A Volatile Lactone of Hymenoscyphus pseudoalbidus,Pathogen of European Ash Dieback,Inhibits Host Germination

The largely unknown secondary metabolism of the plant pathogenic fungus Hymenoscyphus pseudoalbidus was investigated by use of the CLSA method. A set of volatile lactones was identified by GC/MS. The lactones were synthesized and used in bioassays in which one of the compounds was found to be a strong germination inhibitor for ash seeds, causing necroses in the plant tissue.     

Microwave assisted preparation of efficient activated carbon from grapevine rhytidome for the removal of methyl violet from aqueous solution

Grapevine rhytidome (the outer layer of bark on trunk), as an abundant and low-cost precursor, was used to prepare granular activated carbon with high surface area for the removal of methyl violet from aqueous solution. Microwave heating source was used to reduce the treatment time and energy consumption. To optimize the preparation, the effects of the different parameters, such as phosphoric acid concentration, acid/precursor weight ratio, impregnation time, microwave power, radiation time, and oven heating time on the ability of the samples for removal of methyl violet were studied. The obtained activated carbon was characterized by N₂ adsorption/desorption, SAXS, TEM and SEM methods. The adsorption of methyl violet onto the activated carbon was studied from both equilibrium and kinetic point of view and the results were compared with the commercial granular activated carbon. The rate of adsorption onto the prepared activated carbon was faster than commercial activated carbon. Different kinetic models were used to analyze the experimental kinetic data. The obtained activated carbon showed higher adsorption capacity (more than twice) for the adsorption of methyl violet in comparison with the commercial one. The equilibrium data were analyzed using different isotherm models. Adsorption was found to be maximum in the pH range 7-9.     

Quantification of abscisic acid in grapevine leaf (Vitis vinifera) by isotope-dilution liquid chromatography–mass spectrometry

A specific, sensitive, precise, and accurate method for the determination of abscisic acid (ABA) in grapevine leaf tissues is described. The method employs high-performance liquid chromatography and electrospray ionization–mass spectrometry (LC-ESI-MS) in selected ion monitoring mode (SIM) to analyze ABA using a stable isotope-labeled ABA as an internal standard. Absolute recoveries ranged from 72% to 79% using methanol/water pH 5.5 (50:50 v/v) as an extraction solvent. The best efficiency was obtained when the chromatographic separation was carried out by using a porous graphitic carbon (PGC) column. The statistical evaluation of the method was satisfactory in the work range. A relative standard deviation (RDS) of < 5.5% and < 6.0% was obtained for intra-batch and inter-batch comparisons, respectively. As for accuracy, the relative error (%Er) was between −2.7 and 4.3%, and the relative recovery ranged from 95% to 107%.     

Use of grapevine cell cultures for the production of phytostilbenes of cosmetic interest

Plant cell cultures constitute pesticide-free sources for obtaining plant secondary metabolites or plant extracts. Additionally, they do not contain any fungal contaminants, mycotoxins or heavy metals providing to the consumer potential health benefits and justifying the development of this technology at an industrial scale. Significant production levels of these secondary metabolites can be obtained through the use of elicitors, which activate plant defense mechanisms. Resveratrol, a well-known grapevine polyphenolic compound which possesses potent antioxidant and antiaging activities as well as a protective action on skin, is a good example of such plant secondary metabolites. Resveratrol and its oligomeric derivatives are used by several companies of cosmetic products but their extraction from vine stems and similar vegetal sources remains difficult. Therefore grapevine cell suspensions could represent interesting systems for the large-scale bioproduction of those compounds. Here we present an update of the methods used for the production of phytostilbenes by using grapevine cell cultures and the results obtained.     

Design and validation of an STR hexaplex assay for DNA profiling of grapevine cultivars

Although the analysis of length polymorphism at STR loci has become a method of choice for grape cultivar identification, the standardization of methods for this purpose lags behind that of methods for DNA profiling in human and animal forensic genetics. The aim of this study was thus to design and validate a grapevine STR protocol with a practically useful level of multiplexing. Using free bioinformatics tools, published primer sequences, and nucleotide databases, we constructed and optimized a primer set for the simultaneous analysis of six STR loci (VVIi51, scu08vv, scu05vv, VVMD17, VrZAG47, and VrZAG83) by multiplex PCR and CE with laser‐induced fluorescence, and tested it on 90 grape cultivars. The new protocol requires subnanogram quantities of the DNA template and enables automated, high‐throughput genetic analysis with reasonable discriminatory power. As such, it represents a step toward further standardization of grape DNA profiling.     

Anthocyanin identification and composition of wild Vitis spp. accessions by using LC–MS and LC–NMR

The composition and concentration of anthocyanins of grape berry skins were analyzed in order to assess phenotypic variation between four grape wine varieties belonging to 4 different species: Vitis vinifera, Vitis amurensis, Vitis cinerea and Vitis X champinii. High-performance liquid chromatography coupled to mass spectrometry (LC–MS) and NMR spectroscopy (LC–NMR) were used to separate and identify the structure of anthocyanins present in these species. Combination of LC–MS and LC–NMR data resulted in the identification of 33 anthocyanins. In particular, newly reported cis isomers of p-coumaric-derivatives were identified (petunidin-, peonidin- and malvidin-3-(6-p-coumaroyl)-5-diglucoside). In V. cinerea and V. vinifera, anthocyanins were monoglucoside derivatives whereas in V. amurensis and V. X champinii, both mono- and diglucoside derivatives were identified. Malvidin-, delphinidin- and petunidin-derivatives were, respectively, the most abundant components in V. cinerea and V. vinifera, V. amurensis and V. X champinii.     

Metabolisation of eutypine by plant tissues: an HPLC determination

Eutypine, 4-hydroxy-3-(3-methyl-3-butene-1-ynyl) benzaldehyde, is a toxin produced by Eutypa lata, the causal agent of eutypa dieback of grapevine. The tolerance of some grapevine cultivars to the disease has been ascribed to the potential reduction of eutypine into its corresponding non-toxic alcohol, eutypinol. In the present study, eutypine biotransformation in different tissues of grapevine was investigated by HPLC and LC-MS. Grape callus tissues were able to biotransform eutypine into eutypinol within the first 3 h of culture. The grape plantlets cultured in vitro can also transform eutypine into eutypinol. Grape plantlet leaves do not have any effect on the uptake of eutypine, which goes through the tissues following a concentration gradient. Results revealed that the toxicity of eutypine in grape tissues is an active process showing that eutypinol is rapidly metabolised into other compounds. The use of micro-cuttings and in vitro plants showed that eutypine strongly accumulates in the bottom part of the diseased plant stems.     

Efficient green extraction of polyphenols from post-harvested agro-industry vegetal sources in Piedmont

A study of the polyphenols content and antioxidant capacity of grapevine waste and hazelnut skins (roasted material) from post-harvest products that originate from Piedmont (Italy) has been carried out and the results herein presented. Ultrasound-assisted extraction (UAE) and microwave-assisted extraction (MAE) were used to achieve process intensification in shorter extraction times, with lower environmental impact and higher selectivity compared to classic maceration. Besides classic solvents, the aqueous β-cyclodextrin solution (1.5%) showed to be an excellent extraction medium for grapevine waste. Total phenolic content (TP) from grapevine waste ranged from 18.23 ± 2.4 to 198 ± 3 mg gallic acid equivalents (GAE)/g dry weight, while total antioxidant capacity (TAC) expressed as EC₅₀ ranged from 0.0902 ± 0.08 mg/mL to 0.0041 ± 0.02 mg/mL. For hazelnut skins, TP ranged from 61.68 ± 0.8 to 200.79 ± 3.0 mg GAE/g dry weight, while TAC ranged from 0.0021 ± 0.0004 to 0.0002 ± 0.0001 mg/mL extract. We have shown that, compared to maceration, the use of UAE and MAE methods can enhance polyphenols recovery and antioxidant capacity.     

Antimalarials and Phytotoxins from Botryosphaeria dothidea Identified from a Seed of Diseased Torreya taxifolia

The metabolic pathways in the apicoplast organelle of Plasmodium parasites are similar to those in plastids in plant cells and are suitable targets for malaria drug discovery. Some phytotoxins released by plant pathogenic fungi have been known to target metabolic pathways of the plastid; thus, they may also serve as potential antimalarial drug leads. An EtOAc extract of the broth of the endophyte Botryosphaeria dothidea isolated from a seed collected from a Torreya taxifolia plant with disease symptoms, showed in vitro antimalarial and phytotoxic activities. Bioactivity-guided fractionation of the extract afforded a mixture of two known isomeric phytotoxins, FRT-A and flavipucine (or their enantiomers, sapinopyridione and (-)-flavipucine), and two new unstable γ-lactam alkaloids dothilactaenes A and B. The isomeric mixture of phytotoxins displayed strong phytotoxicity against both a dicot and a monocot and moderate cytotoxicity against a panel of cell lines. Dothilactaene A showed no activity. Dothilactaene B was isolated from the active fraction, which showed moderate in vitro antiplasmodial activity with high selectivity index. In spite of this activity, its instability and various other biological activities shown by related compounds would preclude it from being a viable antimalarial lead.     

Isolation and Identification of Pennogenin Tetraglycoside from Cestrum nocturnum (Solanaceae) and Its Antifungal Activity against Fusarium kuroshium,Causal Agent of Fusarium Dieback

Antifungal assay-guided fractionation of the methanolic crude extract of Cestrum nocturnum (Solanaceae), popular known as ‘lady of the night’, led the isolation and identification of the steroidal saponin named pennogenin tetraglycoside, which was identified for the first time in this plant species by spectroscopic means. The crude extract, fractions and pennogenin tetraglycoside exhibited mycelial growth inhibition of Fusarium solani and F. kuroshium. F. solani is a cosmopolitan fungal phytopathogen that affects several economically important crops. However, we highlight the antifungal activity displayed by pennogenin tetraglycoside against F. kuroshium, since it is the first plant natural product identified as active for this phytopathogen. This fungus along with its insect symbiont known as Kuroshio shot hole borer (Euwallacea kuroshio) are the causal agents of the plant disease Fusarium dieback that affects more than 300 plant species including avocado (Persea americana) among others of ecological relevance. Scanning electron microscopy showed morphological alterations of the fungal hyphae after exposure with the active fractions and 12 phenolic compounds were also identified by mass spectrometry dereplication as part of potential active molecules present in C. nocturnum leaves.