**4. Discussion**

This study was aimed at investigating the di fferent ability of selected *F. verticillioides* strains isolated from maize kernels harvested in five Mediterranean countries to in vitro biosynthesize fumonisins as well as at characterizing their genetic structure to assess possible variabilities among them. So far, various studies have been conducted to analyze the ability of di fferent *F. verticillioides* strains from diverse geographic areas to biosynthesize fumonisins. In several investigations, a large percentage of strains able to produce detectable amounts of these mycotoxins were usually found. However, the presence of strains that were not able to biosynthesize measurable levels of fumonisins was also reported. In this research, the majority of the strains isolated from maize grains in Italy, Spain, Tunisia and Iran, analyzed in this study, produced detectable levels of fumonisins (91%, 100%, 94% and 94% respectively; Figure 2), while the remaining part showed a lack of ability to produce measurable amounts of these mycotoxins. Similar percentages of fumonisin-producing strains (> 80%) were also detected in other *F. verticillioides* populations isolated from maize in Croatia [68], Spain [15,69], Italy [50], Iran [22], Egypt [17], Brazil [41,44,49], Korea [70], USA [71], Argentina [55,72] and from durum wheat in Argentina [2].

Conversely, in this study, only 46% of the analyzed Egyptian strains showed the ability to biosynthesize detectable amounts of fumonisins (Figure 2). Similarly to other studies, low incidences of producing strains were also recorded in other *F. verticillioides* populations such as those isolated from maize in Croatia (55%) [73], Taiwan (66%) [74] and Spain (36%) [14].

In general, the producing strains analyzed in this study biosynthesized fumonisin analogues following the "typical" gradient: FB1 > FB2 > FB3. A predominance of FB1 compared to the other analyzed fumonisin analogues was recovered also in other *F. verticillioides* populations such as those isolated from maize in Spain [15,75], Italy [76], Iran [22], Brazil [44,49], Argentina [55,72], Egypt [17], South Korea and South Africa [39]. In this study, no *F. verticillioides* strains producing more FB2 or FB3 than FB1 were recorded. Conversely, these types of strains were observed in *F. verticillioides* populations isolated from durum wheat in Argentina [2] and from maize and sorghum cultivated in the United States [77].

As known, fumonisin production within the *F. verticillioides* species could quantitatively vary due to the di fferent biosynthetic ability of the di fferent strains [24,40]. Also in this study, variability of fumonisin production among strains isolated in the same country was found, highlighting that mycotoxigenic diversity occurred within the five investigated *F. verticillioides* populations. Variability among *F. verticillioides* strains isolated from maize in the same country was commonly detected in many surveys in other parts of the world [2,8,15,17,22,44,49,55,73–75].

Variability in fumonisin production was also recorded among *F. verticillioides* strains isolated from di fferent countries [30,39,71]. Also in this study, di fferences in fumonisin production among strains of di fferent geographic origin were detected. In particular, the Spanish and Egyptian strains analyzed in this study showed a high level of mycotoxigenic variability, being the populations with the highest and the lowest fumonisin productions, respectively.

Interestingly, these two populations were also those with the highest and lowest percentages of fumonisin-producing (Spain) and non-producing (Egypt) strains. Conversely, the other three investigated populations of *F. verticillioides* (isolated from Italy, Tunisia and Iran) considered in this study did not show a significant variability of fumonisin production. In agreemen<sup>t</sup> with the results of Vogelgsang et al. [78], it is important to consider that in vitro results cannot be fully extrapolated to in vivo conditions because there are several factors influencing *Fusarium* infections and secondary metabolite production in the field. However, in vitro results could provide important information, which may be useful to understand intra-population variability within a single country as well as inter-population variability among di fferent countries.

In this study, the mycotoxigenic characterization of *F. verticillioides* strains from di fferent geographic origins was coupled to the study of the genetic structure of these populations. The genetic diversity of *F. verticillioides* has been studied using multiple techniques, including AFLP and RAPD methods [50,53,79]. Recently, however, direct sequencing of specific genomic regions has become more popular because of its high discrimination power and accuracy. The *FUM1* gene has already beeeen proven to be useful to assess species diversity inside the FFSC, serving as a source of phylogenetic and chemotypic markers [47], showing often higher levels of polymorphisms than constitutively expressed genes [e.g., *beta tubulin* (*tub2*) or *translation elongation factor 1*α (*tef-1*α)].

Our previous studies suggested there might be high levels of intraspecific genetic uniformity inside *F. verticillioides* populations, particularly when compared to the high diversity of the closely related species *F. proliferatum* [61,62,80,81]. The use of the *FUM1* gene sequence analysis allowed for discrimination of subpopulations likely related to the host species of origin. We assumed that a similar rule would be valid for *F. verticillioides*; therefore, we added some pea- and pineapple-derived strains to the analysis (Figure 4). It was also possible that geographical di fferences between populations would become visible.

However, in the present study we could not confirm this hypothesis. In fact, this was in accordance to previous findings, which did not reveal significant di fferences between *F. verticillioides* strains from di fferent hosts [61]. This was also confirmed by the sequence analysis of the intergenic regions between *FUM6* and *FUM7* as well as *FUM7* and *FUM8* genes (results not shown), which were previously used for polymorphism screening [47]. The most likely explanation for this situation may be the endophytic type of growth observed for this pathogen in maize, which combined with the extensive seed material transfer between countries and continents made the population uniform across the world. Another possibility is that *FUM* cluster integrity and structure undergoes much more strict selection pressure in *F. verticillioides* than in *F. proliferatum*. This may implicate that fumonisin production by *F. verticillioides* is more essential to complete its life cycle than it is for *F. proliferatum*. This issue was already reported by Glenn et al. [82] but never confirmed for *F. proliferatum*.

The only outlier obtained in this study was a group of four strains (F10, F12, F13 and F36) isolated from Egypt (Figure 4), which was distinct from the remaining strains. Only one of these strains (F13) produced fumonisins in detectable amounts (Table 1). They should be further studied to explain their genetic diversity.
