Changes in the Phytochemical Profile and Antioxidant Properties of Prunus persica Fruits after the Application of a Commercial Biostimulant Based on Seaweed and Yeast Extract.

Plant biostimulants are formulations that are experiencing great success from the perspective of sustainable agriculture. In this work, we evaluated the effect derived from the application of a biostimulant based on algae and yeast extracts (Expando®) on the agronomic yield and nutraceutical profile of two different cultivars ("Sugar Time" and "West Rose") of Prunus persica (peach). Although, at the agronomic level, significant effects on production yields were not recorded, the biostimulant was able to reduce the ripening time, increase the fruit size, and make the number of harvestable fruits homogeneous. From a nutraceutical point of view, our determinations via spectrophotometric (UV/Vis) and chromatographic (HPLC-DAD-MS/MS) analysis showed that the biostimulant was able to boost the content of bioactive compounds in both the pulp (5.0 L/ha: +17%; 4.0 L/ha: +12%; 2.5 L/ha: +11%) and skin (4.0 L/ha: +38%; 2.5 L/ha: +15%). These changes seem to follow a dose-dependent effect, also producing attractive effects on the antioxidant properties of the fruits harvested from the treated trees. In conclusion, the biostimulant investigated in this work proved to be able to produce more marketable fruit in a shorter time, both from a pomological and a functional point of view.

Melatonin and Phytomelatonin: Chemistry, Biosynthesis, Metabolism, Distribution and Bioactivity in Plants and Animals-An Overview.

Melatonin is a ubiquitous indolamine, largely investigated for its key role in the regulation of several physiological processes in both animals and plants. In the last century, it was reported that this molecule may be produced in high concentrations by several species belonging to the plant kingdom and stored in specialized tissues. In this review, the main information related to the chemistry of melatonin and its metabolism has been summarized. Furthermore, the biosynthetic pathway characteristics of animal and plant cells have been compared, and the main differences between the two systems highlighted. Additionally, in order to investigate the distribution of this indolamine in the plant kingdom, distribution cluster analysis was performed using a database composed by 47 previously published articles reporting the content of melatonin in different plant families, species and tissues. Finally, the potential pharmacological and biostimulant benefits derived from the administration of exogenous melatonin on animals or plants via the intake of dietary supplements or the application of biostimulant formulation have been largely discussed.

Arabidopsis thaliana ITS sequence-specific DNA extraction by ion-tagged oligonucleotides coupled with a magnetic ionic liquid.

This study reports a follow-up investigation on the capture of specific DNA sequences using ion-tagged oligonucleotides (ITOs) and magnetic ionic liquids (MIL). Five allylimidazolium salts bearing octyl substituents ([AOIM+]-ITOs) were used for the selective extraction of the internal transcribed spacer region (ITS) from Arabidopsis thaliana. In this work, the ability of the [AOIM+]-ITOs to enhance the extraction of longer target sequences (~ 700 bp) of plant origin was shown. Moreover, the independence of the probe binding position and the importance of complementarity to the target region for the extraction performance were demonstrated. To test the specificity of the ITOs, the same experiments were performed using the ITS region from another plant species, with a lower target capture for the probes which were specific for the A. thaliana sequence. Finally, extraction in the presence of interferences (heterogenous DNA, primary and secondary metabolites, proteins) provided interesting and insightful results. This work illustrates the feasibility and versatility of these probes when coupled to MILs for rapid, cost-effective, and environmentally sensitive sample preparation in the extraction of specific target sequences from different origins. Graphical abstract.

Reduction of the geomagnetic field delays Arabidopsis thaliana flowering time through downregulation of flowering-related genes.

Variations in magnetic field (MF) intensity are known to induce plant morphological and gene expression changes. In Arabidopsis thaliana Col-0, near-null magnetic field (NNMF, i.e., <100 nT MF) causes a delay in the transition to flowering, but the expression of genes involved in this response has been poorly studied. Here, we showed a time-course quantitative analysis of the expression of both leaf (including clock genes, photoperiod pathway, GA20ox, SVP, and vernalization pathway) and floral meristem (including GA2ox, SOC1, AGL24, LFY, AP1, FD, and FLC) genes involved in the transition to flowering in A. thaliana under NNMF. NNMF induced a delayed flowering time and a significant reduction of leaf area index and flowering stem length, with respect to controls under geomagnetic field. Generation experiments (F1 - and F2 -NNMF) showed retention of flowering delay. The quantitative expression (qPCR) of some A. thaliana genes expressed in leaves and floral meristem was studied during transition to flowering. In leaves and flowering meristem, NNMF caused an early downregulation of clock, photoperiod, gibberellin, and vernalization pathways and a later downregulation of TSF, AP1, and FLC. In the floral meristem, the downregulation of AP1, AGL24, FT, and FLC in early phases of floral development was accompanied by a downregulation of the gibberellin pathway. The progressive upregulation of AGL24 and AP1 was also correlated to the delayed flowering by NNMF. The flowering delay is associated with the strong downregulation of FT, FLC, and GA20ox in the floral meristem and FT, TSF, FLC, and GA20ox in leaves. Bioelectromagnetics. 39:361-374, 2018. © 2018 The Authors. Bioelectromagnetics Published by Wiley Periodicals, Inc.

Direct Contact - Sorptive Tape Extraction coupled with Gas Chromatography - Mass Spectrometry to reveal volatile topographical dynamics of lima bean (Phaseolus lunatus L.) upon herbivory by Spodoptera littoralis Boisd.

BACKGROUND: The dynamics of plant volatile (PV) emission, and the relationship between damaged area and biosynthesis of bioactive molecules in plant-insect interactions, remain open questions. Direct Contact-Sorptive Tape Extraction (DC-STE) is a sorption sampling technique employing non adhesive polydimethylsiloxane tapes, which are placed in direct contact with a biologically-active surface. DC-STE coupled to Gas Chromatography - Mass Spectrometry (GC-MS) is a non-destructive, high concentration-capacity sampling technique able to detect and allow identification of PVs involved in plant responses to biotic and abiotic stresses. Here we investigated the leaf topographical dynamics of herbivory-induced PV (HIPV) produced by Phaseolus lunatus L. (lima bean) in response to herbivory by larvae of the Mediterranean climbing cutworm (Spodoptera littoralis Boisd.) and mechanical wounding by DC-STE-GC-MS. RESULTS: Time-course experiments on herbivory wounding caused by larvae (HW), mechanical damage by a pattern wheel (MD), and MD combined with the larvae oral secretions (OS) showed that green leaf volatiles (GLVs) [(E)-2-hexenal, (Z)-3-hexen-1-ol, 1-octen-3-ol, (Z)-3-hexenyl acetate, (Z)-3-hexenyl butyrate] were associated with both MD and HW, whereas monoterpenoids [(E)-ß-ocimene], sesquiterpenoids [(E)-nerolidol] and homoterpenes (DMNT and TMTT) were specifically associated with HW. Up-regulation of genes coding for HIPV-related enzymes (Farnesyl Pyrophosphate Synthase, Lipoxygenase, Ocimene Synthase and Terpene Synthase 2) was consistent with HIPV results. GLVs and sesquiterpenoids were produced locally and found to influence their own gene expression in distant tissues, whereas (E)-ß-ocimene, TMTT, and DMNT gene expression was limited to wounded areas. CONCLUSIONS: DC-STE-GC-MS was found to be a reliable method for the topographical evaluation of plant responses to biotic and abiotic stresses, by revealing the differential distribution of different classes of HIPVs. The main advantages of this technique include: a) in vivo sampling; b) reproducible sampling; c) ease of execution; d) simultaneous assays of different leaf portions, and e) preservation of plant material for further "omic" studies. DC-STE-GC-MS is also a low-impact innovative method for in situ PV detection that finds potential applications in sustainable crop management.

Plant defences against ants provide a pathway to social parasitism in butterflies.

Understanding the chemical cues and gene expressions that mediate herbivore-host-plant and parasite-host interactions can elucidate the ecological costs and benefits accruing to different partners in tight-knit community modules, and may reveal unexpected complexities. We investigated the exploitation of sequential hosts by the phytophagous-predaceous butterfly Maculinea arion, whose larvae initially feed on Origanum vulgare flowerheads before switching to parasitize Myrmica ant colonies for their main period of growth. Gravid female butterflies were attracted to Origanum plants that emitted high levels of the monoterpenoid volatile carvacrol, a condition that occurred when ants disturbed their roots: we also found that Origanum expressed four genes involved in monoterpene formation when ants were present, accompanied by a significant induction of jasmonates. When exposed to carvacrol, Myrmica workers upregulated five genes whose products bind and detoxify this biocide, and their colonies were more tolerant of it than other common ant genera, consistent with an observed ability to occupy the competitor-free spaces surrounding Origanum. A cost is potential colony destruction by Ma. arion, which in turn may benefit infested Origanum plants by relieving their roots of further damage. Our results suggest a new pathway, whereby social parasites can detect successive resources by employing plant volatiles to simultaneously select their initial plant food and a suitable sequential host.

Separation of early and late responses to herbivory in Arabidopsis by changing plasmodesmal function.

Herbivory results in an array of physiological changes in the host that are separable from the associated physical damage. We have made the surprising observation that an Arabidopsis line (pdko3) mutated in genes encoding plasmodesmal proteins is defective in some, but not all, of the typical plant responses to herbivory. We tested the responses of plasma transmembrane potential (Vm) depolarization, voltage gated K(+) channel activity, cytosolic calcium [Ca2+]cyt and reactive oxygen species (ROS) (H2 O2 and NO) release, shoot-to-root signaling, biosynthesis of the phytohormone jasmonic acid (JA) and the elicitation of volatile organic compounds (VOCs). Following herbivory and the release of factors present in insect oral secretions (including a putative ß-galactofuranose polysaccharide), both the pdko3 and wild type (WT) plants showed a increased accumulation of [Ca2+]cyt , NO and H2 O2 . In contrast, unlike WT plants, the mutant line showed an almost complete loss of voltage gated K(+) channel activity and Vm depolarization, a loss of shoot-induced root-Vm depolarization, a loss of activation and regulation of gene expression of the JA defense pathway, and a much diminished release and altered profile of VOCs. The mutations in genes for plasmodesmal proteins have provided valuable genetic tools for the dissection of the complex spectrum of responses to herbivory and shown us that the responses to herbivory can be separated into a calcium-activated oxidative response and a K(+) -dependent Vm-activated jasmonate response associated with the release of VOCs.

Bioactive Compounds and Antioxidant Properties with Involved Mechanisms of Eugenia involucrata DC Fruits.

In this study, the phytochemical profile and the antioxidative properties of Eugenia involucrata fruits were evaluated. Spectrophotometric assays indicated that these berries are a rich source of polyphenols with very high radical-scavenging and metal-reducing activities. High-performance liquid chromatography-Orbitrap analysis was able to carry out the annotation of 36 different compounds, mainly belonging to the flavonol, flavan-3-ol, and anthocyanin families. Antioxidant activity of the fruit extract was evaluated in a cell-based lipid peroxidation model. Obtained data showed that the extract, at very low concentration, was able to prevent oxidative damage in HepG2 cells exposed to oxidative stimuli. Moreover, the evaluation of the gene expression of the most important antioxidant enzymes suggested that the observed antioxidant protection in cells also involves an improvement in enzymatic antioxidant defenses. Finally, the collected data show that E. involucrata fruits are a good source of natural antioxidant molecules and provide evidence of their potential application in the nutraceutical field.

The application of a biostimulant based on tannins affects root architecture and improves tolerance to salinity in tomato plants.

Roots have important roles for plants to withstand adverse environmental conditions, including salt stress. Biostimulant application was shown to enhance plant resilience towards abiotic stresses. Here, we studied the effect of a tannin-based biostimulant on tomato (Solanum lycopersicum L.) grown under salt stress conditions. We investigated the related changes at both root architecture (via imaging and biometric analysis) and gene expression (RNA-Seq/qPCR) levels. Moreover, in order to identify the main compounds potentially involved in the observed effects, the chemical composition of the biostimulant was evaluated by UV/Vis and HPLC-ESI-Orbitrap analysis. Sixteen compounds, known to be involved in root development and having a potential antioxidant properties were identified. Significant increase of root weight (+ 24%) and length (+ 23%) was observed when the plants were grown under salt stress and treated with the biostimulant. Moreover, transcriptome analysis revealed that the application of the biostimulant upregulated 285 genes, most of which correlated to root development and salt stress tolerance. The 171 downregulated genes were mainly involved in nutrient uptake. These data demonstrated that the biostimulant is able not only to restore root growth in salty soils, but also to provide the adequate plant nourishment by regulating the expression of essential transcription factors and stress responsive genes.

Microbial Biostimulants as Response to Modern Agriculture Needs: Composition, Role and Application of These Innovative Products.

An increasing need for a more sustainable agriculturally-productive system is required in order to preserve soil fertility and reduce soil biodiversity loss. Microbial biostimulants are innovative technologies able to ensure agricultural yield with high nutritional values, overcoming the negative effects derived from environmental changes. The aim of this review was to provide an overview on the research related to plant growth promoting microorganisms (PGPMs) used alone, in consortium, or in combination with organic matrices such as plant biostimulants (PBs). Moreover, the effectiveness and the role of microbial biostimulants as a biological tool to improve fruit quality and limit soil degradation is discussed. Finally, the increased use of these products requires the achievement of an accurate selection of beneficial microorganisms and consortia, and the ability to prepare for future agriculture challenges. Hence, the implementation of the microorganism positive list provided by EU (2019/1009), is desirable.

The Application of a Plant Biostimulant Based on Seaweed and Yeast Extract Improved Tomato Fruit Development and Quality.

Plant biostimulants are under investigation as innovative products to improve plant production and fruit quality, without resulting in environmental and food contaminations. Here, the effects of the application of Expando, a biostimulant based on seaweed and yeast extracts, on plant productivity, fruit ripening times, and fruit quality of Solanum lycopersicum var. Micro-Tom were evaluated. After biostimulant treatment, a two-week reduction of ripening times and a concomitant enhancement of the production percentage during the earliest ripening times, in terms of both fruit yield (+110%) and size (+85%), were observed. Concerning fruit quality, proximate analysis showed that tomatoes treated with the biostimulant had better nutritional composition compared to untreated samples, since both the quality of unsatured fatty acids (C16:3ω3: +328%; C18:2ω6: -23%) and micronutrients essential for human health (Fe: +14%; Cu: +21%; Zn: +24%) were increased. From a nutraceutical point of view, despite strong changes in bioactive compound profile not being observed, an increase of the antioxidant properties was recorded in fruits harvested by plants treated with the biostimulant (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS): +38%; 2,2-diphenyl-1-picrylhydrazyl (DPPH): +11%). In conclusion, the biostimulant application was able to reduce the ripening times and fruit size, while slightly increasing nutritional and nutraceutical values, leading to more marketable tomato fruits.

A Biostimulant Seed Treatment Improved Heat Stress Tolerance During Cucumber Seed Germination by Acting on the Antioxidant System and Glyoxylate Cycle.

Seed enhancement technologies have the potential to improve germination and seedling growth under environmental stress. The effects of KIEM®, an innovative biostimulant based on lignin derivatives and containing plant-derived amino acids and molybdenum, were investigated on cucumber (Cucumis sativus L.) seed germination. To determine the metabolic targets of this product, biometric, transcriptional and biochemical analyses were carried out on both non-treated and KIEM®-treated seeds incubated for 24 and 48 h under standard (28°C) and heat stress (35°C) conditions. The application of the biostimulant as a seed treatment increased the percent germination (+6.54%) and fresh biomass (+13%) at 48 h, and decreased the content of H2O2 in treated seeds at 28°C (-70%) and at 35°C (-80%). These changes in biometric and biochemical properties were accompanied by changes in expression levels of the genes coding for ROS-producing (RBOH) and scavenging (SOD, CAT, GST) enzymes and their specific activity. In general, the treatment with KIEM® in heat-stress condition appeared to stimulate a higher accumulation of three scavenger gene transcripts: CuZnSOD (+1.78), MnSOD (+1.75), and CAT (+3.39), while the FeSOD isoform was dramatically downregulated (0.24). Moreover, the amount of non-protein thiols, important antioxidant molecules, was increased by the biostimulant after 48 h (+20%). Taken together these results suggest that KIEM® acts through mitigation of the effects of the oxidative stress. Moreover, after 48 h, the pre-sowing treatment with KIEM® increased the transcription levels (+1.5) and the activity of isocitrate lyase (+37%), a key enzyme of the glyoxylate cycle, suggesting a potential effect of this product in speeding up the germination process. Finally, the chemical characterization of KIEM® identified five essential and three non-essential amino acids, and others bioactive compounds, including five organic and inorganic acids that might be potentially involved in its activity. Based on these data, insights on the potential mechanism of action of the biostimulant, suggested that there are broader applications as a product able to increase seed tolerance to different abiotic stress typical of adverse environmental conditions.

Exogenous polyamines elicit herbivore-induced volatiles in lima bean leaves: involvement of calcium, H2O2 and Jasmonic acid.

We investigated the role of polyamines (PAs) in lima bean (Phaseolus lunatus) leaves on the production of herbivorous mite (Tetranychus urticae)-induced plant volatiles that attract carnivorous natural enemies of the herbivores. To do this, we focused on the effects of the exogenous PAs [cadaverine, putrescine, spermidine and spermine (Spm)] on the production of volatiles, H(2)O(2) and jasmonic acid (JA) and the levels of defensive genes, cytosolic calcium and reactive oxygen species (ROS). Among the tested PAs, Spm was the most active in inducing the production of volatile terpenoids known to be induced by T. urticae. An increase in JA levels was also found after Spm treatment, indicating that Spm induces the biosynthesis of JA, which has been shown elsewhere to regulate the production of some volatile terpenoids. Further, treatment with JA and Spm together resulted in greater volatile emission than that with JA alone. In a Y-tube olfactometer, leaves treated with Spm + JA attracted more predatory mites (Phytoseiulus persimilis) than those treated with JA alone. After treatment with Spm + JA, no effects were found on the enzyme activity of polyamine oxidase and copper amine oxidase. However, induction of calcium influx and ROS production, and increased enzyme activities and gene expression for NADPH oxidase complex, superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase and glutathione peroxidase were found after treatment with Spm + JA. These results indicate that Spm plays an important role in the production of T. urticae-induced lima bean leaf volatiles.

Development of an innovative and sustainable one-step method for rapid plant DNA isolation for targeted PCR using magnetic ionic liquids.

BACKGROUND: Nowadays, there is an increasing demand for fast and reliable plant biomolecular analyses. Conventional methods for the isolation of nucleic acids are time-consuming and require multiple and often non-automatable steps to remove cellular interferences, with consequence that sample preparation is the major bottleneck in the bioanalytical workflow. New opportunities have been created by the use of magnetic ionic liquids (MILs) thanks to their affinity for nucleic acids. RESULTS: In the present study, a MIL-based magnet-assisted dispersive liquid-liquid microextraction (maDLLME) method was optimized for the extraction of genomic DNA from Arabidopsis thaliana (L.) Heynh leaves. MILs containing different metal centers were tested and the extraction method was optimized in terms of MIL volume and extraction time for purified DNA and crude lysates. The proposed approach yielded good extraction efficiency and is compatible with both quantitative analysis through fluorimetric-based detection and qualitative analysis as PCR amplification of multi and single locus genes. The protocol was successfully applied to a set of plant species and tissues. CONCLUSIONS: The developed MIL-based maDLLME approach exhibits good enrichment of nucleic acids for extraction of template suitable for targeted PCR; it is very fast, sustainable and potentially automatable thereby representing a powerful tool for screening plants rapidly using DNA-based methods.

Intra-specific variation in the little-known Mediterranean plant Ptilostemon casabonae (L.) Greuter analysed through phytochemical and biomolecular markers.

Ptilostemon casabonae (L.) Greuter is a Mediterranean endemism traditionally used for its health-giving properties. Little is known about this species, therefore this study provides additional information about the phytochemical and biomolecular patterns of this plant, to have a combined fingerprint as a taxonomic tool. Several P. casabonae specimens were therefore collected from three different sites, two from Sardinia (Italy) and one from Corsica and the hydroalcoholic extracts of their aerial parts were investigated through HPLC-PDA-MS/MS analysis to study the phenolic composition. Quercetin, luteolin, kaempferol, apigenin and diosmetin O-glycosides, and caffeoylquinic acid derivatives were found as main components. Samples from the three sites showed similar phenolic profiles, although statistical analyses highlighted some quantitative differences for several compounds. The biomolecular analysis included amplification and sequencing of ITS, 5S-rRNA-NTS and psbA regions. No difference was found in the nucleotides among the P. casabonae samples from different geographical origins; however, a comparison with other Ptilostemon species sequences from Genbank, revealed an interspecific variability of ITS and psbA regions. The combination of the results of the phytochemical and biomolecular studies provide information on P. casabonae useful to depict this little-known plant, which can also be applied for future investigations and to obtain a fingerprint of it. Moreover, the stability of the phenolic profile within the species affords to identify a set of specialised metabolites useful for its chemotaxonomic characterization. At the same time, the stability of the biomolecular profile of P. casabonae, and the identification of sequences specific for this species, enables to identify useful biomolecular markers to distinguish it unequivocally.

The microbial community of Vetiver root and its involvement into essential oil biogenesis.

Vetiver is the only grass cultivated worldwide for the root essential oil, which is a mixture of sesquiterpene alcohols and hydrocarbons, used extensively in perfumery and cosmetics. Light and transmission electron microscopy demonstrated the presence of bacteria in the cortical parenchymatous essential oil-producing cells and in the lysigen lacunae in close association with the essential oil. This finding and the evidence that axenic Vetiver produces in vitro only trace amounts of oil with a strikingly different composition compared with the oils from in vivo Vetiver plants stimulated the hypothesis of an involvement of these bacteria in the oil metabolism. We used culture-based and culture-independent approaches to analyse the microbial community of the Vetiver root. Results demonstrate a broad phylogenetic spectrum of bacteria, including alpha-, beta- and gamma-Proteobacteria, high-G+C-content Gram-positive bacteria, and microbes belonging to the Fibrobacteres/Acidobacteria group. We isolated root-associated bacteria and showed that most of them are able to grow by using oil sesquiterpenes as a carbon source and to metabolize them releasing into the medium a large number of compounds typically found in commercial Vetiver oils. Several bacteria were also able to induce gene expression of a Vetiver sesquiterpene synthase. These results support the intriguing hypothesis that bacteria may have a role in essential oil biosynthesis opening the possibility to use them to manoeuvre the Vetiver oil molecular structure.

Chamazulene Attenuates ROS Levels in Bovine Aortic Endothelial Cells Exposed to High Glucose Concentrations and Hydrogen Peroxide.

Endothelial cells surround the lumen of blood vessels and modulate many physiological processes, including vascular tone, blood fluidity, inflammation, immunity and neovascularization. Many pathological conditions, including hyperglycemia, may alter endothelial function through oxidative stress, leading to impaired nitric oxide bioavailability and to the onset of an inflammatory state. As widely shown in the last decade, dietary intervention could represent a good strategy to control endothelial dysfunction and atherosclerosis. In particular, extensive research in the field of antioxidant natural derivatives has been conducted. In this study, we evaluated the capability of Chamazulene (Cham), an azulene compound from chamomile essential oil, to attenuate ROS levels in bovine aortic endothelial cells (BAECs) stressed with either high glucose or H2O2. Cell viability at different concentrations of Cham was evaluated through the WST-1 assay, while ROS production acutely induced by High Glucose (HG, 4.5 g/L) treatment or H2O2 (0.5 mM) for 3 h, was quantified with 2'-7'-Dichlorofluorescein diacetate (DCFH-DA) probe using confocal microscopy and flow cytometry. Our results showed a reduction in ROS produced after simultaneous treatment with High Glucose or H2O2 and Cham, thus suggesting an in vitro antioxidant activity of the compound. On the whole, this study shows for the first time the potential role of Cham as a scavenging molecule, suggesting its possible use to prevent the rise of endothelial ROS levels and the consequent vascular damage.

Characterization of four wild edible Carduus species from the Mediterranean region via phytochemical and biomolecular analyses.

Carduus species (Compositae) are widely distributed in the Mediterranean area, and traditionally used for both food and medicinal purposes. The hydroalcoholic extracts of four wild edible Carduus species collected in Sardinia (Carduus argyroa Biv., Carduus nutans subsp. macrocephalus (Desf.) Nyman, Carduus pycnocephalus L., Carduus cephalanthus Viv.) were analyzed and characterized by HPLC-PDA-MS/MS and PCR-RFLP of the nrDNA internal transcribed spacer (ITS). Flavonoids and caffeoylquinic acid derivatives were the predominant classes of secondary metabolites characterizing the extracts. The ITS region was sequenced in parallel, and a PCR-RFLP method was applied with three selective restriction enzymes. Statistical analyses, on both chemical and biomolecular results, revealed that individuals clustered according to their taxonomic classification. The combination of the two techniques discriminates the four species within the genus, giving further information on these little-investigated plants, traditionally used in the Mediterranean area and in Sardinia.

PCR and PCR-RFLP of the 5S-rRNA-NTS region and salvinorin A analyses for the rapid and unequivocal determination of Salvia divinorum.

Salvia divinorum Epling & Játiva-M. is a perennial herb belonging to the Lamiaceae family; its active ingredient, the neoclerodane diterpene salvinorin A, is a psychotropic molecule that produces hallucinations. A comparative evaluation of S. divinorum fresh and dried leaves, S. officinalis fresh leaves, and dried powdered leaves claimed to be S. divinorum was done. HPLC-MS data confirmed the presence of salvinorin A in both S. divinorun leaf extracts and the powdered leaves, whereas no salvinorin A was found in S. officinalis. The non-transcribed spacer (NTS) in the 5S-rRNA gene of all leaf samples and the dried powdered leaves was amplified by PCR using a pair of primers located at the 3' and 5' ends of the coding sequence of 5S-rRNA gene. The resulting PCR products (about 500bp for S. divinorum and 300bp for S. officinalis) were gel purified, subcloned into pGEM-T Easy vector and sequenced. By aligning the isolated nucleotide sequences, great diversities were found in the spacer region of the two species. Specific S. divinorum primers were designed on the sequence of the 5S-rRNA gene spacer region. In addition, a PCR-restriction fragment length polymorphism (PCR-RFLP) method was applied using NdeI and TaqI restriction enzymes. An NdeI site, absent in S. officinalis, was found in S. divinorum NTS region at 428-433bp. For TaqI, multiple sites (161-164, 170-173, and 217-220bp) were found in S. officinalis, whereas a unique site was found in S. divinorum (235-238bp). The results of this work show that the combined use of analytical chemical (HPLC-MS) and molecular (DNA fingerprinting) methods lead to the precise and unequivocal identification of S. divinorum.