Molecular Methods for the Simultaneous Detection of Tomato Fruit Blotch Virus and Identification of Tomato Russet Mite, a New Potential Virus-Vector System Threatening Solanaceous Crops Worldwide.

Tomato fruit blotch virus (ToFBV) (Blunervirus solani, family Kitaviridae) was firstly identified in Italy in 2018 in tomato plants that showed the uneven, blotchy ripening and dimpling of fruits. Subsequent High-Throughput Sequencing (HTS) analysis allowed ToFBV to be identified in samples collected in Australia, Brazil, and several European countries, and its presence in tomato crops was dated back to 2012. In 2023, the virus was found to be associated with two outbreaks in Italy and Belgium, and it was included in the EPPO Alert list as a potential new threat for tomato fruit production. Many epidemiologic features of ToFBV need to be still clarified, including transmission. Aculops lycopersici Massee (Acariformes: Eriophyoidea), the tomato russet mite (TRM), is a likely candidate vector, since high population densities were found in most of the ToFBV-infected tomato cultivations worldwide. Real-time RT-PCR tests for ToFBV detection and TRM identification were developed, also as a duplex assay. The optimized tests were then transferred to an RT-ddPCR assay and validated according to the EPPO Standard PM 7/98 (5). Such sensitive, reliable, and validated tests provide an important diagnostic tool in view of the probable threat posed by this virus-vector system to solanaceous crops worldwide and can contribute to epidemiological studies by simplifying the efficiency of research. To our knowledge, these are the first molecular methods developed for the simultaneous detection and identification of ToFBV and TRM.

Carlavirus Species Infecting Hop Plants in Italy: Molecular Identification and Phylogenetic Analyses of the Detected Isolates.

Hop (Humulus lupulus L.) is a minor ingredient in the beer production but has a strong influence on the beer quality due to the high chemical complexity of the cones used in brewing. One of the major factors that can severely affect the chemical composition of the hop cones and their marketability is the presence of viral infections in the plant. Amongst the five major hop viruses, three belong to the Carlavirus genus: hop mosaic virus (HpMV), hop latent virus (HpLV), and American hop latent virus (AHLV). The occurrence of carlaviruses on hop germplasm in Italy was firstly recorded in 2017 but, in that context, a generic detection was only performed and no information on the infecting Carlavirus species was provided. To fill this gap, 51 hop samples previously found infected by carlaviruses were analysed by RT-PCR employing primer pairs specific for the coat protein (CP) of HpMV, HpLV and AHLV, respectively. HpLV resulted largely prevalent as it was detected in 96.1% of tested samples whereas for HpMV and AHLV an infection rate of 5.9% and 3.9% was recorded, respectively. CP nucleotide sequences from 13 selected virus isolates were obtained and analysed; moreover, the complete genome sequence of 7 isolates was obtained by using high throughput sequencing (HTS). Phylogenetic analysis showed close relationships among isolates from different geographical origin, including European and non-European countries, according to the worldwide movement of hop germplasm due to global trade. This is the first report of HpMV, HpLV and AHLV on hop germplasm in Italy.

Development and Validation of a Duplex RT-qPCR for Detection of Peach Latent Mosaic Viroid and Comparison of Different Nucleic-Acid-Extraction Protocols.

Peach latent mosaic viroid (PLMVd) is an important pathogen that causes disease in peaches. Control of this viroid remains problematic because most PLMVd variants are symptomless, and although there are many detection tests in use, the reliability of PCR-based methods is compromised by the complex, branched secondary RNA structure of the viroid and its genetic diversity. In this study, a duplex RT-qPCR method was developed and validated against two previously published single RT-qPCRs, which were potentially able to detect all known PLMVd variants when used in tandem. In addition, in order to simplify the sample preparation, rapid-extraction protocols based on the use of crude sap or tissue printing were compared with commercially available RNA purification kits. The performance of the new procedure was evaluated in a test performance study involving five participant laboratories. The new method, in combination with rapid-sample-preparation approaches, was demonstrated to be feasible and reliable, with the advantage of detecting all different PLMVd isolates/variants assayed in a single reaction, reducing costs for routine diagnosis.

In Vivo Antiphytoviral and Aphid Repellency Activity of Essential Oils and Hydrosols from Mentha suaveolens and Foeniculum vulgare to Control Zucchini Yellow Mosaic Virus and Its Vector Aphis gossypii.

In recent years, natural compounds have gained attention in many fields due to their wide-range biological activity. In particular, essential oils and their associated hydrosols are being screened to control plant pests, exerting antiviral, antimycotic and antiparasitic actions. They are more quickly and cheaply produced and are generally considered safer for the environment and non-target organisms than conventional pesticides. In this study, we report the evaluation of the biological activity of two essential oils and their corresponding hydrosols obtained from Mentha suaveolens and Foeniculum vulgare in the control of zucchini yellow mosaic virus and its vector, Aphis gossypii, in Cucurbita pepo plants. The control of the virus was ascertained with treatments applied either concurrently with or after virus infection; choice tests were performed to verify repellency activity against the aphid vector. The results indicated that treatments could decrease virus titer as measured using real-time RT-PCR, while the experiments on the vector showed that the compounds effectively repelled aphids. The extracts were also chemically characterized using gas chromatography-mass spectrometry. Mentha suaveolens and Foeniculum vulgare hydrosol extracts mainly comprised fenchone and decanenitrile, respectively, while essential oils analysis returned a more complex composition, as expected.

In vivo Antiphytoviral Activity of Essential Oils and Hydrosols From Origanum vulgare, Thymus vulgaris, and Rosmarinus officinalis to Control Zucchini Yellow Mosaic Virus and Tomato Leaf Curl New Delhi Virus in Cucurbita pepo L.

In the last decades, the interest in biological activity of natural compounds has been growing. In plant protection, essential oils have been reported to exhibit antiviral, antimycotic, and antiparasitic activities, and are regarded as promising for the formulation of safe antimicrobial agents. Attention has also been focused on hydrosols, the by-products of hydro-distillation of essential oils. Their production is easy, fast, and cheap, and they seem to arise less concern for human health than essential oils. Plant viruses represent a major concern for agricultural crops since no treatment compound is available for virus control. This work was aimed at evaluating the antiphytoviral effectiveness of treatments with three essential oils and corresponding hydrosols extracted from Origanum vulgare, Thymus vulgaris, and Rosmarinus officinalis on Cucurbita pepo plants infected by zucchini yellow mosaic virus or tomato leaf curl New Delhi virus. Treatments were applied either concurrently or after virus inoculation to ascertain an inhibition or curative activity, respectively. Symptoms were observed and samplings were performed weekly. Virus titer and expression levels of phenylalanine ammonia lyase gene (PAL) were measured on treated and untreated infected plants by real-time PCR. PAL gene plays an important role in plant defense response as it is involved in tolerance/resistance to phytopathogens. Results indicated that treatments were effective against tomato leaf curl New Delhi virus whether applied simultaneously with the inoculation or after. A major inhibition was observed with O. vulgare essential oil and hydrosol, resulting in 10-4-fold decrease of virus titer 3 weeks after treatment. Curative activity gave maximum results with all three essential oils and T. vulgaris and R. officinalis hydrosols, recording from 10-2-fold decrease to virus not detected 4 weeks after treatment. An induction of PAL gene expression was recorded at 12 d.p.i. and then was restored to the levels of untreated control. This allows to hypothesize an early plant defense response to virus infection, possibly boosted by treatments. Plant extracts' composition was characterized by gas chromatography-mass spectrometry. Phenols were largely main components of O. vulgare and T. vulgaris extracts (carvacrol and thymol, respectively), while extracts from R. officinalis were based on monoterpene hydrocarbons (essential oil) and oxygenated monoterpenes (hydrosol).

WRKY Gene Family Drives Dormancy Release in Onion Bulbs.

Onion (Allium cepa L.) is an important bulb crop grown worldwide. Dormancy in bulbous plants is an important physiological state mainly regulated by a complex gene network that determines a stop of vegetative growth during unfavorable seasons. Limited knowledge on the molecular mechanisms that regulate dormancy in onion were available until now. Here, a comparison between uninfected and onion yellow dwarf virus (OYDV)-infected onion bulbs highlighted an altered dormancy in the virus-infected plants, causing several symptoms, such as leaf striping, growth reduction, early bulb sprouting and rooting, as well as a lower abscisic acid (ABA) level at the start of dormancy. Furthermore, by comparing three dormancy stages, almost five thousand four hundred (5390) differentially expressed genes (DEGs) were found in uninfected bulbs, while the number of DEGs was significantly reduced (1322) in OYDV-infected bulbs. Genes involved in cell wall modification, proteolysis, and hormone signaling, such as ABA, gibberellins (GAs), indole-3-acetic acid (IAA), and brassinosteroids (BRs), that have already been reported as key dormancy-related pathways, were the most enriched ones in the healthy plants. Interestingly, several transcription factors (TFs) were up-regulated in the uninfected bulbs, among them three genes belonging to the WRKY family, for the first time characterized in onion, were identified during dormancy release. The involvement of specific WRKY genes in breaking dormancy in onion was confirmed by GO enrichment and network analysis, highlighting a correlation between AcWRKY32 and genes driving plant development, cell wall modification, and division via gibberellin and auxin homeostasis, two key processes in dormancy release. Overall, we present, for the first time, a detailed molecular analysis of the dormancy process, a description of the WRKY-TF family in onion, providing a better understanding of the role played by AcWRKY32 in the bulb dormancy release. The TF co-expressed genes may represent targets for controlling the early sprouting in onion, laying the foundations for novel breeding programs to improve shelf life and reduce postharvest.

Effects of Organic Biostimulants Added with Zeolite on Zucchini Squash Plants Infected by Tomato Leaf Curl New Delhi Virus.

The use of organic substances in integrated pest management can contribute to human- and environment-safe crop production. In the present work, a combination of organic biostimulants (Fullcrhum Alert and BioVeg 500) and an inorganic corroborant (Clinogold, zeolite) was tested for the effects on the plant response to the quarantine pest tomato leaf curl New Delhi virus (ToLCNDV). Biostimulants were applied to healthy and infected greenhouse-grown zucchini plants, and the vegetative parameters and viral titer were evaluated. Although no antiviral effects were observed in terms of both virus replication and symptom expression, these biostimulants were shown to influence plant fitness. A significant increase in biomass and in leaf, flower, and fruit production was induced in both healthy and infected plants. Biostimulants also enhanced the production of metabolites commonly involved in plant response to virus infection, such as carbohydrates, phenylpropanoids and free amino acids. These results encourage new field trials to evaluate the actual productivity of infected plants after treatments and the possible application of organic biostimulants in agriculture.

Development and Validation of a One-Step Reverse Transcription Real-Time PCR Assay for Simultaneous Detection and Identification of Tomato Mottle Mosaic Virus and Tomato Brown Rugose Fruit Virus.

Tobamovirus species represent a threat to solanaceous crops worldwide, due to their extreme stability and because they are seed borne. In particular, recent outbreaks of tomato brown rugose fruit virus in tomato and pepper crops led to the establishment of prompt control measures, and the need for reliable diagnosis was urged. Another member of the genus, tomato mottle mosaic virus, has recently gained attention due to reports in different continents and its common features with tomato brown rugose fruit virus. In this study, a new real-time RT-PCR detection system was developed for tomato brown rugose fruit virus and tomato mottle mosaic virus on tomato leaves and seeds using TaqMan chemistry. This test was designed to detect tomato mottle mosaic virus by amplifying the movement protein gene in a duplex assay with the tomato brown rugose fruit virus target on the CP-3'NTR region, which was previously validated as a single assay. The performance of this test was evaluated, displaying analytical sensitivity 10-5-10-6-fold dilution for seeds and leaves, respectively, and good analytical specificity, repeatability, and reproducibility. Using the newly developed and validated test, tomato brown rugose fruit virus detection was 100% concordant with previously performed analyses on 106 official samples collected in 2021 from different continents.

Inter-Laboratory Comparison of RT-PCR-Based Methods for the Detection of Tomato Brown Rugose Fruit Virus on Tomato.

In 2020, a test performance study (TPS) for the specific detection of tomato brown rugose fruit virus (ToBRFV) was organized in the frame of the H2020 Valitest project. Since no validated tests were available, all the protocols reported in the literature were at first screened, performing preliminary studies in accordance with the EPPO standard PM 7/98 (4). Five molecular tests, two conventional RT-PCR and three real-time RT-PCR were found to be suitable and were included in the TPS. Thirty-four laboratories from 18 countries worldwide took part in TPS, receiving a panel of 22 blind samples. The panel consisted of sap belonging to symptomatic or asymptomatic leaves of Solanum lycopersicum and Capsicum annuum. The results returned by each laboratory were analyzed and diagnostic parameters were assessed for each test: reproducibility, repeatability, analytical sensitivity, diagnostic sensitivity and diagnostic specificity. All the evaluated tests resulted in being reliable in detecting ToBRFV and were included in an EPPO Standard PM 7/146-Diagnostics.

Metabolites response to onion yellow dwarf virus (OYDV) infection in 'Rossa di Tropea' onion during storage: a 1 H HR-MAS NMR study.

BACKGROUND: Plant viral infections induce changes in metabolic components in the host plant, with potential effects on compositional, organoleptic and storability features of agricultural products. Identification of modulated metabolites may provide clues concerning pathways implementing responses in plant-pathogen interactions. A time course study of metabolic fingerprinting of onion yellow dwarf virus (OYDV)-infected versus healthy 'Rossa di Tropea' onion bulbs was performed using proton high-resolution magic angle spinning nuclear magnetic resonance (1 H HR-MAS NMR) and ultra-performance liquid chromatography (UPLC), providing an overview of the metabolic state of the bulb in response to OYDV infection during storage. RESULTS: Metabolites accumulated/depleted upon infection were identified, belonging to flavonoid, saccharide, amino acid and organic acid classes. A decrease in quercetin glucosides content and antioxidant activity was observed in infected bulbs; some amino acids (Arg, Asn, Phe, Val) accumulated, while others were depleted (Leu); for some metabolites, a bimodal time-course was observed during storage (Glc, Lys). Virus interference on metabolic pathways, and the effects of the metabolic shift on edible product storability, organoleptic and nutritional quality were discussed. CONCLUSIONS: OYDV infection induces a metabolic shift in 'Rossa di Tropea' onion during bulb storage, involving several pathways and affecting storability and organoleptic and nutritional quality of bulbs at marketable stage. © 2020 Society of Chemical Industry.

Kinetics of drug release from a hyaluronan-steroid conjugate investigated by NMR spectroscopy.

The methylprednisolone steroid ester of hyaluronan was hydrolyzed under physiological conditions in vitro, and the kinetics of drug release was investigated by NMR spectroscopy. Transverse relaxation times are correlated with the molecular rotational freedom, which undergoes large changes for methylprednisolone when released. Multi-exponential decays were observed, which together with the corresponding population gave valuable insights into the conformational changes that occur in the biopolymer during hydrolysis. The biomaterial exists in aqueous solution in two conformations, 'collapsed' and 'water-exposed', in equilibrium. Under physiological conditions, the methylprednisolone is completely released within 48 h. Transverse relaxation times proved to be an appropriate tool for monitoring the drug release in vitro.

Investigating the interactions of hyaluronan derivatives with biomolecules. The use of diffusional NMR techniques.

[Chemical structure: see text] The interactions between a biomaterial and biomolecules present in body fluids often determine the fate of the biomaterial. This paper presents a study on hyaluronan (HA)-containing materials (in soluble or colloidal form) that focuses on their interactions with lipids and proteins and for the first time uses PFG NMR as an analytical technique for probing these events. The interactions of HA-based polymers with phospholipids (DPPC and DPPG liposomes) are shown to depend both on charge and hydrophobicity factors. Despite the difference in behavior between albumin (substantially non-adhesive) and fibrinogen (adhesive), the interactions of the polymers with proteins do not seem to be based on hydrophobic effects but on surface polar interactions.

Expression and characterization of human-elastin-repeat-based temperature-responsive protein polymers for biotechnological purposes.

Rapid progress has been made in the design and synthesis of oligomers and polymers that emulate the properties of natural proteins. Molecular bioengineering offers the chance to design and produce artificial polymeric proteins with tailored polymeric properties. The elastin-like polypeptides are a well-defined family of polymers with noteworthy characteristic based on the VPGVG repeated motif of bovine elastin. In the human homologue, the most regular sequence is represented by the repetition of the VAPGVG hexapeptidic motif. On the basis of this sequence, a synthetic gene has been designed, cloned and expressed in Escherichia coli to obtain artificial protein polymers. The rapid one-step in-frame cloning of any biologically active sequence can be achieved directly in the expression vector, allowing further improvement of the potential of the resulting product.

Characterization of methylprednisolone esters of hyaluronan in aqueous solution: conformation and aggregation behavior.

Methylprednisolone steroid esters of hyaluronan differing in degree of functionalization and molecular weight were investigated in aqueous solution. Conformation and aggregation phenomena were elucidated by means of circular dichroism, viscometry, rheology, and nuclear magnetic resonance, mainly by (1)H pulsed field gradient (PFG) NMR, which allows the determination of the diffusion coefficient of the species under investigation. The functionalization of hyaluronan with the steroid induces a reduction of the molecular volume, as a consequence of intramolecular hydrophobic interactions. For concentrated samples we have observed the coexistence of unimolecular collapsed chains and of aggregates, the latter disappearing upon dilution. The methylprednisolone ester of lower molecular weight hyaluronan has a larger molecular volume than its higher molecular weight analogue, even though still smaller than the underivatized polymer. This effect can be explained with the reduced flexibility of the polymer backbone probably impairing intramolecular interactions.

Synthesis and partial characterization of hydrogels obtained via glutaraldehyde crosslinking of acetylated chitosan and of hyaluronan derivatives.

Hydrophilic networks based on functionalized hyaluronic acid and on partially acetylated chitosan, respectively, have been obtained. In the case of hyaluronic acid (HA), primary amino functionalities have been introduced along the polysaccharide chains. The ensuing derivatives, i.e., HA-lysine (HA-K), HA-diamino pentane (HA-DAP), and HA-glycine-lysine (HA-GK), have been characterized by high field NMR spectroscopy. NMR 2D-DOSY experiments have allowed us to optimize the purification procedure. Chitosan was made soluble in water by partial acetylation. Cross-linking reactions have been performed using glutaraldehyde. The obtained networks have been qualitatively characterized by means of (13)C CP-MAS NMR technique. The hydrogels have been characterized also in terms of water uptake.

New gelatin-based hydrogels via enzymatic networking.

New types of hydrogels have been obtained starting from high bloom purified gelatin A, alone or in mixtures with hyaluronan and with a hyaluronan derivative bearing primary amino groups, by transglutaminase-catalyzed cross-linking. The reticulation process, carried out adopting two different temperature protocols, and the ensuing materials have been characterized in terms of rheologically estimated gel times, equilibrium swelling in water and in phosphate buffer solution (PBS), and rigidity modulus. Main structural and conformational factors governing the physicochemical properties and the possible application of the new hydrogels are discussed.