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Kaishu Ling - One of the best experts on this subject based on the ideXlab platform.

  • Pepino Mosaic Virus genotype shift in north america and development of a loop mediated isothermal amplification for rapid genotype identification
    Virology Journal, 2013
    Co-Authors: Kaishu Ling, Rugang Li, Michael Bledsoe
    Abstract:

    Pepino Mosaic, once an emerging disease a decade ago, has become endemic on greenhouse tomatoes worldwide in recent years. Three distinct genotypes of Pepino Mosaic Virus (PepMV), including EU, US1 and CH2 have been recognized. Our earlier study conducted in 2006–2007 demonstrated a predominant EU genotype in Canada and United States. The objective of the present study was to monitor the dynamic of PepMV genetic composition and its current status in North America. Through yearly monitoring efforts in 2009–2012, we detected a dramatic shift in the prevalent genotype of PepMV from the genotype EU to CH2 in North America since early 2010, with another shift from CH2 to US1 occurring in Mexico only two years later. Through genetic diversity analysis using the coat protein gene, such genotype shifting of PepMV in North America was linked to the positive identification of similar sequence variants in two different commercial tomato seed sources used for scion and rootstock, respectively. To allow for a quick identification, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) system was developed and demonstrated to achieve a rapid identification for each of the three genotypes of PepMV, EU, US1 and CH2. Through systemic yearly monitoring and genetic diversity analysis, we identified a linkage between the field epidemic isolates and those from commercial tomato seed lots as the likely sources of initial PepMV inoculum that resulted in genetic shifting as observed on greenhouse tomatoes in North America. Application of the genotype-specific RT-LAMP system would allow growers to efficiently determine the genetic diversity on their crops.

  • effectiveness of chemo and thermotherapeutic treatments on Pepino Mosaic Virus in tomato seed
    Plant Disease, 2010
    Co-Authors: Kaishu Ling
    Abstract:

    Ling, K.-S. 2010. Effectiveness of chemo- and thermotherapeutic treatments on Pepino Mosaic Virus in tomato seed. Plant Dis. 94:325-328. Pepino Mosaic Virus (PepMV) is a seedborne Virus of importance in greenhouse tomato. The ease of mechanical transmission of PepMV from contaminated tomato seed to seedlings makes commercial tomato seed a potential source of initial Virus inoculum. The objective of this study was to evaluate the effectiveness of chemo- and thermotherapeutic treatments on PepMV using seed from a commercial hybrid tomato seed lot. The effect of various seed treatments was evaluated through bioassay with mechanical inoculation of the treated seed extract on indicator plants (Nicotiana benthamiana). Three commonly used seed-treatment chemicals and two thermotreatment methods were evaluated. The most effective chemical was the commercial bleach solution (0.5 and 1.0% sodium hypochlorite), followed by trisodium phosphate. On the other hand, hydrochloric acid treatments were not effective. Under the stated chemotherapeutic treatment conditions, the rate of seed germination was not significantly affected. With thermotherapeutic treatments, although hot water soaking for 2 h at 55°C deactivated Virus infectivity, it also resulted in a detrimental effect on seed germination. However, treatment with dry-heat baking (72 or 80°C for 48 to 72 h) was effective in reducing PepMV infection with minimum impact on seed germination.

  • Pepino Mosaic Virus on tomato seed Virus location and mechanical transmission
    Plant Disease, 2008
    Co-Authors: Kaishu Ling
    Abstract:

    ABSTRACT In just a few years, Pepino Mosaic Virus (PepMV) has become a major threat to greenhouse tomato production around the world. Although tomato seed is suspected to spread the disease, its importance as an initial Virus inoculum for PepMV has not been established. To determine the potential for seed transmission, a tomato seed lot highly contaminated with PepMV was used for large-scale seedling grow-out tests. None of 10,000 grow-out seedlings was infected as determined by symptom expression, enzyme-linked immunosorbent assay (ELISA), or infectivity assay on Nicotiana benthamiana. Even though PepMV was not seed transmitted on tomato, the Virus was effectively transmitted to tomato and N. benthamiana seedlings through mechanical transmission with seed extract. To examine the exact location where PepMV particles accumulated on the tomato seed, seed coats and embryos were carefully isolated and tested separately by ELISA, real-time RT-PCR, and bioassay on N. benthamiana. PepMV was detected in the seed ...

  • genetic composition of Pepino Mosaic Virus population in north american greenhouse tomatoes
    Plant Disease, 2008
    Co-Authors: Kaishu Ling, William M Wintermantel, Michael Bledsoe
    Abstract:

    ABSTRACT In just a few short years, Pepino Mosaic disease has quickly become endemic in greenhouse tomatoes around the world. Although three genotypes of Pepino Mosaic Virus (PepMV) were identified in the United States, genetic composition of PepMV in greenhouse tomato crops in North America has not been determined. In this study, genetic variability and population structure of PepMV were evaluated through nucleotide sequence comparison and phylogenetic analysis of two genomic regions (helicase domain and TGB2-3) derived from 91 cDNA clones that were derived from 31 field-collected samples. These samples were collected from several major greenhouse tomato facilities in five states in the United States and two provinces in Canada. All four major genotypes of PepMV (EU, US1, US2, and CH2) were found in North America. Three distinct genotypes (EU, US1, and US2) were found in mixed infection in samples collected from Arizona and Colorado, two genotypes (EU and CH2) in Texas, and a single genotype (EU) in Alab...

  • development of a one step immunocapture real time taqman rt pcr assay for the broad spectrum detection of Pepino Mosaic Virus
    Journal of Virological Methods, 2007
    Co-Authors: Kaishu Ling, Patrick W Wechter, Ramon Jordan
    Abstract:

    A real-time reverse-transcription polymerase chain reaction (RT-PCR) was developed for efficient detection of genetically diverse Pepino Mosaic Virus (PepMV) isolates. The novel detection system was designed to use a duo-primer system targeting the conserved region in the triple gene block 2 (TGB2) gene with a single conserved TaqMan TM probe to broaden its reaction to cover all available PepMV strains. This duo-primer real-time RT-PCR assay was evaluated against US1, US2, Ch1, Ch2 and 25 field isolates collected from six major commercial tomato greenhouse facilities in U.S. and Canada in 2006. Under optimum reaction conditions, sensitivity of the detection was as low as 100 fg of purified viral RNA. This assay was also evaluated for its efficiency in detecting PepMV in various levels of contaminated seed samples. Using immunocapture sample preparation, real-time RT-PCR was able to detect PepMV in one infested seed in 1000. This level of sensitivity indicated that the one-step immunocapture duo-primer TaqMan TM real-time RT-PCR developed in the present study could be used for routine seed health assays. Published by Elsevier B.V.

Maja Ravnikar - One of the best experts on this subject based on the ideXlab platform.

  • filling the gaps in diagnostics of Pepino Mosaic Virus and potato spindle tuber viroid in water and tomato seeds and leaves
    Plant Pathology, 2017
    Co-Authors: Nataša Mehle, Ion Gutierrezaguirre, Polona Kogovsek, Nejc Racki, Tjasa Jakomin, Petra Kramberger, Maja Ravnikar
    Abstract:

    Waterborne and seedborne Pepino Mosaic Virus (PepMV) and Potato spindle tuber viroid (PSTVd) pose serious threats to tomato production due to seed transmission and mechanical transmission, coupled with their long-term stability outside the host plant. Therefore, rapid and sensitive diagnostic procedures are needed to prevent the spread of these quarantine pathogens. In particular, water and seed contamination are difficult to detect and confirm without efficient concentration methods. This study presents procedures that improve detection of PSTVd from tomato seeds and leaf tissue, and PepMV from water and tomato leaf tissue. For efficient concentration of PepMV from water samples, a procedure was optimized using convective interaction media monolithic chromatography columns, which provides concentration by three orders of magnitude. For concentration of PSTVd from seed extracts, an easy-to-use and efficient method was developed based on RNA binding to positively charged anion-exchange resin beads that provides up to 100-fold more sensitive detection in comparison with procedures without a concentration step. This thus allows confirmation of RT-qPCR results with sequencing of RT-PCR products in samples with low viroid levels. In addition, reverse-transcription loop-mediated isothermal amplification assays for detection of PSTVd and PepMV were optimized and adapted to both laboratory and on-site testing requirements. This allows rapid detection of these pathogens in crude leaf homogenates, in under 30 min. These procedures of concentration and detection are shown to be efficient and to fill the gaps in diagnostics of PepMV and PSTVd, especially when these pathogens are present at low levels in difficult matrices such as water and seeds.

  • Host range and symptomatology of Pepino Mosaic Virus strains occurring in Europe
    European Journal of Plant Pathology, 2015
    Co-Authors: Dag-ragnar Blystad, R A A Van Der Vlugt, Dimitrinka Hristova, Henryk Pospieszny, Ana Alfaro-fernández, María Del Carmen Córdoba, Gábor Bese, Nataša Mehle, Maja Ravnikar, Laura Tomassoli
    Abstract:

    Pepino Mosaic Virus (PepMV) has caused great concern in the greenhouse tomato industry after it was found causing a new disease in tomato in 1999. The objective of this paper is to investigate alternative hosts and compare important biological characteristics of the three PepMV strains occurring in Europe when tested under different environmental conditions. To this end we compared the infectivity and symptom development of three, well characterized isolates belonging to three different PepMV strains, EU-tom, Ch2 and US1, by inoculating them on tomato, possible alternative host plants in the family Solanaceae and selected test plants. The inoculation experiments were done in 10 countries from south to north in Europe. The importance of alternative hosts among the solanaceous crops and the usefulness of test plants in the biological characterization of PepMV isolates are discussed. Our data for the three strains tested at 10 different European locations with both international and local cultivars showed that eggplant is an alternative host of PepMV. Sweet pepper is not an important host of PepMV, but potato can be infected when the right isolate is matched with a specific cultivar. Nicotiana occidentalis 37B is a useful indicator plant for PepMV studies, since it reacts with a different symptomatology to each one of the PepMV strains.

  • survival and transmission of potato Virus y Pepino Mosaic Virus and potato spindle tuber viroid in water
    Applied and Environmental Microbiology, 2014
    Co-Authors: Nataša Mehle, Ion Gutierrezaguirre, Duska Delic, N Prezelj, U Vidic, Maja Ravnikar
    Abstract:

    ABSTRACT Hydroponic systems and intensive irrigation are used widely in horticulture and thus have the potential for rapid spread of water-transmissible plant pathogens. Numerous plant Viruses have been reported to occur in aqueous environments, although information on their survival and transmission is minimal, due mainly to the lack of effective detection methods and to the complexity of the required transmission experiments. We have assessed the role of water as a source of plant infection using three mechanically transmissible plant pathogens that constitute a serious threat to tomato and potato production: Pepino Mosaic Virus (PepMV), potato Virus Y (PVY), and potato spindle tuber viroid (PSTVd). PepMV remains infectious in water at 20 ± 4°C for up to 3 weeks, PVY (NTN strain) for up to 1 week, and PSTVd for up to 7 weeks. Experiments using a hydroponic system show that PepMV (Ch2 genotype) and PVY (NTN strain) can be released from plant roots into the nutrient solution and can infect healthy plants through their roots, ultimately spreading to the green parts, where they can be detected after a few months. In addition, tubers developed on plants grown in substrate watered with PSTVd-infested water were confirmed to be the source of viroid infection. Our data indicate that although well-known pathways of Virus spread are more rapid than water-mediated infection, like insect or mechanical transmission through leaves, water is a route that provides a significant bridge for rapid Virus/viroid spread. Consequently, water should be taken into account in future epidemiology and risk assessment studies.

  • cross protection or enhanced symptom display in greenhouse tomato co infected with different Pepino Mosaic Virus isolates
    Plant Pathology, 2010
    Co-Authors: Inge M. Hanssen, Anneleen Paeleman, Bart Lievens, A.c.r.c. Vanachter, Maja Ravnikar, I Gutierrezaguirre, K Goen, Lieve Wittemans, Bart P. H. J. Thomma
    Abstract:

    The potential of three mild Pepino Mosaic Virus (PepMV) isolates, belonging to the CH2, EU and LP genotypes, to protect a tomato (Solanum lycopersicum) crop against an aggressive challenge isolate of the CH2 genotype was assessed in greenhouse trials and PepMV symptoms were rated at regular time points. After challenge infection, enhanced symptom display was recorded in plants that were pre-inoculated with a protector isolate belonging to a different genotype (EU, LP) from the challenge isolate. A quantitative genotype-specific TaqMan assay revealed that in these plants, the accumulation of the challenge isolate only temporarily slowed down. By contrast, efficient cross-protection was obtained using the mild isolate of the CH2 genotype, and in this case the challenge isolate was barely detectable in the pre-inoculated plants. These results suggest that the interaction between PepMV isolates largely depends on RNA sequence homology and that post-transcriptional gene silencing plays an important role in cross-protection.

  • real time quantitative pcr based sensitive detection and genotype discrimination of Pepino Mosaic Virus
    Journal of Virological Methods, 2009
    Co-Authors: Ion Gutierrezaguirre, Nataša Mehle, R A Mumford, Duska Delic, Kristina Gruden, Maja Ravnikar
    Abstract:

    Over the last decade a new Virus disease caused by Pepino Mosaic Virus (PepMV) has been threatening the tomato industry worldwide. Reliable detection is vitally important to aid disease control. Methods must be both sensitive and capable of detecting the range of distinct genotypes that have been identified. The development of five new reverse transcription real-time quantitative PCR (RT-qPCR) assays is described, which allow the detection of all known PepMV genotypes. The performance of the assays was evaluated on Peruvian, European tomato, Ch2 and US1 PepMV genotypes and optimised for both two- and one-step RT-qPCR detection formats. One-step RT-qPCR detected PepMV European tomato genotype particles at least two orders of magnitude more sensitively than ELISA. The method detected as little as one naturally infected seed among 5000 uninfected seeds. The genotype-specificity of the five assays was compared using PepMV isolates representing all of the different genotypes. The following genotype combinations were all discriminated successfully: European tomato-Peruvian, Ch2, and US1. In addition to its application for diagnostic purposes, the genotype-specificity and the quantitative potential of the method, makes it very useful for epidemiological studies or for studies evaluating resistance of plants to Virus infection.

H Pospieszny - One of the best experts on this subject based on the ideXlab platform.

  • ultrastructural insights into tomato infections caused by three different pathotypes of Pepino Mosaic Virus and immunolocalization of viral coat proteins
    Micron, 2015
    Co-Authors: Julia Minicka, H Pospieszny, Katarzyna Otulak, Grazyna Garbaczewska, Beata Hasiowjaroszewska
    Abstract:

    This paper presents studies on an ultrastructural analysis of plant tissue infected with different pathotypes of Pepino Mosaic Virus (PepMV) and the immunolocalization of viral coat proteins. Because the PepMV Virus replicates with a high mutation rate and exhibits significant genetic diversity, therefore, isolates of PepMV display a wide range of symptoms on infected plants. In this work, tomato plants of the Beta Lux cultivar were inoculated mechanically with three pathotypes representing the Chilean 2 (CH2) genotype: mild (PepMV-P22), necrotic (PepMV-P19) and yellowing (PepMV-P5-IY). The presence of viral particles in all infected plants in the different compartments of various cell types (i.e. spongy and palisade mesophyll, sieve elements and xylem vessels) was revealed via ultrastructural analyses. For the first time, it was possible to demonstrate the presence of crystalline inclusions, composed of Virus-like particles. In the later stage of PepMV infection (14 dpi) various pathotype-dependent changes in the structure of the individual organelles (i.e. mitochondria, chloroplasts) were found. The strongest immunogold labeling of the viral coat proteins was also observed in plants infected by necrotic isolates. A large number of viral coat proteins were marked in the plant conductive elements, both xylem and phloem.

  • the genetic determinants of symptoms induction by Pepino Mosaic Virus in tomato
    Acta Horticulturae, 2015
    Co-Authors: Beata Hasiowjaroszewska, Julia Minicka, N Borodynko, H Pospieszny
    Abstract:

    The Pepino Mosaic Virus (PepMV) is one of the most important pathogens infecting tomato crops worldwide. Four major genotypes have been identified so far: Peruvian (LP), European (EU), Chilean 1 (CH1) and Chilean 2 (CH2). In Poland isolates representing the EU and CH2 genotypes have been found. The isolates belonging to the CH2 genotype induce a wide range of symptoms from very mild Mosaic or even a lack of symptoms, through severe yellowing, up to necrosis of leaves. Therefore, the characterization of different genetic variants of PepMV is important to clarify processes of disease emergence caused by this Virus. The necrotic and yellowing pathotypes representing the Chilean 2 genotype have been extensively studied. Development of infectious clones facilitates manipulation of the viral genome by site-directed mutagenesis. The studies performed made it possible to identify the viral genetic determinants responsible for the induction of different symptoms on infected plants. The introduction of single nucleotide substitutions in particular positions of triple gene block 3 (TGB3) resulted in changes of symptoms on tomato plants. Moreover, the mutations affect significantly viral accumulation, which has been measured by real-time RT-PCR. Single nucleotide substitutions introduced to the genome of genetically different European isolates also had an effect on biological properties and accumulation of the Virus. Identified mutations in the viral genome are responsible for the development of symptoms and Virus accumulation, and may play an important role during infection.

  • cross protection between different pathotypes of Pepino Mosaic Virus representing chilean 2 genotype
    Acta Scientiarum Polonorum-hortorum Cultus, 2014
    Co-Authors: Beata Hasiowjaroszewska, Julia Minicka, H Pospieszny
    Abstract:

    Viral cross-protection in plants is a phenomenon, where a mild Virus isolate can protect plants against damage caused by a severe challenge isolate of the same Virus. It has been used on a large scale in cases where no resistant plants are available. We exam- ined differences in cross-protection between pathotypes of Pepino Mosaic Virus represent- ing Chilean 2 genotype. The potential of a mild PepMV-P22 isolate to protect tomato against more aggressive challenge isolates causing yellowing and necrotic symptoms was established. The challenge isolates were PepMV-P5-IY (yellowing), PepMV-P19 (ne- crotic) and PepMV-P22 K67E (artificial necrotic mutant of PepMV-P22 which differ from PepMV-P22 only by a point mutation). Efficient cross-protection was obtained using mild PepMV-P22 against PepMV-P5-IY. After a challenge inoculation with PepMV-P19 or PepMV-P22 K67E symptoms severity were significantly reduced in comparison to non-protected plants; however, necrotic symptoms appeared two months after coinfection. The real-time PCR analysis revealed that the level of accumulation of the necrotic isolate in tomato plants was even 5-7 times higher than that of PepMV-P22.

  • single mutation converts mild pathotype of the Pepino Mosaic Virus into necrotic one
    Virus Research, 2011
    Co-Authors: Beata Hasiowjaroszewska, Paulina Jackowiak, Marek Figlerowicz, N Borodynko, H Pospieszny
    Abstract:

    Abstract Pepino Mosaic Virus (PepMV) is a member of the Flexiviridae family, genus PotexVirus . PepMV isolates from the same genotype differ in the symptoms they induce in Datura inoxia and Solanum lycopersicum . Necrotic isolates cause necrosis on these plants whereas mild isolates are very often asymptomatic or induce mild Mosaic. Sequence analysis of the genomes of mild (PepMV-P22) and necrotic (PepMV-P19) strains revealed that they differ by several nonsynonymous mutations. Eleven mutations are located in the RdRp coding region and one mutation is placed in the TGB3 coding region. To investigate whether these mutations affect the symptom induction, both PepMV-P22 and PepMV-P19 were mutagenized. As a result we found that the genetic determinant responsible for necrosis induction was amino acid 67 of TGB3. This showed that one point mutation is sufficient to alter the Virus from mild to aggressive in tomato and D. inoxia .

  • tridimensional model structure and patterns of molecular evolution of Pepino Mosaic Virus tgbp3 protein
    Virology Journal, 2011
    Co-Authors: Beata Hasiowjaroszewska, H Pospieszny, S. F. Elena, Anna Czerwoniec
    Abstract:

    Background: Pepino Mosaic Virus (PepMV) is considered one of the most dangerous pathogens infecting tomatoes worldwide. The Virus is highly diverse and four distinct genotypes, as well as inter-strain recombinants, have already been described. The isolates display a wide range on symptoms on infected plant species, ranging from mild Mosaic to severe necrosis. However, little is known about the mechanisms and pattern of PepMV molecular evolution and about the role of individual proteins in host-pathogen interactions. Methods: The nucleotide sequences of the triple gene block 3 (TGB3) from PepMV isolates varying in symptomatology and geographic origin have been analyzed. The modes and patterns of molecular evolution of the TGBp3 protein were investigated by evaluating the selective constraints to which particular amino acid residues have been subjected during the course of diversification. The tridimensional structure of TGBp3 protein has been modeled de novo using the Rosetta algorithm. The correlation between symptoms development and location of specific amino acids residues was analyzed. Results: The results have shown that TGBp3 has been evolving mainly under the action of purifying selection operating on several amino acid sites, thus highlighting its functional role during PepMV infection. Interestingly, amino acid 67, which has been previously shown to be a necrosis determinant, was found to be under positive selection. Conclusions: Identification of diverse selection events in TGB3p3 will help unraveling its biological functions and is essential to an understanding of the evolutionary constraints exerted on the PotexVirus genome. The estimated tridimensional structure of TGBp3 will serve as a platform for further sequence, structural and function analysis and will stimulate new experimental advances.

Miguel A Aranda - One of the best experts on this subject based on the ideXlab platform.

  • second generation of Pepino Mosaic Virus vectors improved stability in tomato and a wide range of reporter genes
    Plant Methods, 2019
    Co-Authors: Fabiola Ruizramon, Raquel Navarro Sempere, Eduardo Mendezlopez, Amelia M Sanchezpina, Miguel A Aranda
    Abstract:

    Background Vectors based on plant Viruses are important tools for functional genomics, cellular biology, plant genome engineering and molecular farming. We previously reported on the construction of PepGFP2a, a viral vector based on Pepino Mosaic Virus (PepMV) which expressed GFP efficiently and stably in plants of its experimental host Nicotiana benthamiana, but not in its natural host tomato. We have prepared a new set of PepMV-based vectors with improved stability that are able to express a wide range of reporter genes, useful for both N. benthamiana and tomato.

  • in situ hybridization for the localization of two Pepino Mosaic Virus isolates in mixed infections
    Journal of Virological Methods, 2019
    Co-Authors: Cristina Gomezaix, Cristina Alcaide, Pedro Sanchez Gomez, Miguel A Aranda, M A Sanchezpina
    Abstract:

    Abstract In situ hybridization (ISH) is an informative and relatively accessible technique for the localization of viral genomes in plant tissue and cells. However, simultaneous visualization of related plant Viruses in mixed infections may be limited by the nucleotide similarity in the genomes and the single chromogenic detection over the same sample preparation. To address this issue, we used two Pepino Mosaic Virus isolates and performed ISH over consecutive serial cross-sections of paraffin-embedded leaf samples of single and mixed infected Nicotiana benthamiana plants. Moreover, the probe design was optimized to reduce cross-hybridisation, and co-localization was based on the overlapping of consecutive cross-sections from mixed infected leaves; thus, our results showed that both Pepino Mosaic Virus isolates co-localized in the same leaf tissue. In turn, both isolates were localized in the cytoplasm of the same cells. These results provide valuable information for studying mixed infections in plants by using a simple ISH procedure that is accessible to any pathology laboratory.

  • biodistribution of filamentous plant Virus nanoparticles Pepino Mosaic Virus versus potato Virus x
    Biomacromolecules, 2019
    Co-Authors: Duc H T Le, Miguel A Aranda, Eduardo Mendezlopez, Chao Wang, Ulrich Commandeur, Nicole F Steinmetz
    Abstract:

    Nanoparticles with high aspect ratios have favorable attributes for drug delivery and bioimaging applications based on their enhanced tissue penetration and tumor homing properties. Here, we investigated a novel filamentous viral nanoparticle (VNP) based on the Pepino Mosaic Virus (PepMV), a relative of the established platform Potato Virus X (PVX). We studied the chemical reactivity of PepMV, produced fluorescent versions of PepMV and PVX, and then evaluated their biodistribution in mouse tumor models. We found that PepMV can be conjugated to various small chemical modifiers including fluorescent probes via the amine groups of surface-exposed lysine residues, yielding VNPs carrying payloads of up to 1600 modifiers per particle. Although PepMV and PVX share similarities in particle size and shape, PepMV achieved enhanced tumor homing and less nonspecific tissue distribution compared to PVX in mouse models of triple negative breast cancer and ovarian cancer. In conclusion, PepMV provides a novel tool for n...

  • stable and broad spectrum cross protection against Pepino Mosaic Virus attained by mixed infection
    Frontiers in Plant Science, 2018
    Co-Authors: Jesus Aguero, Cristina Gomezaix, Raquel Navarro Sempere, Julio Garciavillalba, Jorge Garcianunez, Yolanda Hernando, Miguel A Aranda
    Abstract:

    While recent Pepino Mosaic Virus (PepMV; genus PotexVirus, family Alphaflexiviridae) epidemics seem to be predominantly caused by isolates of the Ch2 genotype, PepMV epidemics in Spain are caused by both Ch2 and EU isolates that co-circulate, representing a challenge in terms of control, including cross-protection. In this work, we hypothesized that mixed infections with two mild isolates of the EU and Ch2 genotypes (PepMV-Sp13 and -PS5, respectively) may be useful in PepMV cross-protection in Spanish epidemics, providing protection against a broad range of aggressive isolates. Thus, we performed a range of field trials and an experimental evolution assay to determine the phenotypic and genetic stability of PepMV-Sp13 and -PS5 mixed infections, as well as their cross-protective efficiency. Our results showed that (i) the phenotype of PepMV-Sp13 and -PS5 mixed infections was mild and did not change significantly when infecting different tomato cultivars or under different environmental conditions, (ii) PepMV-Sp13 and -PS5 mixed infections provided more efficient protection against aggressive EU and Ch2 isolates than single infections, and (iii) PepMV-Sp13 and -PS5, either in single or in mixed infections, were at least as genetically stable as other PepMV isolates occurring naturally in PepMV epidemics.

  • Pepino Mosaic Virus rna dependent rna polymerase pol domain is a hypersensitive response like elicitor shared by necrotic and mild isolates
    Phytopathology, 2016
    Co-Authors: Raquel Navarro Sempere, Beata Hasiowjaroszewska, Cristina Gomezaix, Pedro Sanchez Gomez, M A Sanchezpina, Fabiola Ruizramon, Miguel A Aranda
    Abstract:

    Pepino Mosaic Virus (PepMV) is an emerging pathogen that represents a serious threat to tomato production worldwide. PepMV-induced diseases manifest with a wide range of symptoms, including systemic necrosis. Our results showed that PepMV accumulation depends on the Virus isolate, tomato cultivar, and environmental conditions, and associates with the development of necrosis. Substitution of lysine for glutamic acid at position 67 in the triple gene block 3 (TGB3) protein, previously described as a necrosis determinant, led to increased Virus accumulation and was necessary but not sufficient to induce systemic necrosis. Systemic necrosis both in tomato and Nicotiana benthamiana shared hypersensitive response (HR) features, allowing the assessment of the role of different genomic regions on necrosis induction. Overexpression of both TGB3 and the polymerase domain (POL) of the RNA-dependent RNA polymerase (RdRp) resulted in necrosis, although only local expression of POL triggered HR-like symptoms. Our resul...

N Borodynko - One of the best experts on this subject based on the ideXlab platform.

  • Detection of Pepino Mosaic Virus isolates from tomato by one-step reverse transcription loop-mediated isothermal amplification
    Archives of Virology, 2013
    Co-Authors: Beata Hasiów-jaroszewska, N Borodynko
    Abstract:

    Loop-mediated isothermal amplification (LAMP) of DNA is a novel technique for the amplification of DNA under isothermal conditions. For the first time, we applied this method to develop a simple and sensitive tool for the detection of Pepino Mosaic Virus (PepMV). PepMV is an emerging pathogen that causes yield and quality losses in tomato crops. Specific RT-LAMP primers for PepMV detection were designed based on triple gene block sequences. The reaction was performed in a single tube at 65 °C for 30 min. The RT-LAMP assay is easy to perform and inexpensive, and it may be applied in the rapid and specific diagnosis of PepMV.

  • Characterization of the necrosis determinant of the European genotype of Pepino Mosaic Virus by site-specific mutagenesis of an infectious cDNA clone
    Archives of Virology, 2012
    Co-Authors: Beata Hasiów-jaroszewska, N Borodynko
    Abstract:

    Mild and necrotic isolates have been described for the European (EU) genotype of Pepino Mosaic Virus (PepMV), an important pathogen of tomato worldwide. In this study, we produced various infectious cDNA clones of an EU isolate with point mutations introduced by site-directed mutagenesis. Our results showed that the genetic determinant responsible for necrosis induction on tomato and Datura inoxia was amino acid 67 of TGBp3. This amino acid residue also acts as necrosis determinant in PepMV isolates belonging to the Chilean 2 genotype. This demonstrates that a single point mutation plays a role in necrosis induction by PepMV, irrespective of genotype.

  • Quasispecies nature of Pepino Mosaic Virus and its evolutionary dynamics
    Virus Genes, 2010
    Co-Authors: Beata Hasiów-jaroszewska, N Borodynko, Paulina Jackowiak, Marek Figlerowicz, Henryk Pospieszny
    Abstract:

    Genetic variability is an essential feature of RNA Viruses. It allows them to adapt to the ever-changing environmental conditions. Important biological properties of the Viruses, their infectivity, adaptability, and host range, may also depend on the level of quasispecies diversity. Here, we present the analysis of the genetic polymorphism of Pepino Mosaic Virus (PepMV). The examined populations were isolated from the naturally infected tomato plants ( Solanum lycopersicum ). In order to determine the complexity of the PepMV populations, the number of different viral variants and their genetic diversity was established. Moreover, phylogenetic trees were created to depict relations between the identified variants. For the first time we have shown that the PepMV exists as a quasispecies. The observed level of genetic variability allows PepMV for a quick and flexible adaptation to different hosts. Our results suggest that the level of PepMV variability possibly influences the course of infection.

  • new necrotic isolates of Pepino Mosaic Virus representing the ch2 genotype
    Journal of Phytopathology, 2009
    Co-Authors: Beata Hasiowjaroszewska, H Pospieszny, N Borodynko
    Abstract:

    New necrotic isolates of Pepino Mosaic Virus (PepMV) were found in 2007 infecting greenhouse tomato plants in Poland. The isolates differ from previously identified PepMV isolates in host range and symptomatology. They induce severe necrosis on tomato plants (Solanum lycopersicum) and local necrotic lesions on Datura inoxia. Phylogenetic analysis, based on three distinct regions, triple gene block 1, the coat protein gene and a part of polymerase gene, revealed that the new necrotic isolates share high nucleotide sequence identity with isolates of the Ch2 genotype. This is the first report describing a necrotic type of PepMV of the Ch2 genotype.

  • Infectious RNA transcripts derived from cloned cDNA of a Pepino Mosaic Virus isolate
    Archives of Virology, 2009
    Co-Authors: Beata Hasiów-jaroszewska, N Borodynko, Henryk Pospieszny
    Abstract:

    For the first time, a full-length cDNA clone of the RNA genome of Pepino Mosaic Virus (PepMV) was constructed. RNA was extracted from purified virions of isolate PepMV-Pa and used for cDNA synthesis. The full-length cDNA was produced as one 6.4-kb fragment representing the entire PepMV genome. This fragment was ligated into the pCR^® - XL-TOPO^® vector downstream of T7 RNA polymerase promoter, which was included in the 5′ primer sequence used for RT-PCR. The PepMV-Pa RNA transcripts obtained were infectious in different host plants, causing symptoms indistinguishable from those of the wild-type isolate. The presence and authenticity of the progeny Virus were verified by ELISA, RT-PCR and nucleotide sequencing.