*2.1. Fungal Strains*

A total of 90 *F. verticillioides* strains (Table 1) isolated from single maize kernels harvested from different fields in five Mediterranean countries (22 from Italy, 9 from Spain, 16 from Tunisia, 28 from Egypt and 15 from Iran) were used in this study (Figure 1). Isolation operations were carried out in the country of origin where all strains were properly stored in fungal collections. The investigated strains had not been extensively subcultured, thus avoiding possible alterations in fumonisin production. Some of the Italian strains used in this work had been already investigated in a previous study [50] and were included to further characterize them in a wider geographical context (Figure 1).

**Strain ID Origin Fumonisin Production (**μ**g**/**g) \* Fumonisin B1 Fumonisin B2 Fumonisin B3 Total Fumonisins \*\*,§** PG 21C Italy nd † - nd - nd - nd - - PG 39B Italy nd - nd - nd - nd - - ITEM 9313 Italy 0.03 (±0.01) nd - nd - 0.03 (±0.01) a ITEM 9319 Italy 0.16 (±0.08) 0.03 (±0.01) 0.05 (±0.02) 0.24 (±0.11) ab PG 60A1 Italy 0.20 (±0.02) 0.04 (±0.01) 0.05 (±0.01) 0.29 (±0.02) b ITEM 9330 Italy 0.30 (±0.08) 0.05 (±0.01) 0.06 (±0.01) 0.41 (±0.09) ab ITEM 9320 Italy 0.63 (±0.60) 0.10 (±0.10) 0.08 (±0.07) 0.81 (±0.77) ab ITEM 9300 Italy 0.65 (±0.37) 0.11 (±0.06) 0.11 (±0.05) 0.87 (±0.48) ab PG 28A Italy 1.01 (±0.40) 0.22 (±0.10) 0.25 (±0.08) 1.49 (±0.58) ab ITEM 9318 Italy 1.03 (±0.68) 0.22 (±0.15) 0.35 (±0.24) 1.59 (±1.07) ab PG 22A Italy 1.67 (±1.52) 0.24 (±0.23) 0.25 (±0.22) 2.16 (±1.97) ab PG 20A Italy 2.81 (±1.50) 0.66 (±0.35) 0.40 (±0.16) 3.87 (±2) abc ITEM 9310 Italy 6.56 (±3.09) 2.46 (±1.19) 0.68 (±0.29) 9.69 (±4.56) abcd PG 5A Italy 6.99 (±0.89) 2.35 (±0.37) 0.85 (±0.06) 10.19 (±1.27) cd ITEM 9309 Italy 7.70 (±3.45) 2.23 (±1) 0.80 (±0.30) 10.74 (±4.74) abcd PG 76A1 Italy 8.78 (±4.50) 2.32 (±1.29) 1.24 (±0.60) 12.34 (±6.39) abcde PG 30B Italy 10.36 (±1.25) 2.95 (±0.45) 1.26 (±0.26) 14.57 (±1.92) d ITEM 9329 Italy 10.71 (±2.32) 3.04 (±0.71) 0.84 (±0.16) 14.59 (±3.16) cd PG 35A Italy 13.30 (±6.96) 4.39 (±2.26) 1.78 (±0.80) 19.47 (±10) abcde PG 58A1 Italy 19.39 (±5.28) 7.51 (±1.73) 2.16 (±0.15) 29.07 (±7.05) abcde ITEM 10027 Italy 23.64 (±1.57) 7.22 (±0.44) 2.49 (±0.05) 33.35 (±1.99) e PG 36B Italy 23.87 (±0.44) 5.63 (±1.56) 4.23 (±0.19) 33.73 (±1.49) e 03-2/A Spain 0.24 (±0.17) nd - nd - 0.24 (±0.17) FVMM 3-2 Spain 0.78 (±0.29) 0.03 (±0.03) 0.01 (±0.01) 0.82 (±0.33) a C1-2 SEV Spain 2.24 (±1.19) 0.53 (±0.42) 0.01 - 2.77 (±1.61) ab FVMM 2-1 Spain 2.60 (±1.60) 0.55 (±0.46) 0.24 (±0.13) 3.38 (±2.17) ab FVMM AD 2-4 Spain 6.38 (±3.28) 1.61 (±0.91) 0.20 (±0.05) 8.19 (±4.19) ab 03-5/B SEV.1 Spain 6.63 (±1.08) 1.31 (±0.31) 0.31 (±0.05) 8.24 (±1.43) b 03-5/B SEV Spain 7.70 (±3.57) 1.81 (±0.92) 1.06 (±0.66) 10.57 (±5.01) ab FVMM 1-1 Spain 15.63 (±4.19) 4.68 (±1.25) 1.77 (±0.33) 22.08 (±5.74) ab 0-C-1-3 2/2 Spain 56.12 (±5.31) 10.67 (±1.35) 3.04 (±0.21) 69.84 (±6.57) c M16 Tunisia nd - nd - nd - nd - - M11 Tunisia 0.29 (±0.07) 0.04 (±0.02) nd - 0.33 (±0.09) a M19 Tunisia 0.30 (±0.07) 0.03 (±0.02) 0.11 (±0.03) 0.45 (±0.11) a M12 Tunisia 0.56 (±0.23) 0.12 (±0.05) 0.06 (±0.02) 0.74 (±0.30) ab M15 Tunisia 0.47 (±0.17) 0.06 (±0.02) 0.27 (±0.08) 0.80 (±0.28) ab M20 Tunisia 0.92 (±0.13) nd - 0.01 - 0.93 (±0.13) b M17 Tunisia 0.91 (±0.21) 0.12 (±0.03) 0.55 (±0.12) 1.58 (±0.36) ab M5 Tunisia 2.55 (±1.43) 0.27 (±0.26) nd - 2.83 (±1.69) ab M2 Tunisia 3.21 (±1.32) 0.61 (±0.31) 0.01 - 3.82 (±1.63) ab M8 Tunisia 3.53 (±1.80) 1.01 (±0.58) 0.01 - 4.55 (±2.39) abc M7 Tunisia 3.80 (±3.05) 0.77 (±0.75) 0.40 (±0.32) 4.97 (±4.11) abc M22 Tunisia 6.85 (±3.59) 1.15 (±0.40) 2.07 (±0.47) 10.07 (±4.45) abc M21 Tunisia 7.10 (±4.93) 1.47 (±1.24) 1.72 (±1.09) 10.29 (±7.24) abc M1 Tunisia 8.82 (±1.28) 2.16 (±0.35) 0.68 (±0.23) 11.66 (±1.81) c M14 Tunisia 10.50 (±0.10) 1.72 (±0.12) 1.07 (±0.10) 13.28 (±0.18) c M10Tunisia11.07(±1.71)2.48(±0.55)0.04(±0.03)13.59(±2.23)c

**Table 1.** Strain ID, country of origin and fumonisin B1, fumonisin B2 and fumonisin B3 production (μg/g)with standard errors (±SE) by *Fusarium verticillioides* strains isolated from maize kernels harvested in five Mediterranean countries and analyzed in this study.


**Table 1.** *Cont.*

\* values represent the average (±SE) of three biological replicates. \*\* sum of fumonisin B1, fumonisin B2 and fumonisin B3. † nd: not detected (<0.002 μg/g for fumonisin B1 and <0.001 μg/g for fumonisin B2 and fumonisin B3). § within the same country of origin, means followed by different letters are significantly different (*p* < 0.05).

**Figure 1.** Countries of origin (red dots) of the *Fusarium verticillioides* strains used in this study. Map downloaded from www.google.com/maps and modified by the authors.

### *2.2. Confirmation of F. verticillioides Identity by PCR Assays*

To preliminarily confirm the identity of the 90 *F. verticillioides* strains used in this study, species-specific PCR assays were conducted. All strains were grown on Potato Dextrose Agar (PDA (Biolife Italiana, Milan, Italy)) at 22 ◦C for 14 d in the dark. DNA was extracted as described by Beccari et al. [56,57]. PCR assays were carried out with the specific primers VERT1 (GTCAGAATCCATGCCAGAACG) and VERT2 (CACCCGCAGCAATCCATCAG) [58]. A single PCR protocol was optimized using a total reaction of 20 μL. Each reaction contained 9.2 μL of sterile water for molecular biology (5prime, Hilden, Germany), 1.5 μL of cresol red (Sigma-Aldrich, Saint Louis, MO, USA), 2 μL of 10X PCR buffer (Microtech, Pozzuoli, Naples, Italy), 1.2 μL of magnesium chloride (Microtech), 2 μL of 10 mM DNTP mix (Microtech), 1 μL of 10 μM forward and reverse primers, 0.1 μL of 5 U/μL Taq polymerase (Microtech) and 2 μL of template DNA. The PCR cycle consisted of an initial denaturation step at 94 ◦C for 2 min, followed by 30 cycles of denaturation (94 ◦C for 35 s), annealing (60 ◦C for 30 s), extension (72 ◦C for 2 min) and a final extension at 72 ◦C for 5 min. PCR assays contained a positive control (template DNA of *F. verticillioides*) and a negative control with no DNA added. The amplification was performed in a T-100 thermal cycler (Bio Rad, Hercules, CA, USA). All PCR fragments were separated by electrophoresis by applying a tension of 110 V for about 1 h. Electrophoretic runs were visualized using an UV Image analyzer (Euroclone, Pero, Milan, Italy).

### *2.3. Determiantion of Fumonisin Biosynthesis by F. verticillioides In Vitro*
