Microscopy coupled with DNA barcoding-based analysis as diagnostic tools to identify toxic plants

Federica Betuzzi ¹, Camilla Micaletti ¹, Elena Capuzzi ¹, Enrico Bajona ², Jessica Frigerio ³, Laura Cornara ¹

Department of Earth, Environment and Life Sciences (DISTAV), University of Genoa, Corso Europa 26, 16132 Genova, Italy ¹, Department of Agricultural, Food and Forest Sciences (SAAF), University of Palermo, Viale delle Scienze 4, 90128 Palermo, Italy ², Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza Della Scienza 2, 20126 Milan, Italy ³

Plant poisoning can be related to the: (i) misidentification between edible and toxic wild herbs due to morphological similarities; (ii) contamination of crops by weeds; (iii) use of toxic plants in traditional herbal medicine. Species belonging to Solanaceae family contain tropane alkaloids and are often involved in human poisoning. For example, in southern Italy, leaves of Mandragora autumnalis Bertol. can be confused by inexpert pickers with those of Spinacia oleracea L., Borago officinalis L., Beta vulgaris L., and accidentally collected for culinary uses. Moreover, M. autumnalis can grow near cultivated fields, so its leaves can be mechanically harvested and occasionally present in commercial frozen vegetables. Despite its known toxicity, mandrake root and rhizome are present in herbal market stalls of Morocco for their aphrodisiac properties or as cold remedy. However, this practice is potentially dangerous for public health. In particular, the presence of mandrake is sometimes indicated in artisanal “Thè Royal du Maroc”, a typical beverage of the Maghreb area. Since mandrake is used in form of root cut to prepare this herbal tea, its botanical identification is very difficult even for experts. Moreover, some species are used as a substitute for or to counterfeit mandrake, such as Alpinia officinarum Hance, Acorus calamus L., Withania somnifera (L.) Dunal, Sceletium tortuosum L. Considering this, we carried out a micro-morphological characterization of mandrake leaves and rhizome to identify their diagnostic features. The leaves were compared with those of spinach and beet, while the rhizome was compared with those of A. officinarum, A. calamus and an unknown rhizome found in an artisanal sample of “Thè Royal du Maroc”. Species-specific primers were designed for A. officinarum, A. calamus, and M. autumnalis with the aim of identifying these species within the “Thè Royal du Maroc” mixture. The primer pairs were designed based on specific marker standards of DNA barcoding (matK, psbA-trnH) and initially tested on pure samples to verify their efficiency. Results showed that mandrake, spinach and beet leaves had distinguishing features such as different kinds and distribution of covering and glandular trichomes. Instead, the rhizome fragments present in the herbal mixture called “Thè Royal du Maroc” showed anatomical characteristics very similar to those of A. officinarum, such as numerous vascular bundles scattered throughout the cortex and stele, without any particular orientation. These bundles were collateral endarch and surrounded by a bundle sheath. On the contrary, mandrake rhizome had a structure typical of other Solanaceae members, showing a thin layer of cork surrounding the cortex, a phloem region, a radiate xylem, and a central pith with scattered groups of intraxylary phloem. Regarding the DNA barcoding-based analyses, an amplicon was obtained for leaf mixes of the target species created in the laboratory but not for “Thè Royal du Maroc”, suggesting that in dried rhizomes DNA can be degraded. Microscopy is a first tool for identifying toxic plants in complex mixtures. DNA barcoding is useful for confirming or not the presence of certain species, but its effectiveness depends on DNA quantity and quality and plant material.

Main author career stage: PhD student

Contribution type: Poster

First choice session: 4. Structure, physiology, and development

Second choice session: 5. Genetics, genomics, and bioinformatics