Differential Detection of the Tobamoviruses Tomato Mosaic Virus (ToMV) and Tomato Brown Rugose Fruit Virus (ToBRFV) Using CRISPR-Cas12a

CRISPR/Cas12a-based detection is a novel approach for the efficient, sequence-specific identification of viruses. Here we adopt the use of CRISPR/Cas12a to identify the tomato brown rugose fruit virus (ToBRFV), a new and emerging tobamovirus which is causing substantial damage to the global tomato industry. Specific CRISPR RNAs (crRNAs) were designed to detect either ToBRFV or the closely related tomato mosaic virus (ToMV). This technology enabled the differential detection of ToBRFV and ToMV. Sensitivity assays revealed that viruses can be detected from 15–30 ng of RT-PCR product, and that specific detection could be achieved from a mix of ToMV and ToBRFV. In addition, we show that this method can enable the identification of ToBRFV in samples collected from commercial greenhouses. These results demonstrate a new method for species-specific detection of tobamoviruses. A future combination of this approach with isothermal amplification could provide a platform for efficient and user-friendly ways to distinguish between closely related strains and resistance-breaking pathogens.

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Differential detection of Tomato mosaic virus (ToMV) and Tomato brown rugose fruit virus (ToBRFV) using CRISPR-Cas12

10.1101/2021.03.16.435580 ◽

2021 ◽

Author(s):

Dan Alon ◽

Hagit Hak ◽

Menachem Bornstein ◽

Gur Pines ◽

Ziv Spiegelman

Keyword(s):

Mosaic Virus ◽

Plant Viruses ◽

Tomato Mosaic Virus ◽

Differential Detection ◽

Specific Detection ◽

Guide Rnas ◽

Novel Approach ◽

Pcr Product ◽

Species Specific ◽

User Friendly

CRISPR/Cas12-based detection is a novel approach for efficient, sequence-specific identification of viruses. Here we adopt the use of CRISPR/Cas12a to identify the Tomato brown rugose fruit virus (ToBRFV), a new and emerging Tobamovirus causing substantial damage to the global tomato industry. Specific guide RNAs (gRNAs) were designed to detect either ToBRFV or the closely related Tomato mosaic virus (ToMV). This technology enabled the differential detection of ToBRFV and ToMV. Sensitivity assays revealed that viruses can be detected from 15-30 ng of RT-PCR product, and that specific detection could be achieved from a mix of ToMV and ToBRFV. In addition, we show that this method enabled the identification of ToBRFV in samples collected from commercial greenhouses. These results demonstrate a new method for species-specific detection of plant viruses. This could provide a platform for the development of efficient and user-friendly ways to distinguish between closely related strains and resistance-breaking pathogens.

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

Plant Disease ◽

10.1094/pd-90-1108a ◽

2006 ◽

Vol 90 (8) ◽

pp. 1108-1108 ◽

Cited By ~ 3

Author(s):

C. Córdoba ◽

A. García-Rández ◽

N. Montaño ◽

C. Jordá

Keyword(s):

Mosaic Virus ◽

Seed Transmission ◽

Tomato Mosaic Virus ◽

Capsicum Chinense ◽

Rt Pcr ◽

Plant Host ◽

First Report ◽

Coat Protein Region ◽

Pcr Product ◽

First Time

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

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

Plant Disease ◽

10.1094/pdis-91-8-1060b ◽

2007 ◽

Vol 91 (8) ◽

pp. 1060-1060 ◽

Cited By ~ 17

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.

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Molecular and Biological Characterization of Tomato mottle mosaic virus and Development of RT-PCR Detection

Plant Disease ◽

10.1094/pdis-10-16-1504-re ◽

2017 ◽

Vol 101 (5) ◽

pp. 704-711 ◽

Cited By ~ 7

Author(s):

Xuelian Sui ◽

Yi Zheng ◽

Rugang Li ◽

Chellappan Padmanabhan ◽

Tongyan Tian ◽

...

Keyword(s):

New York ◽

South Carolina ◽

Mosaic Virus ◽

Genetic Relationships ◽

The United States ◽

Biological Properties ◽

Tomato Mosaic Virus ◽

Rt Pcr ◽

The World ◽

Species Specific

Tomato mottle mosaic virus (ToMMV) was first identified in 2013 as a novel tobamovirus infecting tomatoes in Mexico. In just a few years, ToMMV has been identified in several countries around the world, including the United States. In the present study, we characterized the molecular, serological, and biological properties of ToMMV and developed a species-specific RT-PCR to detect three tomato-infecting tobamoviruses: Tobacco mosaic virus (TMV), Tomato mosaic virus (ToMV), and ToMMV. Previously, ToMMV has been reported in Florida and New York. In this study, we made two new reports on the occurrences of ToMMV on tomatoes in California and South Carolina. Their complete genome sequences were obtained and their genetic relationships to other tobamoviruses evaluated. In host range studies, some differential responses in host plants were also identified between ToMMV and ToMV. To alleviate cross-serological reactivity among the tomato-infecting tobamoviruses, a new multiplex RT-PCR was developed to allow for species-specific detection and identification of TMV, ToMV, and ToMMV. In addition, we observed resistance breaking by ToMMV on selected tomato cultivars that were resistant to ToMV. This has caused serious concerns to tomato growers worldwide. In conclusion, the characterization in molecular and biological properties of ToMMV would provide us with fundamental knowledge to manage this emerging virus on tomato and other solanaceous crops in the U.S. and around the world.

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Bidens mottle virus Identified in Tropical Soda Apple in Florida

Plant Disease ◽

10.1094/pdis-91-7-0905a ◽

2007 ◽

Vol 91 (7) ◽

pp. 905-905 ◽

Cited By ~ 3

Author(s):

C. A. Baker ◽

I. Kamenova ◽

R. Raid ◽

S. Adkins

Keyword(s):

Host Range ◽

Mosaic Virus ◽

Plant Viruses ◽

Mottle Virus ◽

Tomato Mosaic Virus ◽

Enzyme Linked Immunosorbent Assay ◽

Rt Pcr ◽

Pcr Product ◽

Potyvirus Species ◽

Tropical Soda Apple

Tropical soda apple (TSA) (Solanum viarum Dunal), a plant native to South America, was first identified in Florida in 1988 (4). It rapidly became a noxious weed in pastures throughout the state and it is known to be a reservoir for Cucumber mosaic virus, Potato leafroll virus, Potato virus Y (PVY), Tobacco etch virus (TEV), Tomato mosaic virus, and Tomato mottle virus, viruses that infect important vegetable crops in Florida (3). During a routine survey of Florida weeds during May of 2004, a TSA plant with chlorotic, young leaves found near Okeechobee, FL was determined to be infected with a potyvirus by using a commercially available enzyme linked immunosorbent assay kit (Agdia, Elkhart, IN). The results of a host range study indicated this potyvirus was neither PVY nor TEV. The virus caused local lesions in Chenopodium amaranticolor and systemic symptoms in C quinoa, Coreopsis sp. (C. A. Baker, unpublished), Helianthus annus, Nicotiana benthamiana, Petunia × hybrida, Verbena hybrida, and Zinnia elegans. It did not infect Gomphrena globosa, N. glutinosa, Pisum sativum, or Phaseolus vulgaris (1). Cylindrical inclusions consistent with those observed in plants infected with Bidens mottle virus (BiMoV) were observed in Z. elegans. Immunodiffusion tests with antiserum to BiMoV (Department of Plant Pathology, University of Florida) gave a reaction of identity with leaf extracts of the symptomatic zinnia, a known sample of BiMoV originally isolated from Bidens pilosa and a recent isolate of BiMoV from lettuce in Belle Glade, FL (C. A. Baker and R. Raid, unpublished). A partial polyprotein gene fragment (GenBank Accession No. EF467235) was amplified from total RNA of an inoculated C. quinoa plant by reverse transcription (RT)-PCR with previously described degenerate potyvirus primers (2). Analysis of the RT-PCR product sequence confirmed the host range results and indicated that the potyvirus infecting TSA was neither PVY nor TEV. However, the nucleotide and deduced amino acid sequences of a 247-bp portion of the RT-PCR product were 94 and 98% identical, respectively, with the coat protein sequence (GenBank Accession No. AF538686) of Sunflower chlorotic spot virus (SCSV). SCSV is a tentative potyvirus species described from Taiwan that is not yet recognized as an accepted species by the International Committee on Taxonomy of Viruses. On the basis of our concurrent host range, inclusion body, and serological data, it is likely that SCSV is in actuality the previously described and currently accepted potyvirus species BiMoV, for which no previous sequence data existed. As part of a comprehensive viral disease management plan, it is recommended that TSA plants growing in and around lettuce-production areas be controlled along with other weed hosts of this virus. References: (1) A. A. Brunt et al., eds. Plant Viruses Online: Descriptions and Lists from the VIDE Database. Version: 20 at http://biology.anu.edu.au/Groups/MES/vide/ , 1996. (2) A. Gibbs and A. J. Mackenzie. Virol. Methods 63:9, 1997. (3) R. J. McGovern et al. Int. J. Pest Manag. 40:270, 1994. (4) J. J. Mullahey et al. Weed Technol. 7:783, 1993.

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Tropical soda apple mosaic virus Identified in Solanum capsicoides in Florida

Plant Disease ◽

10.1094/pdis-91-9-1204a ◽

2007 ◽

Vol 91 (9) ◽

pp. 1204-1204 ◽

Cited By ~ 2

Author(s):

S. Adkins ◽

G. McAvoy ◽

E. N. Rosskopf

Keyword(s):

Sequence Analysis ◽

Mosaic Virus ◽

Sandwich Elisa ◽

Tomato Mosaic Virus ◽

Rt Pcr ◽

Degenerate Primers ◽

Lee County ◽

Cp Gene ◽

Local Lesions ◽

Tropical Soda Apple

Red soda apple (Solanum capsicoides All.), a member of the Solanaceae, is a weed originally from Brazil (3). It is a perennial in southern Florida and is characterized by abundant prickles on stems, petioles, and leaves. Prickles on stems are more dense than those on its larger, noxious weed relative, tropical soda apple (Solanum viarum Dunal), and the mature red soda apple fruits are bright red in contrast to the yellow fruits of tropical soda apple (2). Virus-like foliar symptoms of light and dark green mosaic were observed on the leaves of a red soda apple in a Lee County cow pasture during a tropical soda apple survey during the fall of 2004. The appearance of necrotic local lesions following inoculation of Nicotiana tabacum cv. Xanthi nc with sap from the symptomatic red soda apple leaves suggested the presence of a tobamovirus. Tropical soda apple mosaic virus (TSAMV), a recently described tobamovirus isolated from tropical soda apple in Florida, was specifically identified by a double-antibody sandwich-ELISA (1). An additional six similarly symptomatic red soda apple plants were later collected in the Devils Garden area of Hendry County. Inoculation of N. tabacum cv. Xanthi nc with sap from each of these symptomatic plants also resulted in necrotic local lesions. Sequence analysis of the TSAMV coat protein (CP) gene amplified from total RNA by reverse transcription (RT)-PCR with a mixture of upstream (SolA5′CPv = 5′-GAACTTWCAGAAGMAGTYGTTGATGAGTT-3′; SolB5′CPv = 5′-GAACTCACTGARRMRGTTGTTGAKGAGTT-3′) and downstream (SolA3′CPvc = 5′-CCCTTCGATTTAAGTGGAGGGAAAAAC-3′; SolB3′CPvc = 5′-CGTTTMKATTYAAGTGGASGRAHAAMCACT-3′) degenerate primers flanking the CP gene of Solanaceae-infecting tobamoviruses confirmed the presence of TSAMV in all plants from both locations. Nucleotide and deduced amino acid sequences of the 483-bp CP gene were both 98 to 99% identical to the original TSAMV CP gene sequences in GenBank (Accession No. AY956381). TSAMV was previously identified in tropical soda apple in these two locations in Lee and Hendry counties and three other areas in Florida (1). Sequence analysis of the RT-PCR products also revealed the presence of Tomato mosaic virus in the plant from Lee County. To our knowledge, this represents the first report of natural TSAMV infection of any host other than tropical soda apple and suggests that TSAMV may be more widely distributed in solanaceous weeds than initially reported. References: (1) S. Adkins et al. Plant Dis. 91:287, 2007. (2) N. Coile. Fla. Dep. Agric. Consum. Serv. Div. Plant Ind. Bot. Circ. 27, 1993. (3) U.S. Dep. Agric., NRCS. The PLANTS Database. National Plant Data Center. Baton Rouge, LA. Published online, 2006.

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First Report of Tomato Brown Rugose Fruit Virus Infecting Tomato Crop in Saudi Arabia

Plant Disease ◽

10.1094/pdis-05-21-1065-pdn ◽

2021 ◽

Author(s):

Ahmed Sabra ◽

Mohammed Ali Al Saleh ◽

I. M. Alshahwan ◽

Mahmoud A. Amer

Keyword(s):

Saudi Arabia ◽

Mosaic Virus ◽

Solanum Lycopersicum ◽

Tomato Mosaic Virus ◽

Enzyme Linked Immunosorbent Assay ◽

Rt Pcr ◽

First Report ◽

Pcr Products ◽

Dark Green ◽

Leaf Symptoms

Tomato (Solanum lycopersicum L.) is the most economically important member of family Solanaceae and cultivated worldwide and one of the most important crops in Saudi Arabia. The aim of this study is screening of the most common viruses in Riyadh region and identified the presence of tomato brown rugose fruit virus (ToBRFV) in Saudi Arabia. In January 2021, unusual fruit and leaf symptoms were observed in several greenhouses cultivating tomatoes commercially in Riyadh Region, Saudi Arabia. Fruit symptoms showed irregular brown spots, deformation, and yellowing spots which render the fruits non-marketable, while the leaf symptoms included mottling, mosaic with dark green wrinkled and narrowing. These plants presented the symptoms similar to those described in other studies (Salem et al., 2015, Luria et al., 2017). A total 45 Symptomatic leaf samples were collected and tested serologically against suspected important tomato viruses including: tomato chlorosis virus, tomato spotted wilt virus, tomato yellow leaf curl virus, tomato chlorotic spot virus, tomato aspermy virus, tomato bushy stunt virus, tomato black ring virus, tomato ringspot virus, tomato mosaic virus, pepino mosaic virus and ToBRFV using Enzyme linked immunosorbent assay (ELISA) test (LOEWE®, Biochemica, Germany), according to the manufacturers' instructions. The obtained results showed that 84.4% (38/45) of symptomatic tomato samples were infected with at least one of the detected viruses. The obtained results showed that 55.5% (25/45) of symptomatic tomato samples were found positive to ToBRFV, three out of 25 samples (12%) were singly infected, however 22 out of 45 (48.8%) had mixed infection between ToBRFV and with at least one of tested viruses. A sample with a single infection of ToBRFV was mechanically inoculated into different host range including: Chenopodium amaranticolor, C. quinoa, C. album, C. glaucum, Nicotiana glutinosa, N. benthamiana, N. tabacum, N. occidentalis, Gomphrena globosa, Datura stramonium, Solanum lycopersicum, S. nigrum, petunia hybrida and symptoms were observed weekly and the systemic presence of the ToBRFV was confirmed by RT-PCR and partial nucleotide sequence. A Total RNA was extracted from DAS-ELISA positive samples using Thermo Scientific GeneJET Plant RNA Purification Mini Kit. Reverse transcription-Polymerase chain reaction (RT-PCR) was carried out using specific primers F-3666 (5´-ATGGTACGAACGGCGGCAG-3´) and R-4718 (5´-CAATCCTTGATGTG TTTAGCAC-3´) which amplified a fragment of 1052 bp of Open Reading Frame (ORF) encoding the RNA-dependent RNA polymerase (RdRp). (Luria et al. 2017). RT-PCR products were analyzed using 1.5 % agarose gel electrophoresis. RT-PCR products were sequenced in both directions by Macrogen Inc. Seoul, South Korea. Partial nucleotide sequences obtained from selected samples were submitted to GenBank and assigned the following accession numbers: MZ130501, MZ130502, and MZ130503. BLAST analysis of Saudi isolates of ToBRFV showed that the sequence shared nucleotide identities ranged between 98.99 % to 99.50 % among them and 98.87-99.87 % identity with ToBRFV isolates from Palestine (MK881101 and MN013187), Turkey (MK888980, MT118666, MN065184, and MT107885), United Kingdom (MN182533), Egypt (MN882030 and MN882031), Jordan (KT383474), USA (MT002973), Mexico (MK273183 and MK273190), Canada (MN549395) and Netherlands (MN882017, MN882018, MN882042, MN882023, MN882024, and MN882045). To our knowledge, this is the first report of occurrence of ToBRFV infecting tomato in Saudi Arabia which suggests its likely introduction by commercial seeds from countries reported this virus and spread in greenhouses through mechanical means. The author(s) declare no conflict of interest. Keywords: Tomato brown rugose fruit virus, tomato, ELISA, RT-PCR, Saudi Arabia References: Luria N, et al., 2017. PLoS ONE 12(1): 1-19. Salem N, et al., 2015. Archives of Virology 161(2): 503-506. Fig. 1. Symptoms caused by ToBRFV showing irregular brown spots, deformation, yellowing spots on fruits (A, B, C) and bubbling and mottling, mosaic with dark green wrinkled and narrowing on leaf (D).

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First Report of Alfalfa mosaic virus in Viburnum lucidum in Spain

Plant Disease ◽

10.1094/pdis-92-7-1132b ◽

2008 ◽

Vol 92 (7) ◽

pp. 1132-1132 ◽

Cited By ~ 2

Author(s):

M. C. Cebrián ◽

M. C. Córdoba-Sellés ◽

A. Alfaro-Fernández ◽

J. A. Herrera-Vásquez ◽

C. Jordá

Keyword(s):

Mosaic Virus ◽

Natural Infection ◽

Alfalfa Mosaic Virus ◽

The United States ◽

Rt Pcr ◽

Water Control ◽

First Report ◽

Pcr Product ◽

Pcr Assays ◽

The Mediterranean

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

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Natural Occurrence of Viruses in Lycopersicon spp. in Ecuador

Plant Disease ◽

10.1094/pd-89-1244c ◽

2005 ◽

Vol 89 (11) ◽

pp. 1244-1244 ◽

Cited By ~ 6

Author(s):

S. Soler ◽

C. López ◽

F. Nuez

Keyword(s):

Mosaic Virus ◽

Potato Virus ◽

Potato Virus X ◽

Tomato Mosaic Virus ◽

Enzyme Linked Immunosorbent Assay ◽

Natural Occurrence ◽

Single Plant ◽

Rt Pcr ◽

Specific Primers ◽

In The Wild

The Andean region is home of important genetic diversity for the genus Lycopersicon. A survey of three asymptomatic populations of L. hirsutum, 17 of L. parviflorum, 188 of L. pimpinellifolium, and four cultivated populations of L. esculentum was made in nine departments of Ecuador. Samples were analyzed serologically for Tomato spotted wilt virus (TSWV), Tomato mosaic virus (ToMV), Tobacco mosaic virus (TMV), Cucumber mosaic virus (CMV), Potato virus Y (PVY), Potato virus X (PVX), Groundnut ringspot virus (GRSV), Tomato chlorosis spot virus (TCSV), and Pepino mosaic virus (PepMV). Samples positive as determined using double-antibody sandwich enzyme-linked immunosorbent assay (absorbance values three times higher than negative controls) were analyzed using reverse transcription-polymerase chain reaction (RT-PCR) with virus-specific primers. L pimpinellifolium was the only species of the four found to be infected with viruses. In the department of Manabí, ToMV was detected in 15 of 16 plants from one population, but only a single plant was infected with PepMV. In this department, PepMV was also detected in a single-plant population that corresponded to a volunteer plant found in the wild and TSWV was detected in another plant. In Esmeraldas and Guayas, two single-plant populations were found infected with PepMV and CMV, respectively. TMV, PVY, PVX, GRSV, and TCSV were not detected in this survey. Specific primers were selected for ToMV (To1/To2, genome coordinates 3498-3518/4902-4922, AJ417701), PepMV (Pe1/Pe2 genome coordinates 5030-5050/5913-5935, AJ606359), CMV (Cm1/Cm2 genome coordinates 541-561/1756-1779, D00356), and TSWV (Ts1/Ts2 genome coordinates 4078-4101/4738-4769, AF208498). Amplicons of the expected size were obtained using RT-PCR and then cloned and sequenced. DNA fragments of ToMV, PepMV, and TSWV showed identities greater than 99% with respective sequences in the GenBank database. The highest identity of the CMV DNA fragment was 92% with an isolate from Indonesia (AB042292). The occurrence of viruses such as CMV, ToMV, and TSWV in coastal Ecuador was not surprising. However, infected plants were not found among the samples collected in the departments of Azuay, Carchí, El Oro, Imbabura, Loja, and Pichincha in eastern Ecuador. L. chilense, L. chmielewskii, L. parviflorum, and L. peruvianum were previously reported as natural hosts of PepMV in central and southern Peru (2), and the virus was also detected in L. esculentum in Chile (1). Our results show that PepMV now occurs in wild L. pimpinellifolium populations along the Pacific coast of the South American continent and that it must have efficient means of transmission, although no specific vectors have as yet been identified for this virus. To our knowledge, this is the first report of PepMV in Ecuador and L. pimpinellifolium as a natural host of PepMV. References: (1) M. Muñoz et al. Fitopatología 37:67, 2002. (2) S. Soler et al. J. Phytopathol. 150:49, 2002.

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First Report of Peanut mottle virus Infecting Soybean in South Korea

Plant Disease ◽

10.1094/pdis-04-14-0356-pdn ◽

2014 ◽

Vol 98 (9) ◽

pp. 1285-1285 ◽

Cited By ~ 5

Author(s):

S. Lim ◽

Y.-H. Lee ◽

D. Igori ◽

F. Zhao ◽

R. H. Yoo ◽

...

Keyword(s):

Nucleotide Sequence ◽

South Korea ◽

Mosaic Virus ◽

Soybean Mosaic Virus ◽

Nucleotide Sequences ◽

Mottle Virus ◽

Post Inoculation ◽

Rt Pcr ◽

First Report ◽

Pcr Product

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

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