Analysis of Hop Stunt Viroid Diversity in Grapevine (Vitis vinifera L.) in Slovakia: Coexistence of Two Particular Genetic Groups.

The hop stunt viroid (HSVd) is a widespread subviral pathogen infecting a broad spectrum of plant hosts including grapevine (Vitis vinifera L.). Despite its omnipresence in virtually all grapevine growing areas around the world, molecular data characterizing HSVd populations are missing from Slovakia. Analysis of the complete nucleotide sequences of 19 grapevine variants revealed the existence of two genetic HSVd groups in Slovakia (internally named the "6A" and "7A" groups based on the particular stretch of adenines at nucleotide positions 39-44/45, respectively). Despite their sampling at different times in various unrelated vineyards, the 6A and 7A groups are characterized by low intra-group divergence (~0.3 and 0.2%, respectively). On the other hand, inter-group divergence reached 2.2% due to several mutations, seven of which were found to be group-specific and mainly (except for one) located in the region of the pathogenic domain. Interestingly, in addition to their frequent co-existence within the same geographical location, the mixed infection of the 6A and 7A type sequence variants was also unequivocally and repeatedly proven within single grapevine plants. The RNA secondary structure analysis of representative isolates from each of these two genetic groups indicated a potential compensatory explanation of such mutations. These group-specific sites could be pointing towards the evolutionary selection linked to the necessity of the viroid to retain its structural conformational integrity, crucial for its functional biochemical ability to interact with specific grapevine cellular host factors required for HSVd propagation.

Elicitation of Hyoscyamine Production in Datura stramonium L. Plants Using Tobamoviruses.

Datura stramonium L. produces tropane alkaloids, and the hyoscyamine is dominant among them. Hyoscyamine is produced by hairy root cultures in vitro derived from native plants or plants with the genetically modified biosynthetic pathway for hyoscyamine. A common procedure is extraction from cultivated plants. Elicitors for increased production can be used in both cases. Live viruses are not well known for use as elicitors, therefore, D. stramonium plants grown in soil were artificially infected with the tobamoviruses Pepper mild mottle virus (PMMoV), Tomato mosaic virus (ToMV), and Tobacco mosaic virus (TMV). Differences in the content of hyoscyamine were between capsules and roots of infected and non-infected plants. Elicitation increased content of hyoscyamine in capsules 1.23-2.34 times, compared to the control. The most effective viruses were PMMoV and ToMV (isolate PV143), which increased content to above 19 mg/g of fresh weight of a capsule. The effect of each virus elicitor was expressed also in hyoscyamine content in roots. Elicited plants contained 5.41-16.54 times more hyoscyamine in roots compared to non-elicited plants. The most effective elicitor was ToMV SL-1, which raised production above 20 mg/g fresh weight of roots. It has been shown that tobamoviruses can be used as biotic elicitors.

The Vapor Phase of Selected Essential Oils and Their Antifungal Activity In Vitro and In Situ against Penicillium commune, a Common Contaminant of Cheese.

This study aimed to determine the in vitro and in situ antifungal activity of (14) selected essential oils (EOS), namely clove, thyme, red thyme, litsea, eucalyptus, niaouli, fennel, anise, cumin, basil, rosemary, sage, bergamot mint, and marjoram, by vapor contact against the growth of two strains of Penicillium commune (KMi-183 and KMi-402). Furthermore, to exclude the negative effect of EOs on the lactic acid bacteria (LABs) (Streptococcus spp.) on cheeses, their influence was monitored. Next, the sensory evaluation of cheese treated by EOs was evaluated. The results show that litsea and clove EOs were the most effective in the vapor phase against both tested strains. These EOs were characterized by the highest amount of α- (40.00%) and ß-Citral (34.35%) in litsea and eugenol (85.23%) in clove. The antitoxicogenic activity of less effective (in growth inhibition) EOs on cyclopiazonic acid (CPA) production by the tested strains was also observed. The growth of Streptococcus spp. (ranging from 8.11 to 9.69 log CFU/g) was not affected by the EOs in treated cheese. Even though the evaluators recognized some EOs in sensory evaluation by the triangle test, they did not have a negative effect on the taste and smell of the treated cheeses and were evaluated as edible. The antifungal activity of EOs against several types of microscopic fungi and their effect on the sensory properties of treated foods needs to be further tested to achieve the most effective protection of foods from their direct contaminants.

Evaluation of New Polyclonal Antibody Developed for Serological Diagnostics of Tomato Mosaic Virus.

Plant viruses threaten agricultural production by reducing the yield, quality, and economical benefits. Tomato mosaic virus (ToMV) from the genus Tobamovirus causes serious losses in the quantity and quality of tomato production. The management of plant protection is very difficult, mainly due to the vector-less transmission of ToMV. Resistant breeding generally has low effectiveness. The most practical approach is the use of a rapid diagnostic assay of the virus' presence before the symptoms occur in plants, followed by the eradication of virus-infected plants. Such approaches also include serological detection methods (ELISA and Western immunoblotting), where antibodies need to be developed for an immunochemical reaction. The development and characterization of polyclonal antibodies for the detection of ToMV with appropriate parameters (sensitivity, specificity, and cross-reactivity) were the subjects of this study. A new polyclonal antibody, AB-1, was developed in immunized rabbits using the modified oligopeptides with antigenic potential (sequences are revealed) derived from the coat protein of ToMV SL-1. the developed polyclonal antibody. AB-1, showed higher sensitivity when compared with commercially available analogs. It also detected ToMV in infected pepper and eggplant plants, and detected another two tobamoviruses (TMV and PMMoV) and ToMV in soil rhizosphere samples and root residues, even two years after the cultivation of the infected tomato plant.

High-Throughput Sequencing Discloses the Cucumber Mosaic Virus (CMV) Diversity in Slovakia and Reveals New Hosts of CMV from the Papaveraceae Family.

Cucumber mosaic virus (CMV; Cucumovirus, Bromoviridae) is an omnipresent virus characterized by a large host range and high genetic variability. Using high-throughput sequencing, we have characterized near complete genomes of 14 Slovak CMV variants from different plant hosts. Of these, three variants originated from the Papaveraceae species (oilseed poppy, common poppy and great celandine), previously poorly described as CMV natural hosts. Based on a BLAST search and phylogenetic analysis, the Slovak CMV isolates can be divided into two genetically different Groups, Ia and II, respectively. The SL50V variant, characterized by a divergent RNA2 sequence, potentially represents a reassortant variant. In four samples (T101, SL50V, CP2, MVU2-21), the presence of satellite CMV RNA was identified along with CMV. Although mechanically transmitted to experimental cucumber plants, the role of satellite RNA in the symptomatology observed could not be established due to a complex infection of original hosts with different viruses.

Biotic and Abiotic Elicitors of Stilbenes Production in Vitis vinifera L. Cell Culture.

The in vitro cell cultures derived from the grapevine (Vitis vinifera L.) have been used for the production of stilbenes treated with different biotic and abiotic elicitors. The red-grape cultivar Váh has been elicited by natural cellulose from Trichoderma viride, the cell wall homogenate from Fusarium oxysporum and synthetic jasmonates. The sodium-orthovanadate, known as an inhibitor of hypersensitive necrotic response in treated plant cells able to enhance production and release of secondary metabolite into the cultivation medium, was used as an abiotic elicitor. Growth of cells and the content of phenolic compounds trans-resveratrol, trans-piceid, δ-viniferin, and ɛ-viniferin, were analyzed in grapevine cells treated by individual elicitors. The highest accumulation of analyzed individual stilbenes, except of trans-piceid has been observed after treatment with the cell wall homogenate from F. oxysporum. Maximum production of trans-resveratrol, δ- and ɛ-viniferins was triggered by treatment with cellulase from T. viride. The accumulation of trans-piceid in cell cultures elicited by this cellulase revealed exactly the opposite effect, with almost three times higher production of trans-resveratrol than that of trans-piceid. This study suggested that both used fungal elicitors can enhance production more effectively than commonly used jasmonates.

Molecular Characterization of Potato Virus Y (PVY) Using High-Throughput Sequencing: Constraints on Full Genome Reconstructions Imposed by Mixed Infection Involving Recombinant PVY Strains.

In recent years, high throughput sequencing (HTS) has brought new possibilities to the study of the diversity and complexity of plant viromes. Mixed infection of a single plant with several viruses is frequently observed in such studies. We analyzed the virome of 10 tomato and sweet pepper samples from Slovakia, all showing the presence of potato virus Y (PVY) infection. Most datasets allow the determination of the nearly complete sequence of a single-variant PVY genome, belonging to one of the PVY recombinant strains (N-Wi, NTNa, or NTNb). However, in three to-mato samples (T1, T40, and T62) the presence of N-type and O-type sequences spanning the same genome region was documented, indicative of mixed infections involving different PVY strains variants, hampering the automated assembly of PVY genomes present in the sample. The N- and O-type in silico data were further confirmed by specific RT-PCR assays targeting UTR-P1 and NIa genomic parts. Although full genomes could not be de novo assembled directly in this situation, their deep coverage by relatively long paired reads allowed their manual re-assembly using very stringent mapping parameters. These results highlight the complexity of PVY infection of some host plants and the challenges that can be met when trying to precisely identify the PVY isolates involved in mixed infection.

Procedures for DNA Extraction from Opium Poppy (Papaver somniferum L.) and Poppy Seed-Containing Products.

Several commonly used extraction procedures and commercial kits were compared for extraction of DNA from opium poppy (Papaver somniferum L.) seeds, ground seeds, pollen grains, poppy seed filling from a bakery product, and poppy oil. The newly developed extraction protocol was much simpler, reduced the cost and time required for DNA extraction from the native and ground seeds, and pollen grains. The quality of extracted DNA by newly developed protocol was better or comparable to the most efficient ones. After being extended by a simple purification step on a silica membrane column, the newly developed protocol was also very effective in extracting of poppy DNA from poppy seed filling. DNA extracted from this poppy matrix was amplifiable by PCR analysis. DNA extracted from cold-pressed poppy oil and suitable for amplifications was obtained only by methods developed previously for olive oil. Extracted poppy DNA from all tested matrices was analysed by PCR using primers flanking a microsatellite locus (156 bp) and two different fragments of the reference tubulin gene (553 bp and 96 bp). The long fragment of the reference gene was amplified in DNA extracted from native seeds, ground seeds, and pollen grains. Poppy DNA extracted from the filling of bakery product was confirmed only by amplification of short fragments (96 bp and 156 bp). DNA extracted from cold-pressed poppy oil was determined also only by amplification of these two short fragments.

Plant Viruses Infecting Solanaceae Family Members in the Cultivated and Wild Environments: A Review.

Plant viruses infecting crop species are causing long-lasting economic losses and are endangering food security worldwide. Ongoing events, such as climate change, changes in agricultural practices, globalization of markets or changes in plant virus vector populations, are affecting plant virus life cycles. Because farmer's fields are part of the larger environment, the role of wild plant species in plant virus life cycles can provide information about underlying processes during virus transmission and spread. This review focuses on the Solanaceae family, which contains thousands of species growing all around the world, including crop species, wild flora and model plants for genetic research. In a first part, we analyze various viruses infecting Solanaceae plants across the agro-ecological interface, emphasizing the important role of virus interactions between the cultivated and wild zones as global changes affect these environments on both local and global scales. To cope with these changes, it is necessary to adjust prophylactic protection measures and diagnostic methods. As illustrated in the second part, a complex virus research at the landscape level is necessary to obtain relevant data, which could be overwhelming. Based on evidence from previous studies we conclude that Solanaceae plant communities can be targeted to address complete life cycles of viruses with different life strategies within the agro-ecological interface. Data obtained from such research could then be used to improve plant protection methods by taking into consideration environmental factors that are impacting the life cycles of plant viruses.

Diacylglycerol Acetyltransferase Gene Isolated from Euonymus europaeus L. Altered Lipid Metabolism in Transgenic Plant towards the Production of Acetylated Triacylglycerols.

Euonymus species from the Celastraceae family are considered as a source of unusual genes modifying the oil content and fatty acid composition of vegetable oils. Due to the possession of genes encoding enzyme diacylglycerol acetyltransferase (DAcT), Euonymus plants can synthesize and accumulate acetylated triacyglycerols. The gene from Euonymus europaeus (EeDAcT) encoding the DAcT was identified, isolated, characterized, and modified for cloning and genetic transformation of plants. This gene has a unique nucleotide sequence and amino acid composition, different from orthologous genes from other Euonymus species. Nucleotide sequence of original EeDAcT gene was modified, cloned into transformation vector, and introduced into tobacco plants. Overexpression of EeDAcT gene was confirmed, and transgenic host plants produced and accumulated acetylated triacylglycerols (TAGs) in immature seeds. Individual transgenic plants showed difference in amounts of synthesized acetylTAGs and also in fatty acid composition of acetylTAGs.

Progress in the genetic engineering of cereals to produce essential polyunsaturated fatty acids.

Essential polyunsaturated fatty acids with more than two double bonds and length of carbon chain 18-22 must be taken in the diet to prevent diseases and imbalances caused by their deficiency. Terrestrial sources of polyunsaturated fatty acids are limited to only a few plant species whose large-scale cultivation is not possible and the production of their seeds and oil is ineffective. The complete biosynthetic pathway of fatty acids is known in organisms, including plants. After the first gene encoding the enzyme catalysing the initial steps of PUFA biosynthesis (ω-3 desaturase, Δ6-desaturase) were isolated, isolation of other genes encoding relevant enzymes of the PUFA pathway from different donor organisms followed. Genetic transformations of model plants by the desaturase- and elongase-encoding genes opened the way for the genetic engineering of oilseed crop species. Some of the developed transgenic plants produced PUFAs, including eicosapentaenoic and docosahexaenoic acids. Seed oils extracted from them were similar to fish oil. Tools of the synthetic biology can be applied in modifications of the PUFA pathway and also in overcoming of limitations when the gene and its expression product are absent in the pathway. Such progress in cereals (barley, wheat, maize) has been made only recently, when the first successful modifications of the ω-3 and ω-6 PUFA pathways succeeded. This review focuses on genetic modifications of the PUFA biosynthetic pathway in cereals in relation to the status reached in model plants and oilseed crops.

Experimental Infection of Different Tomato Genotypes with Tomato mosaic virus Led to a Low Viral Population Heterogeneity in the Capsid Protein Encoding Region.

The complete genome sequence of a Slovak SL-1 isolate of Tomato mosaic virus (ToMV) was determined from the next generation sequencing (NGS) data, further confirming a limited sequence divergence in this tobamovirus species. Tomato genotypes Monalbo, Mobaci and Moperou, respectively carrying the susceptible tm-2 allele or the Tm-1 and Tm-2 resistant alleles, were tested for their susceptibility to ToMV SL-1. Although the three tomato genotypes accumulated ToMV SL-1 to similar amounts as judged by semi-quantitative DAS-ELISA, they showed variations in the rate of infection and symptomatology. Possible differences in the intra-isolate variability and polymorphism between viral populations propagating in these tomato genotypes were evaluated by analysis of the capsid protein (CP) encoding region. Irrespective of genotype infected, the intra-isolate haplotype structure showed the presence of the same highly dominant CP sequence and the low level of population diversity (0.08-0.19%). Our results suggest that ToMV CP encoding sequence is relatively stable in the viral population during its replication in vivo and provides further demonstration that RNA viruses may show high sequence stability, probably as a result of purifying selection.