First Report of Tobacco rattle virus Causing Corky Ringspot in Potatoes Grown in Minnesota and Wisconsin

In July 2007, potato tubers cv. Russet Burbank (RB) with necrotic arcs and spots were detected in three fields in Buffalo County, Wisconsin and one field in Benson County, Minnesota. Umatilla Russet (UR) potatoes harvested from the west half of a field in Swift County, MN had similar, but visually distinct necrotic lesions. Portions of one field in Minnesota were abandoned, and the stored potato crop from two fields in Wisconsin was rejected by processors, representing a total crop loss due to tuber necrosis. Tuber symptoms displayed in both cultivars resembled those described for corky ringspot caused by Tobacco rattle virus (TRV) (4). Total RNA was isolated from necrotic tuber tissue crushed in liquid nitrogen and extracted using the Total RNA Isolation Kit (Promega Corp., Madison, WI). These extracts were tested for the presence of TRV by reverse transcription (RT)-PCR using primers complementary to nucleotides 6555 to 6575 and identical to nucleotides 6113 to 6132 within the 3′ terminal open reading frame of TRV RNA-1 (3). The expected 463-bp fragments were amplified from RB tubers. Nucleotide sequences from a Wisconsin and Minnesota isolate (GenBank Accession Nos. EU569290 and EU569291, respectively) were 99 to 100% identical to the corresponding region in a published TRV sequence (GenBank Accession No. AF055912). A 396-bp fragment was amplified from UR tubers and sequence data (GenBank Accession No. EU569292) indicated a unique 63 nucleotide sequence was substituted for a 129 nucleotide sequence spanning residues 227 to 357 of the 463-bp amplicon from the RB TRV isolates. Seven fragments were sequenced from different UR tubers and the 396-bp fragment was identical among them. The sequence outside the substituted region had 92% identity to the published TRV sequence. Amplification of the full-length TRV RNA2 using primers 179/180 located in the 5′ and 3′ untranslated regions (2) was successful for 28 and 0% of the RB and UR samples, respectively, suggesting that the RNA2 is not present in these strains or has undergone significant mutation. TRV-infected sap from both potato cultivars was mechanically transmitted to tobacco cv. Samsun NN and these plants subsequently tested positive for TRV by ELISA using ATCC antiserum PVAS 820. Ninety tubers exhibiting mild to severe symptoms of TRV were planted in the greenhouse. Each tuber was bisected laterally; necrotic tissue was removed from one half of the tuber and tested for the presence of TRV using RT-PCR protocols described above for RNA1. The remaining half was bisected horizontally and both sections were planted. Foliage from each emerged plant was subsequently also tested by RT-PCR for TRV RNA1. All RB tubers from Wisconsin tested positive for TRV, but only 7 of 24 emerged plants tested positive. Only 72% of the UR tubers and 4 of 25 emerged plants tested positive. TRV has been confirmed in California, Colorado, Florida, Idaho, Michigan (1), Oregon, and Washington. To our knowledge, this is the first report of corky ringspot in potato caused by TRV in Minnesota and Wisconsin. References: (1) W. W. Kirk et al. Plant Dis. 92:485, 2008. (2) S. A. MacFarlane. J. Virol. Methods. 56:91, 1996. (3) D. J. Robinson. J. Virol. Methods 40:57, 1992. (4) S. A. Slack. Tobacco rattle virus. Page 71 in: Compendium of Potato Diseases. 2nd ed. W. R. Stevenson et al., eds. The American Phytopathological Society, St. Paul, MN, 2001.

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First Report of Tobacco rattle virus Associated with Corky Ringspot in Potatoes Grown in North Dakota

Plant Disease ◽

10.1094/pdis-94-1-0130b ◽

2010 ◽

Vol 94 (1) ◽

pp. 130-130 ◽

Cited By ~ 6

Author(s):

N. David ◽

I. Mallik ◽

N. C. Gudmestad

Keyword(s):

North Dakota ◽

Consensus Sequence ◽

Tobacco Rattle Virus ◽

Alfalfa Mosaic Virus ◽

Rna Isolation ◽

Rt Pcr ◽

Total Rna ◽

First Report ◽

Tuber Necrosis ◽

Corky Ringspot

Tobacco rattle virus (TRV) belongs to the genus Tobravirus and causes a stem mottle of potato (Solanum tuberosum) foliage and necrotic arcs and rings in tubers referred to as corky ringspot. This virus is generally transmitted by a number of species of stubby-root nematode. The virus is widespread and has been reported in California, Colorado, Florida, Idaho, Michigan, Oregon, Washington, Minnesota, and Wisconsin (2). In the spring of 2009, we received potato tubers of cv. Russet Burbank with internal necrotic arcs very similar to those caused by TRV from potato storages located in Grand Forks and Dickey counties of North Dakota. Total RNA was extracted from the necrotic lesions of two tubers from each location using the Total RNA Isolation kit (Promega Corp., Madison WI). These extracts were tested for TRV by reverse transcription (RT)-PCR using primers complementary to nucleotides 6555 to 6575 (Primer A) and identical to nucleotides 6113 to 6132 (Primer B) within the 3′ terminus of TRV-SYM RNA-1 (GenBank Accession No. X06172) (3). The expected 463-bp amplicons from two separate tuber samples from each county were cloned (TOPO Cloning; Invitrogen, Carlsbad, CA) and sequenced. The sequences obtained from the four clones at both locations were found to be identical to each other and were 99% identical to the corresponding regions of TRV isolates from Michigan and Florida (GenBank Accession Nos. EU315226.1 and AF055912.1, respectively). Since sequences from all four clones were identical, only one of the sequences was submitted to Genbank (Accession No. GQ223114) and thus represents a consensus sequence. The extracts also tested positive in RT-PCR with a second set of primers corresponding to sequences in TRV RNA-2 yielding a 3.8-kbp amplicon (1). No evidence was found by RT-PCR for several other viruses that cause tuber necrosis in potato (Potato mop top virus, Tomato spotted wilt virus, Alfalfa mosaic virus, and tuber necrosis strains of Potato virus Y). The virus was mechanically transmitted by inoculating sap from symptomatic tubers from both counties to tobacco cv. Samsun NN, which showed typical bright yellow patches and spots on leaves 2 weeks postinoculation. TRV was confirmed in tobacco by RT-PCR from total RNA extracted from tobacco leaves with both sets of the aforementioned primers. To our knowledge, this is the first report of TRV in North Dakota and the first report of corky ringspot disease of potato in this state. References: (1) J. M. Crosslin et al. Virus Res. 96:99, 2003. (2) N. C. Gudmestad et al. Plant Dis. 92:1254, 2008. (3) D. J. Robinson. J. Virol. Methods 40:57, 1992.

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First Report of Corky Ringspot Caused by Tobacco rattle virus on Potatoes (Solanum tuberosum) in Michigan

Plant Disease ◽

10.1094/pdis-92-3-0485b ◽

2008 ◽

Vol 92 (3) ◽

pp. 485-485 ◽

Cited By ~ 7

Author(s):

W. W. Kirk ◽

S. L. Gieck ◽

J. M. Crosslin ◽

P. B. Hamm

Keyword(s):

Tobacco Rattle Virus ◽

The United States ◽

Rt Pcr ◽

Agricultural Commodity ◽

Reading Frame ◽

First Report ◽

Elisa Testing ◽

Internal Necrosis ◽

Corky Ringspot ◽

Storage Facilities

Potato is the fourth most important agricultural commodity in Michigan and is grown for table stock, chip processing, and seed. Tubers are either processed or fresh packed immediately following harvest or sent to storage. Tubers of potato cv. FL1879 representing two separate fields in Huron County were removed from separate storage facilities in February and March of 2007, and substantial internal necrosis was observed in 1 to 2% of the tubers. Symptoms included arcs similar to those caused by Tobacco rattle virus (TRV). This virus is a member of the genus Tobravirus and is transmitted by a number of species of stubby-root nematodes (Paratrichodorus or Trichodorus spp.). Stubby-root nematodes have been reported previously from Michigan (1). To confirm the presence of TRV, nucleic acid extractions were made from these symptomatic tubers (3). Samples were initially tested for TRV by reverse transcription (RT)-PCR using primers specific to the 16 kDa open reading frame on genomic RNA-1 (2). Samples from both storage facilities were positive. The 463-bp RT-PCR product, amplified with these primers, was sequenced (GenBank Accession No. EU315227) and was 99.6% identical to the corresponding region of two TRV isolates from Florida and Washington (GenBank Accession Nos. AF055912 and EU315226, respectively). In addition, sap from cv. FL1879 tuber samples was used to transmit the virus mechanically to tobacco cv. Samsun NN, which produced typical TRV symptoms following inoculation, and sap extracts from the tobacco leaves also tested positive with antiserum specific to TRV upon subsequent ELISA testing. Corky ringspot can result in substantial losses, with entire potato fields being rejected because of internal tuber damage. Once found, fields must be considered permanently at risk to this disease due to the large host range of both the virus and the nematode vector. This disease has been previously found in the United States in California, Colorado, Florida, Idaho, Washington, Oregon, and it is likely present in Indiana. To our knowledge, this is the first report of corky ringspot and TRV on potato in Michigan. References: (1) G. W. Bird and N. Knobloch. Plant Dis. Rep. 60:76, 1976. (2) J. M. Crosslin and P. E. Thomas. Am. Potato J. 72:605, 1995. (3) G. G. Presting et al. Phytopathology 85:436, 1995.

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First Report of Tobacco rattle virus in Sedum in Minnesota

Plant Disease ◽

10.1094/pdis-94-3-0374a ◽

2010 ◽

Vol 94 (3) ◽

pp. 374-374 ◽

Cited By ~ 2

Author(s):

B. E. Lockhart ◽

S. L. Mason

Keyword(s):

Nucleotide Sequence ◽

Tobacco Rattle Virus ◽

Leaf Tissue ◽

Garden Soil ◽

Rt Pcr ◽

Electron Microscopic ◽

Arabis Mosaic Virus ◽

First Report ◽

Virus Like Particles ◽

Rigid Rod

Sedums (Sedum spp.; Crassulaceae) are perennial landscape plants that are grown widely because they are drought tolerant and winter hardy. Plants of Sedum ‘Matrona’ showing faint foliar ringspot symptoms were collected at a nursery retail outlet in St. Paul, MN in July 2008 and tested for possible viral infection by transmission electron microscopic (TEM) examination of negatively stained, partially purified leaf tissue extracts (1). The only virus-like particles observed were rigid, rod-shaped particles similar to those of Tobacco rattle virus (TRV) and other tobraviruses. A random sample of 100 measurements showed particles 20 nm in diameter with two modal lengths of 115 nm and 175 nm. These virus-like particles were confirmed to be those of TRV by immunosorbent electron microscopy (1) using antiserum to TRV (ATCC PVAS 75) and by reverse transcription (RT)-PCR using total RNA extracted with the RNeasy Kit (Qiagen, Valencia, CA) and primers that yield a 462-bp amplicon from TRV RNA 1 (4). An amplicon of the expected size was obtained by RT-PCR and its nucleotide sequence (GenBank Accession No. GQ268817) had 95 to 99% identity to published TRV sequences (AAW13192 and AAB48382). Two additional amplicons generated by RT-PCR from separate plants were identical in size and nucleotide sequence to the first. On the basis of virion morphology, serological relatedness, and sequence identity, the virus associated with mild ringspot symptoms in sedum was identified as an isolate of TRV. To our knowledge, this represents the first report of TRV incidence in sedum. Although Arabis mosaic virus is the only other virus reported to occur in sedum (2), we have observed numerous, flexuous filamentous 750 to 800 nm virus-like particles in partially purified extracts of a range of sedums showing mild mosaic and/or vein-clearing symptoms in Minnesota. Similar virus-like particles were not observed by TEM in partially purified extracts from TRV-infected ‘Matrona’ plants, suggesting that they did not contribute to the symptoms observed. We have reported previously (3) the occurrence of TRV in a variety of widely grown perennial ornamentals that provide potential sources of inoculum for spread of this virus by nematode vectors (Trichodorus and Paratrichodorus spp.) that occur commonly in garden soil, and Sedum is now added to the list of potential TRV reservoir plants. References: (1) Y. S. Ahlawat et al. Plant Dis. 80:590, 1996. (2) A. Gera et al. Acta Hortic. 722:175, 2006. (3) B. E. Lockhart et al. Plant Dis. 79:1249, 1995. (4) D. J. Robinson. J. Virol. Methods 40:57, 1992.

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First Report of Tobacco etch virus Infection in Coleus in the United States

Plant Disease ◽

10.1094/pdis-94-7-0921c ◽

2010 ◽

Vol 94 (7) ◽

pp. 921-921 ◽

Cited By ~ 5

Author(s):

B. E. L. Lockhart ◽

S. L. Mason ◽

D. A. Johnson ◽

D. S. Mollov

Keyword(s):

Electron Microscopy ◽

Nucleotide Sequence ◽

Leaf Tissue ◽

Tobacco Etch Virus ◽

Nucleotide Sequence Analysis ◽

Rt Pcr ◽

Total Rna ◽

First Report ◽

Virus Like Particles ◽

Orange Leaf

Virus-like disease symptoms consisting of foliar and veinal necrosis similar to those caused by Coleus vein necrosis virus (CVNV) (2) were observed in plants of coleus (Coleus blume Benth.) ‘Rustic Orange’ obtained from retail greenhouse outlets in Missouri and Minnesota. Flexuous, filamentous, 750 to 770 nm virus-like particles (vlps) were observed by transmission electron microscopy in negatively stained partially purified leaf tissue extracts from symptomatic ‘Rustic Orange’ leaf tissue. No other virus-like particles were observed and none were detected in extracts from asymptomatic leaves. These vlps were longer than those of CVNV (640 nm) (2) and were not detected by immunosorbent electron microscopy (ISEM) using antibodies to CVNV (2). Degenerate potyvirus primers PNIbF1 (5′GGBAAYAATAGTGGNCAACC3′) and PCPR1 (5′GGGGAGGTGCCGTTCTCDATRCACCA3′) (1) and total RNA extracted from ‘Rustic Orange’ leaf tissue with a Qiagen RNeasy Kit were used for reverse transcription-PCR with Ready-To-Go RT-PCR Beads (GE Healthcare). A 950-bp amplicon was obtained from total RNA from diseased but not from healthy leaf tissue. The nucleotide sequence of the amplicon (GenBank Accession No. GQ268818) had levels of identity to published Tobacco etch virus (TEV) sequences comprising portions of the nuclear inclusion body (NIb) and coat protein (CP) gene regions ranging from 89% (L38714) to 93% (M15239, M11458). The identity of the virus occurring in ‘Rustic Orange’ was further confirmed by ISEM. Virions were trapped and decorated by antibodies to TEV (ATCC PVAS 32). Systemically infected leaf tissue from Datura stramonium in which the coleus TEV isolate was propagated was used to mechanically inoculate Carborundum-dusted leaves of virus-free test plants of ‘Rustic Orange’ (Park Seed, Greenwood, SC). Inoculated plants developed foliar necrosis symptoms similar to those observed originally, and the presence of TEV was confirmed by ISEM and RT-PCR and nucleotide sequence analysis as described above. To our knowledge, this is the first report of a disease of coleus caused by TEV. Many of approximately 30 ‘Rustic Orange’ plants in one nursery in Minnesota showed similar necrotic foliar symptoms and randomly selected plants tested positive for TEV by ISEM. This suggests that TEV infection in this variety may be spread by vegetative propagation from infected stock plants. References: (1) Y.-C. Hsu et al. J. Virol. Methods 128:54. 2005. (2) D. S. Mollov et al. Plant Dis. 91:754. 2007.

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First report of groundnut ringspot virus infecting Zinnia sp. in Brazil

Plant Disease ◽

10.1094/pdis-05-21-1028-pdn ◽

2021 ◽

Author(s):

Felipe Franco de Oliveira ◽

Gabriel Madoglio Favara ◽

Camila Geovana Ferro ◽

Heron Delgado Kraide ◽

Eike Yudi Nishimura Carmo ◽

...

Keyword(s):

Nucleotide Sequence ◽

Nucleotide Sequences ◽

Ringspot Virus ◽

Enzyme Linked Immunosorbent Assay ◽

Rt Pcr ◽

Concentric Ring ◽

Leaf Extracts ◽

Total Rna ◽

First Report ◽

Two Samples

Zinnia sp. is a genus belonging to Asteraceae family, originated in Mexico and adapted to a warm-hot climate (Hemmati and Mehrnoosh, 2017). Several types of zinnias with different flower color and forms are cultivated in Brazil (Min et al., 2020 and Souza Jr. et al., 2020). Characteristic symptoms of infection caused by orthotospovirus, including chlorotic spots and concentric rings on the leaves, were observed in two plants of Zinnia sp. of a florist located in the city of Piracicaba, State of São Paulo, Brazil. Orthotospovirus-like particles were observed by transmission electron microscope in leaf extracts from both plants, stained negatively with 1% uranyl acetate. By analyzing ultrathin sections of infected leaf tissues, particles of 80-100 nm in diameter were found in the lumen of the endoplasmic reticulum and nucleocapsid aggregates in the cytoplasm. Total RNA extracted separately from the leaves of both samples, using the Purelink Viral DNA / RNA kit (Thermo Fisher Scientific), was used to detect the virus by reverse transcription polymerase chain reaction (RT-PCR), using the universal primers for orthotospovirus BR60, complementary to the 3’ end of the non-translated region of the S RNA (position 1 to 15 nt), and BR65, matching the nucleocapsid gene (N) (position 433 to 453 nt), generating and amplicon of 453 nt (Eiras et al., 2001). Amplicons of the expected size were obtained for the two samples. An amplicon was purified with the Wizard SV Gel and PCR Clean-Up System kit (Promega) and sequenced in both directions at Macrogen Inc (South Korea). The nucleotide sequence (GenBank MW629018) showed 99.29-99.76% identity with nucleotide sequences of the orthotospovirus groundnut ringspot virus (GRSV) isolates (GenBank MH686229 and KY400110). Leaf extracts from symptomatic plants were also analyzed by plate-trapped antigen-enzyme-linked immunosorbent assay (PTA-ELISA), using polyclonal antiserum produced against the GRSV nucleocapsid protein (Esquivel et al., 2019). The absorbance values obtained for the extracts of the two symptomatic plants of Zinnia sp. (1.3 and 1.7) were twice as high as the value obtained for the healthy plant extract (0.5). Leaf extract of symptomatic Zinnia sp. was inoculated mechanically onto leaves of healthy plants of Zinnia sp., Capsicum annuum cv. Dara, Cucumis sativus, Cucurbita pepo cv. Caserta, Chenopodium amaranticolor, Datura stramonium, Nicotiana tabacum cv. Turkish and Solanum lycopersicum cv. Compack. At 5 days post inoculation (dpi), inoculated leaves of D. stramonium reacted with local lesions, and at 9 dpi, newly developed leaves of inoculated S. lycopersicum plants showed necrotic spot and concentric ring symptoms, whereas C. annuum exhibited concentric rings at 10 dpi. Inoculated zinnia plants showed systemic chlorotic spot and concentric ring symptoms at 20 dpi, indistinguishable from those observed under natural infection. The other inoculated plant species were not symptomatic, nor the virus was detected. PTA-ELISA and RT-PCR confirmed infection with GRSV in symptomatic plants. The amplicons generated by RT-PCR of total RNA extracted from an experimentally infected plant of C. annuum and D. stramonium, and two plants of Zinnia sp. were sent for nucleotide sequencing. The obtained nucleotide sequences (MW629019, MW629020, MW629021, MW629022) shares 100% identity with the nucleotide sequence corresponding to the original GRSV isolate (MW629018) identified in Zinnia sp. This is the first report of the natural occurrence of GRSV in Zinnia sp. in Brazil. Studies on incidence and damage are needed to recommend alternatives for management.

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First Report of Tomato spotted wilt virus Causing Potato Tuber Necrosis in Texas

Plant Disease ◽

10.1094/pdis-93-8-0845a ◽

2009 ◽

Vol 93 (8) ◽

pp. 845-845 ◽

Cited By ~ 6

Author(s):

J. M. Crosslin ◽

I. Mallik ◽

N. C. Gudmestad

Keyword(s):

North Dakota ◽

Tomato Spotted Wilt Virus ◽

Tobacco Rattle Virus ◽

Potassium Phosphate ◽

Mechanical Transmission ◽

Rt Pcr ◽

First Report ◽

Tuber Necrosis ◽

Tomato Spotted Wilt ◽

Wilt Virus

In the summer of 2008, potato (Solanum tuberosum L.) tubers (cvs. FL1867, FL2053, and FL1922) from commercial fields near Dalhart, TX were observed with distinct external erumpent rings and severe internal discolorations including blotches, spots, and dry, cork-like tissue. The presence of rings suggested the possible involvement of one or more viruses. Nucleic acid from seven of eight symptomatic tubers received in Washington (cvs. FL1867 and FL1922) tested positive for Tomato spotted wilt virus (TSWV) by reverse transcription (RT)-PCR with primers TSWV 1 and 2 (3). Similarly, tubers (cvs. FL1867 and FL2053) received in North Dakota tested positive for TSWV with forward (S1983) and reverse (S2767) primers of Tsompana et al. (4). The 777-bp amplicon obtained with primers TSWV 1 and 2 and the 803-bp amplicon obtained with primers S1983 and S2767 were cloned and three clones of each were sequenced. Analysis of the consensus sequences and BLAST comparisons confirmed the Washington and North Dakota sequences were indeed TSWV in origin and were each 98 to 99% identical to the corresponding nucleocapsid region of a number of TSWV isolates and most closely related to an isolate detected in eastern black nightshade from Colorado (GenBank No. AY777475). The deduced amino acid sequences of the 777-bp nucleocapsid open reading frame differed from AY777475 at only two residues in each of the Washington and North Dakota sequences. The Washington and North Dakota derived sequences were deposited with GenBank (Nos. FJ882069 and FJ882070, respectively). None of the eight symptomatic tubers tested positive for Tobacco rattle virus (TRV), Alfalfa mosaic virus (AMV), or the necrotic strains of Potato virus Y (PVY) by RT-PCR. Mechanical transmission tests were conducted by grinding symptomatic tissue of a TSWV-positive FL1867 tuber in 10 volumes of 30 mM potassium phosphate buffer, pH 8.0, containing 10 mM of sodium diethyldithiocarbamate and 10 mM of sodium thioglycollate and rub inoculated onto Carborundum-dusted leaves of Samsun NN tobacco. Approximately 10 days after inoculation, chlorotic-necrotic rings were present on the inoculated leaves and circular necrotic lesions developed on the upper leaves. Dark stem lesions were also present on inoculated tobacco, and after 3 weeks, the upper leaves developed severe, spreading lesions. Tissue from the symptomatic tobacco tested positive for TSWV by RT-PCR (primers TSWV 1 and 2) and also with a TSWV-specific ImmunoStrip (Agdia, Inc., Elkhart, IN), but tested negative for TRV, AMV, and PVY by RT-PCR. TSWV has been reported on field-grown potatoes in North Carolina (1) and has been reported on potatoes in Australia (2) and in other parts of the world (referenced in 1). To our knowledge, this is the first report associating TSWV with tuber necrosis on potatoes in Texas. References: (1) J. A. Abad et al. Am. J. Potato Res. 82:255, 2005. (2) L. J. Latham and R. A. C. Jones. Aust. J. Agric. Res. 48:359, 1997. (3) R. Navarre et al. Am. J. Potato Res. 86:88, 2009. (4) M. Tsompana et al. Mol. Ecol. 14:53, 2005.

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

Plant Disease ◽

10.1094/pdis-06-13-0668-pdn ◽

2014 ◽

Vol 98 (5) ◽

pp. 698-698 ◽

Cited By ~ 1

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.

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Fast, Precise, and Reliable Multiplex Detection of Potato Viruses by Loop-Mediated Isothermal Amplification

International Journal of Molecular Sciences ◽

10.3390/ijms21228741 ◽

2020 ◽

Vol 21 (22) ◽

pp. 8741

Author(s):

Güven Edgü ◽

Lena Julie Freund ◽

Stefanie Hartje ◽

Eckhard Tacke ◽

Hans-Reinhard Hofferbert ◽

...

Keyword(s):

Disease Management ◽

Management Practices ◽

Tobacco Rattle Virus ◽

Simultaneous Detection ◽

Isothermal Amplification ◽

Diagnostic Methods ◽

Tuber Quality ◽

Loop Mediated Isothermal Amplification ◽

Tuber Necrosis ◽

Corky Ringspot

Potato is an important staple food crop in both developed and developing countries. However, potato plants are susceptible to several economically important viruses that reduce yields by up to 50% and affect tuber quality. One of the major threats is corky ringspot, which is a tuber necrosis caused by tobacco rattle virus (TRV). The appearance of corky ringspot symptoms on tubers prior to commercialization results in ≈ 45% of the tubers being downgraded in quality and value, while ≈ 55% are declared unsaleable. To improve current disease management practices, we have developed simple diagnostic methods for the reliable detection of TRV without RNA purification, involving minimalized sample handling (mini), subsequent improved colorimetric loop-mediated isothermal amplification (LAMP), and final verification by lateral-flow dipstick (LFD) analysis. Having optimized the mini-LAMP-LFD approach for the sensitive and specific detection of TRV, we confirmed the reliability and robustness of this approach by the simultaneous detection of TRV and other harmful viruses in duplex LAMP reactions. Therefore, our new approach offers breeders, producers, and farmers an inexpensive and efficient new platform for disease management in potato breeding and cultivation.

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Total RNA‐Isolation of Abdominal Hernia of Rats for Quantitative Real‐Time Reverse Transcription (RT) PCR Assays

Preparative Biochemistry & Biotechnology ◽

10.1080/10826060701774387 ◽

2007 ◽

Vol 38 (1) ◽

pp. 87-93

Author(s):

Christian Morsczeck ◽

Michael Korenkov ◽

Manfred Nagelschmidt ◽

Domonkos Feher ◽

Jörg Michael Schierholz

Keyword(s):

Real Time ◽

Reverse Transcription ◽

Rna Isolation ◽

Abdominal Hernia ◽

Rt Pcr ◽

Total Rna ◽

Pcr Assays ◽

Total Rna Isolation

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First Report of Little cherry virus 2 in Flowering and Sweet Cherry Trees in China

Plant Disease ◽

10.1094/pdis-10-10-0766 ◽

2011 ◽

Vol 95 (11) ◽

pp. 1484-1484 ◽

Cited By ~ 4

Author(s):

W.-L. Rao ◽

F. Li ◽

R.-J. Zuo ◽

R. Li

Keyword(s):

Sweet Cherry ◽

Sequence Data ◽

Community Gardens ◽

Economic Losses ◽

Helicase Domain ◽

Rt Pcr ◽

Viral Origin ◽

First Report ◽

Flowering Cherry ◽

Cherry Trees

Many viruses infect Prunus spp. and cause diseases on them. During a survey of stone fruit trees in 2008 and 2009, flowering cherry (Prunus serrulata) and sweet cherry (P. avium) trees with foliar chlorosis and reddening, stem deformity, and tree stunting were observed in private orchards in Anning and Fumin counties of Yunnan Province. Some sweet cherry trees with severe symptoms yielded small and few fruits and had to be removed. Leaf samples were collected from 68 flowering cherry and 30 sweet cherry trees, either symptomatic or asymptomatic, from private orchards and community gardens in Kunming and counties Anning, Chenggong, Fumin, Jinning, Ludian and Yiliang. Total nucleic acids were extracted with a CTAB extraction method and tested by reverse transcription (RT)-PCR assay using virus-specific primers. Little cherry virus 2 (LChV-2), Cherry virus A (CVA), Prunus necrotic ringspot virus (PNRSV), and Prune dwarf virus (PDV) were detected and infection rates were 68.4, 16.3, 9.2, and 7.1%, respectively. Infection of LChV-2 was common in all counties except Ludian where the orchards were healthy. Of 68 infected trees, 29 were found to be infected with LChV-2 and CVA, PDV or PNRSV. LChV-2 was detected in this study by RT-PCR using a pair of novel primers, LCV2-1 (5′-TTCAATATGAGCAGTGTTCCTAAC-3′) and LCV2-4 (5′-ACTCGTCTTGTGACATACCAGTC-3′), in 59 flowering cherry (87%) and 8 sweet cherry (27%) trees, respectively. The primer pair was designed according to alignment of three available LChV-2 sequences (GenBank Nos. NC_005065, AF416335, and AF333237) to amplify the partial RNA-dependent RNA polymerase gene (ORF1b) of 781 bp. The amplicons of selected samples (Anning26 and Yiliang60) were sequenced directly and sequences of 651 bp (GenBank No. HQ412772) were obtained from both samples. Pairwise comparisons and phylogenetic analysis of the sequences show that the two isolates are identical to one another and share 92 to 96% at the amino acid (aa) sequence level to those of other isolates available in the GenBank database. The sequence data confirm that these isolates are a strain of LChV-2 and genetic variation among different strains is relatively high (2). Biological and serological assays are not available for the LChV-2 detection; therefore, the LChV-2 infections of these trees were further confirmed by RT-PCR using primer pair LCV2-5 (5′-TGTTTGTGTCATGTTGTCGGAGAAG-3′) and LCV2-6 (5′-TGAATACCCGAGAACAAGGACTC-3′), which amplified the helicase domain (ORF1a) of ~451 bp. The amplicons from samples Anning26 and Yiliang60 were cloned and sequenced. The 408-bp sequences (excluding primer sequences) were 92 to 98% identical at the aa sequence level to those of other isolates, confirming again their viral origin. LChV-2 (genus Ampelovirus, family Closteroviridae) (4) has been associated with little cherry disease (LChD), a widespread viral disease of sweet and sour cherries (1,3). The virus is transferred between geographic areas mainly by propagated materials. Ornamental and sweet cherries are important crops in China and LChD has the potential to cause significant economic losses. Thus, certified clean stock should be used to establish new orchards. To our knowledge, this is the first report of LChV-2 in cherries in China. References: (1) N. B. Bajet et al. Plant Dis. 92:234, 2008. (2) W. Jelkmann et al. Acta Hortic. 781:321, 2008. (3) B. Komorowska and M. Cieslińska, Plant Dis. 92:1366, 2008. (4) M. E. Rott and W. Jelkmann. Arch. Virol. 150:107, 2005.

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