This review describes the development of the bioassay as a means of quantifying plant viruses, with particular attention to tobamovirus. It delves into various models used to establish a correlation ...between virus particle concentration and the number of induced local lesions (the infectivity dilution curve), including the Poisson, Furumoto and Mickey, Kleczkowski, Growth curve, and modified Poisson models. The parameters of each model are described, and their application or performance in the context of the tobacco mosaic virus is explored. This overview highlights the enduring value of the infectivity dilution curve in tobamovirus quantification, providing valuable insights for researchers or practitioners of bioassays and theoreticians of modeling.
(ToBRFV) is a highly infectious tobamovirus that causes severe disease in tomato (
L.) crops. In Italy, the first ToBRFV outbreak occurred in 2018 in several provinces of the Sicily region. ToBRFV ...outbreak represents a serious threat for tomato crops in Italy and the Mediterranean Basin.
Molecular and biological characterisation of the Sicilian ToBRFV ToB-SIC01/19 isolate was performed, and a sensitive and specific Real-time RT-PCR TaqMan minor groove binder probe method was developed to detect ToBRFV in infected plants and seeds. Moreover, four different sample preparation procedures (immunocapture, total RNA extraction, direct crude extract and leaf-disk crude extract) were evaluated.
The Sicilian isolate ToB-SIC01/19 (6,391 nt) showed a strong sequence identity with the isolates TBRFV-P12-3H and TBRFV-P12-3G from Germany, Tom1-Jo from Jordan and TBRFV-IL from Israel. The ToB-SIC01/19 isolate was successfully transmitted by mechanical inoculations in
L. and
L., but no transmission occurred in
L. The developed real-time RT-PCR, based on the use of a primer set designed on conserved sequences in the open reading frames3, enabled a reliable quantitative detection. This method allowed clear discrimination of ToBRFV from other viruses belonging to the genus
, minimising false-negative results. Using immunocapture and total RNA extraction procedures, the real-time RT-PCR and end-point RT-PCR gave the same comparable results. Using direct crude extracts and leaf-disk crude extracts, the end-point RT-PCR was unable to provide a reliable result. This developed highly specific and sensitive real-time RT-PCR assay will be a particularly valuable tool for early ToBRFV diagnosis, optimising procedures in terms of costs and time.
Reactions of plants in 173 wild tomato accessions belonging to
Solanum habrochaites
and
S. peruvianum
were studied by inoculation with a tobamovirus, tomato brown rugose fruit virus (ToBRFV). Around ...10–50% of plants in nine accessions of
S. habrochaites
and one of
S. peruvianum
were demonstrated to be highly resistant. Resistant plants showed no symptoms at 22–24 °C, and no virus could be detected in their inoculated and newly developed leaves using bioassays and RT-qPCR. ToBRFV-resistant plants were also resistant to tobacco mosaic virus and tomato mosaic virus. The susceptible wild tomatoes were infected systemically with ToBRFV showing different severity of symptoms. When resistant plants inoculated with ToBRFV were incubated in a plant growth chamber at a temperature of 33 °C, they expressed mosaic and deformation symptoms, indicating that the resistance was broken at elevated temperature. However, when these plants were transferred to the greenhouse at 24 °C, their newly emerged leaves showed no symptoms, and the virus could not be detected in the new leaves. Cleft grafting was done with scions from a resistant plant of
S. habrochaites
LA1739 into ToBRFV-infected susceptible tomato rootstock. The scions became infected and showed mosaic symptoms indicating that the resistance was ineffective after grafting. Sequences comparison of Solyc08g075630 loci of nine resistant accessions showed high heterogenity. Only one resistant plant of
S. habrochaites
carried an allele almost identical to the resistance gene reported previously. All other resistant plants may have probably unknown gene(s) of resistance to ToBRFV.
Since the first report of the tobamovirus tomato brown rugose fruit virus (ToBRFV) in 2014, it has become globally distributed. Its rapid spread has been primarily attributed to seed-borne ...transmission. Here, the seed-borne nature of ToBRFV transmission was investigated in different cultivars of tomato, bell pepper, and eggplant. In situ hybridization to localize the virus in reproductive organs of ToBRFV-infected tomato plants revealed that the virus was not present in shoot apices, flower buds, or in ovules during flower opening, indicating the virus may be restricted to the outer integument and transported in the vascular bundles during seed development. However, during early fruit development, the virus was present in the integuments in the ovule. Seeds of tomato cultivars with or without tobamovirus resistance gene
Tm-2
2
transmitted the virus to the progeny seedlings at rates that reflected the ineffectiveness of the gene against ToBRFV. Seeds of bell peppers transmitted ToBRFV at higher rates than tomato seeds, but a bell pepper cultivar that has resistance gene
L
3
was not systemically infected, and its seeds did not harbor the virus. Three eggplant cultivars were systemically infected with ToBRFV but without showing any obvious symptoms, and even though ToBRFV was present in their seeds, the seedlings were not infected. ToBRFV was detected in the seed coats of contaminated tomato and bell pepper seeds, but not in eggplant seed coats. These results indicate mechanistic differences in seed-borne transmission among the three Solanaceae crops.
(ToBRFV) is a new damaging plant virus of great interest from both an economical and research point of view. ToBRFV is transmitted by contact, remains infective for months, and to-date, no resistant ...cultivars have been developed. Due to the relevance of this virus, new effective, sustainable, and operator-safe antiviral agents are needed. Thus, 4-hydroxybenzoic acid was identified as the main product of the alkaline autoxidation at high temperature of the methanolic extract of the leaves of
, known for antiviral activity. The autoxidized extract and 4-hydroxybenzoic acid were assayed in in vitro experiments, in combination with a mechanical inoculation test of tomato plants. Catechinic acid, a common product of rearrangement of catechins in hot alkaline solution, was also tested. Degradation of the viral particles, evidenced by the absence of detectable ToBRFV RNA and the loss of virus infectivity, as a possible consequence of disassembly of the virus coat protein (CP), were shown. Homology modeling was then applied to prepare the protein model of ToBRFV CP, and its structure was optimized. Molecular docking simulation showed the interactions of the two compounds, with the amino acid residues responsible for CP-CP interactions. Catechinic acid showed the best binding energy value in comparison with ribavirin, an anti-tobamovirus agent.
The tobamoviruses tomato brown rugose fruit virus (ToBRFV) and cucumber green mottle mosaic virus (CGMMV) have caused severe crop damages worldwide. Soil-mediated dispersion of the mechanically ...transmitted tobamoviruses constitute a major hindrance toward mitigating disease spread in crops carefully planted under sanitized conditions. Tobamoviruses are viable for months in soil and plant debris and for more than a year adhere to clay. However, a low percentage of infectious foci occur in soil following a tobamovirus-infected growing cycle, rendering disinfection studies of several contaminated plots inconclusive for large-scale crop productions. We have therefore formulated a rigorous platform for studying disinfectant efficacy in greenhouses by pouring a virus inoculum to planting pits prior to disinfectant treatment and by truncating seedling roots before planting, which was otherwise conducted under sanitized conditions. We have found that chlorine-based Taharan was significantly efficient in preventing disease spread of ToBRFV and CGMMV in tomato and cucumber plants, respectively. KlorBack was often as good as Taharan. In addition, a formulation of chlorinated tri-sodium phosphate used at a nonphytotoxic 3% concentration showed disinfection efficiency similar to Taharan effect on ToBRFV infection only. Our study provided a small-scale platform for disinfectant efficacy evaluation necessary for application in tobamovirus-contaminated soil, which commonly occurs in commercial tomato and cucumber greenhouses.
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.
Tomato brown rugose fruit virus (ToBRFV) was identified in Israel during October 2014 in tomato plants (
). These plants, carrying the durable resistance gene against tomato mosaic virus,
, displayed ...severe disease symptoms and losses to fruit yield and quality. These plants were found infected with a tobamovirus similar to that discovered earlier in Jordan. This study was designed to screen and identify tomato genotypes resistant or tolerant to ToBRFV. The identified resistance and tolerance traits were further characterized virologically and genetically. Finally, DNA markers linked to genes controlling these traits were developed as tools to expedite resistance breeding. To achieve these objectives, 160 genotypes were screened, resulting in the identification of an unexpectedly high number of tolerant genotypes and a single genotype resistant to the virus. A selected tolerant genotype and the resistant genotype were further analyzed. Analysis of genetic inheritance revealed that a single recessive gene controls tolerance whereas at least two genes control resistance. Allelic test between the tolerant and the resistant genotype revealed that these two genotypes share a locus controlling tolerance, mapped to chromosome 11. This locus displayed a strong association with the tolerance trait, explaining nearly 91% of its variation in segregating populations. This same locus displayed a statistically significant association with symptom levels in segregating populations based on the resistant genotype. However, in these populations, the locus was able to explain only ~41% of the variation in symptom levels, confirming that additional loci are involved in the genetic control of the resistance trait in this genotype. A locus on chromosome 2, at the region of the
gene, was finally found to interact with the locus discovered on chromosome 11 to control resistance.
Tomato Brown Rugose Fruit Virus (ToBRFV) is a plant pathogen that infects important
crop species and can dramatically reduce tomato crop yields. The ToBRFV has rapidly spread around the globe due to ...its ability to escape detection by antiviral host genes which confer resistance to other tobamoviruses in tomato plants. The development of robust and reproducible methods for detecting viruses in the environment aids in the tracking and reduction of pathogen transmission. We detected ToBRFV in municipal wastewater influent (WWI) samples, likely due to its presence in human waste, demonstrating a widespread distribution of ToBRFV in WWI throughout Ontario, Canada. To aid in global ToBRFV surveillance efforts, we developed a tiled amplicon approach to sequence and track the evolution of ToBRFV genomes in municipal WWI. Our assay recovers 95.7% of the 6393 bp ToBRFV RefSeq genome, omitting the terminal 5' and 3' ends. We demonstrate that our sequencing assay is a robust, sensitive, and highly specific method for recovering ToBRFV genomes. Our ToBRFV assay was developed using existing ARTIC Network resources, including primer design, sequencing library prep, and read analysis. Additionally, we adapted our lineage abundance estimation tool, Alcov, to estimate the abundance of ToBRFV clades in samples.
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