scholarly journals First Report of Sweet Pepper (Capsicum annuum) as a Natural Host Plant for Tomato chlorosis virus

Plant Disease ◽  
2004 ◽  
Vol 88 (2) ◽  
pp. 224-224 ◽  
Author(s):  
G. Lozano ◽  
E. Moriones ◽  
J. Navas-Castillo
Keyword(s):  
Capsicum Annuum ◽  
Sweet Pepper ◽  
Natural Host ◽  
Molecular Hybridization ◽  
Rt Pcr ◽  
Total Rna ◽  
First Report ◽  
Pcr Product ◽  
Tomato Chlorosis Virus ◽  
Specific Symptoms

Since 1997, epidemics of a tomato yellowing disease have occurred in the Málaga and Almería provinces of southern Spain. These epidemics have been associated with infections of Tomato chlorosis virus (ToCV) (genus Crinivirus, family Closteroviridae) (2). During the past few years, an increasing incidence of the disease was observed and it spread to new areas including eastern Spain and the Balearic and Canary Islands (G. Lozano, E. Moriones, and J. Navas-Castillo, unpublished results and [1]). In 1999, plants of sweet pepper (Capsicum annuum L.) exhibiting symptoms of interveinal yellowing, mild upward leaf curling, and stunting were observed in greenhouses of Almería that were heavily infested with the whitefly, Bemisia tabaci. Symptomatic plants were tested for the presence of the begomovirus, Tomato yellow leaf curl virus, a virus previously reported in sweet pepper (3) by molecular hybridization or polymerase chain reaction (PCR). Some of these plants tested positive. Total RNA extracts from the symptomatic plants were also analyzed for the presence of tomato criniviruses using reverse transcription (RT)-PCR with primers MA59 and MA60 for the HSP70h gene (2). A PCR DNA product of the expected size (587 bp) was obtained from several samples. The cloning and sequencing of the PCR product obtained from one of these samples confirmed the presence of ToCV, with a sequence 100% identical to the equivalent region of the first ToCV isolated from tomato in Málaga (2). Total RNA extracts from plants that tested positive using RT-PCR were also positive with molecular hybridization using a probe for the HSP70h gene of ToCV. To our knowledge, this is the first report of sweet pepper as a natural host of a tomato crinivirus, which may have important epidemiological consequences in regions where both crops are grown. Association between ToCV infection and specific symptoms observed in sweet pepper plants is under study. References: (1) M. I. Font et al. Bol. San. Veg. Plagas 29:109, 2003. (2) J. Navas-Castillo et al. Plant Dis. 84:835, 2000. (3) J. Reina et al. Plant Dis. 83:1176, 1999.

scholarly journals First Report of Tomato chlorosis virus Infecting Sweet Pepper in Costa Rica

Plant Disease ◽  
2011 ◽  
Vol 95 (11) ◽  
pp. 1482-1482 ◽  
Author(s):  
J. A. Vargas ◽  
E. Hernández ◽  
N. Barboza ◽  
F. Mora ◽  
P. Ramírez
Keyword(s):  
Costa Rica ◽  
The United States ◽  
Sweet Pepper ◽  
Nutritional Deficiencies ◽  
Total Rna ◽  
First Report ◽  
Leaf Curling ◽  
Pcr Products ◽  
Tomato Chlorosis Virus ◽  
Pepper Plants

In September 2008, a survey of whiteflies and whitefly-borne viruses was performed in 11 pepper-growing greenhouses in the province of Cartago, Costa Rica. During this survey, the vast majority of sweet pepper (Capsicum annuum cv. Nataly) plants showed interveinal chlorosis, enations, necrosis, and mild upward leaf curling. Large populations of whiteflies were present and they were found to be composed only of Trialeurodes vaporariorum. Total RNA from frozen plant samples was extracted with TRI Reagent (Molecular Research Inc., Cincinnati, OH). RevertAid H Minus Reverse Transcriptase Kit (Fermentas, Hanover, MD) was used for reverse transcription of the total RNA extract, with cDNA synthesis directed using random primers. A real-time PCR assay was performed to detect Tomato chlorosis virus (ToCV) (genus Crinivirus, family Closteroviridae) using the SYBR Green PCR Master Mix (Applied Biosystems, Carlsbad, CA). Three sets of primers were used to confirm the presence of ToCV in the samples: TocQ875F/TocQ998R primer set directed to a fragment of 123 bp of the HSP gene (3); ToCVp22RQF (5′-TGGATCTCACTGGTTGCTTG-3′)-ToCVp22RQR (5′-TAGTGTTTCAGCGCCAACAG-3′) primer pair that amplifies a 198-bp segment of the ToCV p22 gene (R. Hammond, E. Hernandez, J. Guevara, J. A. Vargas, A. Solorzano, R. Castro, N. Barboza, F. Mora, and P. Ramirez, unpublished) and the ToCVCPmRQF (5′-CATTGGTTGGGGATTACGTC-3′)-ToCVCPmRQR (5′-TCTCAGCCTTGACTTGAGCA-3′) primer pair designed to amplify a 170-bp portion of the ToCV CPm gene (R. Hammond, E. Hernandez, J. Guevara, J. A. Vargas, A. Solorzano, R. Castro, N. Barboza, F. Mora and P. Ramirez, unpublished). Fifteen symptomatic samples per greenhouse were tested for a total of 165 sweet pepper plants. From this total, seven samples from four different greenhouses produced amplification of PCR products with all three sets of primers. One of the seven samples showed mild chlorosis, but others were highly chlorotic with different levels of upward leaf curling. None of the other samples showed amplification with any of the primer sets; the symptoms on these plants could have been due to nutritional deficiencies or infection by viruses. Sequence analysis of the 460-bp HSP PCR products, produced using previously reported primers (3), and 150-bp fragment of the P22 revealed 100% sequence identity with a tomato isolate of ToCV from the United States (GenBank Accession No. AY903448). Because of the low number of samples infected with ToCV and the high incidence of symptoms, DNA extraction and a begomovirus PCR detection assay was performed using primer pair AV494/AC1048 (4). Negative results were obtained for all samples. To our knowledge, this is the first report of ToCV infecting sweet pepper plants in Costa Rica and the third one worldwide. ToCV has also been found to be infecting tomato in open field and greenhouses (1) and other weeds in greenhouses including Ruta chalepensis (Rutaceae), Phytolacca icosandra (Phytolaccaceae), Plantago major (Plantaginaceae), and Brassica sp. (Brassicaceae) (2) in the same region of Costa Rica, suggesting that it has adapted to the conditions of the area and poses an important threat to the vegetable production. References: (1) R. M. Castro et al. Plant Dis. 93:970, 2009. (2) A. Solorzano-Morales et al. Plant Dis. 95:497, 2011. (3) W. M. Wintermantel et al. Phytopathology 98:1340, 2008. (4) S. Wyatt and J. Brown. Phytopathology 86:1288, 1996.

scholarly journals First Report of Tomato chlorosis virus in Potato in Brazil

Plant Disease ◽  
2012 ◽  
Vol 96 (4) ◽  
pp. 593-593 ◽  
Author(s):  
D. M. S. Freitas ◽  
I. Nardin ◽  
N. Shimoyama ◽  
J. A. C. Souza-Dias ◽  
J. A. M. Rezende
Keyword(s):  
Leaf Roll ◽  
Rt Pcr ◽  
Specific Primers ◽  
Total Rna ◽  
First Report ◽  
Interveinal Chlorosis ◽  
Potato Plants ◽  
The World ◽  
Tomato Chlorosis Virus ◽  
Biotype B

Potato plants (Solanum tuberosum cv. Ágata) exhibiting symptoms of leaf roll and interveinal chlorosis, especially on older leaves, were found in a commercial crop in the County of Cristalina, State of Goiás, Brazil in June 2011. The crop was severely infested by whitefly Bemisia tabaci biotype B. Four potato tubers from symptomatic plants were indexed for the presence of the following viruses: Tomato chlorosis virus (ToCV), Potato leaf roll virus (PLRV), Tomato severe rugose virus (ToSRV), and Potato virus Y (PVY). Total RNA was extracted separately from each tuber and used for reverse transcription (RT)-PCR using the HS-11/HS-12 primer pair, which amplifies a fragment of 587 bp from the highly conserved region of the heat shock protein (HSP-70) homolog gene reported for ToCV. The RT-PCR product was subsequently tested by nested-PCR for detection of ToCV with specific primers ToC-5/ToC-6 (2). Amplicons of 463 bp, amplified from total RNA separately extracted from three tubers, were purified and directly sequenced. Comparisons among the three consensus sequences of 448 bp (GenBank Accession Nos. JQ288896, JQ288897, and JQ288898) revealed respectively, 98, 100, and 100% identity with the reported sequence of a tomato isolate of ToCV from Brazil (GenBank Accession No. EU868927) (1). For ToSRV detection, total DNA was extracted from two tubers and a fragment of approximately 820 bp was amplified by PCR with specific primers (3). PLRV and PVY were indexed in two and three tubers, respectively, by double-antibody sandwich-ELISA (SASA, Edinburg, Scotland). Virus-free B. tabaci biotype B were separately transferred to potato and tomato leaves infected with ToCV for an acquisition access period of 24 h. Groups of 30 viruliferous whitefly were transferred to four, young, sprout-grown potato plants cv. Ágata (two plants per virus isolate) for 24-h inoculation access period. After 37 days of inoculation, one plant inoculated with the potato and tomato isolates of ToCV, respectively exhibited symptoms of leaf roll and interveinal chlorosis on order leaves, which were similar to that induced by PLRV. Experimental infection of potato plants with ToCV, which induced leaf roll symptoms resembling PLRV infection, was reported in the United States by Wisler et al. (4). The potato isolate of ToCV was also transmitted by B. tabaci to one of two inoculated tomato plants. The presence of ToCV in all inoculated plants was detected by nested-RT-PCR as described above. To our knowledge, this is the first report on detection of ToCV in field potato plants in the world. Considering that ToCV occurs in innumerous countries around the world, it is transmitted by a cosmopolitan insect, and it induces symptoms similar to PLRV, this finding triggers an alert to field dependent seed-potato multiplication, virus inspector, and certification system. References: (1) J. C. Barbosa et al. Plant Dis. 92:1709, 2008. (2) C. I. Dovas et al. Plant Dis. 86:1345, 2002. (3) F. R. Fernandes et al. Trop. Plant Pathol. 35:43, 2010. (4) G. C. Wisler et al. Plant Dis. 82:270, 1998.

scholarly journals First Report of Tomato chlorosis virus (ToCV) in Tomato Crops in Saudi Arabia

Plant Disease ◽  
2014 ◽  
Vol 98 (11) ◽  
pp. 1590-1590 ◽  
Author(s):  
M. A. Al-Saleh ◽  
I. M. Al-Shahwan ◽  
M. T. Shakeel ◽  
M. A. Amer ◽  
C. G. Orfanidou ◽  
...  
Keyword(s):  
Saudi Arabia ◽  
Open Field ◽  
Rt Pcr ◽  
Degenerate Primers ◽  
Crop Losses ◽  
Total Rna ◽  
Indicator Plants ◽  
First Report ◽  
Access Period ◽  
Tomato Chlorosis Virus

During January 2014, open field and greenhouse tomato (Solanum lycopersicum L.) crops in the peripheral areas of Riyadh region (Al-Aflaj, Al-Kharj, Al-Waseel, and Al-Dalam), Saudi Arabia, were surveyed. In all surveyed tomato crops, yellowing symptoms were observed on the lower leaves, possibly infected by a whitefly transmitted crinivirus (family Closteroviridae) such as Tomato chlorosis virus (ToCV) and/or Tomato infectious chlorosis virus (TICV). Dense population of whiteflies (Bemisia tabaci G.) were present in all affected plants. Incidence of the yellowing disease varied between four greenhouses and three open field tomato crops, but in the majority of the tomato crops surveyed, symptoms typical of Begomovirus infection such as severe stunting, degeneration, upward cupping, distortion and interveinal yellowing of upper leaves, and flower abortion were also observed. Tomato yellow leaf curl virus (TYLCV) is endemic in Saudi Arabia causing severe crop losses (1). Twenty-six leaf samples from 24 symptomatic and two asymptomatic plants from four fields (three greenhouses and one open field crop) were collected and were processed in the lab at King Saud University. Whitefly transmission on tomato indicator plants was carried out using B. tabaci to fulfill Koch's postulates. Two hundred virus-free B. tabaci adults were confined to one of the collected symptomatic tomato sample singly infected with ToCV for a 48-h acquisition access period, followed by a 48-h inoculation access period on five healthy tomato plants Hybrid Super Strain B, using 40 whiteflies per plant. Crinivirus detection following transmission was conducted by RT-PCR. Total RNA was extracted from 26 collected leaf samples using the Total RNA Purification Kit and analyzed by SCRIPT One–Step RT-PCR Kit (Jena Bioscience). First, the degenerate primers HS-11/HS12 were used for amplification of a 587-bp fragment of the HSP70 gene of ToCV and TICV (3). Second, the RT-PCR product was subjected to a nested PCR using specific primers TIC-3/TIC-4 and TOC-5/TOC-6, for the detection of both TICV and ToCV, respectively (2). Finally, degenerate primers (AV494/AC1048) were used for detection of begomoviruses (4). No fragment was amplified by TIC-3/TIC-4 primer whereas TOC-5/TOC-6 amplified a size of 463 bp in all 24 symptomatic tested samples, including one mixed infection with TYLCV detected by AV494/AC1048. Asymptomatic samples did not produce any amplicon regarding TICV, ToCV, and Begomovirus detection. The amplicons of four positive fragments, each from one field, were further sequenced in both directions and all obtained sequences (KJ433488, KJ433489, KJ433490, and KJ433491) analyzed with BLAST and revealed 99% identity with the most closely deposited sequences in NCBI from Japan (AB513442) and Brazil (JQ952601). In the transmission tests, ToCV was detected to all tomato indicator plants which revealed yellowing symptoms 6 weeks post inoculation, whereas no transmission was obtained when non-viruliferous whitefly adults fed on two asymptomatic tomato leaves. To our knowledge, this is the first report of ToCV infecting tomato crops in Saudi Arabia. Further studies are being carried out to study epidemiology and genetic diversity of this virus associated with yellowing diseases of tomato in different regions of Saudi Arabia. This finding is important for the tomato crops and possibly other virus hosts as may cause serious epidemics and crop losses. References: (1) A. M. Ajlan et al. Arab J. Biotech. 10:179, 2007. (3) C. I. Dovas et al. Plant Dis. 86:1345, 2002. (2) J. Navas-Castillo et al. Plant Dis. 84:835, 2000. (4) S. D. Whyatt and J. K. Brown. Phytopathology 86:1288, 1996.

scholarly journals First Report of Tomato chlorosis virus Infecting Tomato in Georgia

Plant Disease ◽  
2011 ◽  
Vol 95 (7) ◽  
pp. 881-881 ◽  
Author(s):  
S. Sundaraj ◽  
R. Srinivasan ◽  
C. G. Webster ◽  
S. Adkins ◽  
K. Perry ◽  
...  
Keyword(s):  
Nucleic Acid Hybridization ◽  
N Gene ◽  
Natural Occurrence ◽  
Rt Pcr ◽  
Specific Primers ◽  
Tomato Production ◽  
First Report ◽  
Interveinal Chlorosis ◽  
Cp Gene ◽  
Tomato Chlorosis Virus

Tomato yellow leaf curl virus (TYLCV) and Tomato spotted wilt virus (TSWV) are prevalent in field-grown tomato (Solanum lycopersicum) production in Georgia. Typical TYLCV symptoms were observed during varietal trials in fall 2009 and 2010 to screen genotypes against TYLCV at the Coastal Plain Experiment Station, Tifton, GA. However, foliar symptoms atypical of TYLCV including interveinal chlorosis, purpling, brittleness, and mottling on upper and middle leaves and bronzing and intense interveinal chlorosis on lower leaves were also observed. Heavy whitefly (Bemisia tabaci (Gennadius), B biotype) infestation was also observed on all tomato genotypes. Preliminary tests (PCR and nucleic acid hybridization) in fall 2009 indicated the presence of TYLCV, TSWV, Cucumber mosaic virus, and Tomato chlorosis virus (ToCV); all with the exception of ToCV have been reported in Georgia. Sixteen additional symptomatic leaf samples were randomly collected in fall 2010 and the preliminary results from 2009 were used to guide testing. DNA and RNA were individually extracted using commercially available kits and used for PCR testing for ToCV, TYLCV, and TSWV. Reverse transcription (RT)-PCR with ToCV CP gene specific primers (4) produced approximately 750-bp amplicons from nine of the 16 leaf samples. Four of the nine CP gene amplicons were purified and directly sequenced in both directions. The sequences were 99.4 to 100.0% identical with each other (GenBank Accession Nos. HQ879840 to HQ879843). They were 99.3 to 99.5%, 97.2 to 97.5%, and 98.6 to 98.9% identical to ToCV CP sequences from Florida (Accession No. AY903448), Spain (Accession No. DQ136146), and Greece (Accession No. EU284744), respectively. The presence of ToCV was confirmed by amplifying a portion of the HSP70h gene using the primers HSP-1F and HSP-1R (1). RT-PCR produced approximately 900-bp amplicons in the same nine samples. Four HSP70h gene amplicons were purified and directly sequenced in both directions. The sequences were 99.4 to 99.7% identical to each other (Accession Nos. HQ879844 to HQ879847). They were 99.2 to 99.5%, 98.0 to 98.4%, and 98.9 to 99.3% identical to HSP70h sequences from Florida (Accession No. AY903448), Spain (Accession No. DQ136146), and Greece (Accession No. EU284744), respectively. TYLCV was also detected in all 16 samples by PCR using degenerate begomovirus primers PAL1v 1978 and PARIc 496 (3) followed by sequencing. TSWV was also detected in two of the ToCVinfected samples by RT-PCR with TSWV N gene specific primers (2) followed by sequencing. To our knowledge, this is the first report of the natural occurrence of ToCV in Georgia. Further studies are required to quantify the yield losses from ToCV alone and synergistic interactions between ToCV in combination with TSWV and/or TYLCV in tomato production in Georgia. References: (1) T. Hirota et al. J. Gen. Plant Pathol. 76:168, 2010. (2) R. K. Jain et al. Plant Dis. 82:900, 1998. (3) M. R. Rojas et al. Plant Dis. 77:340, 1993. (4) L. Segev et al. Plant Dis. 88:1160, 2004.

scholarly journals Squash vein yellowing virus Identified in Watermelon (Citrullus lanatus) in Indiana

Plant Disease ◽  
2007 ◽  
Vol 91 (8) ◽  
pp. 1056-1056 ◽  
Author(s):  
D. S. Egel ◽  
S. Adkins
Keyword(s):  
Citrullus Lanatus ◽  
Amino Acid Sequences ◽  
Rt Pcr ◽  
Strain Vector ◽  
First Report ◽  
Pcr Product ◽  
Field Samples ◽  
Health Progress ◽  
Vine Decline ◽  
Yellowing Virus

During September 2006, moderate vine decline symptoms including vine collapse and wilt and root rot were observed on numerous watermelon plants growing in a commercial field in Sullivan County, Indiana. No symptoms were observed on the fruit. Six plants displaying typical vine decline symptoms were collected and assayed for potyvirus infection and subsequently for Squash vein yellowing virus (SqVYV) and Papaya ringspot virus type W (PRSV-W). SqVYV is a whitefly-transmitted member of the Potyviridae, recently shown to cause watermelon vine decline in Florida (1,4). Plants infected with SqVYV in Florida are also frequently infected with PRSV-W, although SqVYV is sufficient for watermelon vine decline. The six field samples harbored one or more potyviruses as determined by ELISA (Agdia, Elkhart, IN). Mechanical inoculation of squash (Cucurbita pepo) and watermelon with sap from three of the field samples induced mosaic symptoms in both that are typical of potyviruses. Vein yellowing in squash and plant death in watermelon typical of SqVYV (1) later developed in plants inoculated with one field sample. A coat protein gene fragment was amplified by reverse transcription (RT)-PCR with SqVYV primers (1) from total RNA of five of the six field samples and also from the symptomatic, inoculated plants. Nucleotide and deduced amino acid sequences of a 957-bp region of the RT-PCR product (primer sequences deleted prior to analysis) were 100% identical to SqVYV (GenBank accession No. DQ812125). PRSV-W also was identified in two of the five SqVYV-infected field samples by ELISA (Agdia) and by sequence analysis of a 3′ genome fragment amplified by RT-PCR with previously described degenerate potyvirus primers (3). No evidence for infection by other potyviruses was obtained. To our knowledge, this is the first report of SqVYV in Indiana and the first report of the virus anywhere outside of Florida. The whitefly (Bemisia tabaci, B strain) vector of SqVYV is relatively uncommon in Indiana and the cold winter temperatures make it unlikely that any SqVYV-infected watermelon vines or whiteflies will overseason, necessitating reintroductions of virus and vector each season. We feel that the moderate and restricted occurrence of SqVYV in Indiana observed in September 2006 should pose little or no threat to commercial watermelon production in Indiana and should not cause growers to alter their growing practices. The occurrence of SqVYV in Indiana does not appear to explain the similar symptoms of mature watermelon vine decline (MWVD) that has been observed in Indiana since the 1980s. In contrast with the insect vectored SqVYV, MWVD seems to be caused by a soilborne biological agent (2). References: (1) S. Adkins et al. Phytopathology 97:145, 2007. (2) D. S. Egel et al. Online publication. doi:10.1094/PHP-2000-1227-01-HN. Plant Health Progress, 2000. (3) A. Gibbs and A. Mackenzie. J. Virol. Methods 63:9, 1997. (4) P. Roberts et al. Citrus Veg. Mag. December 12, 2004.

scholarly journals First Report of Alfalfa mosaic virus in Viburnum lucidum in Spain

Plant Disease ◽  
2008 ◽  
Vol 92 (7) ◽  
pp. 1132-1132 ◽  
Author(s):  
M. C. Cebrián ◽  
M. C. Córdoba-Sellés ◽  
A. Alfaro-Fernández ◽  
J. A. Herrera-Vásquez ◽  
C. Jordá
Keyword(s):  
Mosaic Virus ◽  
Natural Infection ◽  
Alfalfa Mosaic Virus ◽  
The United States ◽  
Rt Pcr ◽  
Water Control ◽  
First Report ◽  
Pcr Product ◽  
Pcr Assays ◽  
The Mediterranean

Viburnum sp. is an ornamental shrub widely used in private and public gardens. It is common in natural wooded areas in the Mediterranean Region. The genus includes more than 150 species distributed widely in climatically mild and subtropical regions of Asia, Europe, North Africa, and the Americas. In January 2007, yellow leaf spotting in young plants of Viburnun lucidum was observed in two ornamental nurseries in the Mediterranean area of Spain. Symptoms appeared sporadically depending on environmental conditions but normally in cooler conditions. Leaf tissue from 24 asymptomatic and five symptomatic plants was sampled and analyzed by double-antibody sandwich (DAS)-ELISA with specific polyclonal antibodies against Tomato spotted wilt virus (TSWV) (Loewe Biochemica, Sauerlach, Germany) and Alfalfa mosaic virus (AMV) (SEDIAG S.A.S, Longvic, France). All symptomatic plants of V. lucidum were positive for Alfalfa mosaic virus (AMV). The presence of AMV was tested in the 29 samples by one-step reverse transcription (RT)-PCR with the platinum Taq kit (Invitrogen Life Technologies, Barcelona, Spain) using primers derived from a partial fragment of the coat protein gene of AMV (2). The RT-PCR assays produced an expected amplicon of 700 bp in the five symptomatic seropositive samples. No amplification product was observed when healthy plants or a water control were used as a template in the RT-PCR assays. One PCR product was purified (High Pure PCR Product Purification Kit; Roche Diagnostics, Mannheim, Germany) and directly sequenced (GenBank Accession No. EF427449). BLAST analysis showed 96% nucleotide sequence identity to an AMV isolate described from Phlox paniculata in the United States (GenBank Accession No. DQ124429). This virosis has been described as affecting Viburnum tinus L. in France (1). To our knowledge, this is the first report of natural infection of Viburnum lucidum with AMV in Spain, which might have important epidemiological consequences since V. lucidum is a vegetatively propagated ornamental plant. References: (1) L. Cardin et al. Plant Dis. 90:1115, 2006. (2) Ll. Martínez-Priego et al. Plant Dis. 88:908, 2004.

scholarly journals First Report of Tomato chlorosis virus in Tomato in Costa Rica

Plant Disease ◽  
2009 ◽  
Vol 93 (9) ◽  
pp. 970-970 ◽  
Author(s):  
R. M. Castro ◽  
E. Hernandez ◽  
F. Mora ◽  
P. Ramirez ◽  
R. W. Hammond
Keyword(s):  
Costa Rica ◽  
Sequence Analysis ◽  
Nucleotide Sequence Analysis ◽  
Rt Pcr ◽  
Greenhouse Whitefly ◽  
Specific Primers ◽  
First Report ◽  
Sequence Identity ◽  
Pcr Products ◽  
Tomato Chlorosis Virus

In early 2007, severe yellowing and chlorosis symptoms were observed in field-grown and greenhouse tomato (Solanum lycopersicum L.) plants in Costa Rica. Symptoms resembled those of the genus Crinivirus (family Closteroviridae), and large populations of whiteflies, including the greenhouse whitefly Trialeurodes vaporariorum (Westwood), were observed in the fields and on symptomatic plants. Total RNA was extracted from silica gel-dried tomato leaf tissue of 47 representative samples (all were from symptomatic plants) using TRI Reagent (Molecular Research Inc., Cincinnati, OH). Reverse transcription (RT)-PCR reactions were performed separately with each of the four primer sets with the Titan One-Tube RT-PCR Kit (Roche Diagnostics Corp., Chicago IL). Specific primers used for the detection of the criniviruses, Tomato chlorosis virus (ToCV) and Tomato infectious chlorosis virus (TICV), were primer pair ToCV-p22-F (5′-ATGGATCTCACTGGTTGCTTGC-3′) and ToCV-p22-R (5′-TTATATATCACTCCCAAAGAAA-3′) specific for the p22 gene of ToCV RNA1 (1), primer pair ToCVCPmF (5′-TCTGGCAGTACCCGTTCGTGA-3′) and ToCVCPmR (5′-TACCGGCAGTCGTCCCATACC-3′) designed to be specific for the ToCV CPm gene of ToCV RNA2 (GenBank Accession No. AY903448) (2), primer pair ToCVHSP70F (5′-GGCGGTACTTTCGACACTTCTT-3′) and ToCVHSP70R (5′-ATTAACGCGCAAAACCATCTG-3′) designed to be specific for the Hsp70 gene of RNA2 of ToCV (GenBank Accession No. EU284744) (1), and primer pair TICV-CP-F and TICV-CP-R specific for the coat protein gene of TICV (1). Amplified DNA fragments (582 bp) were obtained from nine samples, four from the greenhouse and five from the open field, with the ToCV-p22 specific primers and were cloned into the pCRII TOPO cloning vector (Invitrogen, Carlsbad, CA). Nucleotide sequence analysis of all purified RT-PCR products verified their identity as ToCV, sharing 99.5 to 100% sequence identity among themselves and 96% to 98% sequence identity with previously reported ToCV p22 sequences from Florida (Accession No. AY903447), Spain (Accession No. DQ983480), and Greece (Accession No. EU284745). The presence of ToCV in the samples was confirmed by additional amplification and sequence analysis of the CPm (449-bp fragment) and Hsp70 (420-bp fragment) genes of ToCV RNA2 and sharing 98 to 99% sequence homology to Accession Nos. AY903448 and EU284774, respectively. One representative sequence of the p22 gene of the Costa Rican isolate was deposited at GenBank (Accession No. FJ809714). No PCR products were obtained using either the TICV-specific primers nor from healthy tomato tissue. The ToCV-positive samples were collected from a region in the Central Valley around Cartago, Costa Rica. To our knowledge, this is the first report of ToCV in Costa Rica. The economic impact on tomato has not yet been determined. Studies are underway to determine the incidence of ToCV in Costa Rica field-grown and greenhouse tomatoes. References: (1) A. R. A. Kataya et al. Plant Pathol. 57:819, 2008. (2) W. M. Wintermantel et al. Arch. Virol. 150:2287, 2005.

scholarly journals First Report of Tobacco mild green mosaic virus in Capsicum chinense in Venezuela

Plant Disease ◽  
2006 ◽  
Vol 90 (8) ◽  
pp. 1108-1108 ◽  
Author(s):  
C. Córdoba ◽  
A. García-Rández ◽  
N. Montaño ◽  
C. Jordá
Keyword(s):  
Mosaic Virus ◽  
Seed Transmission ◽  
Tomato Mosaic Virus ◽  
Capsicum Chinense ◽  
Rt Pcr ◽  
Plant Host ◽  
First Report ◽  
Coat Protein Region ◽  
Pcr Product ◽  
First Time

In July 2003, noticeable deformations of leaves were observed on a local variety of Capsicum chinense, also called ‘Aji dulce’, from a pepper plantation located in Venezuela, (Monagas State). ‘Aji dulce’ is a basic ingredient of the Venezuelan gastronomy with an estimated cultivated area of 2,000 ha. The seeds of this local pepper are obtained by the growers who reproduce and multiply their own seeds every year. Seeds of affected plants were sent to our laboratory, and a group of approximately 100 seeds was sown in a controlled greenhouse that belongs to the Polytechnic University of Valencia, Spain. Three months later, obvious curling and bubbling developed on the leaves of the plants. Extracts of symptomatic plants tested negative for Tomato mosaic virus (ToMV), Tobacco mosaic virus (TMV), Pepper mild mottle virus (PMMV), and Tobacco etch virus (TEV) by double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA) with policlonal antibodies specific to each virus (Loewe Biochemica GMBH, Sauerlach, Germany; Phyto-Diagnostics, INRA, France). Total RNA was isolated from 0.5 g of original seed sent from Venezuela and from 25 samples of leaves of plants grown in the greenhouse with an RNeasy Plant Mini Kit (Qiagen Sciences, Germantown, Maryland). The RNA isolated was used in reverse transcription-polymerase chain reaction (RT-PCR) with specific primers for Tobacco mild green mosaic virus (TMGMV) (1) predicted to amplify a 530 bp of the coat protein region. From all samples, a RT-PCR product of the expected size was obtained and then sequenced. BLAST analysis of one sequence (GenBank Accession No. DQ460731) showed high levels of identity with TMGMV isolates, with more than 99% nucleotide identity with the DSMZ PV-112 isolate (GenBank Accession No. AJ429096). The symptomatology observed on pepper plants, the TMGMV RT-PCR assay, and the consensus of sequenced regions with TMGMV lead us to conclude that TMGMV was the causal agent of the diseased C. chinense plants. Although TMGMV has a wide plant host range occurring worldwide (1), to our knowledge, this is not only the first time TMGMV has been detected in Venezuela, but also the first report of TMGMV in C. chinense in Venezuela and the first reliable probe of the TMGMV seed transmission. Reference: (1) J. Cohen et al. Ann. Appl. Biol. 138:153, 2001.

scholarly journals First report of cucurbit chlorotic yellows virus infecting cucumber in South Korea

Plant Disease ◽  
2021 ◽  
Author(s):  
Hae-Ryun Kwak ◽  
Hui-Seong Byun ◽  
Hong-Soo Choi ◽  
Jong-Woo Han ◽  
Chang-Seok Kim ◽  
...  
Keyword(s):  
Coat Protein ◽  
South Korea ◽  
East Asia ◽  
Rna Extraction ◽  
Rt Pcr ◽  
Specific Primers ◽  
Total Rna ◽  
First Report ◽  
Chlorotic Mottle ◽  
Cucurbit Chlorotic Yellows Virus

In October 2018, cucumber plants showing yellowing and chlorotic mottle symptoms were observed in a greenhouse in Chungbuk, South Korea. The observed symptoms were similar to those caused by cucurbit aphid-borne yellows virus (CABYV), which has been detected on cucumber plants in the region since it was reported on melon in Korea in 2015 (Lee et al 2015). To identify the potential agents causing these symptoms, 28 samples from symptomatic leaves and fruit of cucumber plants were subjected to total RNA extraction using the Plant RNA Prep Kit (Biocubesystem, Korea). Reverse transcription polymerase chain (RT-PCR) was performed on total RNA using CABYV specific primers and protocols (Kwak et al. 2018). CABYV was detected in 17 of the 28 samples, while 11 symptomatic samples tested negative. In order to identify the cause of the symptoms, RT-PCR was performed using cucurbit chlorotic yellows virus (CCYV) and cucurbit yellow stunting disorder virus (CYSDV) specific primers (Wintermantel et al. 2019). Eight of the 28 samples were positive using the CCYV specific primers while seven samples were infected with only CCYV and one contained a mixed infection of CABYV with CCYV. None of the samples tested positive for CYSDV. The expected 373 nt amplicons of CCYV were bi-directionally sequenced, and BLASTn analysis showed that the nucleotide sequences shared 98 to 100% identity with CCYV isolates from East Asia, including NC0180174 from Japan. Two pairs of primers for amplification of the complete coat protein and RNA-dependent RNA polymerase (RdRp) genes (Wintermantel et al., 2019) were used to amplify the 753bp coat protein and 1517bp RdRp genes, respectively. Amplicons of the expected sizes were obtained from a CCYV single infection and ligated into the pGEM T- Easy vector (Promega, WI, USA). Three clones from each amplicon were sequenced and aligned using Geneious Prime and found to have identical sequences (Genbank accession nos. MW033300, MW033301). The CP and RdRp sequences demonstrated 99% nucleotide and 100% amino acid identity with the respective genes and proteins of the CCYV isolates from Japan. This study documents the first report of CCYV in Korea. Since CCYV was first detected on melon in Japan, it has been reported in many other countries including those in East Asia, the Middle East, Southern Europe, North Africa, and recently in North America. CCYV has the potential to become a serious threat to production of cucurbit crops in Korea, particularly due to the increasing prevalence of the whitefly, Bemisia tabaci, in greenhouse production systems. It will be important to continue monitoring for CCYV and determine potential alternate hosts in the region to manage and prevent further spread of CCYV in Korea.

scholarly journals First Report of Peanut mottle virus Infecting Soybean in South Korea

Plant Disease ◽  
2014 ◽  
Vol 98 (9) ◽  
pp. 1285-1285 ◽  
Author(s):  
S. Lim ◽  
Y.-H. Lee ◽  
D. Igori ◽  
F. Zhao ◽  
R. H. Yoo ◽  
...  
Keyword(s):  
Nucleotide Sequence ◽  
South Korea ◽  
Mosaic Virus ◽  
Soybean Mosaic Virus ◽  
Nucleotide Sequences ◽  
Mottle Virus ◽  
Post Inoculation ◽  
Rt Pcr ◽  
First Report ◽  
Pcr Product

In July 2013, soybean (Glycine max) plants at the research field in Daegu, South Korea, showed virus-like symptoms, such as mosaic, mottle, yellowing, and stunting. Overall, there were approximately 1% of soybean plants that showed these symptoms. Sixteen soybean samples were collected based on visual symptoms and subjected to laboratory characterization. Total RNA was extracted from each sample with the Tri Reagent (Molecular Research Center, Cincinnati, OH) and cDNA was synthesized using random N25 primer with RevertAid Reverse Transcriptase (Thermo Scientific, Waltham, MA), according to the manufacturers' instructions. All samples were tested by PCR with Prime Taq Premix (2X) (GeNet Bio, Daejeon, Korea) and primer sets specific to Soybean mosaic virus (SMV; 5′-CATATCAGTTTGTTGGGCA-3′ and 5′-TGCCTATACCCTCAACAT-3′), Peanut stunt virus (PSV; 5′-TGACCGCGTGCCAGTAGGAT-3′ and 5′-AGGTDGCTTTCTWTTGRATTTA-3′), Soybean yellow mottle mosaic virus (SYMMV; 5′-CAACCCTCAGCCACATTCAACTAT-3′ and 5′-TCTAACCACCCCACCCGAAGGATT-3′), and Soybean yellow common mosaic virus (SYCMV; 5′-TTGGCTGAGAGGAGTGGCTT-3′ and 5′-TGCGGTCGTGTAGTCAGTG-3′). Among 16 samples tested, five were positive for SMV and two for SYMMV. Three samples were found infected by both SMV and SYMMV and four by both SMV and PSV. Since two of the symptomatic samples were not infected by viruses described above, a pair of primers specific to Peanut mottle virus (PeMoV; 5′-GCTGTGAATTGTTGTTGAGAA-3′ and 5′-ACAATGATGAAGTTCGTTAC-3′) was tested (1). All 16 samples were subjected to RT-PCR with primers specific to PeMoV. Four were found positive for PeMoV. Two of them were already found infected with SYMMV. In order to identify the complete nucleotide sequences of PeMoV coat protein (CP), another primer set (5′-TGAGCAGGAAAGAATTGTTTC-3′ and 5′-GGAAGCGATATACACACCAAC-3′) was used. RT-PCR product was cloned into RBC TA Cloning Vector (RBC Bioscience, Taipei, Taiwan) and the nucleotide sequence of the insert was determined by Macrogen (Seoul, Korea). CP gene of the PeMoV (GenBank Accession No. KJ664838) showed the highest nucleotide sequence identity with PeMoV isolate Habin (KF977830; 99% identity), and the highest amino acid identity with GenBank Accession No. ABI97347 (100% identity). In order to fulfill Koch's postulates, several G. max cv. Williams 82 were inoculated with the extracts of PeMoV-infected leaf tissue. At 14 days post-inoculation, plants showed systemic mottle symptoms. These symptomatic plants were subjected to RT-PCR, and the nucleotide sequences of the PCR product were found identical to that of the virus in the inoculum. To our knowledge, this is the first report of soybean-infecting PeMoV, a member of the genus Potyvirus in the family Potyviridae, in South Korea. Reference: (1) R. G. Dietzgen et al. Plant Dis. 85:989, 2001.

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