scholarly journals First Report of Tomato spotted wilt virus in Brugmansia suaveolens in Korea

Plant Disease ◽  
2014 ◽  
Vol 98 (9) ◽  
pp. 1283-1283
Author(s):  
S.-K. Choi ◽  
I.-S. Cho ◽  
G.-S. Choi ◽  
J.-Y. Yoon
Keyword(s):  
Mosaic Virus ◽  
Tomato Spotted Wilt Virus ◽  
Korean Isolate ◽  
Rt Pcr ◽  
First Report ◽  
Tomato Spotted Wilt ◽  
Pcr Product ◽  
Brugmansia Suaveolens ◽  
Plant Pathol ◽  
Wilt Virus

Brugmansia suaveolens, also known as angel's trumpet, is a semi-woody shrub or a small tree. Because flowers of B. suaveolens are remarkably beautiful and sweetly fragrant, B. suaveolens is grown as ornamentals outdoors year-round in the tropics and subtropics, and as potted plants in temperate regions (1). In February 2013, virus-like symptoms including mosaic symptoms followed by distortion of leaves were observed in a potted B. suaveolens in a nursery in Chung-Nam Province, Korea. Symptomatic leaves were analyzed for the presence of several ornamental viruses including Cucumber mosaic virus (CMV), Tobacco mosaic virus (TMV), Tomato bush stunt virus (TBSV), and Tomato spotted wilt virus (TSWV) by immune-strip diagnostic kits that were developed by our laboratory. Positive controls and extract from healthy leaves of B. suaveolens as a negative control were included in each immune-strip assay. TSWV was detected serologically from the naturally infected B. suaveolens, but CMV, TBSV, and TMV were not detected from the B. suaveolens. The presence of TSWV (named TSWV-AT1) was confirmed by commercially available double-antibody sandwich (DAS)-ELISA kits (Agdia, Elkhart, IN). TSWV-AT1 was mechanically transmitted from the ELISA-positive B. suaveolens to Capsicum annuum and Nicotiana glutinosa, respectively. Inoculated C. annuum showed chlorotic rings in the inoculated leaves and inoculated N. glutinosa produced mosaic and systemic necrosis in the inoculated leaves after 7 days inoculation, respectively, which were consistent with symptoms caused by TSWV (2). To confirm further TSWV-AT1 infection, reverse transcription (RT)-PCR was performed using the One-Step RT-PCR (Invitrogen, Carlsbad, CA) with TSWV-specific primers, TSWV-NCP-For and TSWV-NCP-Rev (3), designed to amplify a 777-bp cDNA of the nucleocapsid protein (NCP) gene. Total RNAs from naturally infected B. suaveolens, symptomatic C. annuum, and N. glutinosa were extracted using RNeasy Plant Mini Kit (Qiagen, Valencia, CA). Total RNAs obtained from a Korean isolate of TSWV (Accession No. JF730744) and healthy B. suaveolens were used as positive and negative controls, respectively. The expected size of the RT-PCR product was amplified from symptomatic B. suaveolens, C. annuum, and N. glutinosa but not from healthy leaves of B. suaveolens. The amplified RT-PCR product from TSWV-AT1 was directly sequenced using BigDye Termination kit (Applied Biosystems, Foster City, CA). Multiple alignment of the TSWV-AT1 NCP sequence (AB910533) with NCP sequences of other TSWV isolates using MEGA5 software (4) revealed 99.0% aa identity with an Korean TSWV isolate (AEB33895) originating from tomato. These results provide additional confirmation of TSWV-AT1 infection. It is known that high-value ornamentals may act also as reservoirs for TSWV that can infect other ornamentals and cultivated crops, because TSWV has a very broad host range (2). Elaborate inspections for TSWV and other viruses are necessary for production of healthy B. suaveolens, since the popularity and economic importance of this ornamental plant is increasing. To our knowledge, this is the first report of TSWV in B. suaveolens in Korea. References: (1) Anonymous. OEPP/EPPO Bull. 34:271, 2004. (2) G. Parrella et al. J. Plant Pathol. 85:227, 2003. (3) B.-N. Chung et al. Plant Pathol. J. 28:87, 2012. (4) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011.

scholarly journals First Report of Tomato spotted wilt virus on Brugmansia sp. in Serbia

Plant Disease ◽  
2013 ◽  
Vol 97 (6) ◽  
pp. 850-850 ◽  
Author(s):  
D. Nikolić ◽  
I. Stanković ◽  
A. Vučurović ◽  
D. Ristić ◽  
K. Milojević ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Tomato Spotted Wilt Virus ◽  
N Gene ◽  
Broad Host Range ◽  
Rt Pcr ◽  
First Report ◽  
Tomato Spotted Wilt ◽  
Plant Pathol ◽  
Negative Controls ◽  
Wilt Virus

Brugmansia (Brugmansia spp.), also known as Angel's trumpet, is a perennial shrub in the Solanaceae that is a popular landscape plant in the tropics and subtropics, and potted plant in temperate regions. In April 2012, virus-like symptoms including chlorotic leaf patterns and curling followed by necrosis and distortion of leaves were observed on five outdoor-grown brugmansia plants in a private garden in Mackovac, Rasina District, Serbia. Symptomatic leaves were tested for the presence of several common ornamental viruses including Tomato spotted wilt virus (TSWV), Impatiens necrotic spot virus (INSV), Cucumber mosaic virus (CMV), and Tobacco mosaic virus (TMV) by commercial double-antibody sandwich (DAS)-ELISA diagnostic kits (Bioreba AG, Reinach, Switzerland). Commercial positive and negative controls and extract from healthy brugmansia leaves were included in each ELISA. TSWV was detected serologically in all five brugmansia samples and all tested samples were negative for INSV, CMV, and TMV. The virus was mechanically transmitted from an ELISA-positive sample (41-12) to five plants of each Petuina × hybrida and Nicotiana glutinosa. Inoculated P. × hybrida plants showed local necrotic lesions and N. glutinosa showed mosaic and systemic necrosis 4 and 12 days post-inoculation, respectively, which were consistent with symptoms caused by TSWV (1). For further confirmation of TSWV infection, reverse transcription (RT)-PCR was performed with the OneStep RT-PCR (Qiagen, Hilden, Germany) using a set of TSWV-specific primers, TSWV CP-f and TSWV CP-r (4), designed to amplify a 738-bp fragment of the nucleocapsid protein (N) gene. Total RNAs from naturally infected brugmansia and symptomatic N. glutinosa plants were extracted using the RNeasy Plant Mini Kit (Qiagen). Total RNAs obtained from the Serbian tobacco isolate of TSWV (GenBank Accession No. GQ373173) and healthy brugmansia plants were used as positive and negative controls, respectively. The expected size of the RT-PCR product was amplified from symptomatic brugmansia and N. glutinosa but not from healthy tissues. The amplified product derived from the isolate 41-12 was sequenced directly after purification with the QIAquick PCR Purification kit (Qiagen), deposited in GenBank (JX468080), and subjected to sequence analysis by MEGA5 software (3). Sequence comparisons revealed that the Serbian isolate 41-12 shared the highest nucleotide identity of 99.9% (99.5% amino acid identity) with an Italian TSWV isolate P105/2006RB (DQ915946) originating from pepper. To our knowledge, this is the first report of TSWV on brugmansia in Serbia. Due to the increasing popularity and economic importance of brugmansia as an ornamental crop, thorough inspections and subsequent testing for TSWV and other viruses are needed. This high-value ornamental plant may act also as reservoir for the virus that can infect other ornamentals and cultivated crops, considering that TSWV has a very broad host range (2). References: (1) Anonymous. OEPP/EPPO Bull. 34:271, 2004. (2) G. Parrella et al. J. Plant Pathol. 85:227, 2003. (3) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011. (4) A. Vučurović et al. Eur. J. Plant Pathol. 133:935, 2012.

scholarly journals First Report of Tomato spotted wilt virus Infection of Tomatillo in Georgia

Plant Disease ◽  
2000 ◽  
Vol 84 (10) ◽  
pp. 1155-1155 ◽  
Author(s):  
J. C. Díaz-Pérez ◽  
H. R. Pappu
Keyword(s):  
Tomato Spotted Wilt Virus ◽  
Plant Size ◽  
Coding Region ◽  
Rt Pcr ◽  
First Report ◽  
Nucleocapsid Gene ◽  
Tomato Spotted Wilt ◽  
Pcr Product ◽  
The University ◽  
Wilt Virus

During the 2000 spring season, tomatillo (Physalis ixocarpa) plants showing chlorotic streaks on leaves were observed in an experimental plot of the University of Georgia's Coastal Plain Experiment Station in Tift County, GA. Leaf samples from 192 plants were collected. These included plants that had chlorotic streaks and those without obvious symptoms. Samples were tested by ELISA using a commercially available Tomato spotted wilt virus (TSWV) detection kit (Agdia Inc., Elkhart, IN). TSWV was found in 10 samples that had chlorotic streaks on leaves, and the remaining plants with no obvious symptoms were negative for TSWV. Infected plants were found in both cultivars, Verde Puebla and Toma Verde. The presence of the virus had no apparent effect on plant size or fruit appearance. TSWV infection of the ELISA-positive samples was further verified by immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) (1). The primer pair (5′-ATGTCTAAGGTTAAGCTC-3′ and 5′ TTAAGCAAGTTCTGTGAG-3′) represented the first and last 18 bases of the coding region of the nucleocapsid gene of TSWV, respectively, and produced approximately 800-bp PCR product (1). IC-RT-PCR gave a single DNA band of expected size and no amplification was found in the uninfected control. This is the first report of TSWV on tomatillo in Georgia. Reference: (1) R. K. Jain et al. Plant Dis. 82:900, 1998.

scholarly journals First Report of Tomato spotted wilt virus in Blackberry Lily in North America

Plant Disease ◽  
2003 ◽  
Vol 87 (1) ◽  
pp. 102-102 ◽  
Author(s):  
S. Adkins ◽  
L. Breman ◽  
C. A. Baker ◽  
S. Wilson
Keyword(s):  
North America ◽  
Tomato Spotted Wilt Virus ◽  
Amino Acid Sequences ◽  
South Florida ◽  
N Gene ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
First Report ◽  
Tomato Spotted Wilt ◽  
Wilt Virus

Blackberry lily (Belamcanda chinensis (L.) DC.) is an herbaceous perennial in the Iridaceae characterized by purple-spotted orange flowers followed by persistent clusters of black fruit. In July 2002, virus-like symptoms including chlorotic ringspots and ring patterns were observed on blackberry lily leaves on 2 of 10 plants in a south Florida ornamental demonstration garden. Inclusion body morphology suggested the presence of a Tospovirus. Tomato spotted wilt virus (TSWV) was specifically identified by serological testing using enzyme-linked immunosorbent assay (Agdia, Elkhart, IN). Sequence analysis of a nucleocapsid (N) protein gene fragment amplified by reverse transcription-polymerase chain reaction (RT-PCR) with primers TSWV723 and TSWV722 (1) from total RNA confirmed the diagnosis. Nucleotide and deduced amino acid sequences of a 579 base pair region of the RT-PCR product were 95 to 99% and 95 to 100% identical, respectively, to TSWV N-gene sequences in GenBank. Since these 2-year-old plants were grown on-site from seed, they were likely inoculated by thrips from a nearby source. Together with a previous observation of TSWV in north Florida nursery stock (L. Breman, unpublished), this represents, to our knowledge, the first report of TSWV infection of blackberry lily in North America although TSWV was observed in plants of this species in Japan 25 years ago (2). References: (1) S. Adkins, and E. N. Rosskopf. Plant Dis. 86:1310, 2002. (2) T. Yamamoto and K.-I. Ohata. Bull. Shikoku Agric. Exp. Stn. 30:39, 1977.

scholarly journals First Report of Tomato spotted wilt virus on Chrysanthemum in Serbia

Plant Disease ◽  
2013 ◽  
Vol 97 (1) ◽  
pp. 150-150 ◽  
Author(s):  
I. Stanković ◽  
A. Bulajić ◽  
A. Vučurović ◽  
D. Ristić ◽  
K. Milojević ◽  
...  
Keyword(s):  
Tomato Spotted Wilt Virus ◽  
Disease Incidence ◽  
N Gene ◽  
Impatiens Necrotic Spot Virus ◽  
Maximum Parsimony Tree ◽  
Rt Pcr ◽  
First Report ◽  
Tomato Spotted Wilt ◽  
Negative Controls ◽  
Wilt Virus

In July 2011, greenhouse-grown chrysanthemum hybrid plants (Chrysanthemum × morifolium) with symptoms resembling those associated with tospoviruses were observed in the Kupusina locality (West Bačka District, Serbia). Disease incidence was estimated at 40%. Symptomatic plants with chlorotic ring spots and line patterns were sampled and tested by double antibody sandwich (DAS)-ELISA using polyclonal antisera (Bioreba AG, Reinach, Switzerland) against the two of the most devastating tospoviruses in the greenhouse floriculture industry: Tomato spotted wilt virus (TSWV) and Impatiens necrotic spot virus (INSV) (2). Commercial positive and negative controls and extracts from healthy chrysanthemum tissue were included in each ELISA. TSWV was detected serologically in 16 of 20 chrysanthemum samples and all tested samples were negative for INSV. The virus was mechanically transmitted from ELISA-positive chrysanthemum samples to five plants each of both Petunia × hybrida and Nicotiana tabacum ‘Samsun’ using chilled 0.01 M phosphate buffer (pH 7) containing 0.1% sodium sulfite. Inoculated plants produced local necrotic spots and systemic chlorotic/necrotic concentric rings, consistent with symptoms caused by TSWV (1). The presence of TSWV in ELISA-positive chrysanthemum plants and N. tabacum‘Samsun’ was further confirmed by conventional reverse transcription (RT)-PCR. Total RNAs were extracted with an RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). RT-PCR was performed with the One-Step RT-PCR Kit (Qiagen) using primers TSWVCP-f/TSWVCP-r specific to the nucleocapsid protein (N) gene (4). A Serbian isolate of TSWV from tobacco (GenBank Accession No. GQ373173) and RNA extracted from a healthy chrysanthemum plant were used as positive and negative controls, respectively. An amplicon of the correct predicted size (738-bp) was obtained from each of the plants assayed, and that derived from chrysanthemum isolate 529-11 was purified (QIAqick PCR Purification Kit, Qiagen) and sequenced (JQ692106). Sequence analysis of the partial N gene, conducted with MEGA5 software, revealed the highest nucleotide identity of 99.6% (99% amino acid identity) with 12 TSWV isolates deposited in GenBank originating from different hosts from Italy (HQ830186-87, DQ431237-38, DQ398945), Montenegro (GU355939-40, GU339506, GU339508), France (FR693055-56), and the Czech Republic (AJ296599). The consensus maximum parsimony tree obtained on a 705-bp partial N gene sequence of TSWV isolates available in GenBank revealed that Serbian TSWV isolate 529-11 from chrysanthemum was clustered in the European subpopulation 2, while the Serbian isolates from tomato (GU369723) and tobacco (GQ373172-73 and GQ355467) were clustered in the European subpopulation 1 denoted previously (3). The distribution of TSWV in commercial chrysanthemum crops is wide (2). To our knowledge, this is the first report of TSWV infecting chrysanthemum in Serbia. Since chrysanthemum popularity and returns have been rising rapidly, the presence of TSWV may significantly reduce quality of crops in Serbia. References: (1) Anonymous. OEPP/EPPO Bull. 34:271, 2004. (2) Daughtrey et al. Plant Dis. 81:1220, 1997. (3) I. Stanković et al. Acta Virol. 55:337, 2011. (4) A. Vučurović et al. Eur. J. Plant Pathol. 133:935, 2012.

scholarly journals Dieback and Wilting Caused by Tomato spotted wilt virus in Arctotis × hybrida in Italy

Plant Disease ◽  
2013 ◽  
Vol 97 (10) ◽  
pp. 1387-1387
Author(s):  
G. Parrella ◽  
B. Greco ◽  
L. Cavicchi ◽  
M. G. Bellardi
Keyword(s):  
South Korea ◽  
Tomato Spotted Wilt Virus ◽  
Protein Gene ◽  
Cultivated Plants ◽  
Rt Pcr ◽  
Tomato Spotted Wilt ◽  
Local Lesions ◽  
Tomato Isolate ◽  
Plant Pathol ◽  
Wilt Virus

In winter 2012, some potted plants of African daisy (Arctotis × hybrida L., family Asteraceae) cv. Hannah, propagated by rooted stem cuttings and cultivated for commercial purposes in a greenhouse located at Albenga (Liguria region, Italy), were noticed for a rapid dieback, generalized reddening, following by an irreversible wilting. Around 130 plants on a total of 3,000 cultivated plants showed symptoms (4 to 5%). One gram of fresh leaves, each collected from three different symptomatic plants, was ground in 4 ml of cold (∼5°C) sodium phosphate 0.03 M buffer, containing 0.2% sodium diethyldithiocarbamate, 75 mg/ml of active charcoal, and traces of carborundum (600 mesh). The inoculum was rubbed on healthy indicator herbaceous plants and inoculated plants were maintained in an insect-proof greenhouse with natural illumination and temperatures of 24/18°C day/night. Healthy and buffer inoculated plants were also included in the test and used as negative control in the subsequent serological and molecular analysis. Sap-inoculated plants showed the following symptoms after 1 to 3 weeks: necrotic local lesions in Chenopodium amaranticolor and C. quinoa, yellowing and stunting following by systemic necrosis and death of the plants in tomato (Solanum lycopersicum cv. San Marzano), necrotic local lesions following by systemic necrotic patterns and leaf deformation in tobacco (Nicotiana tabacum cv. Xanthi nc.) and N. glutinosa, necrotic local lesions in petunia (Petunia × hybrida cv. Pink Beauty). No symptoms were recorded on buffer inoculated plants. Leaf samples from both symptomatic hosts and the three original symptomatic African daisy plants were tested by double-antibody sandwich-ELISA with polyclonal antisera against Cucumber mosaic virus (CMV) and tospoviruses (Tospovirus broad-spectrum, Serogroups I, II, and III, Bioreba AG, Switzerland). Positive reaction was obtained with Tospo-groups antibodies, but not with the CMV ones. Total RNA was extracted from infected leaves of African daisy with the RNeasy Plant Mini Kit (Qiagen, Valencia, CA) and subjected to reverse transcription (RT)-PCR by using the tospovirus universal primers BR60/BR65 that amplify part of the nucleocapsid protein gene (1). Target amplicons of 454 bp were produced for all samples tested. The PCR products were cloned and sequenced on both strands (one clone per amplicon cloned). The resulting sequences were 100% identical, so a single sequence was deposited in GenBank (HF913777). The sequence showed highest homology (99%) with the Tomato spotted wilt virus (TSWV) tomato isolate NJ-JN from South Korea (HM581936). The identity of the virus infecting African daisy was further confirmed by sequencing amplicons obtained by RT-PCR using primers partially covering the movement protein gene of TSWV (2). The sequence obtained (HF913776) showed the highest homology (99%) with three TSWV isolates: a tomato isolate from Spain (AY744493), a pepper isolate from South Korea (AB663306), and again the tomato NJ-JN isolate from South Korea (HM581936). To our knowledge, this is the first natural report of TSWV infecting African daisy plants. Moreover, since this ornamental is often cultivated with other flowering plants, it can act as reservoir for the virus that can infect other ornamentals and crops, considering that TSWV has a very broad host range (3). This result also represents the first finding of TSWV in the genus Arctotis, family Asteraceae, the greater botanical family of TSWV hosts (3). References: (1) M. Eiras et al. Fitopatol. Bras. 26:170, 2001. (2) M. M. Finetti et al. J. Plant Pathol. 84:145, 2002. (3) G. Parrella et al. J. Plant Pathol. 85:227. 2003.

scholarly journals First Report of Tomato spotted wilt virus in Lettuce Crops in Saudi Arabia

Plant Disease ◽  
2014 ◽  
Vol 98 (11) ◽  
pp. 1591-1591 ◽  
Author(s):  
M. A. Al-Saleh ◽  
I. M. Al-Shahwan ◽  
M. A. Amer ◽  
M. T. Shakeel ◽  
M. H. Ahmad ◽  
...  
Keyword(s):  
Saudi Arabia ◽  
Tomato Spotted Wilt Virus ◽  
Post Inoculation ◽  
Rt Pcr ◽  
Specific Primers ◽  
Indicator Plants ◽  
First Report ◽  
Tomato Spotted Wilt ◽  
Local Lesions ◽  
Wilt Virus

A survey for viruses in open field lettuce crops was carried out in March 2014 in the Al-Uyaynah area, central region of Saudi Arabia. In one plot, more than 50% of the lettuce plants (Lactuca sativa; hybrid: Romaine), with the majority of the affected plants in the edges of the plot, were showing virus-like symptoms such as necrotic lesions, necrosis of the lamina of the younger leaves, and leaf curling, indicating a possible infection by a Tospovirus, possibly Tomato spotted wilt virus (TSWV). Most of them were dead when the field was visited again 3 weeks later. Samples from 10 symptomatic and two asymptomatic plants were collected. Five of the samples from symptomatic and two from asymptomatic plants were mechanically inoculated onto Nicotiana benthamiana and N. glutinosa (three indicator plants of each species were used for each sample) using 0.1 M phosphate buffer (pH 7) containing 0.01M Na2SO3 mM. All the symptomatic lettuce samples were also tested serologically using polyclonal antisera (3) against TSWV, CMV, and by using monoclonal antibodies against potyviruses. Moreover, total RNA was extracted (1) and detection of TSWV was also attempted with reverse transcription (RT)-PCR using species specific primers (4) for a 276-bp fragment of the L RNA segment. In both serological and molecular methods, positive and negative controls were included. All the mechanically inoculated plants with tissue from the symptomatic lettuce plants of N. benthamiana showed chlorotic local lesions followed by systemic top necrosis 2 to 3 weeks post inoculation. Similarly, all inoculated N. glutinosa plants showed necrotic local lesions followed by systemic chlorosis. However, all the indicator plants mechanically inoculated with tissue from asymptomatic lettuce plants gave no reaction. All the symptomatic lettuce samples reacted positively, while asymptomatic samples reacted negatively in ELISA tests with TSWV antiserum and the presence of the virus was further confirmed by RT-PCR by using specific primers (method A) (4). PCR products of two randomly selected positive samples were directly sequenced and BLAST analysis of the obtained sequences (Accession Nos. KJ701035 and KJ701036) revealed 99% nucleotide and 100% amino acid identity with the deposit sequence in NCBI from South Korea (KC261947). Regarding mechanical inoculation, 10 days post-inoculation, both indicator plants showed typical symptoms of TSWV infection, such as necrotic local lesions, systemic necrotic patterns, and leaf deformation. None of the symptomatic plants was found to be infected with either CMV or potyvirus. To our knowledge, this is the first report of TSWV naturally infecting lettuce in Saudi Arabia; therefore, insect vector and weed management are necessary measures to control the virus spread to other crops such as tomato and pepper (2). References: (1) E. Chatzinasiou et al. J. Virol. Meth. 169:305, 2010. (2) E. K. Chatzivassiliou. Plant Dis. 92:1012, 2008. (3) E. K. Chatzivassiliou et al. Phytoparasitica 28:257, 2000. (4) R. A. Mumford et al. J. Virol. Meth. 46:303, 1994.

scholarly journals First Report of Tomato spotted wilt virus Infecting African Clover (Trifolium tembense) in Georgia

Plant Disease ◽  
2009 ◽  
Vol 93 (2) ◽  
pp. 202-202 ◽  
Author(s):  
N. A. Barkley ◽  
D. L. Pinnow ◽  
M. L. Wang ◽  
G. A. Pederson
Keyword(s):  
Tomato Spotted Wilt Virus ◽  
Plant Genetic Resources ◽  
Germplasm Collection ◽  
Rt Pcr ◽  
First Report ◽  
Nucleocapsid Gene ◽  
Weed Host ◽  
Tomato Spotted Wilt ◽  
Wilt Virus ◽  
Das Elisa

Tomato spotted wilt virus (TSWV; family Bunyaviridae, genus Tospovirus), which is vectored by several species of thrips (order Thysanoptera, family Thripidae), causes a destructive disease that affects many economically important host plants such as tomatoes, peppers, and peanuts. Controlling the spread of this disease is challenging, and currently, only limited strategies are available to prevent and/or control its dissemination, including early diagnosis, destruction of infected material, and elimination of the vector. TSWV has been previously reported in subterranean clover (Trifolium subterraneum), white clover (T. repens), and various unidentified wild clovers (Trifolium spp.) in North America and Australia (1,3), but never before in an African species. T. tembense (Fresen.), an herbaceous annual African clover that is mainly used for grazing, is part of the national germplasm collection housed at the Plant Genetic Resources Conservation Unit in Griffin, GA. TSWV was found naturally infecting several accessions of this species being grown for regeneration in a greenhouse during 2008. Initial putative identification of the virus was done by visual inspection of host symptoms that included ringspots, necrotic and chlorotic local lesions, sometimes mild systemic wilting, and eventually an overall decline of healthy tissue in the infected plants. This was subsequently confirmed by double-antibody sandwich (DAS)-ELISA and reverse transcription (RT)-PCR. Primers (5′-ATGTCTAAGGTTAAGCTC-3′ forward and 5′-TTAAGCAAGTTCTGTGAG-3′ reverse) targeted the nucleocapsid gene of TSWV and amplified an expected product of approximately 800 bp (2). No product was amplified in any of the negative controls. Twenty-six individuals representing twelve plant accessions (PI 517788, 517790, 517792, 517793, 517809, 517832, 517842, 517845, 517851, 517871, 517876, and 517889) were screened for TSWV. Two to three individuals were targeted from each accession. Samples were chosen on the basis of the availability of leaf tissue to perform two diagnostic assays, ELISA and RT-PCR. Samples chosen for this study were all naturally infected by thrips. All but four individuals representing two plant accessions tested positive for the virus. The RT-PCR data substantiated the DAS-ELISA results and confirmed the suspected infection. More than 26% of the positive samples naturally infected by TSWV were further characterized by purifying and sequencing (bidirectionally) the RT-PCR product on an automated CEQ 8000 sequencer (Beckman Coulter, Fullerton, CA). The resulting sequences were aligned and edited using AlignIR (LI-COR, Lincoln, NE). More than 700 bp of sequence data (GenBank Accession No. FJ183743–FJ183746) was compiled and they displayed 98% identity with deposited TSWV nucleocapsid gene sequences in GenBank, with no similarity to any other targets. To our knowledge, this is the first report of TSWV infection in T. tembense. Accessions potentially resistant to TSWV within this species were identified and need to be further substantiated. T. tembense is a wild, native clover in Africa and could serve as a weed host for infection of nearby agronomically important crops. References: (1) I. Bitterlich and L. S. MacDonald. Can. Plant Dis. Surv. 73:137, 1993. (2) R. J. Holguín-Peña and E. O. Rueda-Puente. Plant Dis. 91:1682, 2007. (3) C. R. Wilson. Plant Pathol. 47:171, 1998.

scholarly journals First Report of Tomato torrado virus in Tomato in the Canary Islands, Spain

Plant Disease ◽  
2007 ◽  
Vol 91 (8) ◽  
pp. 1060-1060 ◽  
Author(s):  
A. Alfaro-Fernández ◽  
C. Córdoba-Sellés ◽  
M. C. Cebrián ◽  
J. A. Sánchez-Navarro ◽  
A. Espino ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Canary Islands ◽  
Polyclonal Antibodies ◽  
Tomato Mosaic Virus ◽  
Rt Pcr ◽  
Water Control ◽  
First Report ◽  
Pcr Product ◽  
Negative Controls ◽  
Wilt Virus

In 2003, greenhouse-grown tomato crops (Lycopersicon esculentum Mill.) in the Canary Islands (Spain) were observed showing an initial yellowing in defined areas at the base of the leaflet that later developed into necrotic spots or an extensive necrotic area progressing from the base to tip. Fruits were also affected, showing necrotic areas and often developing cracking. Generally, the plants that were affected seemed to be burnt, their growth was reduced, and the production level was seriously damaged. Similar symptoms have been observed in Murcia (Spain) since 2001, which have been recently associated with Tomato torrado virus (ToTV) infection (2). Twenty-two tomato samples showing “torrado disease” symptoms were collected from different greenhouses between 2003 and 2006 in Las Palmas (Canary Islands, Spain). To verify the identity of the disease, double-antibody sandwich (DAS)-ELISA was performed on leaf and fruit extracts of symptomatic plants using polyclonal antibodies specific to Potato virus Y (PVY), Tomato mosaic virus (ToMV), Tomato spotted wilt virus (TSWV) (Loewe Biochemica, Sauerlach, Germany), and Pepino mosaic virus (PepMV) (DSMZ, Braunschweig, Germany). Total RNA was extracted from the 22 tomato samples with the RNAwiz Extraction kit (Ambion, Huntingdon, United Kingdom) and tested using one-step reverse-transcription (RT)-PCR with the SuperScript Platinum Taq kit (Invitrogen Life Technologies, Barcelona, Spain) with primers specific to PepMV (1) and ToTV (2). All analyses included healthy tomato plants as negative controls. Five of the twenty-two tomato samples were positive for PepMV and negative for the other viruses tested by serological analysis. However, all 22 samples were positive in RT-PCR performed with the primers specific to ToTV segment RNA2. The RT-PCR assay to detect ToTV produced an amplicon of the expected size (580 bp). No amplification product was observed when healthy plants or a water control were used as a template in the RT-PCR reaction. The ToTV RT-PCR product was purified (High Pure PCR Product Purification kit, Roche Diagnostics, Mannheim, Germany) and sequenced. BLAST analysis of one sequence (GenBank Accession No. EF436286) showed 99% identity to ToTV RNA2 sequence (GenBank Accession No. DQ388880). To our knowledge, this is the first report of ToTV in the Canary Islands. References: (1) I. Pagán et al. Phytopathology 96:274, 2006. (2) M. Verbeek et al. Online Publication. doi:10.1007/s00705-006-0917-6. Arch. Virol., 2007.

Development of Reverse Transcription Thermostable Helicase-Dependent DNA Amplification for the Detection of Tomato Spotted Wilt Virus

2016 ◽  
Vol 99 (6) ◽  
pp. 1596-1599 ◽  
Author(s):  
Xinghai Wu ◽  
Chanfa Chen ◽  
Xizhi Xiao ◽  
Ming Jun Deng
Keyword(s):  
Reverse Transcriptase ◽  
Mosaic Virus ◽  
Reverse Transcription ◽  
Tomato Spotted Wilt Virus ◽  
Tomato Mosaic Virus ◽  
N Gene ◽  
Impatiens Necrotic Spot Virus ◽  
Rt Pcr ◽  
Tomato Spotted Wilt ◽  
Wilt Virus

Abstract A protocol for the reverse transcription-helicase-dependent amplification (RT–HDA) of isothermal DNA was developed for the detection of tomato spotted wilt virus (TSWV). Specific primers, which were based on the highly conserved region of the N gene sequence in TSWV, were used for the amplification of virus's RNA. The LOD of RT–HDA, reverse transcriptase-loop-mediated isothermal amplification (RT-LAMP), and reverse transcriptase-polymerase chain reaction (RT-PCR) assays were conducted using 10-fold serial dilution of RNA eluates. TSWV sensitivity in RT–HDA and RT-LAMP was 4 pg RNA compared with 40 pg RNA in RT-PCR. The specificity of RT–HDA for TSWV was high, showing no cross-reactivity with other tomato and Tospovirus viruses including cucumber mosaic virus (CMV), tomato black ring virus (TBRV), tomato mosaic virus (ToMV), or impatiens necrotic spot virus (INSV). The RT–HDA method is effective for the detection of TSWV in plant samples and is a potential tool for early and rapid detection of TSWV.

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