Several secondary metabolite gene clusters in the genomes of ten Penicillium spp. raise the risk of multiple mycotoxin occurrence in chestnuts.

Penicillium spp. produce a great variety of secondary metabolites, including several mycotoxins, on food substrates. Chestnuts represent a favorable substrate for Penicillium spp. development. In this study, the genomes of ten Penicillium species, virulent on chestnuts, were sequenced and annotated: P. bialowiezense. P. pancosmium, P. manginii, P. discolor, P. crustosum, P. palitans, P. viridicatum, P. glandicola, P. taurinense and P. terrarumae. Assembly size ranges from 27.5 to 36.8 Mb and the number of encoded genes ranges from 9,867 to 12,520. The total number of predicted biosynthetic gene clusters (BGCs) in the ten species is 551. The most represented families of BGCs are non ribosomal peptide synthase (191) and polyketide synthase (175), followed by terpene synthases (87). Genome-wide collections of gene phylogenies (phylomes) were reconstructed for each of the newly sequenced Penicillium species allowing for the prediction of orthologous relationships among our species, as well as other 20 annotated Penicillium species available in the public domain. We investigated in silico the presence of BGCs for 10 secondary metabolites, including 5 mycotoxins, whose production was validated in vivo through chemical analyses. Among the clusters present in this set of species we found andrastin A and its related cluster atlantinone A, mycophenolic acid, patulin, penitrem A and the cluster responsible for the synthesis of roquefortine C/glandicoline A/glandicoline B/meleagrin. We confirmed the presence of these clusters in several of the Penicillium species conforming our dataset and verified their capacity to synthesize them in a chestnut-based medium with chemical analysis. Interestingly, we identified mycotoxin clusters in some species for the first time, such as the andrastin A cluster in P. flavigenum and P. taurinense, and the roquefortine C cluster in P. nalgiovense and P. taurinense. Chestnuts proved to be an optimal substrate for species of Penicillium with different mycotoxigenic potential, opening the door to risks related to the occurrence of multiple mycotoxins in the same food matrix.

Efficacy of Essential Oil Vapours in Reducing Postharvest Rots and Effect on the Fruit Mycobiome of Nectarines.

Nectarines can be affected by many diseases, resulting in significant production losses. Natural products, such as essential oils (EOs), are promising alternatives to pesticides to control storage rots. This work aimed to test the efficacy of biofumigation with EOs in the control of nectarine postharvest diseases while also evaluating the effect on the quality parameters (firmness, total soluble solids, and titratable acidity) and on the fruit fungal microbiome. Basil, fennel, lemon, oregano, and thyme EOs were first tested in vitro at 0.1, 0.5, and 1.0% concentrations to evaluate their inhibition activity against Monilinia fructicola. Subsequently, an in vivo screening trial was performed by treating nectarines inoculated with M. fructicola, with the five EOs at 2.0% concentration by biofumigation, performed using slow-release diffusers placed inside the storage cabinets. Fennel, lemon, and basil EOs were the most effective after storage and were selected to be tested in efficacy trials using naturally infected nectarines. After 28 days of storage, all treatments showed a significant rot reduction compared to the untreated control. Additionally, no evident phytotoxic effects were observed on the treated fruits. EO vapors did not affect the overall quality of the fruits but showed a positive effect in reducing firmness loss. Metabarcoding analysis showed a significant impact of tissue, treatment, and sampling time on the fruit microbiome composition. Treatments were able to reduce the abundance of Monilinia spp., but basil EO favored a significant increase in Penicillium spp. Moreover, the abundance of other fungal genera was found to be modified.

Theoretically Predicted and Experimentally Detected Chirality of Dibenzocyclooctynes and Their Triazole Adducts with Azides.

Dibenzocyclooctynes have emerged as promising scaffolds for bioorthogonal ligation. An important structural aspect that has not been addressed so far relates to their chirality. Herein, we explore, by theoretical and experimental methods, this structural aspect that has been neglected so far. First, computational analysis is conducted, and the results are used as a guide for the experimental investigation. Next, an array of different experiments (high-performance liquid chromatography (HPLC) on chiral columns, chiroptical spectroscopy, and X-ray diffraction) for structure elucidation is scrutinized in concert. Finally, this work demonstrates the chirality and the stereodynamic behavior of dibenzocyclooctynes and their triazole derivatives with simple azides and also uncovers their conformational behavior.

Efficacy and Mechanisms of Action of Essential Oils' Vapours against Blue Mould on Apples Caused by Penicillium expansum.

Biofumigation with slow-release diffusers of essential oils (EOs) of basil, oregano, savoury, thyme, lemon, and fennel was assessed for the control of blue mould of apples, caused by Penicillium expansum. In vitro, the ability of the six EOs to inhibit the mycelial growth was evaluated at concentrations of 1.0, 0.5, and 0.1%. EOs of thyme, savoury, and oregano, at all three concentrations, and basil, at 1.0 and 0.5%, were effective in inhibiting the mycelial growth of P. expansum. In vivo, disease incidence and severity were evaluated on 'Opal' apples artificially inoculated with the pathogen and treated at concentrations of 1.0% and 0.5% of EOs. The highest efficacy in reducing blue mould was observed with EOs of lemon and oregano at 1.0% after 60 days of storage at 1 ± 1 °C (incidence of rot, 3 and 1%, respectively) and after a further 14 days of shelf-life at 15 ± 1 °C (15 and 17%). Firmness, titratable acidity, and total soluble solids were evaluated at harvest, after cold storage, and after shelf-life. Throughout the storage period, no evident phytotoxic effects were observed. The EOs used were characterised through GC-MS to analyse their compositions. Moreover, the volatile organic compounds (VOCs) present in the cabinets were characterised during storage using the SPME-GC-MS technique. The antifungal effects of EOs were confirmed both in vitro and in vivo and the possible mechanisms of action were hypothesised. High concentrations of antimicrobial and antioxidant compounds in the EOs explain the efficacy of biofumigation in postharvest disease control. These findings provide new insights for the development of sustainable strategies for the management of postharvest diseases and the reduction of fruit losses during storage.

Preparation of a high-density vinyl silica gel to anchor cysteine via photo-click reaction and its applications in hydrophilic interaction chromatography.

Modification of surface silanols is a topic of interest in the preparation of organo-functionalized silica particles. Herein, two novel contributions, mainly focused on separation science, were presented: i) the horizontal polymerization on silica surface by using the vinyl-triacetoxy silane and ii) a preparation of cysteine-based stationary phase via photo-click thiol-ene coupling. In the first derivatization step, the vinyl-triacetoxy silane was employed instead of conventional trichloro vinyl one. The one-step synthetic procedure needed imidazole as an activating agent in addition to the silica hydration. Modified silica particles offer a high loading of vinyl fragments and an extensive passivation of silanols such as to not require a subsequent end-capping procedure. The structural morphology of media was deeper characterized by combining infrared spectroscopy, solid-state nuclear magnetic resonance, and elemental analysis. A first application, the photo-click cysteine-based material was prepared by photo-click reaction and the stationary phase was employed in the separation of some conventional targets by hydrophilic interaction chromatography.

Essential Oils Reduce Grey Mould Rot of Apples and Modify the Fruit Microbiome during Postharvest Storage.

Botrytis cinerea is the causal agent of grey mould rot of apples. The efficacy of biofumigation with thyme (Thymus vulgaris), savoury (Satureja montana), and basil (Ocimum basilicum) essential oils (EOs) at 1%, 0.5%, and 0.1% concentrations were tested against B. cinerea. In vitro, the results showed 100% growth inhibition at 1% concentration for all oils. Subsequent biofumigation experiments on apples of cultivar 'Opal' with 1% EOs showed that, after 60 d storage, thyme and savoury EOs significantly reduced grey mould rot incidence (average incidence 2% for both treatments) compared to the control (7%). Analyses of quality indicated slightly higher fruit firmness for 1% thyme at 30 d and slightly higher titratable acidity for 1% thyme and savoury at 60 d. Sampling of the atmosphere inside the cabinets was performed to characterize and quantify the volatile components of EOs released through biofumigation. Though thymol and p-cymene were the main components of thyme EO, the antimicrobial activity was mainly due to the presence of thymol and, to a lower extent, of carvacrol. In savoury EO, carvacrol and p-cymene were the main components, whereas in basil EO, linalool and estragole were mainly present. Metabarcoding analyses showed that the epiphytic microbiome had higher richness and evenness compared to their endophytic counterpart. By the end of shelf-life, treatments with thyme EO reduced B. cinerea abundance compared to the inoculated control for both endophytes (from 36.5% to 1.5%) and epiphytes (from 7.0% to 0.7%), while favouring a significant increase in Penicillium species both in endophytes (from 0.2% to 21.5%) and epiphytes (from 0.5% to 18.6%). Results indicate that thyme EO (1%) and savoury EO (1%) are equally effective in hampering grey mould rot development in vivo.

Exploring the Assembly of Resorc[4]arenes for the Construction of Supramolecular Nano-Aggregates.

Many biologically active compounds feature low solubility in aqueous media and, thus, poor bioavailability. The formation of the host-guest complex by using calixarene-based macrocycles (i.e., resorcinol-derived cyclic oligomers) with a good solubility profile can improve solubilization of hydrophobic drugs. Herein, we explore the ability of resorc[4]arenes to self-assemble in polar solutions, to form supramolecular aggregates, and to promote water-solubility of an isoflavone endowed with anti-cancer activity, namely Glabrescione B (GlaB). Accordingly, we synthesized several architectures featuring a different pattern of substitution on the upper rim including functional groups able to undergo acid dissociation (i.e., carboxyl and hydroxyl groups). The aggregation phenomenon of the amphiphilic resorc[4]arenes has been investigated in a THF/water solution by UV-visible spectroscopy, at different pH values. Based on their ionization properties, we demonstrated that the supramolecular assembly of resorc[4]arene-based systems can be modulated at given pH values, and thus promoting the solubility of GlaB.

On-column quantification of amino functionalities bonded to solid porous matrices packed within high performance liquid chromatography columns.

Stationary phases (SPs) based on silica matrices functionalized with amino groups linked to their surface through alkyl chains of various length have found remarkable success in performing HILIC separations, showing really effective resolution towards a wide typology of compounds of biological interest, such as carbohydrates, nucleosides, purine and pyrimidine bases. Recently, we developed an operationally simple procedure, named DNBA-M, non-destructive for the analysed SP, designed to quantify the density of basic groups (typically amino groups) chemically bonded to the surface of porous solids. In the present study the DNBA-M procedure has been suitably modified to allow the quantification of any typology of amino groups present on silica matrices packed into HPLC columns. The new approach, named OC-DNBA-M, has been successfully validated through analysis of two HPLC columns packed with aminopropyl-silica matrices. Afterwards, it was also demonstrated as the OC-DNBA-M procedure may allow the effective and in-depth analysis of the structural composition characterizing SPs packed inside HPLC columns, in which amino-groups have been differently and only partially involved in following ureidic functionalizations. It was also proved how the analysed columns can be readily re-employed for the chromatographic applications for which they have been designed, without appreciable deterioration of the respective discrimination abilities.

Modular and conservative procedure for the quantification of amino functionalities bonded to solid porous matrices.

Nowadays solid materials in which amino groups are linked to silica matrices through alkyl chains of different length (C18, C8, C4) are successfully employed in CO2 capture and storage technologies, as well as in a variety of chromatographic applications. In particular, their use as stationary phases finds remarkable success in performing HILIC separations and, in general, in the effective resolution of important compound classes (e.g. mixtures of mono- or oligo-saccharides). In this study an original and operationally simple procedure designed to quantify the density of basic groups (typically amino groups) chemically bonded to the surface of porous solids, which also allows a full recovery of the analysed material, is presented. The method is based on the preventive acid-base reaction of the basic groups linked to the solid by 3,5-dinitrobenzoic acid (DNBA). The quantification of the basic functionalities is then performed by an UV-spectrophotometric retro-titration of the thus salified solid matrix (or, alternatively, by HPLC approach), resorting to a preventive either acid or basic displacement of DNBA from the matrix. The uncertainty of the density measurements is assessed by 13%.