
B. Sreekanth ◽  
V. Srinivasa Rao
Keyword(s):  
Coat Protein ◽  
Molecular Characterization ◽  
Andhra Pradesh ◽  
Disease Incidence ◽  
Tobacco Streak Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Streak Virus ◽  
Rt Pcr ◽  
Cp Gene ◽  
Stem Necrosis

Background: Peanut stem necrosis disease (PSND) caused by Tobacco streak virus (TSV) is a major constraint for groundnut production in Andhra Pradesh (A.P.). However, studies on prevalence and spread of the disease confined to only few districts of A.P. with this background current study focused on incidence and spread of the disease in entire state of A.P. Further an isolate of TSV occurring in A.P. characterized on the basis of genetic features by comparing with other TSV isolates originated from different hosts and locations from world.Methods: Roving survey was conducted during kharif 2017-18 in groundnut growing districts of Andhra Pradesh (A.P.) for peanut stem necrosis disease incidence. Groundnut plants showing PSND symptoms were collected and tested with direct antigen coating enzyme linked immunosorbent assay (DAC-ELISA). Groundnut samples found positive by ELISA once again tested by reverse transcription polymerase chain reaction (RT-PCR). The representative TSV-GN-INDVP groundnut isolate from Prakasham district was maintained on cowpea seedlings by standard sap inoculation method in glasshouse for further molecular characterization. The Phylogenetic tree for coat protein (CP) gene was constructed using aligned sequences with 1000 bootstrap replicates following neighbor-joining phylogeny.Result: Thirty-eight (52.7%) of seventy-two groundnut samples collected from different locations in A.P were given positive reaction to TSV by DAC-ELISA. For the first time, PSND incidence observed in coastal districts (Krishna, Guntur, Sri Pottisriramulu Nellore, Prakasham) of A.P. Maximum PSND incidence recorded from Bathalapalli (22.2%) and the minimum incidence in Mulakalacheruvu (4.1%). The coat protein (CP) gene of TSV-GN-INDVP groundnut isolate was amplified by RT-PCR and it shared maximum per cent nucleotide identity (97.51-98.62%) with TSV isolates from groundnut and other different crops reported in India. All Indian isolates cluster together irrespective of crop and location based on the phylogenetic analysis.

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