Fabrizio De Mattia

Identification of poisonous plants by DNA barcoding approach

The plant exposures are one of the most frequent poisonings reported to poison control centres. The diagnosis of intoxicated patients is usually based on the morphological analysis of ingested plant portions; this procedure requires experience in systematic botany, because the plant identification is based on few evident traits. The objective of this research is to test DNA barcoding approach as a new universal tool to identify toxic plants univocally and rapidly. Five DNA barcode regions were evaluated: three cpDNA sequences (trnH-psbA, rpoB and matK) and two nuclear regions (At103 and sqd1). The performance of these markers was evaluated in three plant groups: (1) a large collection of angiosperms containing different toxic substances, (2) congeneric species showing different degrees of toxicity and (3) congeneric edible and poisonous plants. Based on assessments of PCR, sequence quality and resolution power in species discrimination, we recommend the combination of plastidial and nuclear markers to identify toxic plants. Concerning plastidial markers, matK and trnH-psbA showed consistent genetic variability. However, in agreement with CBOL Plant Working Group, we selected matK as the best marker, because trnH-psbA showed some problems in sequences sizes and alignments. As a final and relevant observation, we also propose the combination of matK with a nuclear marker such as At103 to distinguish toxic hybrids form parental species. In conclusion, our data support the claim that DNA barcoding is a powerful tool for poisonous plant identifications.

Phytotoxic and genotoxic effects of silver nanoparticles exposure on germinating wheat seedlings

We investigated the effects of 1 and 10 mg L(-1) AgNPs on germinating Triticum aestivum L. seedlings. The exposure to 10 mg L(-1) AgNPs adversely affected the seedling growth and induced morphological modifications in root tip cells. TEM analysis suggests that the observed effects were due primarily to the release of Ag ions from AgNPs. To gain an increased understanding of the molecular response to AgNP exposure, we analyzed the genomic and proteomic changes induced by AgNPs in wheat seedlings. At the DNA level, we applied the AFLP technique and we found that both treatments did not induce any significant DNA polymorphisms. 2DE profiling of roots and shoots treated with 10 mg L(-1) of AgNPs revealed an altered expression of several proteins mainly involved in primary metabolism and cell defense.

A DNA Barcoding Approach to Characterize Pollen Collected by Honeybees

In the present study, we investigated DNA barcoding effectiveness to characterize honeybee pollen pellets, a food supplement largely used for human nutrition due to its therapeutic properties. We collected pollen pellets using modified beehives placed in three zones within an alpine protected area (Grigna Settentrionale Regional Park, Italy). A DNA barcoding reference database, including rbcL and trnH-psbA sequences from 693 plant species (104 sequenced in this study) was assembled. The database was used to identify pollen collected from the hives. Fifty-two plant species were identified at the molecular level. Results suggested rbcL alone could not distinguish among congeneric plants; however, psbA-trnH identified most of the pollen samples at the species level. Substantial variability in pollen composition was observed between the highest elevation locality (Alpe Moconodeno), characterized by arid grasslands and a rocky substrate, and the other two sites (Cornisella and Ortanella) at lower altitudes. Pollen from Ortanella and Cornisella showed the presence of typical deciduous forest species; however in samples collected at Ortanella, pollen of the invasive Lonicera japonica, and the ornamental Pelargonium x hortorum were observed. Our results indicated pollen composition was largely influenced by floristic local biodiversity, plant phenology, and the presence of alien flowering species. Therefore, pollen molecular characterization based on DNA barcoding might serve useful to beekeepers in obtaining honeybee products with specific nutritional or therapeutic characteristics desired by food market demands.

DNA Barcoding as an Effective Tool in Improving a Digital Plant Identification System: A Case Study for the Area of Mt. Valerio, Trieste (NE Italy)

Background Identification keys are decision trees which require the observation of one or more morphological characters of an organism at each step of the process. While modern digital keys can overcome several constraints of classical paper-printed keys, their performance is not error-free. Moreover, identification cannot be always achieved when a specimen lacks some morphological features (i.e. because of season, incomplete development or miss-collecting). DNA barcoding was proven to have great potential in plant identification, while it can be ineffective with some closely related taxa, in which the relatively brief evolutionary distance did not produce differences in the core-barcode sequences. Methodology/Principal Findings In this paper, we investigated how the DNA barcoding can support the modern digital approaches to the identification of organisms, using as a case study a local flora, that of Mt. Valerio, a small hill near the centre of Trieste (NE Italy). The core barcode markers (plastidial rbcL and matK), plus the additional trnH-psbA region, were used to identify vascular plants specimens. The usefulness of DNA barcoding data in enhancing the performance of a digital identification key was tested on three independent simulated scenarios. Conclusions/Significance Our results show that the core barcode markers univocally identify most species of our local flora (96%). The trnH-psbA data improve the discriminating power of DNA barcoding among closely related plant taxa. In the multiparametric digital key, DNA barcoding data improves the identification success rate; in our simulation, DNA data overcame the absence of some morphological features, reaching a correct identification for 100% of the species. FRIDA, the software used to generate the digital key, has the potential to combine different data sources: we propose to use this feature to include molecular data as well, creating an integrated identification system for plant biodiversity surveys.

Effects of a complex mixture of therapeutic drugs on unicellular algae Pseudokirchneriella subcapitata

Pharmaceutically-active compounds are regularly and widely released into the aquatic environment in an unaltered form or as metabolites. So far, little is known about their potential detrimental effects on algae populations which can ultimately impact nutrient cycling and oxygen balance. For our analysis, the common microalga Pseudokirchneriella subcapitata (P. subcapitata) was exposed to a mixture of 13 drugs found in Italian wastewaters and rivers. Traces of pharmaceuticals investigated were detected in treated algal cells, except for cyclophosphamide and ranitidine, indicating that these algae are able to absorb pharmaceutical pollutants from the environment. The effects of the treatment were investigated by Amplified Fragment Length Polymorphism (AFLP) assessment of DNA damage and 2-DE proteomic analysis. While no genotoxic effect was detected, proteomic analysis showed that algae are sensitive to the presence of drugs and that, in particular, the chloroplast is affected.

Uptake and effects of a mixture of widely used therapeutic drugs in Eruca sativa L. and Zea mays L. plants

Pharmaceutically active compounds (PACs) are continuously dispersed into the environment due to human and veterinary use, giving rise to their potential accumulation in edible plants. In this study, Eruca sativa L. and Zea mays L. were selected to determine the potential uptake and accumulation of eight different PACs (Salbutamol, Atenolol, Lincomycin, Cyclophosphamide, Carbamazepine, Bezafibrate, Ofloxacin and Ranitidine) designed for human use. To mimic environmental conditions, the plants were grown in pots and irrigated with water spiked with a mixture of PACs at concentrations found in Italian wastewaters and rivers. Moreover, 10× and 100× concentrations of these pharmaceuticals were also tested. The presence of the pharmaceuticals was tested in the edible parts of the plants, namely leaves for E. sativa and grains for Z. mays. Quantification was performed by liquid chromatography mass spectroscopy (LC/MS/MS). In the grains of 100× treated Z. mays, only atenolol, lincomycin and carbamazepine were above the limit of detection (LOD). At the same concentration in E. sativa plants the uptake of all PACs was >LOD. Lincomycin and oflaxacin were above the limit of quantitation in all conditions tested in E. sativa. The results suggest that uptake of some pharmaceuticals from the soil may indeed be a potential transport route to plants and that these environmental pollutants can reach different edible parts of the selected crops. Measurements of the concentrations of these pharmaceuticals in plant materials were used to model potential adult human exposure to these compounds. The results indicate that under the current experimental conditions, crops exposed to the selected pharmaceutical mixture would not have any negative effects on human health. Moreover, no significant differences in the growth of E. sativa or Z. mays plants irrigated with PAC-spiked vs. non-spiked water were observed.

DNA Barcoding for Minor Crops and Food Traceability

This outlook paper addresses the problem of the traceability of minor crops. These kinds of cultivations consist in a large number of plants locally distributed with a modest production in terms of cultivated acreage and quantity of final product. Because of globalization, the diffusion of minor crops is increasing due to their benefit for human health or their use as food supplements. Such a phenomenon implies a major risk for species substitution or uncontrolled admixture of manufactured plant products with severe consequences for the health of consumers. The need for a reliable identification system is therefore essential to evaluate the quality and provenance of minor agricultural products. DNA-based techniques can help in achieving this mission. In particular, the DNA barcoding approach has gained a role of primary importance thanks to its universality and versatility. Here, we present the advantages in the use of DNA barcoding for the characterization and traceability of minor crops based on our previous or ongoing studies at the ZooPlantLab (Milan, Italy). We also discuss how DNA barcoding may potentially be transferred from the laboratory to the food supply chain, from field to table.

Genetic variability of relict Rhododendron ferrugineum L. populations in the Northern Apennines with some inferences for a conservation strategy

Abstract In this study, the genetic diversity of three Rhododendron ferrugineum L Apennine populations (AP1, AP2, and AP3) was analyzed and compared to three populations of the Maritime Alps and six populations of Central-Eastern Alps. Genetic variations across microsatellite markers revealed that the Apennine populations show some clonal individuals and the lowest genetic diversity values (AP1 and AP3 A[18] values are 2.46 and 2.31, respectively), as well as heterozygosity deficiency with respect to the Alpine populations. Genetic relationships among populations (Neis genetic distance) showed that populations from the Central-Eastern Alps and from the Maritime Alps clustered in two separate groups. Unweighted pair group method with arithmetic averages as well as PCA analysis showed a clear separation of the three Apennine populations according to the high FST values detected (AP1–AP2 = 0.427; AP1–AP3 = 0.446; AP2–AP3 = 0.325). Mantel test revealed a significant correlation between genetic and geographical distance matrices (r = 0.314, P = 0.001, 999 permutations). Concerning the relationship between Apennines and alpine population, PCA analysis showed a clear genetic similarity among Maritime Alps populations and individuals of AP1 population. Considering the geographical and ecological peripheral condition of these populations and the high impact of the climatic changes on their habitat, we suggest the combination of in situ and ex situ conservation strategies to preserve the genetic diversity of this species in the Northern Apennines.

Physiological and molecular effects associated with palladium treatment in Pseudokirchneriella subcapitata

Human activities have increased the levels of environmental palladium (Pd) worldwide. Due to the growing evidence of its toxicity, Pd pollution has become the focus of serious concern. Several studies have given an account of the increasing concentration of Pd in aquatic ecosystems. The aim of the current study is to analyze the physiological and molecular effects induced by Pd on freshwater unicellular green algae. To do this, Pseudokirchneriella subcapitata (P. subcapitata) was exposed in vitro to different concentrations (0.1, 0.25 and 0.5 mg l(-1)) of K(2)PdCl(4), a soluble salt of Pd, corresponding to 0.03, 0.075 and 0.15 mg l(-1) of Pd. The uptake and the effects on algal growth and morphology were determined. The main results are that Pd is able to induce damage in P. subcapitata at a concentration of 0.1 mg l(-1) of K(2)PdCl(4), with the damage becoming more evident at a concentration of 0.25 mg l(-1)of K(2)PdCl(4); at a concentration of 0.5 mg l(-1) of K(2)PdCl(4), cellular degeneration occurs. The main cellular target of Pd is the chloroplast, as shown by TEM and proteomic analysis. TEM analysis also showed accumulation of precipitates, probably of Pd, in the chloroplasts, although further experiments are necessary to confirm that these are Pd-precipitates. Amplified fragment length polymorphism analysis (AFLP) demonstrated that Pd, even at the lowest concentration tested, induced randomly distributed DNA changes either directly or indirectly in the algal genome and that oxidative processes were involved.

Chloroplast and nuclear DNA markers to characterize cultivated and spontaneous Ribes

Abstract Nine universal cpSSR markers were tested on a representative group of cultivated accessions and spontaneous ribes populations. All primer combinations, with the exception of ccmp8, underwent amplification. Monomorphic alleles were detected at the ccmp2, ccmp3, ccmp4, ccmp5, ccmp7, ccmp9 primer pairs. Two and four polymorphic alleles were observed at the ccmp6 and ccmp10 loci, respectively. These six alleles combined in five different haplotypes, two of which were found in the cultivated accessions, and three only in the spontaneous populations. AFLP analysis was also performed to better define the relationships among cultivated and spontaneous varieties, and to compare nuclear markers with chloroplast microsatellites. AFLP data were able to distinguish clearly all cultivars, while in spontaneous populations the detected polymorphisms were too low. In conclusion, cpSSR markers can be considered useful in the characterisation of ribes accessions. Thus, the combination of this tool with nuclear markers, such as AFLP, could help in distinguishing and characterising each cultivated accession, and in defining population genetic parameters in the study of spontaneous Ribes species.