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In agriculture, new and sustainable strategies are increasingly demanded to integrate the traditional management of viral diseases based on the use of virus-free propagation materials and resistant or tolerant cultivars and on the control of insect vectors. Among the possible Integrated Pest Management (IPM) approaches, organic biostimulants have shown promising results in enhancing plant tolerance to virus infections by improving plant fitness and productivity and modulating metabolic functions. In this study, the combination of two organic biostimulants, Alert D-Max and Resil EVO Q, composed of seaweed and alfalfa extracts, enzymatic hydrolysates, and micronized zeolite, was applied on the leaves and roots of zucchini squashes, both healthy and infected by zucchini yellow mosaic virus (ZYMV). Four applications were scheduled based on ZYMV inoculation timing, and plant vegetative and reproductive parameters were recorded along with the virus titre and symptom severity. The modulation of the expression of specific genes potentially involved in pattern-triggered immunity (PTI), systemic acquired resistance (SAR), and oxidative stress defence pathways was also investigated. Besides increasing the general fitness of the healthy plants, the biostimulants significantly improved the production of flowers and fruits of the infected plants, with a potential positive impact on their productivity. The repeated biostimulant applications also led to a one-tenth reduction in ZYMV titre over time and induced a progressive slowdown of symptom severity. Genes associated with SAR and PTI were up-regulated after biostimulant applications, suggesting the biostimulant-based priming of plant defence mechanisms. Due to the observed beneficial effects, the tested biostimulant mix can be an effective component of the IPM of cucurbit crops, acting as a sustainable practice for enhancing plant fitness and tolerance to potyviruses.

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.

Psyllids, members of the family Triozidae, represent a potential threat to the cultivation of solanaceous and apiaceous crops worldwide, mainly as vectors of the phloem-restricted bacterium ‘Candidatus Liberibacter solanacearum’ (Lso). The Lso haplotypes C, D and E are known to affect apiaceous crops, such as carrot and celery, in several European countries. In Italy, data on the incidence and natural spread of both Lso and psyllids have not been reported so far. In this study, the presence of the vectors was investigated in a main Italian district for carrot production, the “Altopiano del Fucino” area (Central Italy). Both occasional and regular surveys were carried out on a total of five carrot fields and one potato field in 2021 and 2022. Bactericera trigonica (Hodkinson), which is known to efficiently transmit Lso to carrots, was found to be well-established in the area. High levels of population density were recorded in the summer period (more than 100 adult specimens per trap caught every two weeks) and then sharply decreased after the carrot harvest, confirming the strict association of this psyllid species with crop availability. In 2022, 27.5% of the total tested psyllid samples resulted in being positive for Lso haplotypes D and E, the latter being prevalent. This survey revealed, for the first time in Italy, the presence of B. trigonica adults associated with Lso in carrot crops. Although this study was limited to a few fields located in one area, it provided important evidence of the risks for Lso outbreaks and prompted further research to assess the spread and incidence of the disease in apiaceous cultivations in Italy.

The onion yellow dwarf virus (OYDV) represents a limiting biotic stress in onion (Allium cepa L.); little information is available regarding resistant varieties. In Italy, onion production is limited but represented by a wide diversity of ecotypes. A two-year trial was carried out to test the OYDV-susceptibility/tolerance of different Italian onion cultivars by rating symptom severity and plant growth parameters and assessing post-harvest secondary infections. The cultivar and ecotypes included in the study were characterized by simple sequence repeats (SSR) analysis, and the expression analysis of two genes (Eukaryotic translation initiation factors, EIFs) involved in potyvirus replication was also performed. Two susceptible and one tolerant cultivar were identified based on symptom expression and virus impact on plants. Although differences in growth parameters were limited to the first-year trial, the infection was correlated to a higher incidence of secondary infections in post-harvest, with altered water balance in infected bulbs. This correlation was also demonstrated during the long-term storage of bulbs. SSR analysis identified different clusters and only one gene isoform (EIF4eiso1) showed different expression levels in the OYDV/onion pathosystem. In conclusion, this study defines the genetic profile of Italian onion cultivars and provides evidence on susceptibility/tolerance features which will be useful in the future for the identification of viral resistance traits in onion.

The meadow spittlebug Philaenus spumarius (Hemiptera: Aphrophoridae) is distributed in several habitats worldwide and has been recently recognized as the main vector of Xylella fastidiosa subsp. pauca. This bacterium has been associated with olive quick decline syndrome (OQDS) in the Salento Peninsula (Italy) and is responsible for extensive desiccation and die-off of olive trees. Current OQDS management strategies include the control of P. spumarius populations, mainly through the removal of weed hosts and insecticide treatments. In addition to the mandatory phytosanitary measures, the demand for new strategies compatible with integrated pest management is increasing. In this study, laboratory biological assays were performed to assess the potential toxic effect of vegetal formulations against P. spumarius adults. Two formulations were tested at different concentrations: Form A, an emulsion of 10% hot pepper-infused oil (Capsicum annuum subspecies Cayenna in olive oil) and Arabic gum in an aqueous solution of extracts of Salvia guaranitica, and Form B, an aqueous solution of extracts of Taxus baccata. Both Form A and B showed to be toxic against P. spumarius compared to the water control. The mean percentage of spittlebug mortality obtained with Form A one hour after treatments was dose–dependent; the lethal dose values were 0.13% (LD25), 0.36% (LD50), and 0.85% (LD75). At the same time, no significant differences in mortality rate were observed between the 0.75% treatment and the treatments with deltamethrin (about 90%). The mean percentage of spittlebug mortality obtained with Form B ranged from 21% to 53% one hour after treatment, but these values were significantly lower than those obtained with deltamethrin. The effectiveness of Form A on the P. spumarius population was also evaluated in the field. The averages of captures in the three experimental blocks were 1.8/trap for treated and 7.7/trap for untreated plots, and the spittlebug populations significantly decreased after treatments. Based on these results and the literature data, we hypothesize that the effectiveness of Form A is the result of the synergistic effect of all its components. No symptoms of phytotoxicity were recorded on olive trees treated with Form A, and the number of P. spumarius specimens collected on these plants was much lower than on untreated plants. These results suggest the potential use of Form A in the protection of olive trees. This vegetal formulation can thus be considered as a valid alternative to chemical insecticides for the control of the main vector of X. fastidiosa and could be integrated into a sustainable management system for OQDS.

In Europe and the Mediterranean region, ‘Candidatus Liberibacter solanacearum’ (Lso) is associated with emerging diseases of Apiaceae crops, mainly carrot. Emergency measures for import of carrot seed were set, requiring seed to be heat-treated at 50°C or tested as Lso-negative by PCR. The germination response to heat treatment was assessed for 24 carrot cultivar and hybrid seed lots. Ten parsley, five fennel, and two celery seed lots were also analysed. Of these 41 seed lots, 21 were Lso-infected. Water heat treatment significantly decreased germinability compared to dry heat treatment, indicating that dry heat treatment is a cheaper and less detrimental procedure. However, the dry heat treatment significantly decreased seed germination compared to untreated controls in four of 24 seed lots of carrot, four of ten parsley seed lots, three of five fennel seed lots, and one of two celery seed lots. For parsley, the heat treatment reduced germinability to a lesser extent in Lso-infected than Lso-free seed lots. These data show that heat treatment can affect the germination of Apiaceae seeds to varying degrees, depending on species or variety, the type of heat treatment, and the sanitary status of the seeds.

Among the cucurbit-infecting viruses, watermelon mosaic virus (WMV) and zucchini yellow mosaic virus (ZYMV) (Potyvirus: Potyviridae) are responsible for severe symptoms on cucumber, melon, watermelon, and zucchini cultivations worldwide. In this study, reverse transcription real-time PCR (real-time RT-PCR) and droplet-digital PCR (RT-ddPCR) assays targeting the coat protein (CP) genes of WMV and ZYMV were developed and validated according to the international standards of plant pest diagnosis (EPPO PM 7/98 (5)). First, the diagnostic performance of WMV-CP and ZYMV-CP real-time RT-PCRs was evaluated, and the assays displayed an analytical sensitivity of 10−5 and 10−3, respectively. The tests also showed an optimal repeatability, reproducibility and analytical specificity, and were reliable for the virus detection in naturally infected samples and across a wide range of cucurbit hosts. Based on these results, the real-time RT-PCR reactions were adapted to set up RT-ddPCR assays. These were the first RT-ddPCR assays aiming at the detection and quantification of WMV and ZYMV and showed a high sensitivity, being able to detect until 9 and 8 copies/µL of WMV or ZYMV, respectively. The RT-ddPCRs allowed the direct estimation of the virus concentrations and opened to a broad range of applications in disease management, such as the evaluation of partial resistance in breeding processes, identification of antagonistic/synergistic events, and studies on the implementation of natural compounds in the integrated management strategies.

Biochar is a rich carbon product obtained by pyrolysis of biomass under a limited supply of oxygen. It is composed mainly of aromatic molecules, but its agronomic value is hard to evaluate and difficult to predict due to its great variable characteristics depending on the type of starting biomass and the conditions of pyrolysis. Anyway, it could be used as soil amendment because it increases the soil fertility of acidic soils, increases the agricultural productivity, and seems to provide protection against some foliar and soilborne diseases. In this study, the effects of biochar, obtained from olive pruning, have been evaluated on tomato seedlings growth and on their response to systemic agents' infection alone or added with beneficial microorganisms (Bacillus spp. and Trichoderma spp.). First, experimental data showed that biochar seems to promote the development of the tomato seedlings, especially at concentrations ranging from 1 to 20% (w/w with peat) without showing any antimicrobial effects on the beneficial soil bacteria at the tomato rhizosphere level and even improving their growth. Thus, those concentrations were used in growing tomato plants experimentally infected with tomato spotted wilt virus (TSWV) and potato spindle tuber viroid (PSTVd). The biochar effect was estimated by evaluating three parameters, namely, symptom expression, number of infected plants, and pathogen quantification, using RT-qPCR technique and −ΔΔCt analysis. Biochar at 10–15% and when added with Trichoderma spp. showed that it reduces the replication of PSTVd and the expression of symptoms even if it was not able to block the start of infection. The results obtained on TSWV-infected plants suggested that biochar could contribute to reducing both infection rate and virus replication. For systemic viral agents, such as PSTVd and TSWV, there are no curative control methods, and therefore, the use of prevention means, as can be assumed the use biochar, for example, in the nursery specialized in horticultural crops, can be of great help. These results can be an encouraging starting point to introduce complex biochar formulates among the sustainable managing strategies of plant systemic diseases.

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.

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.

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.

Bemisia tabaci is a key pest of horticultural, fibre and ornamental crops worldwide, primarily as a vector of plant viruses. In Italy, B. tabaci has established since the 1980s–1990s in southern regions as well as in Sicily and Sardinia. Recent reports of infestations in some areas of central Italy prompted a new survey to assess the whitefly distribution in the country as well as to update the species and haplotype composition of the populations present in southern Italy and in the main islands. The survey confirmed that B. tabaci is nowadays established in central Italy even at more northern latitudes than those noticed before. Most of the specimens collected throughout the country belonged to the Mediterranean (MED) species. The MEDQ1 and Q2 haplogroups were prevailing in open-field and greenhouse cultivations, respectively, except in Sardinia where only Q1 specimens were found on a wide range of crops and weeds. Population genetics analyses showed that several MEDQ1 haplotypes currently occur in Italy and their distribution is unrelated to evident temporal and geographic trends, except for a new genetic variant which seems to have originated in Sardinia. The MED species is known to better adapt to insecticide treatments and high temperatures, and its northward spread in Italy may have been favoured by the intensive agricultural practices and steady increase in both winter and summer temperatures occurring in the last few decades. The extensive presence of B. tabaci in Italy proves that a strict surveillance for possible new outbreaks of whitefly-transmitted viruses should be addressed to a range of sites that are expanding northwards.

The development of 3D printable hydrogels based on the crosslinking between chitosan and gelatin is proposed. Chitosan and gelatin were both functionalized with methyl furan groups. Chemical modification was performed by reductive amination with methyl furfural involving the lysine residues of gelatin and the amino groups of chitosan to generate hydrogels with tailored properties. The methyl furan residues present in both polymers were exploited for efficient crosslinking via Diels-Alder ligation with PEG-Star-maleimide under cell-compatible conditions. The obtained chitosan-gelatin hybrid was employed to formulate hydrogels and 3D printable biopolymers and its processability and biocompatibility were preliminarily investigated.

Abstract   Scaphoideus titanus Ball is a Nearctic leafhopper that was introduced for the first time in Europe probably at the beginning of the 20th century. In Europe, this species is a specialist on cultivated grapevines and is of great economic importance as the vector of Flavescence dor�e (FD), a Grapevine Yellows disease caused by Candidatus Phytoplasma vitis. The Random Amplified Polymorphic DNA (RAPD) technique was employed to obtain genetic information about the diffusion and the structure of S. titanus populations. Two American and 14 European populations were analysed. A total of 188 reproducible bands, obtained from three arbitrary primers, were considered to assess the amount and the pattern of genetic variation within and among leafhopper populations. American populations showed high levels of intra-population polymorphism and dissimilarity and appeared to be the most isolated of all the tested samples. The results confirm the historical role of American samples as the sources for the more recently founded European populations. RAPD analyses revealed a weak genetic structure of European samples that could probably be explained invoking the human role in their diffusion. The non-natural spreading of S. titanus across Europe is in fact attributable to the exchange of grapevine canes and grafts carrying eggs that the insect laid under the bark to overwinter. [ABSTRACT FROM AUTHOR]

A protocol based on the combination of different analytical methodologies is proposed to standardize the experimental conditions for reproducible formulations of hybrid hydrogels. The final hybrid material, based on the combination of gelatin and chitosan functionalized with methylfuran and cross-linked with 4-arm-PEG-maleimide, is able to mimic role, dynamism, and structural complexity of the extracellular matrix. Physical–chemical properties of starting polymers and finals constructs were characterized exploiting the combination of HP-SEC-TDA, UV, FT-IR, NMR, and TGA.

Non-covalent interactions in supramolecular chemistry provide useful systems to understand biological processes, and self-assembly systems are suitable assets to build-up innovative products for biomedical applications. In this field, polyelectrolyte complexes are interesting, especially when polysaccharides are involved, due to their non-toxicity and bio-absorbability. In this work, we investigated a polyelectrolyte formed by hyaluronic acid (HA), a negatively charged linear polysaccharide, with Chitlac (Ch), a positively charged lactose-modified chitosan. The aim of the study was the investigation of a novel Ch–HA polyelectrolyte complex, to understand the interaction between the two polysaccharides and the stability towards enzymatic activity. By means of gel permeation chromatography–triple detector array (GPC–TDA), nuclear magnetic resonance (NMR), dynamic viscosity, Zeta Potential and scanning electron microscopy (SEM), the polyelectrolyte complex properties were identified and compared to individual polysaccharides. The complex showed monodisperse molecular weight distribution, high viscosity, negative charge, and could be degraded by specific enzymes, such as hyaluronidase and lysozyme. The results suggest a close interaction between the two polysaccharides in the complex, which could be considered a self-assembly system.

The evaluation of weight average molecular weight (Mw) and molecular weight distribution represents one of the most controversial aspects concerning the characterization of low molecular weight heparins (LMWHs). As the most commonly used method for the measurement of such parameters is high performance size exclusion chromatography (HP-SEC), the soundness of results mainly depends on the appropriate calibration of the chromatographic columns used. With the aim of meeting the requirement of proper Mw standards for LMWHs, in the present work the determination of molecular weight parameters (Mw and Mn) by HP-SEC combined with a triple detector array (TDA) was performed. The HP-SEC/TDA technique permits the evaluation of polymeric samples by exploiting the combined and simultaneous action of three on-line detectors: light scattering detectors (LALLS/RALLS); refractometer and viscometer. Three commercial LMWH samples, enoxaparin, tinzaparin and dalteparin, a γ-ray depolymerized heparin (γ-Hep) and its chromatographic fractions, and a synthetic pentasaccharide were analysed by HP-SEC/TDA. The same samples were analysed also with a conventional HP-SEC method employing refractive index (RI) and UV detectors and two different chromatographic column set, silica gel and polymeric gel columns. In both chromatographic systems, two different calibration curves were built up by using (i) γ-Hep chromatographic fractions and the corresponding Mw parameters obtained via HP-SEC/TDA; (ii) the whole γ-Hep preparation with broad Mw dispersion and the corresponding cumulative distribution function calculated via HP-SEC/TDA. In addition, also a chromatographic column calibration according to European Pharmacopoeia indication was built up. By comparing all the obtained results, some important differences among Mw and size distribution values of the three LMWHs were found with the five different calibration methods and with HP-SEC/TDA method. In particular, the detection of the lower molecular weight components turned out to be the most critical aspect. Whereas HP-SEC/TDA may underestimate species under 2 KDa when present in low concentration, other methods appeared to emphasize their content.

In a collaborative study involving six laboratories in the USA, Europe, and India the molecular weight distributions of a panel of heparin sodium samples were determined, in order to compare heparin sodium of bovine intestinal origin with that of bovine lung and porcine intestinal origin. Porcine samples met the current criteria as laid out in the USP Heparin Sodium monograph. Bovine lung heparin samples had consistently lower average molecular weights. Bovine intestinal heparin was variable in molecular weight; some samples fell below the USP limits, some fell within these limits and others fell above the upper limits. These data will inform the establishment of pharmacopeial acceptance criteria for heparin sodium derived from bovine intestinal mucosa. The method for MW determination as described in the USP monograph uses a single, broad standard calibrant to characterize the chromatographic profile of heparin sodium on high-resolution silica-based GPC columns. These columns may be short-lived in some laboratories. Using the panel of samples described above, methods based on the use of robust polymer-based columns have been developed. In addition to the use of the USP’s broad standard calibrant for heparin sodium with these columns, a set of conditions have been devised that allow light-scattering detected molecular weight characterization of heparin sodium, giving results that agree well with the monograph method. These findings may facilitate the validation of variant chromatographic methods with some practical advantages over the USP monograph method.

Super paramagnetic iron oxide nanoparticles (SPION) were augmented by both hyaluronic acid (HA) and bovine serum albumin (BSA), each covalently conjugated to dopamine (DA) enabling their anchoring to the SPION. HA and BSA were found to simultaneously serve as stabilizing polymers of Fe3O4·DA-BSA/HA in water. Fe3O4·DA-BSA/HA efficiently entrapped and released the hydrophobic cytotoxic drug paclitaxel (PTX). The relative amount of HA and BSA modulates not only the total solubility but also the paramagnetic relaxation properties of the preparation. The entrapping of PTX did not influence the paramagnetic relaxation properties of Fe3O4·DA-BSA. Thus, by tuning the surface structure and loading, we can tune the theranostic properties of the system.

BACKGROUND: Successful non-viral gene delivery currently requires compromises to achieve useful transfection levels while minimizing toxicity. Despite high molecular weight (MW) branched polyethylenimine (bPEI) is considered the gold standard polymeric transfectant, it suffers from high cytotoxicity. Inversely, its low MW counterpart is less toxic and effective in transfection. Moreover, chitosan is a highly biocompatible and biodegradable polymer but characterized by very low transfection efficiency. In this scenario, a straightforward approach widely exploited to develop effective transfectants relies on the synthesis of chitosan-graft-low MW bPEIs (Chi-g-bPEI(x)) but, despite the vast amount of work that has been done in developing promising polymeric assemblies, the possible influence of the degree of grafting on the overall behavior of copolymers for gene delivery has been largely overlooked. METHODOLOGY/PRINCIPAL FINDINGS: With the aim of providing a comprehensive evaluation of the pivotal role of the degree of grafting in modulating the overall transfection effectiveness of copolymeric vectors, we have synthesized seven Chi-g-bPEI(x) derivatives with a variable amount of bPEI grafts (minimum: 0.6%; maximum: 8.8%). Along the Chi-g-bPEI(x) series, the higher the degree of grafting, the greater the ζ-potential and the cytotoxicity of the resulting polyplexes. Most important, in all cell lines tested the intermediate degree of grafting of 2.7% conferred low cytotoxicity and higher transfection efficiency compared to other Chi-g-bPEI(x) copolymers. We emphasize that, in transfection experiments carried out in primary articular chondrocytes, Chi-g-bPEI(2.7%) was as effective as and less cytotoxic than the gold standard 25 kDa bPEI. CONCLUSIONS/SIGNIFICANCE: This work underlines for the first time the pivotal role of the degree of grafting in modulating the overall transfection effectiveness of Chi-g-bPEI(x) copolymers. Crucially, we have demonstrated that, along the copolymer series, the fine tuning of the degree of grafting directly affected the overall charge of polyplexes and, altogether, had a direct effect on cytotoxicity.