Detection Of Tobacco Mosaic Virus Research Articles

Direct Detection of Tobacco Mosaic Virus in Infected Plants with SERS-Sensing Hydrogels.

The impact of plant pathogens on global crop yields is a major societal concern. The current agricultural diagnostic paradigm involves either visual inspection (inaccurate) or laboratory molecular tests (burdensome). While field-ready diagnostic methods have advanced in recent years, issues remain with detection of presymptomatic infections, multiplexed analysis, and requirement for in-field sample processing. To overcome these issues, we developed surface-enhanced Raman scattering (SERS)-sensing hydrogels that detect pathogens through simple contact with a leaf. In this work, we developed a novel reagentless SERS sensor for the detection of tobacco mosaic virus (TMV) and embedded it in an optimized hydrogel material to produce sensing hydrogels. To test the diagnostic application of our sensing hydrogels, we demonstrate their use to detect TMV infection in tobacco plants. This technology has the potential to shift the current agricultural diagnostic paradigm by offering a field-deployable tool for presymptomatic and multiplexed molecular identification of pathogens.

Triplex Immunostrip Assay for Rapid Diagnosis of Tobacco Mosaic Virus, Tobacco Vein Banding Mosaic Virus, and Potato Virus Y.

Mixed virus infection has increasingly become a problem in the production of Solanaceae crops in recent years; therefore, a fast and accurate detection method is needed. In this study, a novel triplex immunostrip assay was developed for the simultaneous detection of tobacco mosaic virus (TMV), tobacco vein banding mosaic virus (TVBMV), and potato virus Y (PVY). The limits of detection of this novel immunostrip reached 200 ppb (ng/ml), 1 ppm (µg/ml), and 2 ppm for TMV, PVY, and TVBMV particles, respectively. Importantly, no cross-reactivity was observed among TMV, TVBMV, and PVY or to a nontarget virus. When the assay was applied to suspected virus-infected tobacco, tomato, and potato samples collected from fields in Southwest China, samples of single or mixed virus infection were successfully identified. In conclusion, the triplex immunostrip assay provides a fast and easy to use on-site detection method for field epidemiological studies of TMV, TVBMV, and PVY, and for managing diseases that are caused by them.

Development of one-step multiplex RT-PCR assay for rapid simultaneous detection of five RNA viruses and Acidovorax citrulli in major cucurbitaceous crops in China.

Cucurbitaceous fruits and vegetables are important crops. Viral and bacterial diseases cause substantial economic losses to cucurbit crops globally. For rapid detection of these pathogens and improved disease control, a one-step multiplex reverse-transcription polymerase chain reaction (mRT-PCR) system was created. This method allowed for the concurrent detection of Tobacco mosaic virus (TMV), Zucchini yellow mosaic virus (ZYMV), Watermelon mosaic virus (WMV), Cucumber green mottle mosaic virus (CGMMV), Cucumber mosaic virus (CMV), and Acidovorax citrulli. Five pairs of specific primers were created according to the conserved regions around the coat protein (CP) genes of each virus, and one pair was based on the A. citrulli internal transcribed spacer (ITS). To limit false negatives, one pair of primers, created based on the Transcriptional elongation factor 1-α (EF1-α) from the major cucurbitaceous crop species, was put into the mRT-PCR reaction system. Primer concentrations, annealing temperature, extension time, and amplification cycles were optimized. Anticipated fragments of 152bp (TMV), 205bp (ZYMV), 318bp (WMV), 419bp (CGMMV), 529bp (CMV), 662bp (A. citrulli), and 821bp (EF1-α) were amplified by the multiplex RT-PCR system, and their origin was established via DNA sequencing. This method was successfully used to examine field-collected seed samples of cucurbitaceous crops from China. The results demonstrated that the one-step mRT-PCR technique is a quick, efficient, and sensitive assay for the concurrent detection of six pathogens of cucurbits. It provides a method for monitoring and preventing these diseases.

Photoinduced atom transfer radical polymerization combined with click chemistry for highly sensitive detection of tobacco mosaic virus RNA

An electrochemical biosensor for highly sensitive detection of tobacco mosaic virus (TMV) RNA (tRNA) based on click chemistry and photoinduced atom transfer radical polymerization (photoATRP) is developed for the first time. Herein, tRNA is recognized and captured by hairpin DNA immobilized on the gold electrode surface by Au–S self-assembly. Propyl 2-bromoisobutyrate (PBIB), a photoATRP initiator containing an alkyne group, is conjugated to the azide group of hairpin DNA via a Cu(I)-catalyzed azidoalkyl cyclization reaction (CuAAC). Under the irradiation of 470 nm blue light, photoATRP is activated by the photoredox catalyst (eosin Y, EY), resulting in the formation of a large number of electroactive probes (ferrocenylmethyl methacrylate, FMMA), which significantly amplifies the signal. Under the optimal experimental parameters, the strategy has a wide linear detection (0.1 pM–10 nM) (R2 = 0.995) with a limit of detection (LOD) as low as 3.5 fM. In addition, the biosensor also exhibited good selectivity for mismatched bases, excellent stability and reproducibility. Moreover, satisfactory result was achieved when the biosensor was applied to the detection of tRNA from healthy rehmannia total RNA extracts, which demonstrates the great potential of the method in the practical detection of TMV.

Detection of plant virus particles with a capacitive field-effect sensor

Plant viruses are major contributors to crop losses and induce high economic costs worldwide. For reliable, on-site and early detection of plant viral diseases, portable biosensors are of great interest. In this study, a field-effect SiO2-gate electrolyte-insulator-semiconductor (EIS) sensor was utilized for the label-free electrostatic detection of tobacco mosaic virus (TMV) particles as a model plant pathogen. The capacitive EIS sensor has been characterized regarding its TMV sensitivity by means of constant-capacitance method. The EIS sensor was able to detect biotinylated TMV particles from a solution with a TMV concentration as low as 0.025 nM. A good correlation between the registered EIS sensor signal and the density of adsorbed TMV particles assessed from scanning electron microscopy images of the SiO2-gate chip surface was observed. Additionally, the isoelectric point of the biotinylated TMV particles was determined via zeta potential measurements and the influence of ionic strength of the measurement solution on the TMV-modified EIS sensor signal has been studied.

6-pentyl-α-pyrone from Trichoderma koningii induces systemic resistance in tobacco against tobacco mosaic virus

This study aimed at evaluating the ability of 6-pentyl-α-pyrone (6PP) isolated from Trichoderma koningii CTX1172 (AUMC 11520), an endophytic fungal strain to induce systemic resistance in tobacco (Nicotiana tabacum cv. White Burley) plant against tobacco mosaic virus (TMV). 6PP exhibited 10–60% symptoms inhibition at low concentrations (10–30 μg mL−1) achieving 100% biocontrol efficacy at high concentrations (40 and 50 μg mL−1), compared with control. Using DAS-ELISA technique for detection of TMV (expressed by decreasing in absorbance value), the TMV detection was significantly reduced in plants treated with 6PP at low concentrations compared with plants inoculated with TMV only and it was almost undetectable in plants treated with high concentrations, as well as healthy control plants. Application of 6PP not only increased the accumulation of proline as a non-enzymatic antioxidant compound, but also increased the activities of pathogenesis-related enzymes (superoxide dismutase, peroxidase and polyphenol oxidase), indicating that 6PP acts as elicitor for induction of resistance in tobacco against TMV. On the molecular level, plants treated with 6PP also showed augmented and rapid expression of defense-related genes including PR-a, PR-b and PR-10. This study was the first to imply the potential of a pyrone compound in biocontrol of plant viral disease.

First Detection of Tobacco Mosaic Virus in Tobacco Fields in Northern Lebanon.

Tobacco mosaic virus (TMV) is the most known virus in the plant mosaic virus family and is able to infect a wide range of crops, in particular, tobacco, causing a production loss. Herein, and for the first time in Lebanon, we investigated the presence of TMV infection in crops by analyzing 88 samples of tobacco, tomato, cucumber and pepper collected from different regions in North Lebanon. Double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), revealed a potential TMV infection of four tobacco samples out of 88 crop samples collected. However, no tomato, cucumber and pepper samples were infected. The TMV+ tobacco samples were then extensively analyzed by RT-PCR to detect viral RNA using different primers covering all the viral genome. PCR results confirmed those of DAS-ELISA showing TMV infection of four tobacco samples collected from three crop fields of North Lebanon. In only one of four TMV+ samples, we were able to amplify almost all the regions of viral genome, suggesting possible mutations in the virus genome or an infection with a new, not yet identified, TMV strain. Our study is the first in Lebanon, revealing TMV infection in crop fields and highlighting the danger that may affect the future of agriculture.

A multiplex reverse transcription PCR assay for simultaneous detection of six main RNA viruses in tomato plants

Tomato virus diseases occur all around the world, causing serious yield losses. To detect these viruses quickly and provide a basis for disease control, a multiplex reverse transcription polymerase chain reaction system was established for simultaneous detection of Tobacco mosaic virus (TMV), Cucumber mosaic virus (CMV), Tomato spotted wilt virus (TSWV), Tomato chlorosis virus (ToCV), Potato virus Y (PVY) and Potato virus X (PVX) in tomato plants, with 6 pairs of specific primers being designed based on the coat protein (CP) genes of these viruses. Transcriptional elongation factor-1α (EF-1α) from tomato was added to the multiplex RT-PCR reaction system to prevent false negatives. The concentration of the primers, annealing temperature, annealing time, extension time and amplification cycles were optimized. Expected fragments of 159 bp (ToCV), 262 bp (PVY), 362 bp (EF-1α), 430 bp (TMV), 500 bp (TSWV), 600 bp (CMV) and 705 bp (PVX) were amplified by this multiplex RT-PCR system, and their origin was confirmed by DNA sequencing. This method will have a wide application in virus detection of field samples.

Procedure for detecting tobamovirus in tomato and pepper seeds decreases the cost analysis

ABSTRACT The transmission of tobamovirus by tomato and pepper seeds is an important mean of virus introduction in crops. Therefore, detecting its presence in the seed becomes essential for the preventive control of virus diseases. In this study, a method was proposed for the detection of Tobacco mosaic virus (TMV) and Tomato mosaic virus (ToMV) in tomatoes (Solanum lycopersicum) and Pepper mild mottle virus (PMMoV) in pepper (Capsicum annum) seeds. Seed lots with different levels of incidence were analyzed by biological, serological, and molecular methods. Using DAS-ELISA technique, it was possible to detect TMV up to the limit rate of 1:170 (1 contaminated seed: total seeds) and the ToMV up to 1:200 in tomato seeds; PMMoV in pepper seeds was detected up to 1:140. The IC-RT-PCR detected the TMV and ToMV up to the limit of 1:400 and PMMoV up to the limit of 1:300. The assembled lots containing only 1 contaminated seed in 1000 (1:1000) were combined into 30 sub lots for DAS-ELISA analysis and 10 sub lots for IC-RT-PCR analysis. Both techniques, DAS-ELISA and IC-RT-PCR, were efficient to detect the three viruses in all analyzed samples, but the detection of tobamoviruses with RT-PCR and biological tests was not reliable. Based on the results of this study, in which a combination of seeds in sub lots was made to reduce the number of tests performed, it is possible to make significant savings in the cost of the diagnostic methods routinely conducted in official laboratories, with high efficiency and reliability.

Development of a concentration method for detection of tobacco mosaic virus in irrigation water.

Tobacco mosaic virus (TMV) causes significant yield loss in susceptible crops irrigated with contaminated water. However, detection of TMV in water is difficult owing to extremely low concentrations of the virus. Here, we developed a simple method for the detection and quantification of TMV in irrigation water. TMV was reliably detected at concentrations as low as 10 viral copies/μL with real-time PCR. The sensitivity of detection was further improved using polyethylene glycol 6000 (PEG6000, MW 6000) to concentrate TMV from water samples. Among the 28 samples from Shaanxi Province examined with our method, 17 were tested positive after virus concentration. Infectivity of TMV in the original water sample as well as after concentration was confirmed using PCR. The limiting concentration of TMV in water to re-infect plants was determined as 10(2) viral copies/mL. The method developed in this study offers a novel approach to detect TMV in irrigation water, and may provide an effective tool to control crop infection.

Rapid immunohistochemical diagnosis of tobacco mosaic virus disease by microwave-assisted plant sample preparation.

Immunoelectron microscopy is a powerful method to diagnose viral diseases and to study the distribution of the viral agent within plant cells and tissues. Nevertheless, current protocols for the immunological detection of viral diseases with transmission electron microscopy (TEM) in plants take between 3 and 6 days and are therefore not suited for rapid diagnosis of virus diseases in plants. In this study, we describe a method that allows rapid cytohistochemical detection of tobacco mosaic virus (TMV) in leaves of tobacco plants.With the help of microwave irradiation, sample preparation of the leaves was reduced to 90 min. After sample sectioning, virus particles were stained on the sections by immunogold labelling of the viral coat protein, which took 100 min. After investigation with the TEM, a clear visualization of TMV in tobacco cells was achieved altogether in about half a day. Comparison of gold particle density by image analysis revealed that samples prepared with the help of microwave irradiation yielded significantly higher gold particle density as samples prepared conventionally at room temperature.This study clearly demonstrates that microwave-assisted plant sample preparation in combination with cytohistochemical localization of viral coat protein is well suited for rapid diagnosis of plant virus diseases in altogether about half a day by TEM.

Development of a One-Step Immunocapture Real-Time RT-PCR Assay for Detection of Tobacco Mosaic Virus in Soil

Tobacco mosaic virus (TMV) causes significant losses in many economically important crops. Contaminated soils may play roles as reservoirs and sources of transmission for TMV. In this study we report the development of an immunocapture real-time RT-PCR (IC-real-time RT-PCR) assay for direct detection of TMV in soils without RNA isolation. A series of TMV infected leaf sap dilutions of 1:101, 1:102, 1:103, 1:104, 1:105 and 1:106 (w/v, g/mL) were added to one gram of soil. The reactivity of DAS-ELISA and conventional RT-PCR was in the range of 1:102 and 1:103 dilution in TMV-infested soils, respectively. Meanwhile, the detection limit of IC-real-time RT-PCR sensitivity was up to 1:106 dilution. However, in plant sap infected by TMV, both IC-real-time RT-PCR and real-time RT-PCR were up to 1:106 dilution, DAS-ELISA could detect at least 1:103 dilution. IC-real-time RT-PCR method can use either plant sample extracts or cultivated soils, and show higher sensitivity than RT-PCR and DAS-ELISA for detection of TMV in soils. Therefore, the proposed IC-real-time RT-PCR assay provides an alternative for quick and very sensitive detection of TMV in soils, with the advantage of not requiring a concentration or RNA purification steps while still allowing detection of TMV for disease control.

Detection of tobacco mosaic virus and tomato mosaic virus in pepper seeds by enzyme linked immunosorbent assay (ELISA)

Pepper seed samples were tested for the infection of tobacco mosaic virus (TMV) and tomato mosaic virus (ToMV) by enzyme linked immunosorbent assay (ELISA). Out of 26 pepper seed samples tested, 17 were infected with TMV and ToMV in ELISA. About 34.7% of pepper seed samples were found to be healthy. Infections of TMV or ToMV were recorded to be 61.53% and 11.5%, respectively of the total tested seed samples.

A Basic Approach Towards the Development of Bioelectric Bacterial Biosensors for the Detection of Plant Viruses

AbstractThere is a growing need for virus sensors with improved sensitivity and dynamic range for disease diagnosis, pharmaceutical research, agriculture and homeland security. Membrane‐engineered animal cells bearing antibodies against viral antigens have been previously used for biorecognition biosensors for the ultrarapid (3 min), sensitive (1 ng/ml) detection of plant viruses, such as the cucumber mosaic virus. We here report a new approach for the construction of cell‐based sensors for virus detection, based on membrane (antibody)‐engineered bacteria. The novel method was applied for the detection of tobacco mosaic virus (TMV) and cherry leaf roll virus (CLRV) using sensors containing modified Escherichia coli XL‐1Blue MRF’ bacteria. E. coli membranes have been engineered with electro‐inserted, virus‐homologous antibodies. The detection principle was based on the measurement of changes in the bacterial membrane potential as a result of virus–antibody binding. After optimization of the membrane‐engineering process, the virus detection limit for TMV and CLRV with the bacteria‐based biosensor system was 1 pg/ml, representing a 1000‐fold improvement over currently available methods. Although the novel biosensor is still in its proof‐of‐concept stage of development, its sensitivity and speed (assay time: 60–100 s) could make it a very promising tool for high throughput, field‐based virus screening.

Detection of Tobacco mosaic virus and Tomato mosaic virus in pepper and tomato by multiplex RT-PCR

To develop a highly sensitive and rapid protocol for simultaneous detection and differentiation of Tobacco mosaic virus (TMV) and Tomato mosaic virus (ToMV) in pepper and tomato. In this study, we use the multiplex PCR technique to detect dual infection of these two viruses. A multiplex RT-PCR method consisting of one-tube reaction with two primer pairs targeted to replicase genes was developed to simultaneously detect TMV and ToMV in seed samples of pepper and tomato. Specific primers were designed from conserved regions of each of the virus genomes, and their specificity was confirmed by sequencing PCR products. RT-PCR detected up to 10(-6) dilution of total RNA extracted from infected leaves. Multiplex RT-PCR revealed the presence of both TMV and ToMV in three of 18 seed samples of tomato and one of 18 seed samples of pepper. The multiplex PCR assay was a cost effective, quick diagnostic technique, which was helpful in differentiating TMV and ToMV accurately. The multiplex PCR assay described in this study is a valuable tool for plant pathology and basic research studies. This method may facilitate better recognition and distinction of TMV and ToMV in both pepper and tomato.

Evaluation of SDS-Polyacrylamide gel electrophoresis and immunostrip technique for detection of Tobacco mosaic virus and Cucumber mosaic virus in IRAQ

his study was carried out to evaluate the efficiency of electrophoresis on SDS- poly acrylamide slap gel and immunostrip techniques for detection of Tobacco mosaic virus (TMV, genus Tobamovirus) and Cucumber mosaic virus (CMV, genus Cucumovirus, family Bromoviridae), compared with symptoms on diagnostic plants for the two viruses. The results obtained showed that the two methods were effective. The analysis of samples of purified CMV, total proteins from infected cucumber plants, and extracts from infected plants with or without chlorophyll, by electrophoresis on 10% polyacrylamide slap gel containing 0.1% SDS showed two bands of 24 and 26 kd in size, and absent in samples of total protein or extracts of healthy plants. These two proteins represent the coat protein (CP) of CMV. In addition, one 18 kd protein band appeared on SDS- polyacrylamide gel profile which represent the CP of TMV, when samples of purified virus, total protein of infected plants, and plant extracts with or without chlorophyll were analyzed. This band was absent in similar samples from healthy plants. The test of immunostrip specific for CMV showed positive reaction with extracts from melon, cucumber, winter squash, and zucchini infected plants. Similarly, a positive reaction with immunostrip specific for TMV appeared with extracts from tobacco, tomato infected with TMV. No reaction was obtained with healthy plants extract. These results were similar to those obtained from indicator plants for the two viruses.

Rapid detection of tobacco mosaic virus using the reverse transcription loop-mediated isothermal amplification method

Tobacco mosaic virus (TMV) can cause a severe disease that is capable of greatly reducing tobacco quality and yield. In this study, a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detection of TMV. The concentration of Mg(2+), reaction temperature and reaction time of the RT-LAMP were optimized to 5mM, 65°C, and 60min, respectively. The detection limit of the method was 100 times higher than that of RT-PCR. Visual inspection of RT-LAMP amplification demonstrated that positive and negative reactions exhibit distinctly different colours in daylight. Our results demonstrate that the method is stable, sensitive and specific.

Rapid Detection of Tobacco Mosaic Virus from Crude Samples on an Oscillatory-flow Reverse Transcription-Polymerase Chain Reaction Microfluidics

Abstract An oscillatory-flow reverse transcription-polymerase chain reaction (RT-PCR) microfluidics based on the capillary reactor was developed for the detection of tobacco mosaic virus (TMV). A pair of PCR primers was designed according to the TMV movement protein gene. Direct RT-PCR-amplified products, 179-base pair (bp) DNA fragments, from TMV particles have been successfully obtained in this research. 0.025% bovine serum albumin was used for passive and dynamic passivation to improve the PCR efficiency. On this device, the detection limit of 0.01 μg cDNA was achieved at the flow rate of 70 μl min −1 , and the amplification of 179-bp fragments can be successfully performed within 17 min.

Detection of TMV with ODA-H 2O 2-HRP voltammetric enzyme-linked immunoassay system

o-Dianisidine (ODA)-H 2O 2-horseradish peroxidase (HRP) voltammetric enzyme-linked immunoassay system has firstly been used for the detection of tobacco mosaic virus (TMV). HRP catalyzes strongly the oxidation reaction of ODA by H 2O 2, the product of which produces a sensitive second order derivative linear sweep voltammetric peak at potential of −0.56 V (versus SCE) in Britton–Robinson (BR) buffer. HRP activity has been measured with this voltammetric peak and TMV detected through immunoreaction. The detection limit for HRP is 9.25×10 -7 mU l −1 and the linear range is 2.5×10 −6–5.0×10 −4 mU l −1. The detection limit for the clarified TMV is 0.25 ng ml −1 and the highest dilution ratio detected for the infected leaf sap is 1:8×10 5. The sensitivity for TMV detection with this method is higher than that with the enzyme-linked immunosorbent spectrophotometric assay (ELISA) using ODA-H 2O 2-HRP system. The processes of the enzyme-catalyzed reaction and the electro-reduction of the product of the enzyme-catalyzed reaction have been described.

Detection of Tobacco Mosaic Virus by Monoclonal Antibody MAM-1F5

TMV is a single-stranded RNA virus. It causes disease (with mosaic appearance) in many plant species. The most common infected plant is tobacco, which is of economic importance for Turkey. Immunochemical techniques are widely used in the accurate diagnosis of antigens derived from plant pathogenic microorganisms. Although polyclonal sera have served well over the years, the monospecificty of monoclonal antibodies makes them considerably more suitable. In this study, we developed a hiybrid cell (MAM-1F5) producing a monoclonal antibody specific to TMV for use in specific and sensitive serologic detection of TMV. By using an indirect ELISA method based on the monoclonal antibody, we were able to detect the TMV antigen at a 1 µg concentration and observe regional specificity in TMV contamination.