Differential Detection of the Tobamoviruses Tomato Mosaic Virus (ToMV) and Tomato Brown Rugose Fruit Virus (ToBRFV) Using CRISPR-Cas12a.
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ABSTRACT:
SUBMITTER: Alon DM
PROVIDER: S-EPMC8234260 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundTobamoviruses, including tomato brown rugose fruit virus (ToBRFV) on tomato and pepper, and cucumber green mottle mosaic virus (CGMMV) on cucumber and watermelon, have caused many disease outbreaks around the world in recent years. With seed-borne, mechanical transmission and resistant breaking traits, tobamoviruses pose serious threat to vegetable production worldwide. With the absence of a commercial resistant cultivar, growers are encouraged to take preventative measures to manage those highly contagious viral diseases. However, there is no information available on which disinfectants are effective to deactivate the virus infectivity on contaminated hands, tools and equipment for these emerging tobamoviruses. The purpose of this study was to evaluate a collection of 16 chemical disinfectants for their effectiveness against mechanical transmission of two emerging tobamoviruses, ToBRFV and CGMMV.MethodsBioassay was used to evaluate the efficacy of each disinfectant based on virus infectivity remaining in a prepared virus inoculum after three short exposure times (10 s, 30 s and 60 s) to the disinfectant and inoculated mechanically on three respective test plants (ToBRFV on tomato and CGMMV on watermelon). Percent infection of plants was measured through symptom observation on the test plants and the presence of the virus was confirmed through an enzyme-linked immunosorbent assay with appropriate antibodies. Statistical analysis was performed using one-way ANOVA based on data collected from three independent experiments.ResultsThrough comparative analysis of percent infection of test plants, a similar trend of efficacy among 16 disinfectants was observed between the two pathosystems. Four common disinfectants with broad spectrum activities against two different tobamoviruses were identified. Those effective disinfectants with 90-100% efficacy against both tobamoviruses were 0.5% Lactoferrin, 2% Virocid, and 10% Clorox, plus 2% Virkon against CGMMV and 3% Virkon against ToBRFV. In addition, SP2700 generated a significant effect against CGMMV, but poorly against ToBRFV.ConclusionIdentification of common disinfectants against ToBRFV and CGMMV, two emerging tobamoviruses in two different pathosystems suggest their potential broader effects against other tobamoviruses or even other viruses.
| S-EPMC7787650 | biostudies-literature
Project description:Tomato brown rugose fruit virus (ToBRFV) is a Tobamovirus that was first observed in 2014 and 2015 on tomato plants in Israel and Jordan respectively. Since the first description, the virus has been reported from all continents except Oceania and Antarctica, and has been found infecting both tomato and pepper crops. In October 2019, the Dutch National Plant Protection Organization received a ToBRFV infected tomato sample as part of a generic survey targeting tomato pests. Presence of the virus was verified using Illumina sequencing. A follow-up survey was initiated to determine the extent of ToBRFV presence in the Dutch tomato horticulture and identify possible linkages between ToBRFV genotypes, companies and epidemiological traits. Nextstrain was used to visualize these potential connections. By November 2019, 68 companies had been visited of which 17 companies were found to be infected. The 50 ToBRFV genomes from these outbreak locations group in three main clusters, which are hypothesized to represent three original sources. No correlation was found between genotypes, companies and epidemiological traits, and the source(s) of the Dutch ToBRFV outbreak remain unknown. This paper describes a Nextstrain build containing ToBRFV genomes up to and including November 2019. Sharing data with this interactive online tool will enable the plant virology field to better understand and communicate the diversity and spread of this new virus. Organizations are invited to share data or materials for inclusion in the Nextstrain build, which can be accessed at https://nextstrain.nrcnvwa.nl/ToBRFV/20191231.
| S-EPMC7544112 | biostudies-literature
Project description:The tobamovirus tomato brown rugose fruit virus (ToBRFV), a major threat to tomato production worldwide, has recently been documented in mixed infections with the potexvirus pepino mosaic virus (PepMV) CH2 strain in traded tomatoes in Israel. A study of greenhouse tomato plants in Israel revealed severe new viral disease symptoms including open unripe fruits and yellow patched leaves. PepMV was only detected in mixed infections with ToBRFV in all 104 tested sites, using serological and molecular analyses. Six PepMV isolates were identified, all had predicted amino acids characteristic of CH2 mild strains excluding an isoleucine at amino acid position 995 of the replicase. High-throughput sequencing of viral RNA extracted from four selected symptomatic plants showed solely the ToBRFV and PepMV, with total aligned read ratios of 40.61% and 11.73%, respectively, indicating prevalence of the viruses. Analyses of interactions between the co-infecting viruses by sequential and mixed viral inoculations of tomato plants, at various temperatures, showed a prominent increase in PepMV titers in ToBRFV pre-inoculated plants and in mixed-infected plants at 18-25 °C, compared to PepMV-single inoculations, as analyzed by Western blot and quantitative RT-PCR tests. These results suggest that Israeli mild PepMV isolate infections, preceded by ToBRFV, could induce symptoms characteristic of PepMV aggressive strains.
| S-EPMC7472245 | biostudies-literature
Project description:Tomato brown rugose fruit virus (ToBRFV) is a member of Tobamovirus infecting tomato and pepper. Within North America, both the United States and Mexico consider ToBRFV to be a regulated pest. In Canada, the presence of ToBRFV has been reported, but an efficient diagnostic system has not yet been established. Here, we describe the development and assessment of a loop-mediated isothermal amplification (LAMP)-based assay to detect ToBRFV. The LAMP test was efficient and robust, and results could be obtained within 35 min with an available RNA sample. Amplification was possible when either water bath or oven were used to maintain the temperature at isothermal conditions (65°C), and results could be read by visual observation of colour change. Detection limit of the LAMP was eight target RNA molecules. Under the experimental conditions tested, LAMP was as sensitive as qPCR and 100 times more sensitive than the currently used RT-PCR. We recommend this sensitive, efficient LAMP protocol to be used for routine lab testing of ToBRFV.
| S-EPMC7313975 | biostudies-literature
Project description:Nucleotide-binding leucine-rich repeat (NLR) plant immune receptors mediate the recognition and activation of defense signaling pathways in response to intra- and extracellular pathogens. Several NLR such as Tm-2 and Tm-22 have been introgressed into commercial solanaceous varieties to confer protection against different tobamoviruses. Particularly, Tm-22 was used during recent decades to confer resistance against tobacco mosaic virus, tomato mottle mosaic virus and tomato mosaic virus, which recognizes the viral movement protein (MP). However, tomato brown rugose fruit virus(ToBRFV), a novel tobamovirus, can avoid the protection conferred by Tm-22 due to the presence of key substitutions in the MP. The aim of this work was to identify the key amino acid residues involved in the interaction between Tm-22 and ToBRFV MP through bioinformatic analyses, and to identify potential Tm-22 mutations that could generate greater binding affinity. In silico 3D structure prediction, molecular docking, and computational affinity methods were performed. We predicted that R350, H384 and K385 Tm-22 residues are relevant for the interaction with MP, and two mutations (H384W and K385L) were identified as putative sites to increase the affinity of Tm-22 to the MP with the potential elicitation of resistance against ToBRFV.
| S-EPMC9562835 | biostudies-literature
Project description:The reaction of 636 Solanum (sections Lycopersicon and Juglandifolia) accessions were evaluated under greenhouse conditions after mechanical inoculation with a Jordanian isolate of the new tobamovirus tomato brown rugose fruit virus (ToBRFV). Local and systemic infections were assayed by symptoms evaluation and virus detection via biotests and RT-PCR. All cultivated tomatoes (Solanum lycopersicum) and the great majority of wild tomato accessions proved susceptible to ToBRFV. They showed a wide range of symptoms (mosaic, leaf deformations, mottling, shoestring, and stunting). Twenty-six accessions representing S. lycopersicum var. cerasiforme, S. pimpinellifolium, S. habrochaites, and S. chilense were tolerant. High levels of resistance have been demonstrated in three accessions of S. ochrantum, a close relative to wild tomatoes (member of the sect. Juglandifolia) not only to ToBRFV but also to the tobamoviruses, tobacco mosaic virus (TMV) and tomato mosaic virus (ToMV). After mechanical inoculation, the three tobamoviruses could be detected only in inoculated leaves in the accessions LA2160, LA2162, and LA 2166, which remained symptomless. However, two other S. ochrantum accessions PI 473,498 and PI 230,519 reacted unusually. They were demonstrated highly resistant to TMV and ToMV, but proved transiently susceptible to ToBRFV showing mild systemic mosaic followed by total recovery from symptoms and the virus.Supplementary informationThe online version contains supplementary material available at 10.1007/s41348-021-00535-x.
| S-EPMC8501354 | biostudies-literature
Project description:During the growing season of 2021-2022, a total of 145 symptomatic tomato leaf and fruit samples were collected from different locations in Riyadh Region, Saudi Arabia, showing a moderate-to-severe mosaic with dark green wrinkling, blistering, narrowing, and deformation with necrosis spot on tomato leaves, while irregular brown necrotic lesions, deformation, and yellowing spots rendering the fruits non-marketable were observed on tomato fruits. These samples were tested serologically against important tomato viruses using enzyme-linked immunosorbent assay (ELISA), and the obtained results showed that 52.4% of symptomatic tomato samples were found positive for Tomato brown rugose fruit virus (ToBRFV), wherein 12 out of 76 samples were singly infected; however, 64 out of 145 had mixed infection. A sample with a single infection of ToBRFV was used for mechanical inoculation into a range of different host plants; symptoms were observed weekly, and the presence of the ToBRFV was confirmed by ELISA and reverse transcription-polymerase chain reaction (RT-PCR). A total RNA was extracted from selected ELISA-positive samples, and RT-PCR was carried out using specific primers F-3666 and R-4718, which amplified a fragment of 1052 bp. RT-PCR products were sequenced in both directions, and partial genome nucleotide sequences were submitted to GenBank under the following accession numbers: MZ130501, MZ130502, and MZ130503. BLAST analysis of Saudi isolates of ToBRFV showed that the sequence shared nucleotide identities (99-99.5%) among them and 99-100% identity with ToBRFV isolates in different countries. A ToBRFV isolate (MZ130503) was selected for mechanical inoculation and to evaluate symptom severity responses of 13 commonly grown tomato cultivars in Saudi Arabia. All of the tomato cultivars showed a wide range of symptoms. The disease severity index of the tested cultivars ranged between 52% and 96%. The importance ToBRFV disease severity and its expanding host range due to its resistance breaking ability was discussed.
| S-EPMC9692878 | biostudies-literature
Project description:Tomato brown rugose fruit virus (ToBRFV) is an economically important seed and mechanically transmitted pathogen of significant importance to tomato production around the globe. Synergistic interaction with pepino mosaic virus (PepMV), another seed and mechanically transmitted virus, and long-distance dissemination of these two viruses via contaminated tomato fruits through global marketing were previously suggested. In 2019, we detected both viruses in several grocery store-purchased tomatoes in South Florida, USA. In this study, to identify potential sources of inoculum, co-infection status, prevalence, and genomic diversity of these viruses, we surveyed symptomatic and asymptomatic imported tomatoes sold in ten different groceries in four cities in South Florida. According to the product labels, all collected tomatoes originated from Canada, Mexico, or repacking houses in the United States. With high prevalence levels, 86.5% of the collected samples were infected with ToBRFV, 90% with PepMV alone, and 73% were mixed-infected. The phylogenetic study showed no significant correlations between ToBRFV genomic diversity and the tomato label origin. Phylogenetic analysis of PepMV isolates revealed the prevalence of the PepMV strains, Chilean (CH2) and recombinant (US2). The results of this study highlight the continual presence of PepMV and ToBRFV in imported tomatoes in Florida grocery stores.
| S-EPMC10748365 | biostudies-literature
Project description:Tomato is an important vegetable in the United States and around the world. Recently, tomato brown rugose fruit virus (ToBRFV), an emerging tobamovirus, has impacted tomato crops worldwide and can result in fruit loss. ToBRFV causes severe symptoms, such as mosaic, puckering, and necrotic lesions on leaves; other symptoms include brown rugose and marbling on fruits. More importantly, ToBRFV can overcome resistance in tomato cultivars carrying the Tm-22 locus. In this study, we recovered ToBRFV sequences from tomato seeds, leaves, and fruits from the U.S., Mexico, and Peru. Samples were pre-screened using a real-time RT-PCR assay prior to high-throughput sequencing. Virus draft genomes from 22 samples were assembled and analyzed against more than 120 publicly available genomes. Overall, most sequenced isolates were similar to each other and did not form a distinct population. Phylogenetic analysis revealed three clades within the ToBRFV population. Most of the isolates (95%) clustered in clade 3. Genetic analysis revealed differentiation between the three clades indicating minor divergence occurring. Overall, pairwise identity showed limited genetic diversity among the isolates in this study with worldwide isolates, with a pairwise identity ranging from 99.36% and 99.97%. The overall population is undergoing high gene flow and population expansion with strong negative selection pressure at all ToBRFV genes. Based on the results of this study, it is likely that the limited ToBRFV diversity is associated with the rapid movement and eradication of ToBRFV-infected material between countries.
| S-EPMC9784425 | biostudies-literature
Project description:Tomato brown rugose fruit virus (ToBRFV) is a contact-transmitted tobamovirus affecting many tomato growing regions of the world. This study investigated the effects of different glasshouse surfaces on the survival of the virus; the efficacy of different disinfectants; and heat treatment against ToBRFV (surfaces included steel, aluminium, hard plastic, polythene, glass and concrete). A bioassay followed by ELISA was used to check virus viability. ToBRFV survived for at least 7 days on all surfaces tested and on some for at least 6 months. The virus survived for over two hours on hands and gloves. Hand washing was shown to be unreliable for the removal of the virus. Glutaraldehyde and quaternary ammonium compound disinfectants were effective at one hour on all surfaces. Some other disinfectants were effective at one hour of contact time, on all surfaces except concrete. Sodium hypochlorite was partially effective against ToBRFV, even on concrete. A 5 min soak of plastic trays in water at 90 °C was effective at denaturing ToBRFV; however, 5 min at 70 °C was not. Heating infected sap showed the thermal inactivation point to be 90 °C, confirming the hot water treatment results and showing that deactivation was due to the heat treatment and not a washing effect of the water.
| S-EPMC10611295 | biostudies-literature