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

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 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 Alternanthera mosaic virus Infection in Angelonia in the United States

Plant Disease ◽

10.1094/pdis-92-10-1473b ◽

2008 ◽

Vol 92 (10) ◽

pp. 1473-1473 ◽

Cited By ~ 7

Author(s):

B. E. Lockhart ◽

M. L. Daughtrey

Keyword(s):

United States ◽

Nucleotide Sequence ◽

Mosaic Virus ◽

Genomic Sequence ◽

Nutrient Deficiency ◽

Leaf Tissue ◽

The United States ◽

Nucleotide Sequence Analysis ◽

Degenerate Primers ◽

First Report

Stunting, chlorosis, and light yellow mottling resembling symptoms of nutrient deficiency were observed in angelonia (Angelonia angustifolia) in commercial production in New York. Numerous, filamentous particles 520 to 540 nm long and spherical virus particles 30 nm in diameter were observed by transmission electron microscopy (TEM) in negatively stained partially purified extracts of symptomatic Angelonia leaf tissue. Two viruses, the filamentous potexvirus Alternanthera mosaic virus (AltMV) and the spherical carmovirus Angelonia flower break virus (AnFBV) were subsequently identified on the basis of nucleotide sequence analysis of amplicons generated by reverse transcription (RT)-PCR using total RNA isolated from infected leaf tissue. A 584-bp portion of the replicase-encoding region of the AltMV genome was obtained with the degenerate primers Potex 2RC (5′-AGC ATR GNN SCR TCY TG-3′) and Potex 5 (5′-CAY CAR CAR GCM AAR GAT GA-3′) (3). Forward (AnFBV CP 1F-5′-AGC CTG GCA ATC TGC GTA CTG ATA-3′) and reverse (AnFBV CP 1R-5′-AAT ACC GCC CTC CTG TTT GGA AGT-3′) primers based on the published AnFBV genomic sequence (GenBank Accession No. NC_007733) were used to amplify a portion of the viral coat protein (CP) gene. The nucleotide sequence of the amplicon generated using the potexvirus-specific primers (GenBank Accession No. EU679362) was 99% identical to the published AltMV (GenBank Accession No. NC_007731) sequence and the nucleotide sequence of the amplicon obtained using the AnFBV CP primers was 99% identical to the published AnFBV genomic sequence (GenBank Accession No. EU679363). AnFBV occurs widely in angelonia (1) and AltMV has been identified in phlox (2). These data confirm the presence of AltMV and AnFBV in diseased angelonia plants showing stunting and nutrient deficiency-like symptoms and substantiates, to our knowledge, this first report of AltMV in angelonia in the United States. References: (1) S. Adkins et al. Phytopathology 96:460, 2006. (2) J. Hammond et al. Arch. Virol. 151:477, 2006. (3) R. A. A. van der Vlugt and M. Berendeson. Eur. J. Plant Pathol. 108:367, 2002.

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

Plant Disease ◽

10.1094/pdis-92-8-1254c ◽

2008 ◽

Vol 92 (8) ◽

pp. 1254-1254 ◽

Cited By ~ 8

Author(s):

N. C. Gudmestad ◽

I. Mallik ◽

J. S. Pasche ◽

J. M. Crosslin

Keyword(s):

Nucleotide Sequence ◽

Sequence Data ◽

Potato Crop ◽

Tobacco Rattle Virus ◽

Rna Isolation ◽

Rt Pcr ◽

Total Rna ◽

First Report ◽

Tuber Necrosis ◽

Corky Ringspot

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 Cowpea aphid-borne mosaic virus on Sesame in Paraguay

Plant Disease ◽

10.1094/pdis-07-10-0498 ◽

2011 ◽

Vol 95 (5) ◽

pp. 613-613 ◽

Cited By ~ 2

Author(s):

L. Gonzalez Segnana ◽

M. Ramirez de Lopez ◽

A. P. O. A. Mello ◽

J. A. M. Rezende ◽

E. W. Kitajima

Keyword(s):

Electron Microscopy ◽

Nucleotide Sequence ◽

Mosaic Virus ◽

The United States ◽

Causal Agent ◽

Nucleotide Sequence Analysis ◽

Type I ◽

First Report ◽

San Pedro ◽

Cowpea Aphid

Sesame (Sesamum indicum L.) is cultivated mainly in the central region of the Departamento de San Pedro in Paraguay from October to February and the seed are exported to Asia. The crop is grown on 100,000 ha annually and Escoba blanca is the most common cultivar. The crop plays an important socioeconomical role since it is cultivated mostly by small growers. A disease characterized by yellowing and curling down leaves and shortening of the internodes has been observed in almost all sesame-growing areas. It is referred to locally as “ka'are” because the affected sesame plant resembles Chenopodium ambrosioides L. This disease occurred occasionally and was of marginal importance prior to 2005, but during the last five growing seasons the disease incidence has increased substantially, with some growers losing the entire crop. To determine the causal agent, symptomatic leaf samples were collected from five commercial fields near Colonia San Pedro and Choré, Departamento San Pedro in December 2009. Preliminary transmission electron microscopy (TEM; Zeiss EM900) of extracts from symptomatic leaves revealed the presence of elongated flexible particles resembling a potyvirus. Mechanical transmission assays resulted in chlorotic local lesions on C. quinoa and C. amaranticolor, mosaic on Vigna unguiculata and Nicotiana benthamiana, and symptoms on sesame that are similar to those observed in the field. The disease could also be reproduced in sesame by aphid (Myzus persicae) transmission in a nonpersistent manner. TEM examination of leaf sections of these naturally or experimentally infected plants showed the presence of the type I cylindrical inclusions and masses of filamentous particles. Leaf extracts of naturally or experimentally infected sesame and test plants were positive for Cowpea aphid-borne mosaic virus (CABMV) on the basis of plate-trapped antigen (PTA)-ELISA. CABMV as the causal agent of “ka'are” disease of sesame in Paraguay was further confirmed by analyzing part of the nucleotide sequence of CABMV coat protein and 3′ nontranslated region that were obtained directly from reverse transcription-PCR product amplified with PV1-antisense primer (5′-gatttaggtgacactatagt17-3′) and WCIEN-sense primer (5′-atggtttggtgyatygaraat-3′) (1,2). Comparisons of the 676-bp nucleotide sequence of two sesame virus isolates (GenBank Accession Nos. HQ336402 and HQ336403) revealed 92% identity with the corresponding nucleotide sequence of CABMV available in the GenBank (Accession No. AF348210). Thus, all the assays indicated that the “ka'are” disease of sesame in Paraguay is caused by an isolate of CABMV. Several cowpea fields, nearby sesame diseased crops, also contained plants exhibiting mosaic symptoms. Transmission assays, electron microscopy, PTA-ELISA, and nucleotide sequence analysis indicated that they were also infected by CABMV and may play an important role in the epidemiology of this disease on sesame. CABMV isolates from passion fruit and cowpea from Brazil were mechanically transmitted to sesame but induced milder symptoms. CABMV-infected sesame was described in the United States (3), but to our knowledge, this is the first report of a severe disease on sesame caused by this virus in Paraguay. References: (1) A. Gibbs and A. Mackenzie. J. Virol. Methods 63:9, 1997. (2) L. D. C. Mota et al. Plant Pathol. 53:368, 2004. (3) H. R. Pappu et al. Arch. Virol. 142:1919, 1997.

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First Report of Alfalfa mosaic virus Occurrence in Hydrangea in the United States

Plant Disease ◽

10.1094/pdis-07-12-0618-pdn ◽

2013 ◽

Vol 97 (9) ◽

pp. 1258-1258 ◽

Cited By ~ 4

Author(s):

B. Lockhart ◽

D. Mollov ◽

M. Daughtrey

Keyword(s):

Electron Microscopy ◽

United States ◽

Mosaic Virus ◽

Alfalfa Mosaic Virus ◽

Leaf Tissue ◽

The United States ◽

Spherical Particles ◽

Genomic Sequences ◽

Rt Pcr ◽

First Report

In spring of 2012, a previously unrecorded virus-like disease characterized by conspicuous yellow leaf blotching (calico symptoms) was observed in plants of Hydrangea macrophylla in a single location in Southampton, NY. Bacilliform and spherical particles resembling those of Alfalfa mosaic virus (AMV) were observed by transmission electron microscopy (TEM) in partially purified extracts from symptomatic leaf tissue. The identity of the virus was confirmed by immunosorbent electron microscopy (ISEM) (4) using antiserum to AMV (ATCC PVAS 92) that both trapped and decorated the virions. Three primer pairs designed from available AMV RNA 1, RNA 2, and RNA 3 genomic sequences were used to generate amplicons from the hydrangea AMV isolate. Reverse-transcription (RT)-PCR was done using total RNA extracted from symptomatic hydrangea leaf tissue with a Qiagen RNeasy kit, and Ready-to-Go RT-PCR beads (GE Healthcare). Amplicons of 1,049, 1,013, and 658 bp were obtained using the primer pairs AMV1F (5′-ATCCACCGATGCCAGCCTTA)/AMV1R (5′-TTCCGCCTCACTGCTGTCTG), AMV2F (5′-GATCGCCGGAAGTGATCCAG)/AMV2R (5′-TCACCGGAAGCAACAACGAA), and AMV3F (5′-GCCGGTTCTCCAAAGGGTCT)/AMV3R (5′-CGCGTCGAAGTCCAGACAGA), respectively. The PCR products were cloned using a TOPO TA cloning kit (Invitrogen) and three clones of each were sequenced. The sequences obtained from the hydrangea AMV RNA 1 (JX154090), RNA 2 (JX154091), and RNA 3 (JX154092) had 95 to 98% nucleotide sequence identity to homologous genomic sequences of known AMV isolates. To our knowledge, this is the first report of AMV occurrence in H. macrophylla in the United States. This virus has been reported to occur in H. macrophylla in British Columbia (3), but in a previous survey its presence was not detected in hydrangeas in the United States (1). A report of possible AMV infection in H. macrophylla in Italy (2) was based solely on symptomatology and cross-protection tests and therefore cannot be verified. The AMV-infected hydrangea plants were found by ISEM to also contain low concentrations of Hydrangea ringspot virus (HRSV) and Hydrangea chlorotic mottle virus (HdCMV). However, based on previous evidence of single and mixed infections (3), it is unlikely that the calico symptoms observed were influenced by the presence of HRSV and HdCMV. This report is of interest both because AMV, unlike HRSV and HdCMV, causes foliar symptoms that would render hydrangea plant unmarketable, and because the disease can be spread by a number of common aphid species that transmit AMV. It will also serve to alert growers and diagnosticians to the potential threat posed by AMV infection. References: (1) T. C. Allen et al. Acta Hortic. 164:85, 1985. (2) G. Belli. Phytopathol. Mediterr. 7:70, 1968. (3) A. W. Chiko and S. E. Godkin. Plant Dis. 70:541, 1986. (4) B. E. L. Lockhart et al. Phytopathology 82:691, 1992.

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Detection of Bovine Torovirus in Fecal Specimens of Calves with Diarrhea from Ontario Farms

Journal of Clinical Microbiology ◽

10.1128/jcm.36.5.1266-1270.1998 ◽

1998 ◽

Vol 36 (5) ◽

pp. 1266-1270 ◽

Cited By ~ 38

Author(s):

Lynn Duckmanton ◽

Susy Carman ◽

Éva Nagy ◽

Martin Petric

Keyword(s):

Electron Microscopy ◽

Nucleotide Sequence ◽

Nucleotide Sequence Analysis ◽

Rt Pcr ◽

Southern Ontario ◽

Sequence Identity ◽

Reverse Transcription Pcr ◽

Neonatal Calves ◽

Asymptomatic Control

Breda virus (BRV), a member of the genus Torovirus, is an established etiological agent of disease in cattle. BRV isolates have been detected in the stools of neonatal calves with diarrhea in both Iowa and Ohio and in several areas of Europe. However, this virus has been reported only once in Canada. Therefore, a study was performed to determine the extent to which bovine torovirus is present in calves with diarrhea from farms in southern Ontario. A total of 118 fecal samples from symptomatic calves and 43 control specimens from asymptomatic calves were examined by electron microscopy (EM) and reverse transcription-PCR (RT-PCR) for the presence of torovirus. Torovirus RNA was detected in 43 of the 118 diarrheic samples (36.4%) by RT-PCR with primers designed in the conserved 3′ end of the torovirus genome. By EM, torovirus particles were observed in 37 of the 118 specimens (31.4%). All but one of these samples were also positive by RT-PCR. The incidence of torovirus in the asymptomatic control specimens by RT-PCR was only 11.6%. To establish the identity of the particles observed in the diarrheic specimens, five of the amplicons from samples positive by both RT-PCR and EM were cloned and sequenced. Nucleotide sequence analysis revealed that the bovine torovirus found in southern Ontario manifests between 96 and 97% sequence identity to the BRV type 1 strain found in Iowa. This study shows that bovine torovirus is a common virus in the fecal specimens of calves with diarrhea from farms in southern Ontario and thus may be an important pathogen of cattle.

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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 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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First report of Coleus blumei viroid 1 in commercial Coleus blumei in the United States

Plant Disease ◽

10.1094/pdis-05-21-0917-pdn ◽

2021 ◽

Author(s):

Gardenia Orellana ◽

Alexander V Karasev

Keyword(s):

United States ◽

Leaf Tissue ◽

The United States ◽

Universal Primers ◽

Rt Pcr ◽

Universal Primer ◽

Tissue Samples ◽

First Report ◽

Specific Pcr ◽

Coleus Blumei

Coleus scutellarioides (syn. Coleus blumei) is a widely grown evergreen ornamental plant valued for its highly decorative variegated leaves. Six viroids, named Coleus blumei viroid 1 to 6 (CbVd-1 to -6) have been identified in coleus plants in many countries of the world (Nie and Singh 2017), including Canada (Smith et al. 2018). However there have been no reports of Coleus blumei viroids occurring in the U.S.A. (Nie and Singh 2017). In April 2021, leaf tissue samples from 27 cultivars of C. blumei, one plant of each, were submitted to the University of Idaho laboratory from a commercial nursery located in Oregon to screen for the presence of viroids. The sampled plants were selected randomly and no symptoms were apparent in any of the samples. Total nucleic acids were extracted from each sample (Dellaporta et al. 1983) and used in reverse-transcription (RT)-PCR tests (Jiang et al. 2011) for the CbVd-1 and CbVd-5 with the universal primer pair CbVds-P1/P2, which amplifies the complete genome of all members in the genus Coleviroid (Jiang et al. 2011), and two additional primer pairs, CbVd1-F1/R1 and CbVd5-F1/R1, specific for CbVd-1 and CbVd-5, respectively (Smith et al. 2018). Five C. blumei plants (cvs Fire Mountain, Lovebird, Smokey Rose, Marrakesh, and Nutmeg) were positive for a coleviroid based on the observation of the single 250-nt band in the RT-PCR test with CbVds-P1/P2 primers. Two of these CbVd-1 positive plants (cvs Lovebird and Nutmeg) were also positive for CbVd-1 based on the presence of a single 150-nt band in the RT-PCR assay with CbVd1-F1/R1 primers. One plant (cv Jigsaw) was positive for CbVd-1, i.e. showing the 150-nt band in RT-PCR with CbVd1-F1/R1 primers, but did not show the ca. 250-bp band in RT-PCR with primers CbVds-P1/P2. None of the tested plants were positive for CbVd-5, either with the specific, or universal primers. All coleviroid- and CbVd-1-specific PCR products were sequenced directly using the Sanger methodology, and revealed whole genomes for five isolates of CbVd-1 from Oregon, U.S.A. The genomes of the five CbVd-1 isolates displayed 96.9-100% identity among each other and 96.0-100% identity to the CbVd-1 sequences available in GenBank. Because the sequences from cvs Lovebird, Marrakesh, and Nutmeg, were found 100% identical, one sequence was deposited in GenBank (MZ326145). Two other sequences, from cvs Fire Mountain and Smokey Rose, were deposited in the GenBank under accession numbers MZ326144 and MZ326146, respectively. To the best of our knowledge, this is the first report of CbVd-1 in the United States.

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