Definizione di un protocollo per la caratterizzazione morfologica e molecolare del genere Daldinia Ces. & De Not. (Ascomycota) in Sicilia

[automatically translated] The possibility of clarifying the taxonomy of some complexes of species as in the case of daldinia Ces. De Not. (1) using both morphological and molecular investigations, allows an exact definition of taxa on the basis of their biological and ecological characteristics. In a previous study (2), carried out on the basis of teleomorfici and anamorphic characters, two new taxa have been described for Science from Sicily: daldinia martinii M. Stadler, Venturella & Wollweber and D. Raimundi M. Stadler, Venturella & Wollweber. In this contribution, the same entities and other samples from Sicily daldinia were analyzed with different techniques that have allowed both the SEM observation of morphological profiles of ascospores, confirming previous observations (2), both molecular analysis (3) which provides for the extraction of genomic DNA from ascospores; amplification by polymerase chain reaction of specific target of the fungal genome sequences; the sequencing of PCR products and alignment with the sequences deposited in GenBank, the verification of the construction of the phylogenetic relationships and relative dendrogram. For the extraction of genomic DNA from ascospores they were used different protocols, both laboratory and commercial, and to facilitate the lysis of ascospores of daldinia was carried out a pre-treatment with microwave (4), so as to break the resistant structure of the spore wall preserving, at the same time, the integrity of the nucleic acid. The genomic DNA molecules were used as template for subsequent PCR reactions in which they were used the primers ITS, whether universal or "fungal specific". The ITS amplification products were sequenced by using sequencing to MWG-Biotech Custom Services, Germany (http://www.mwg-biotech.com) and the sequences obtained were compared with those present in the database using the BLAST platform. The sequences of the ITS regions, after having been compared with those present in the database, were aligned with Clustal W program and then subjected to cluster analysis with Dnadist programs and Neighbor-joining Philip package. The results have allowed us to: i) identify the most suitable extraction protocol, then the methodology that, after a pre-treatment of spores with microwaves, It has allowed the extraction of genomic DNA in amounts sufficient for subsequent analysis; ii) delineate the most suitable PCR amplification profile, using different primers and by assaying (by resorting to-Mastercycler gradient, Eppendorf) a range of annealing temperatures, in order to define the most specific; iii) determine the degree of homology of the studied samples, and the phylogenetic relationships between them defining its dendrogram obtained from the analysis of the nucleotide sequences.