Epigenetic effects induced by chronic and acute chromium stress treatments in Arabidopsis thaliana identified by the MSAP-Seq.

Chromium (Cr) metal is highly toxic to plants and causes severe damage to their growth, development, and reproduction. Plant exposure to chronic and acute Cr stress treatments results in significant changes at short time in the gene expression profile and at long time in the genomic DNA methylation profile at a transgenerational level and, consequently, in gene expression. These epigenetic modifications and their implications imposed by the Cr stress in plants are not yet completely known. Herein, were identified the epigenetic changes induced by Cr stress treatments in Arabidopsis thaliana plants using the Methylation Sensitive Amplification Polymorphism approach coupled with next-generation sequencing (MSAP-Seq). Arabidopsis plants kept in hoagland solution were kept under chronic and acute Cr stress treatments (termed F0 plants). For chronic stress, plants were treated through hoagland solution with 2.5 µM Cr during the entire cultivation period until seed harvest. Meanwhile, for acute stress, plants were treated with 5 µM Cr during the first three weeks and returned to unstressful conditions until seed harvest. Seeds from F0 plants were sown and re-submitted to the same Cr stress treatments. The seed germination rate was evaluated for F2 plants under different Cr stress treatments (0, 10, 20, and 40 µM) compared to the unstressful control condition. These data showed significant changes in the germination rate of seeds originating from stressed F1 plants compared with unstressful control plants. Given this data, F1 plants kept under these chronic and acute Cr stress treatments and unstressful control condition were evaluated for the transgenerational epigenetic modifications by the MSAP-Seq approach. The MSAP-Seq data showed that several genes were modified in their methylation status as a consequence of chronic and acute Cr stress treatment to activate the plant defense. In particular, RNA processing, protein translation, photorespiration, energy production, transmembrane transport, DNA transcription, plant development, and plant resilience were the major processes modulated by epigenetic mechanisms identified in F1 plants kept under chronic and acute Cr stress. Therefore, collective data suggested that Arabidopsis plants kept under Cr stress regulate their epigenetic status based on DNA methylation to modulate defense and resilience mechanisms.

Impact of airborne iron oxide nanoparticles on Tillandsia usneoides as a model plant to assess pollution in heavy traffic areas.

Due to the increasing evidence of widespread sub-micron pollutants in the atmosphere, the impact of airborne nanoparticles is a subject of great relevance. In particular, the smallest particles are considered the most active and dangerous, having a higher surface/volume ratio. Here we tested the effect of iron oxide (Fe3O4) nanoparticles (IONPs) with different mean diameter and size distribution on the model plant Tillandsia usneoides. Strands were placed in home-built closed boxes and exposed to levels of airborne IONPs reported for the roadside air, i.e. in the order of 107 - 108 items m-2. Plant growth and other morpho-physiological parameters were monitored for two weeks, showing that exposure to IONPs significantly reduced the length increment of the treated strands with respect to controls. A dose-dependence of this impairing effect was found only for particles with mean size of a few tens of nanometers. These were also proved to be the most toxic at the highest concentration tested. The IONP-induced hamper in growth was correlated with altered concentration of macro- and micronutrients in the plant, while no significant variation in photosynthetic activity was detected in treated samples. Microscopy investigation showed that IONPs could adhere to the plant surface and were preferentially located on the trichome wings. Our results report, for the first time, evidence of the negative effects of airborne IONP pollution on plant health, thus raising concerns about related environmental risks. Future research should be devoted to other plant species and pollutants to assess the impact of airborne pollution on plants and devise suitable attenuation practices.

Growth, physiological parameters and DNA methylation in Spirodela polyrhiza (L.) Schleid exposed to PET micro-nanoplastic contaminated waters.

The effects of polyethylene terephthalate micro-nanoplastics (PET-MNPs) were tested on the model freshwater species Spirodela polyrhiza (L.) Schleid., with focus on possible particle-induced epigenetic effects (i.e. alteration of DNA methylation status). MNPs (size âˆ¼ 200-300 nm) were produced as water dispersions from PET bottles through repeated cycles of homogenization and used to prepare N-medium at two environmentally relevant concentrations (∼0.05 g L-1 and ∼0.1 g L-1 of MNPs). After 10 days of exposure, a reduction in fresh and dry weight was observed in treated plants, even if the average specific growth rate for both frond number and area was not altered. Impaired growth was coupled with a MNP-induced decrease of chlorophyll fluorescence parameters (i.e. ΨETo and Piabs, indicators of photochemical efficiency) and starch concentration, as well as with alterations in plant ionomic profile and oxidative status. The methylation-sensitive amplification polymorphism (MSAP) technique was used to assess possible changes in DNA methylation levels induced by plastic particles. The analysis showed unusual hypermethylation in 5'-CCGG sites that could be implicated in DNA protection from dangerous agents (i.e. reactive oxygen species) or in the formation of new epialleles. This work represents the first evidence of MNP-induced epigenetic modifications in the plant world.

Sileneisabellae (Caryophyllaceae), a new campion species from serpentine soils of Albania.

The new species Sileneisabellae is described and illustrated from the Skënderbëut mountain range of central Albania. It grows on the ultramafic mountain slopes around Qafë Shtamë, in the understorey of open Pinusnigra forests and in the rocky grasslands above the forest belt, at 1000-1600 m a.s.l. Sileneisabellae is a serpentine endemic likely belonging to section Elisanthe (Fenzl ex Endl.) Ledeb. and shows affinities with the widespread European species S.noctiflora L. It is sharply distinct from the latter species in habit, stem and leaf pubescence, morphology, and biology of the flowers and length of the carpophore. Moreover, the ecology of the two taxa is also contrasting, being S.noctiflora a synanthropic-ruderal, mostly in lowlands. Weaker similarities were also observed with the south European subalpine taxa of the group of S.vallesia L. of section Auriculatae (Boiss.) Schischk., though these are not likely to reflect a real systematic affinity.

Nanoformulations with exopolysaccharides from cyanobacteria: enhancing the efficacy of bioactive molecules in the Mediterranean fruit fly control.

The increasing demand for food has required intensive use of pesticides which are hazardous to the ecosystem. A valid alternative is represented by biopesticides; however, these molecules are often insoluble in water, and poorly bioavailable. Nanopesticides can be engineered to reach a selected target with controlled release of the active principle. In this work, capsaicin, an irritant alkaloid from hot chili peppers, and hydroxytyrosol, a phenolic compound obtained from extra-virgin olive oil by-products, were loaded into innovative nanocarriers. These were designed ad hoc combining exopolysaccharides from the cyanobacteria Neocyanospira capsulata, and a lipid component, i.e., egg phosphatidylcholine. The polysaccharide was chosen for chemical affinity with the chitin of insect exoskeleton, while the lipids were introduced to modulate the carrier rigidity. The newly formed nanosystems were characterized by physico-chemical techniques and tested for their possible use in pest control programs. The Mediterranean Fruit Fly Ceratitis capitata Wiedemann, 1824 (Diptera, Tephriditae), a pest of the Mediterranean Region causing high economic losses, was used as a model insect. We found that the nanoformulations nanocarriers prepared in this work, were able to increase the ovicidal effect of hydroxytyrosol. Moreover, the formulation encapsulating either hydroxytyrosol or capsaicin were able to reduce the number of females landing on treated apricots.

Can microplastics threaten plant productivity and fruit quality? Insights from Micro-Tom and Micro-PET/PVC.

Solanum lycopersicum L., a crop grown worldwide with a high nutritional value for the human diet, was used to test the impact of microplastics on plant growth, productivity, and fruit quality. Two of the most represented microplastics in soils, polyethylene terephthalate (PET) and polyvinyl chloride (PVC), were tested. Plants were grown in pots with an environmentally realistic concentration of microplastics and, during the whole crop life cycle, photosynthetic parameters, number of flowers and fruits were monitored. At the end of the cultivation, plant biometry and ionome were evaluated, along with fruit production and quality. Both pollutants had negligible effects on shoot traits, with only PVC causing a significant reduction in shoot fresh weight. Despite an apparent low or no toxicity during the vegetative stage, both microplastics decreased the number of fruits and, in the case of PVC, also their fresh weights. The plastic polymer-induced decline in fruit production was coupled with wide variations in fruit ionome, with marked increases in Ni and Cd. By contrast there was a decline in the nutritionally valuable lycopene, total soluble solids, and total phenols. Altogether, our results reveal that microplastics can not only limit crop productivity but also negatively impact fruit quality and enhance the concentration of food safety hazards, thus raising concerns for their potential health risks for humans.

Salt-Affected Rocket Plants as a Possible Source of Glucosinolates.

Soil salinity can have various negative consequences on agricultural products, from their quality and production to their aesthetic traits. In this work, the possibility to use salt-affected vegetables, that otherwise would be discarded, as a source of nutraceuticals was explored. To this aim, rocket plants, a vegetable featuring bioactive compounds such as glucosinolates, were exposed to increasing NaCl concentrations in hydroponics and analysed for their content in bioactive compounds. Salt levels higher than 68 mM produced rocket plants that did not comply with European Union regulations and would therefore be considered a waste product. Anyway, our findings, obtained by Liquid Chromatography-High Resolution Mass Spectrometry, demonstrated a significant increase in glucosinolates levels in such salt-affected plants. opening the opportunity for a second life of these market discarded products to be recycled as glucosinolates source. Furthermore, an optimal situation was found at NaCl 34 mM in which not only were the aesthetic traits of rocket plants not affected, but also the plants revealed a significant enrichment in glucosinolates. This can be considered an advantageous situation in which the resulting vegetables still appealed to the market and showed improved nutraceutical aspects.

Saline Stress Impairs Lipid Storage Mobilization during Germination in Eruca sativa.

Soil salinization become worse in the last decades, leading to reduced crop yields, especially in the Mediterranean basin. Eruca sativa is a common species cultivated in this area with remarkable economic importance. This study aimed at investigating the effect of salinity on this plant, focusing on (i) seedling development in terms of variations in germination and growth parameters and (ii) anatomical and ultra-structural changes in the morphology of cotyledons. For this reason, seeds were treated with different salinity levels ranging from 137 to 548 mM NaCl. Seed germination was delayed by all the concentrations tested, but only above 137 mM seedling growth was impaired. Results showed a high occurrence of lipid bodies within the mesophyll cells of cotyledons of seedlings exposed to salt concentrations above 137 mM, suggesting an impairment in lipid mobilization caused by salinity during plant development. The cotyledons of treated seedlings showed reduced intercellular spaces and ultrastructural changes in chloroplasts and peroxisomes. Moreover, salt-induced autophagic processes were present in samples grown at the highest NaCl levels. Interestingly, at 137 mM NaCl, seedlings showed the highest values of mesophyll thickness and fresh weight, implying a possible mechanism of salt adaptation during germination.

Tannins-lignin mixed nanoformulations for improving the potential of neem oil as fungicide agent.

Sustainability and circular economy are increasingly pushing for the search of natural materials to foster antiparasitic treatments, especially in the case of economically relevant agricultural cultivations, such as grapevine. In this work, we propose to deliver neem oil, a natural biopesticide loaded into novel nanovectors (nanocapsules) which were fabricated using a scalable procedure starting from Kraft lignin and grapeseed tannins. The obtained formulations were characterized in terms of size and Zeta potential, showing that almost all the nanocapsules had size in the suitable range for delivery purposes (mean diameter 150-300 nm), with low polydispersity and sufficient stability to ensure long shelf life. The target microorganisms were three reference fungal pathogens of grapevine (Botrytis cinerea, Phaeoacremonium minimum, Phaeomoniella chlamydospora), responsible for recurrent diseases on this crop: grey mold or berry rot by B. cinerea and diseases of grapevine wood within the Esca complex of diseases. Results showed that grapeseed tannins did not promote inhibitory effects, either alone or in combination with Kraft lignin. On the contrary, the efficacy of neem oil against P. minimum was boosted by more than 1-2 orders of magnitude and the parasite growth inhibition was higher with respect to a widely used commercial pesticide, while no additional activity was detected against P. chlamydospora and B. cinerea.

In vitro and in vivo copper-treated Myrtus communis L.: terpene profiles and evidence for potential cultivation on metal-contaminated soils.

The intensive application of agro-chemicals, and in particular of copper (Cu)-based compounds, causes increasing pollution of agricultural land, with serious risks for human health. Aromatic plants used for purposes other than food, can be considered for the exploitation and/or remediation of metal-polluted substrates, since contamination of the essential oils by these metals was not significant. Myrtle (Myrtus communis L.) is a Mediterranean evergreen shrub whose essential oil has many commercial applications. In this work, the effect of an excess of Cu in respect to control conditions was assessed on M. communis growth and foliar terpene composition. Metal accumulation in roots and shoots was also evaluated for the possible use of this species in phytoremediation. The amount of Cu applied in our experiments minimally affected the terpene profiles of in vitro grown plants, whereas no variations were detected in in vivo plants. The presence of the metal in the soil did not significantly impair plant growth, thus allowing its cultivation on polluted substrates. On the other hand, the amount of Cu in the plant was not enough to result in a significant reduction of Cu levels in the soil. Therefore, myrtle plants proved to be good candidates for the re-vegetation of Cu-contaminated lands.

Fluorescent Labeling of Lignin Nanocapsules with Fluorol Yellow 088.

The microscopic visualization of nanoparticles in plants is crucial to elucidate the mechanisms of their uptake through the cell wall and plasma membrane and to localize the possible sites of their extracellular or intracellular accumulation. Lignin nanocarriers are polymeric hollow nanocapsules able to contain and transport several bioactive substances inside plant tissues. We describe here a method for the preparation of Fluorol Yellow 088-labeled lignin nanocapsules that allow their localization in plant organs and tissues by fluorescence microscopy.

Transgenerational effects of chromium stress at the phenotypic and molecular level in Arabidopsis thaliana.

In this study, we describe the results obtained in a study of the transgenerational phenotypic effects of chromium (Cr) stress on the model plant species Arabidopsis thaliana. The F1 generation derived from parents grown under chronic and medium chronic stress showed significantly higher levels of the maximal effective concentration (EC50) compared with F1 plants generated from unstressed parents. Moreover, F1 plants from Cr-stressed parents showed a higher germination rate when grown in the presence of Cr. F1 plants derived from parents cultivated under chronic Cr stress displayed reduced hydrogen peroxide levels under Cr stress compared to controls. At lower Cr stress levels, F1 plants were observed to activate promptly more genes involved in Cr stress responses than F0 plants, implying a memory effect linked to transgenerational priming. At higher Cr levels, and at later stages, F1 plants modulated significantly fewer genes than F0 plants, implying a memory effect leading to Cr stress adaptation. Several bHLH transcription factors were induced by Cr stress in F1 but not in F0 plants, including bHLH100, ORG2 and ORG3. F1 plants optimized gene expression towards pathways linked to iron starvation response. A model of the transcriptional regulation of transgenerational memory to Cr stress is presented here, and could be applied for other heavy metal stresses.

Interplay of plastic pollution with algae and plants: hidden danger or a blessing?

In the era of plastic pollution, plants have been discarded as a system that is not affected by micro and nanoplastics, but contrary to beliefs that plants cannot absorb plastic particles, recent research proved otherwise. The presented review gives insight into known aspects of plants' interplay with plastics and how plants' ability to absorb plastic particles can be utilized to remove plastics from water and soil systems. Microplastics usually cannot be absorbed by plant root systems due to their size, but some reports indicate they might enter plant tissues through stomata. On the other hand, nanoparticles can enter plant root systems, and reports of their transport via xylem to upper plant parts have been recorded. Bioaccumulation of nanoplastics in upper plant parts is still not confirmed. The prospects of using biosystems for the remediation of soils contaminated with plastics are still unknown. However, algae could be used to degrade plastic particles in water systems through enzyme facilitated degradation processes. Considering the amount of plastic pollution, especially in the oceans, further research is necessary on the utilization of algae in plastic degradation. Special attention should be given to the research concerning utilization of algae with restricted algal growth, ensuring that a different problem is not induced, "sea blooming", during the degradation of plastics.

Plastic is in the air: Impact of micro-nanoplastics from airborne pollution on Tillandsia usneoides (L.) L. (Bromeliaceae) as a possible green sensor.

Due to the increasing evidence of widespread plastic pollution in the air, the impact on plants of airborne particles of polycarbonate (PC), polyethyleneterephthalate (PET), polyethylene (PE), and polyvinylchloride (PVC) was tested by administering pristine and aged airborne micro-nanoplastics (MNPs) to Tillandsia usneoides for two weeks. Here we showed that exposure to pristine MNPs, significantly reduced plant growth with respect to controls. Particularly, PVC almost halved plant development at the end of the treatment, while the other plastics exerted negative effects on growth only at the beginning of the exposure, with final stages comparable to those of controls. Plants exposed to aged MNPs showed significantly decreased growth at early stages with PC, later in the growth with PE, and even later with PET. Aged PVC did not exert a toxic effect on plants. When present, the plastic-mediated reduction in plant growth was coupled with a decrease in photosynthetic activity and alterations in the plant concentration of macro- and micronutrients. The plastic particles were showed to adhere to the plant surface and, preferentially, on the trichome wings. Our results reported, for the first time, evidence of negative effects of airborne plastic pollution on plant health, thus raising concerns for related environmental risks.

Root Exposure to 5-Aminolevulinic Acid (ALA) Affects Leaf Element Accumulation, Isoprene Emission, Phytohormonal Balance, and Photosynthesis of Salt-Stressed Arundo donax.

Arundo donax has been recognized as a promising crop for biomass production on marginal lands due to its superior productivity and stress tolerance. However, salt stress negatively impacts A. donax growth and photosynthesis. In this study, we tested whether the tolerance of A. donax to salinity stress can be enhanced by the addition of 5-aminolevulinic acid (ALA), a known promoter of plant growth and abiotic stress tolerance. Our results indicated that root exposure to ALA increased the ALA levels in leaves along the A. donax plant profile. ALA enhanced Na+ accumulation in the roots of salt-stressed plants and, at the same time, lowered Na+ concentration in leaves, while a reduced callose amount was found in the root tissue. ALA also improved the photosynthetic performance of salt-stressed apical leaves by stimulating stomatal opening and preventing an increase in the ratio between abscisic acid (ABA) and indol-3-acetic acid (IAA), without affecting leaf methanol emission and plant growth. Supply of ALA to the roots reduced isoprene fluxes from leaves of non-stressed plants, while it sustained isoprene fluxes along the profile of salt-stressed A. donax. Thus, ALA likely interacted with the methylerythritol 4-phosphate (MEP) pathway and modulate the synthesis of either ABA or isoprene under stressful conditions. Overall, our study highlights the effectiveness of ALA supply through soil fertirrigation in preserving the young apical developing leaves from the detrimental effects of salt stress, thus helping of A. donax to cope with salinity and favoring the recovery of the whole plant once the stress is removed.

Improving plant-based genotoxicity bioassay through AFLP technique for trace metal-contaminated water: insights from Myriophyllum aquaticum (Vell.) Verdc. and Cd.

In this work, we evaluated whether the species Myriophyllum aquaticum (Vell.) Verdc. can be a promising material for devising reliable eco-toxicological tests for Cd-contaminated waters. Plants of M. aquaticum were exposed to Cd, using different concentrations (1 mg L-1, 2.5 mg L-1, 5 mg L-1, and 10 mg L-1; experiment 1) and exposure times (2.5 mg L-1 for 3 days, 7 days, 14 days, and 21 days; experiment 2). Plant growth and Cd accumulation were monitored during the treatment period, and Cd genotoxicity was assessed by analyzing Cd-induced changes in the AFLP fingerprinting profiles using famEcoRI(TAC)/MseI(ATG) and hexEcoRI(ACG)/MseI(ATG) pairs of primers. Root and shoot growth was reduced already at the lowest Cd concentration used (about 20% reduction for roots and 60% for shoots at 1 mg L-1; experiment 1) and after 7 days (about 50% reduction for roots and 70% for shoots; experiment 2). The primer combinations produced 154 and 191 polymorphic loci for experiments 1 and 2, respectively. Mean genetic diversity (He) reduction among the treatment groups was observed starting from 2.5 mg L-1 (He 0.211 treated vs 0.236 control; experiment 1) and after 3 days (He 0.169 treated vs 0.261 control; experiment 2), indicating that results obtained from AFLP profiles did not match with plant growth measurements. Therefore, our results showed that M. aquaticum proved to be a suitable model system for the investigation of Cd genotoxicity through AFLP fingerprinting profile, whereas the more classic eco-toxicological tests based only on biometric parameters could not correctly estimate the risk associated with undetected Cd genotoxicity.

Photosynthesizing while hyperaccumulating nickel: Insights from the genus Odontarrhena (Brassicaceae).

Nickel-induced changes in photosynthetic activity were investigated in three Ni-hyperaccumulating Odontarrhena species with increasing Ni tolerance and accumulation capacity, O. muralis, O. moravensis, and O. chalcidica. Plantlets were grown in hydroponics at increasing NiSO4 concentrations (0, 0.25, and 1 mM) for one week, and the effects of Ni on growth, metal accumulation, photosynthesis, and nitrogen (N) allocation to components of the photosynthetic apparatus were analysed. Nickel treatments in O. chalcidica, and O. moravensis to a lesser extent, increased not only the photochemical efficiency of photosystem II (PSII) and the CO2 assimilation rate, but also CO2 diffusion from the atmosphere to the carboxylation sites. These two species displayed a specific increase and/or rearrangement of the photosynthetic pigments and a higher leaf N allocation to the photosynthetic components in the presence of the metal. Odontarrhena muralis displayed a decrease in photosynthetic performance at the lowest Ni concentration due to a combination of both stomatal and non-stomatal limitations. Our data represent the first complete investigation of the effects of Ni on the photosynthetic machinery in Ni hyperaccumulating plants. Our findings clearly indicate a stimulatory, hormetic-like, effect of the metal on both biophysics and biochemistry of photosynthesis in the species with the highest hyperaccumulation capacity.

Impact of microplastics on growth, photosynthesis and essential elements in Cucurbita pepo L.

In this study, Cucurbita pepo L., one of the most cultivated, consumed and economically important crop worldwide, was used as model plant to test the toxic effects of the four most abundant microplastics identified in contaminated soils, i.e. polypropylene (PP), polyethylene (PE), polyvinylchloride (PVC), and polyethyleneterephthalate (PET). Cucurbita plants were grown in pots with increasing concentrations of the microplastics, then plant biometry, photosynthetic parameters and ionome of treated vs. untreated samples were compared to evaluate the toxicity of each plastic. All the pollutants impaired root and, especially, shoot growth. Specific and concentration-dependant effects of the different microplastics were found, including reduction in leaf size, chlorophyll content and photosynthetic efficiency, as well as changes in the micro- and macro-elemental profile. Among all the microplastics, PVC was identified as the most toxic and PE as the less toxic material. PVC decreased the dimensions of the leaf lamina, the values of the photosynthetic performance index and the plant iron concentration to a higher extent in respect to the other treatments. Microplastic toxicity exerted on the growth of C. pepo raises concerns about possible yield and economic loss, as well as for risks of a possible transfer into the food chain.

Agronomic performance and food safety of strawberry cultivated on a remediated sediment.

A marine sediment phytoremediated and homogenized by landfarming was tested for its potential recycle as growing media in horticulture. Two strawberry cultivars, Camarosa and Monterey, were grown on remediated sediment alone (TS100), commercial peat/pumice based growing medium (TS0) and a mixture 1:1 in volume of sediment and peat (TS50). Chemical fertility and strawberry production and safety of produced food were monitored for three consecutive productive seasons on the same growing media. During the first year of cultivation, plants grown on sediment-based media showed a significantly lower biomass production and fruit yield compared with peat, mainly due to the sediment low fertility. In the subsequent two years, the plant re-cultivation improved the sediment structure and N mineralization, and on the third cultivation year both strawberry cultivars showed higher fruit productivity and no accumulation of potentially toxic trace metals. The produced fruits did non accumulate high concentrations of trace metals, and risk assessment showed no risks for human health related to the consumption of strawberry produced on sediment-based growing media. We concluded that a phytoremediated sediment could be recycled as an ingredient of soilless growing media for reducing the environmental impact of plant nursery production and posing no risks for human health. These results show that reclaimed sediments could be reconsidered as a component material category in the new EU regulation on fertilizers.

Green and cost-effective synthesis of copper nanoparticles by extracts of non-edible and waste plant materials from Vaccinium species: Characterization and antimicrobial activity.

The aim of this work was the green synthesis of copper nanoparticles (Cu-NPs) using aqueous extracts of (i) bilberry (Vaccinium myrtillus L.) waste residues from the production of fruit juices and (ii) non-edible "false bilberry" fruits (Vaccinium uliginosum L. subsp. gaultherioides). Different cupric salts (CuCl2, Cu(CH3COO)2 and Cu(NO3)2) were used for the synthesis. The formation of stable nanoparticles (CuNPs) was assessed by transmission electron microscopy and the oxidation state of copper in these aggregates was followed by X-ray photoelectron spectroscopy. The polyphenol composition of the extracts was characterized, before and after the synthesis, using spectrophotometric methods (i.e. total soluble polyphenols and total monomeric anthocyanins) and high-performance liquid chromatography coupled with tandem mass spectrometry (i.e. individual anthocyanins). Polyphenol concentration in the extracts was found to decrease after the synthesis, indicating their active participation to the processes, which led to the formation of Cu-NPs. The antimicrobial activity of Cu-NPs, berry extracts, and cupric ion solutions were analysed by broth microdilution and time-kill assays, on prokaryotic and eukaryotic models. The antimicrobial activity of Cu-NPs, especially those derived from bilberry waste residues, appeared to be higher for both Gram-negative and Gram-positive bacteria, and for fungi, compared to the ones of its single components (cupric salts and berry extracts). Therefore, Cu-NPs from the green synthesis here proposed can be considered as a cost-effective sanitization tool with a wide spectrum of action.