Morphological and molecular characterization of distinct species of fungi with potential medicinal interest collected in Sicily

Medicinal mushrooms are very interesting for their pharmacological effects as well as for their nutritional value, antitumor, antiviral, antibacterial activities. Antitumor activities of mushrooms have been extensively investigated due to recent chemotherapeutic application of some drugs derived from natural sources.The aim of this study was the morphological and molecular characterization of nine species of fungi with potential medicinal interest. In particular, we focused our attention on: Bjerkandera adusta (Willd) P. Karst., Ganoderma resinaceum Boud.,Hericium erinaceus (Bull.) Pers., Pleurotus eryngii var elaeoselini Venturella, Zervakis and La Rocca, P. eryngii var. eryngii (DC.) Quél., P. eryng i var. ferulae (Lanzi) Sacc., P. nebrodensis (Inzenga) Quél., P. opuntiae (Durieu & Lév.) Sacc., P. ostreatus (Jacq.) P. Kumm. The basidiomata were collected in Sicily and subsequently were dried and they were stored in the Herbarium SAF of the Department of Agricultural, Food and Forest Sciences (University of Palermo, Italy). The identification was performed by examining the whole basidiomata (cap, gills, stipe, etc.). The microscopic features were observed in water using a Leica microscope DMLB. Basidiospores measurements were based on 50 observations. Nomenclature is referred to Index Fungorum (http://www.indexfungorum.org/ Names/Names.asp). Subsequently to confirm the identification molecular analysis were carried out. The internal transcribed spacer (ITS) region is the primary choice for molecular identification of fungi and the preferred DNA barcoding marker. Total DNA was extracted from dry tissue using the CTAB- based protocol [4]. PCR amplification was carried out with ITS1F/ ITS4 primer pairs targeting the ITS. PCR conditions, were performed as described by Oliveri et al. Obtained PCR products were sequenced in both directions using an ABI PRISM DNA 377 sequencer (Perkin-Elmer, Boston, MA, USA). Nucleotide sequences were compared with sequences of respective reference species retrieved from GenBank. Phylogenetic relationships were inferred by the Minimum Evolution method with 1,000 bootstrap replicates, using the algorithm Kimura-2-parameter. The phylogenetic analysis revealed that all species examined clustered with reference species with an identity > 99%. A particular case is represented by some species of the genus Ganoderma.In fact, this genus includes many species that are diffic lt to characterize both macroscopically and microscopically, with classical examples being G. resinaceum and G. lucidum which may seem identical but which differ for a few imperceptible characters. Therefore, in order to have a clear characterization, it is useful to use molecular analysis and then of Bar-Coding technology, which can be a very useful tool for the characterization of new fungal species. For this purpose, it would be useful, especially for the characterization of fungal species of difficult morphological identification, to analyze, in addition to the ITS1 region, at least one other coding gene sequence, preferably located in genome portion away from the ITS 1. The molecular analysis technology, with particular reference to the conserved genome regions that can be used as a reference for Bar-Coding, is particularly useful, but not alone, in order to classify fungus with very similar morphological characteristics.