2.3.1. *F. verticillioides* Cultures

To determine in vitro fumonisin biosynthesis, cultures of *F. verticillioides* strains were obtained following the protocol indicated by Covarelli et al. [50] with slight modifications. In brief, 15 g of finely ground maize grains and 15 mL of deionized sterile water were added into 100 mL glass flasks (Duran, Mainz, Germany) to obtain the right moisture for allowing fungal development and then autoclaved three times at alternate days. Three flasks (replicates) for each *F. verticillioides* strain were then inoculated with a mycelium plug (0.6 cm diameter) taken from the growing edge of one-week-old pure fungal cultures of each strain developed on PDA at 22 ◦C in the dark. Three flasks (replicates) were used as controls by adding only a PDA plug. Flasks were incubated in the dark at 22 ◦C for 4 w, and developed cultures were then freeze-dried for 24 h using a lyophilizer instrument (Heto Powder Dry LL3000, Thermo Fisher Scientific, Waltham, MA, USA), ground with mortar and pestle and stored at −80 ◦C until further analysis.

### 2.3.2. Fumonisin Extraction and LC-MS/MS Analysis

Each fungal culture was extracted and analyzed in triplicate according to the validated and routine procedure also described by Covarelli et al. [50] with slight modifications. In brief, 1 g of ground sample was extracted with 5 mL of methanol/water (75:25, *v*/*v*) following 60 min shaking. The extract was filtered through filter paper. Prior to liquid chromatography, tandem Mass Spectrometry (LC-MS/MS) analysis, the extract was diluted by default 1:50 with a mixture of methanol/water (60:40), then filtered through 0.45 μm syringe filter. Twenty microliters were injected into the LC-MS/MS apparatus. If fumonisin levels were out of the calibration range, a further dilution (1:500 or 1:5000) was applied to the raw extract and then re-analyzed.

LC-MS/MS analyses were performed on a QTrap MS/MS system, from Applied Biosystems (Foster City, CA, USA), equipped with an Electrospray Ionization (ESI) interface and a 1100 series micro-Liquid Chromatography system comprising a binary pump and a micro-autosampler from Agilent Technologies (Waldbronn, Germany). The analytical column was a Gemini® C18 column (150 × 2 mm, 5 μm particles) (Phenomenex, Torrance, CA, USA), preceded by a Gemini® C18 guard column (4 × 2 mm, 5 μm particles). The column oven was set at 40 ◦C. The flow rate of the mobile phase was 200 μL/min, and the injection volume was 20 μL.

The column e ffluent was directly transferred into the ESI interface, without splitting. Eluent A was water and eluent B was methanol, both containing 0.5% acetic acid. A gradient elution was performed as follows. The percentage of eluent B was increased from 40% to 80% in 10 min, kept constant 3 min, then increased to 100% in 1 min, and kept constant for 4 min. The column was re-equilibrated with 40% eluent B for 7 min. The ESI interface was used in positive ion mode with the following settings: temperature 350 ◦C; curtain gas, nitrogen, 30 psi; nebulizer gas, air, 10 psi; heater gas, air, 30 psi; ion spray voltage +4500 V. The mass spectrometer operated in Multiple Reaction Monitoring (MRM) mode. Mycotoxin quantification was performed by external calibration in neat solvent. The identity of fumonisins was confirmed by comparison with the analytical standard considering chromatography retention time and MRM transitions (ion ratios) in agreemen<sup>t</sup> with the o fficial guidelines for mycotoxin identification by Mass Spectrometry [59]. Detection limits in maize fungal cultures were 0.002 μg/g for FB1 and 0.001 μg/g for FB2 and FB3.

Methanol (HPLC grade) and glacial acetic acid were purchased from Mallinckrodt Baker (Milan, Italy). Ultrapure water was produced by a Millipore Milli-Q system (Millipore, Bedford, MA, USA). Filter papers (Whatman no. 4) were obtained from Whatman International Ltd. (Maidstone, UK). HPLC syringe filters (regenerated cellulose, 0.45 μm) were from Alltech (Deerfield, IL, USA).

### *2.4. Genetic Structure of Di*ff*erent F. verticillioides Populations*

For genetic diversity assessment, all *F. verticillioides* strains were cultured on PDA for 7 d. Mycelia were harvested, homogenized in liquid nitrogen, and genomic DNA was extracted using the method already described by St ˛epie ´n et al. [60]. A pre-validated *FUM1*-specific marker that showed intraspecific polymorphism in *F. verticillioides* and *F. proliferatum* in previous studies [61,62] was used. Briefly, Fum1F1 (CACATCTGTGGGCGATCC)/Fum1R2 (ATATGGCCCCAGCTGCATA) primers were used for *FUM1* gene fragment PCR-based amplification and sequencing according to Wa´skiewicz et al. [61]. Additionally, *FUM6*-*FUM7* and *FUM7*-*FUM8* intergenic regions were amplified using the primers Fum6eF (AGATTTCCCAACAGTGGCAG)/Fum7bR (GTTTGCTTGGTGGAACTGGT) and Fum7eF (ATCCGGTTGAGTTGGACAAG)/Fum8eR (GGAACAGATGCCCATACCAT) according to St ˛epie ´n et al. [47].

The BigDye Terminator kit v. 3.1 (Life Technologies, Carlsbad, CA, USA) was used for fluorescent labeling according to the manufacturer's instructions. DNA fragments were purified using alkaline phosphatase and exonuclease I (Thermo Fisher Scientific)) and precipitated using ice-cold 96% ethanol (Sigma Aldrich, St. Louis, MO, USA). Sequence reading was performed using Applied Biosystems equipment. Sequence reads were analyzed using BioEdit software [63] and aligned using MEGA5 software package [64] using Maximum Parsimony heuristics with standard settings. Based on *FUM1* sequences, the most parsimonious tree was calculated (bootstrap test with 1000 replications).

Sequences were compared to the NCBI GenBank-deposited sequence (*FUM* cluster NCBI (AF155733)) and, in addition, a total of five *F. verticillioides FUM1* sequences (F.v.F1.8.I.I, F.v.10I3 (*Pisum sativum*, Wiatrowo, Poland); F.v.KF3477, F.v.F1M1.1 (*Z. mays*, Poland); F.v.KF3537 (*Ananas comosus*, Costa Rica)) were used as references. A total of four *Fusarium proliferatum FUM1* sequences (15 *F. proliferatum* (*Z. mays*, Iran); *F. proliferatum* Gar3.2, Gar1 and Gar3.0 (*Allium sativum*, Poznan, Poland)) were used as outgroup.
