**3. Results**

### *3.1. Identity Confirmation of F. verticillioides*

DNA extracted from the 90 *F. verticillioides* strains was subject to PCR assays using the species-specific primer pair VERT1/VERT2. As expected, a single fragment of 800 bp amplified in all the samples, thus confirming their identity as *F. verticillioides*.

### *3.2. Fumonisin Biosynthesis by F. verticillioides In Vitro*

Data on the in vitro biosynthesis of FB1, FB2 and FB3 with the calculation of total fumonisins (sum of FB1, FB2 and FB3) by the 90 *F. verticillioides* strains are summarized in Table 1.

In general, this analysis revealed that 80% (*n* = 71) of the *F. verticillioides* strains investigated in this study were able to produce fumonisins at variable levels, while the remaining 20% (*n* = 19) showed undetectable levels (not detected; nd) of fumonisins and were considered, in this experimental condition, as non-producing strains.

Total fumonisins biosynthesized by all positive strains (*n* = 71) varied from 0.03 to 69.84 μg/g (average 7.88 μg/g), with FB1 being the most abundant analogue followed by FB2 and FB3. All positive strains (100%, *n* = 71) produced FB1 in levels ranging from 0.03–56.12 μg/g (average 5.9 μg/g), while 64 of 71 strains (90%) produced FB2 in levels ranging from 0.03–10.67 μg/g (average 1.6 μg/g). Finally, 59 of 71 strains (83%) biosynthesized FB3 in a range from 0.01–4.23 μg/g (average 0.7 μg/g). The average ratios of FB1:total fumonisins, FB2:total fumonisins and FB3:total fumonisins were 0.77, 0.13 and 0.05, respectively. The three fumonisin analogues analyzed in this study (FB1, FB2 and FB3) were simultaneously produced by 81% of positive strains (*n* = 58), while two analogues, FB1 and FB2 as well as FB1 and FB3, were simultaneously biosynthesized by 7% (*n* = 5) and 1% (*n* = 1) of positive strains, respectively. Finally, 7 out of 71 strains (10%) producerd only FB1. No strains biosynthesized FB2 or FB3 only.

In most cases, considering all producing strains (*n* = 71), di fferences in fumonisin production were detected among the strains isolated in the same country.

In detail, 20 out of 22 strains (91%; Figure 2) isolated from maize grains in Italy and analyzed in this study showed the ability to biosynthesize fumonisins in variable levels (Table 1). Total fumonisins biosynthesized by the Italian positive strains (*n* = 20) varied from 0.03 to 33.73 μg/g (average 9.98 μg/g). All fumonisin-producing Italian strains (100%, *n* = 20) biosynthesized FB1 in levels ranging from 0.03–23.87 μg/g (average 5.7 μg/g), while 19 out of 20 strains (95%) produced FB2 and FB3 in levels ranging from 0.03–5.63 μg/g (average 2.20 μg/g) and 0.05–4.23 μg/g (average 0.94 μg/g), respectively. The average ratios of FB1:total fumonisins, FB2:total fumonisins and FB3:total fumonisins were 0.71, 0.18 and 0.10, respectively. The three fumonisin analogues (FB1, FB2 and FB3) were simultaneously produced by 95% of positive Italian strains (*n* = 20), while 1 out of 20 strains (5%) produced only FB1. Strains ITEM 10027 and PG 36B showed a significantly higher biosynthesis of total fumonisins with respect to the other Italian strains (*p* < 0.02), with the exception of strains PG 58A1, PG 35A and PG 76A1 (*p* > 0.07).

Considering the Spanish strains analyzed in this study, all of them (100%, *n* = 9; Figure 2) were able to in vitro biosynthesize di fferent levels of fumonisins. Total fumonisins produced by these strains ranged from 0.24 to 69.84 μg/g (average 14.01 μg/g) with FB1 being the most abundant (range 0.24–56.12 μg/g; average 10.9 μg/g), followed by FB2 (range 0.03–10.67 μg/g; average 2.4 μg/g) and FB3 (range 0.01–3.04 μg/g; average 0.7 μg/g). The average ratios of FB1:total fumonisins, FB2:total fumonisins and FB3:total fumonisins were 0.81, 0.15 and 0.04, respectively. Eight out of 9 strains (89%) simultaneously biosynthesized all three fumonisin analogues, while in 1 out of 9 strains (11%) only FB1 was detected. Strain 0-C-1–3 2/2 showed a significantly higher (*p* < 0.008) production of total fumonisins with respect to the other Spanish strains analyzed in this study.

**Figure 2.** *Fusarium verticillioides* strains (%) isolated from maize kernels harvested in five Mediterranean countries that showed in vitro production of detectable (fumonisin producers) and non-detectable levels (fumonisin non-producers) of total fumonisins. Italy, *n* = 22; Spain, *n* = 9; Tunisia, *n* = 16; Egypt, *n* = 28; Iran, *n* = 15.

Focusing on the Tunisian strains analyzed in this study, 15 out of 16 strains (94%; Figure 2) produced detectable amounts of fumonisins *in vitro*. Total fumonisin levels ranged from 0.33 to 13.59 μg/g, with an average production equal to 5.36 μg/g. Twelve out of 15 strains (80%) biosynthesized all the analogues, while 2 out of 15 strains (13%) produced FB1 and FB2, and the remaining strain (7%; *n* = 1) produced FB1 and FB3. The gradient of production did not differ from that detected for the other strains: FB1 (average 4.01 μg/g) > FB2 (average 0.86 μg/g) > FB3 (average 0.54 μg/g). The average ratios of FB1:total fumonisins, FB2:total fumonisins and FB3:total fumonisins were 0.76, 0.13 and 0.11, respectively. Strains M10, M14 and M1 showed significantly higher total fumonisin biosynthesis with respect to the other Tunisian strains (*p* < 0.02), with the exception of strains M21, M22, M7 and M8 (*p* > 0.05).

The *F. verticillioides* population isolated from maize kernels in Egypt and analyzed in this study showed a low percentage of fumonisin-producing strains (46%, *n* = 13; Figure 2) with an average total fumonisin production of 3.98 μg/g (range 0.22–11.23 μg/g). All producing strains biosynthesized FB1 (range 0.22–7.52 μg/g; average 2.95 μg/g), while 12 out of 13 strains (92%; average 0.77 μg/g) and 10 out of 13 strains (77%; average 0.40 μg/g) showed the ability to biosynthesize FB2 and FB3, respectively. In other words, 77% of producing strains were able to simultaneously produce all three fumonisin analogues, while 15% (*n* = 2) and 8% (*n* = 1) of the Egyptian strains showed the ability to biosynthesize FB1 and FB2 or FB1 alone, respectively. The average ratios of FB1: total fumonisins, FB2:total fumonisins and FB3:total fumonisins were 0.76, 0.17 and 0.09, respectively. The Egyptian strain F3 showed a significantly higher (*p* < 0.01) production of total fumonisins than F39, F29, F8, F4, F28, F9 and F32 strains.

In the *F. verticillioides* population isolated from maize kernels in Iran and anlyized in this study, a total of 14 fumonisin-producing strains were recovered (93%; Figure 2). Total fumonisins biosynthesized by all positive strains (*n* = 14) varied from 0.03 to 39.79 μg/g (average 7.28 μg/g). All producing Iranian strains (100%, *n* = 14) biosynthesized FB1 in levels ranging from 0.03–30.81 μg/g (average 5.57 μg/g), while 11 out of 14 strains (71%) produced FB2 in levels ranging from 0.1–7.23 μg/g (average 0.70 μg/g), and 10 out of 14 strains (64%) biosynthesized FB3 in levels ranging from 0.09–1.75 μg/g (average 0.70 μg/g), respectively. The average ratios of FB1:total fumonisins, FB2:total fumonisins and FB3:total fumonisins were 0.83, 0.14 and 0.07, respectively. The three fumonisin analogues (FB1, FB2 and FB3) were simultaneously produced by 64% of positive Iranian strains (*n* = 9), while 4 out of 14 strains (29%) produced only FB1, and 1 out of 14 strains (7%) biosynthesized FB1 and FB2. The Iranian strain 89 showed a significantly higher total fumonisin biosynthesis than the other strains from the same country (*p* < 0.01), with the exception of strains 5 and 7 (*p* > 0.05).

Taking into account all fumonisin-producing strains of each country analyzed in this study, differences in total fumonisin biosynthesis among countries were also detected (Figure 3). In particular, the Spanish strains used in this study showed a significantly higher total fumonisin production (average 14.01 μg/g) than the Egyptian ones (average 3.98 μg/g) (*p* = 0.02). Also, the total fumonisin productions detected for the Italian (average 9.98 μg/g), Tunisian (average 5.36 μg/g) and Iranian (average 6.79 μg/g) strains were higher than the Egyptian ones and lower than the Spanish ones, even if no significant differences were recorded (*p* > 0.46 and *p* > 0.47, respectively) (Figure 3).

**Figure 3.** Average of total fumonisins (μg/g) biosynthesized by *Fusarium verticillioides* fumonisin-producing strains isolated from maize kernels harvested in each of the five countries analyzed in this study. Means with different letters are significantly different (*p* < 0.05).

### *3.3. Genetic Structure and Variability of F. verticillioides Populations*

We sequenced a portion of a divergent *FUM1* gene to evaluate the diversity among the five populations of *F. verticillioides* originating from various countries. All strains amplified DNA fragments of about 1100 bp in length. Additionally, the *FUM6*-*FUM7* (ca. 550 bp) and *FUM7*-*FUM8* (ca. 500 bp) intergenic regions were sequenced using the primers described previously [47].

The sequences were aligned, the ends trimmed manually using MEGA 5 software, and dendrograms of similarities were calculated. Interestingly, the intergenic regions did not show polymorphisms, which was rather unexpected, since these regions normally accumulated more point mutations than the coding regions. However, this means that the *F. verticillioides* strains characterized in this study, even if originating from different countries, were basically uniform (results not shown).

Therefore, only slightly more polymorphic *FUM1* sequences were analyzed and shown (Figure 4). Apparently, neither geographical origin nor fumonisin production ability were correlated to the genetic diversity of the strain set, as almost all of them grouped together. Only four strains from Egypt (F10, F12, F13 and F36) were distinguished from the remaining strains at a bootstrap value of 60, including our five reference sequences [61] and NCBI GenBank-deposited *FUM* cluster sequences (AF155773) reported by Proctor et al. [45].

**Figure 4.** A most parsimonious tree calculated based on the partial *FUM1* sequences of 90 *Fusarium verticillioides* strains isolated from *Zea mays* of different origins using the maximum parsimony setting, bootstrap set to 50%, and 1000 replications were done. Five reference strains isolated from *Pisum sativum* (F.v. F1.8.I.I; F.v. 10 I 3), *Z. mays* (F.v. KF3477; F.v. F1M1.1) and *Ananas comosus* (F.v. KF3537) were added to the analysis, as well as the NCBI GenBank-deposited *FUM* cluster sequences (AF155773). Four *Fusarium proliferatum* sequences were also included as outgroup (15; Gar3.2; Gar1; Gar3.0).
