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

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

Plants ◽

10.3390/plants10061256 ◽

2021 ◽

Vol 10 (6) ◽

pp. 1256

Author(s):

Dan Mark Alon ◽

Hagit Hak ◽

Menachem Bornstein ◽

Gur Pines ◽

Ziv Spiegelman

Keyword(s):

Mosaic Virus ◽

Tomato Mosaic Virus ◽

Differential Detection ◽

Rt Pcr ◽

Specific Detection ◽

Novel Approach ◽

Pcr Product ◽

Species Specific ◽

User Friendly ◽

Efficient Sequence

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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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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Identification of Tomato Infecting Viruses That Co-Isolate with Nanovesicles Using a Combined Proteomics and Electron-Microscopic Approach

Nanomaterials ◽

10.3390/nano11081922 ◽

2021 ◽

Vol 11 (8) ◽

pp. 1922

Author(s):

Ramila Mammadova ◽

Immacolata Fiume ◽

Ramesh Bokka ◽

Veronika Kralj-Iglič ◽

Darja Božič ◽

...

Keyword(s):

Electron Microscopy ◽

Mosaic Virus ◽

Protein Identification ◽

Plant Viruses ◽

Size Exclusion ◽

Tomato Mosaic Virus ◽

High Yield ◽

Plant Resources ◽

Electron Microscopic ◽

Viral Particles

Plant-derived nanovesicles (NVs) have attracted interest due to their anti-inflammatory, anticancer and antioxidative properties and their efficient uptake by human intestinal epithelial cells. Previously we showed that tomato (Solanum lycopersicum L.) fruit is one of the interesting plant resources from which NVs can be obtained at a high yield. In the course of the isolation of NVs from different batches of tomatoes, using the established differential ultracentrifugation or size-exclusion chromatography methods, we occasionally observed the co-isolation of viral particles. Density gradient ultracentrifugation (gUC), using sucrose or iodixanol gradient materials, turned out to be efficient in the separation of NVs from the viral particles. We applied cryogenic transmission electron microscopy (cryo-TEM), scanning electron microscopy (SEM) for the morphological assessment and LC–MS/MS-based proteomics for the protein identification of the gradient fractions. Cryo-TEM showed that a low-density gUC fraction was enriched in membrane-enclosed NVs, while the high-density fractions were rich in rod-shaped objects. Mass spectrometry–based proteomic analysis identified capsid proteins of tomato brown rugose fruit virus, tomato mosaic virus and tomato mottle mosaic virus. In another batch of tomatoes, we isolated tomato spotted wilt virus, potato virus Y and southern tomato virus in the vesicle sample. Our results show the frequent co-isolation of plant viruses with NVs and the utility of the combination of cryo-TEM, SEM and proteomics in the detection of possible viral contamination.

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Molecular and biological characterization of an isolate of Tomato mottle mosaic virus (ToMMV) infecting tomato and other experimental hosts in a greenhouse in Valencia, Spain

10.1101/063255 ◽

2016 ◽

Cited By ~ 4

Author(s):

S. Ambrós ◽

F. Martínez ◽

P. Ivars ◽

C. Hernández ◽

F. de la Iglesia ◽

...

Keyword(s):

Mosaic Virus ◽

Plant Species ◽

Rna Virus ◽

Diagnostic Techniques ◽

Plant Viruses ◽

Reservoir Host ◽

Molecular Techniques ◽

Tomato Mosaic Virus ◽

Molecular Diagnostic ◽

Molecular Diagnostic Techniques

AbstractTomato is known to be a natural and experimental reservoir host for many plant viruses. In the last few years a new tobamovirus species, Tomato mottle mosaic virus (ToMMV), has been described infecting tomato and pepper plants in several countries worldwide. Upon observation of symptoms in tomato plants growing in a greenhouse in Valencia, Spain, we aimed to ascertain the etiology of the disease. Using standard molecular techniques, we first detected a positive sense single-stranded RNA virus as the probable causal agent. Next, we amplified, cloned and sequenced a ~3 kb fragment of its RNA genome which allowed us to identify the virus as a new ToMMV isolate. Through extensive assays on distinct plant species, we validated Koch’s postulates and investigated the host range of the ToMMV isolate. Several plant species were locally and/or systemically infected by the virus, some of which had not been previously reported as ToMMV hosts despite they are commonly used in research greenhouses. Finally, two reliable molecular diagnostic techniques were developed and used to assess the presence of ToMMV in different plants species. We discuss the possibility that, given the high sequence homology between ToMMV and Tomato mosaic virus, the former may have been mistakenly diagnosed as the latter by serological methods.

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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 a Begomovirus Associated with Tomato Yellow Leaf Curl Disease in Ethiopia

Plant Disease ◽

10.1094/pd-90-0974a ◽

2006 ◽

Vol 90 (7) ◽

pp. 974-974 ◽

Cited By ~ 6

Author(s):

S. L. Shih ◽

S. K. Green ◽

W. S. Tsai ◽

L. M. Lee ◽

J. T. Wang ◽

...

Keyword(s):

Mosaic Virus ◽

Tomato Yellow Leaf ◽

Tomato Mosaic Virus ◽

Enzyme Linked Immunosorbent Assay ◽

Yellow Leaf ◽

First Report ◽

Sequence Identity ◽

Pcr Product ◽

Leaf Curl Disease ◽

Leaf Curl

During December 2003, severe leaf yellowing, leaf curling, and stunting symptoms were observed in tomato (Lycopersicon esculentum) plantings in Melkassa (1,550 m above sea level), Ethiopia. Eleven symptomatic samples were collected and tested for the presence of a begomovirus using polymerase chain reaction (PCR) with the begomovirus-specific degenerate primer pair PAL1v1978/PAR1c715 (3). Samples were also tested for Cucumber mosaic virus (CMV), Potato virus Y (PVY), Tobacco etch virus (TEV), Pepper veinal mottle virus (PVMV), and Tomato mosaic virus (ToMV) using enzyme-linked immunosorbent assay (ELISA). All samples were negative for CMV, PVY, TEV, PVMV, and ToMV. However, the expected 1.4-kb PCR product for begomoviruses was obtained from all samples. DNA-B and DNA-beta were not detectable using PCR with the DNA-B specific primer pairs DNABLC1/DNABLV2 and DNABLC2/ DNABLV2 (2) and the DNA-beta primer pair Beta01/Beta02 (1), respectively. The 1.4-kb PCR product of one sample was cloned and sequenced. On the basis of the sequence of the 1.4-kb DNA product, specific primers were designed to complete the DNA-A sequence. The DNA-A consisted of 2,785 nucleotides (GenBank Accession No. DQ358913) and was found to contain the six predicted open reading frames (ORFs V1, V2, C1, C2, C3, and C4). A BLAST analysis was conducted with geminivirus sequences available in the GenBank database at the National Center for Biotechnology Information (Bethesda, MD), and DNAMAN software (Lynnon Corporation, Quebec, Canada) was used for further comparisons. The DNA-A sequence of the virus associated with yellow leaf curl disease of tomato from Ethiopia showed highest sequence identity (92%) with Tomato yellow leaf curl Mali virus (TYLCMLV; GenBank Accession No. AY502934). On the basis of the DNA-A sequence comparison and the ICTV demarcation of species at 89% sequence identity, the Ethiopian virus is a provisional strain of TYLCMLV described from Mali. To our knowledge, this is the first report of a begomovirus associated with tomato yellow leaf curl disease in Ethiopia. References: (1) R. W. Briddon et al. Mol. Biotechnol. 20:315, 2002. (2) S. K. Green et al. Plant Dis. 85:1286, 2001. (3) M. R. Rojas et al. Plant Dis. 77:340, 1993.

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First Report of Tobacco mild green mosaic virus Infecting Capsicum annuum in Tunisia

Plant Disease ◽

10.1094/pdis-93-7-0761b ◽

2009 ◽

Vol 93 (7) ◽

pp. 761-761 ◽

Cited By ~ 5

Author(s):

M. I. Font ◽

M. C. Córdoba-Sellés ◽

M. C. Cebrián ◽

J. A. Herrera-Vásquez ◽

A. Alfaro-Fernández ◽

...

Keyword(s):

Mosaic Virus ◽

Capsicum Annuum ◽

Polyclonal Antibodies ◽

Plant Viruses ◽

Mottle Virus ◽

Tomato Mosaic Virus ◽

Rt Pcr ◽

Tomato Plants ◽

First Report ◽

Pepper Plants

During the springs of 2007 and 2008, leaf deformations as well as symptoms of mild green and chlorotic mosaic were observed on pepper (Capsicum annuum) plants grown in Monastir (northwest Tunisia) and Kebili (southeast Tunisia). With the support of projects A/5269/06 and A/8584/07 from the Spanish Agency for International Cooperation (AECI), symptomatic leaf samples were analyzed by transmission electron microscopy (TEM) of leaf-dip preparations. Typical tobamovirus-like particles (rigid rods ≈300 nm long) were observed in crude plant extracts. According to literature, at least six tobamoviruses infect peppers: Paprika mild mottle virus (PaMMV); Pepper mild mottle virus (PMMoV); Ribgrass mosaic virus (RMV); Tobacco mild green mosaic virus (TMGMV); Tobacco mosaic virus (TMV); and Tomato mosaic virus (ToMV) (1). Extracts from six symptomatic plants from Monastir and four from Kebili fields tested negative for ToMV, TMV, and PMMoV and tested positive for TMGMV by double-antibody sandwich (DAS)-ELISA using polyclonal antibodies specific to each virus (Loewe Biochemica GMBH, Sauerlach, Germany). To confirm the positive TMGMV results, total RNAs from 10 symptomatic plants that tested positive by ELISA were extracted and analyzed by reverse transcription (RT)-PCR using primers designed to specifically amplify a region of the coat protein gene (CP) of TMGMV (2). The 524-bp TMGMV-CP specific DNA fragment was amplified from all samples, but was not amplified from healthy plants or the sterile water used with negative controls. RT-PCR products were purified and directly sequenced. BLAST analysis of the obtained sequence (GenBank No. EU770626) showed 99 to 98% nucleotide identity with TMGMV isolates PAN-1, DSMZ PV-0113, TMGMV-Pt, and VZ1 (GenBank Nos. EU934035, EF469769, AM262165, and DQ460731, respectively) and less than 69% with PaMMV and PMMoV isolates (GenBank Nos. X72586 and AF103777, respectively). Two TMGMV-positive, singly, infected symptomatic pepper plants collected from Monastir and Kebili were used in mechanical transmissions to new pepper and tomato plants. Inoculated pepper plants exhibited mild chlorosis symptoms and tested positive for TMGMV only; however, inoculated tomato plants cv. Marmande were asymptomatic and tested negative as expected for TMGMV infection (1). To our knowledge, although C. annuum has been shown as a natural host for TMGMV (2), this is the first report of TMGMV in Tunisia. Reference: (1) A. A. Brunt et al. Plant Viruses Online: Descriptions and Lists from the VIDE Database. Version: 20th August 1996. Online publication, 1996. (2) 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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First Report of Tomato torrado virus Infecting Tomato in Single and Mixed Infections with Cucumber mosaic virus in Panama

Plant Disease ◽

10.1094/pdis-93-2-0198a ◽

2009 ◽

Vol 93 (2) ◽

pp. 198-198 ◽

Cited By ~ 20

Author(s):

J. A. Herrera-Vásquez ◽

A. Alfaro-Fernández ◽

M. C. Córdoba-Sellés ◽

M. C. Cebrián ◽

M. I. Font ◽

...

Keyword(s):

Coat Protein ◽

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Potato Virus ◽

Plant Viruses ◽

Tomato Mosaic Virus ◽

Rt Pcr ◽

Tomato Production ◽

Related Sequence ◽

First Report

In February of 2008, in open-field-grown tomato crops (Solanum lycopersicum L.) from the central regions of Coclé, Herrera, Los Santos, and Veraguas of Panama, unusual disease symptoms, including deformation, necrosis, purple margins, interveinal yellowing, downward and upward curling of the leaflets alternately, necrotic lines in sepals and branches, fruits distorted with necrotic lines on the surface, and severe stunting, were observed. Tomato production was seriously damaged. To verify the identity of the disease, five symptomatic tomato plants from four fields of these regions were selected and analyzed by double-antibody sandwich (DAS)-ELISA using specific antibodies to Cucumber mosaic virus (CMV), Potato virus X (PVX), 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 all plants and tested using reverse transcription (RT)-PCR with three pairs of specific primers: one pair designed to amplify 586 bp of the coat protein gene of CMV (CMV-F 5′-CCTCCGCGGATGCTAACTT-3′ and CMV-R 5′-CGGAATCAGACTGGGAGCA-3′) and the other two pairs to Tomato torrado virus (ToTV) that amplify 580 and 574 bp of the polyprotein (4) and coat protein (Vp23) (3) region of RNA2, respectively; and by dot-blot hybridization with a digoxygenin-labeled RNA probe complementary to the aforementioned polyprotein. The serological analysis for PVX, PVY, ToMV, TSWV, and PepMV were negative. ToTV was detected in all samples analyzed. Three of these samples were also positive for CMV by serological and molecular analysis. No differences in symptom expression were observed between plants infected with both viruses or with ToTV alone. RT-PCR products were purified and directly sequenced. BLAST analysis of one CMV sequence (GenBank Accession No. EU934036) showed 98% identity with a CMV sequence from Brazil (most closely related sequence) (GenBank Accession No. AY380812) and 97% with the Fny isolate (CMV subgroup I) (GenBank Accession No. U20668). Two ToTV sequences were obtained (GenBank Accession Nos. EU934037 and FJ357161) and showed 99% and 98% identities with the polyprotein and coat protein region of ToTV from Spain (GenBank Accession No. DQ388880), respectively. CMV is transmitted by aphids and is distributed worldwide with a wide host range (2), while ToTV is transmitted by whiteflies and has only been reported in tomato crops in Spain and Poland and recently on weeds in Spain (1). To our knowledge, this is the first time ToTV has been detected in Panama and the first report of CMV/ToTV mixed infection. References: (1) A. Alfaro-Fernández et al. Plant Dis. 92:831, 2008. (2) A. A. Brunt et al. Plant Viruses Online: Descriptions and Lists from the VIDE Database. Online Publication, 1996. (3) H. Pospieszny et al. Plant Dis. 91:1364, 2007. (4) M. Verbeek et al. Arch. Virol. 152:881, 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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