## 2 Materials and Methods
### Research Methods  
_Phylological analysis._ Two grams of pollen samples were homogenized in 13 mL 70% ethanol, and after vortexing until homogenized they were centrifuged at 3500 rpm for 20 min. After centrifugation, the supernatant was discarded and 13 mL of 50% glycerine-water mixture (1:1) was added and centrifuged at 3500 rpm for 20 min. After centrifugation, the supernatant was poured, the tubes were inverted on blotter paper and the pellet was allowed to dry. The sediment of the tube was taken with the glycerine-gelatine matrix with the help of a dissecting needle and put on the slide, melted on the hot plate and covered with the coverslip. Three preparations were prepared for each sample [20]. Pollen was identified by optical microscopy, using previously published data [21, 22]. _Moisture content_. Moisture content analysis was determined by drying 5 g of a sample in an oven at 105 \({}^{\circ}\)C temperature until constant weight. _Isolation and morphological characterization of fungi._ The dilute plate method was used for isolation of fungi from bee pollen. Ten grams of each bee pollen sample were homogenized into 90 mL of physiological saline solution (NaCl, 8.5g L\({}^{-1}\)) and shaken on a horizontal shaker for ca. 15 min. at 400 rpm. For each sample, 1 mL aliquots of each of the four dilutions were inoculated (in triplicate) in the Potato Dextrose Agar (PDA) (Sigma-Aldrich) culture media. The plates were incubated at 26 \({}^{\circ}\)C for 5 to 7 days and then fungi were counted and recorded as colony-forming units (cfu) per gram of sample. Separate fungal colonies were transferred onto appropriate identification media. The Czapek Dox agar (Biolife, Italy) and Potato Dextrose Agar (PDA) (Sigma-Aldrich) medium were used to isolate and identify individual _Fusarium_ genera and species. The morphological characteristics of isolated colonies were identified based on macroscopic (colony appearance) and microscopic (spores forming) features in accordance with various descriptors [23; 24; 25; 26; 27]. _Mycotoxins analysis._ In this study, the following mycotoxins were investigated by enzyme-linked immunosorbent assay (ELISA): deoxynivalenol (DON), zearalenone (ZEN) and the T-2 toxin (T-2) [28]. The ELISA method is totally quantitative, simple, sensitive and fast; therefore, it can be used for the detection of mycotoxins in various foods [29]. The RIDASCREEN(r) ELISA kits produced by R-Biopharm AG (Darmstadt, Germany) approved by the AOAC Research Institute (Certificate No. 9001) were used for the analysis No R5906, No R5502. Mycotoxin analysis was performed following the manufacturer's instructions. For the preparation of the extracts, pollen samples were ground and then 5 g fraction were weighed. Three extracts were prepared: for the quantification of DON, extraction was carried out in 100 mL distilled water; in the others 100 mL of a methanol: water solution (70:30 \(v/v\)) was added for the quantification of ZEN and 50:50 \(v/v\) for T-2. Then, samples were stirred at 150 rpm at 25 \({}^{\circ}\)C in an orbital shaker ([PERSON](r), BS-1, Sartorius, Goettingen, Germany) for one hour and the samples were filtered using Whatman #1 filter. The filtrate was used directly for the quantification of T-2. For DON and ZEN, an additional dilution was required. Dilutions were 1:4 in distilled water for DON and 1:5 in 70% methanol for ZEN. The method's limits of detection (LoD) were the following: DON--18.5 ug kg\({}^{-1}\), ZEN--17.0 ug kg\({}^{-1}\) and T-2--5.0 ug kg\({}^{-1}\). Samples with results lower than the LoD values were considered not contaminated. The absorbance was determined using an ELISA reader (Stat Fax(r)303 Plus Microstrip Reader, Awareness Technologies, Westport, CT, USA) with a 450 nm absorbance filter. The optical densities (OD) of the samples were compared to the OD of standards through a linear regression, thus obtaining the concentration of mycotoxin present in each sample. Only calibration curves with R\({}^{2}\) greater than 0.99 were considered.