*3.1. Metabolome Analysis*

The investigation on SM production in liquid (CDB, PDB) or solid (MK) media, the latter representing a commonly used substrate to evaluate mycotoxin production in *Fusarium* spp. [28], revealed that up to 206 compounds are synthesized by strain A1021B in axenic cultures. The PCA score plot demonstrated a differential and significant effect of the medium composition on SM production (Figure 1A). Moreover, the assortment of SMs produced in CDB or PDB was less affected by light exposure than by the culturing time (1 vs. 2 weeks). On the other hand, on MK the SM profile was particularly influenced by the former factor, i.e., multiple compounds after one week of growth in darkness were produced. To obtain a simplified representation of the different assortments, a heatmap clustering compounds was generated (Figure 1B) by selecting 29 entities which made it possible to discriminate among the different treatments, selected on PCA. Among them, FA (179.0974 Da) and its derivatives fusarinol (165.1181 Da) and 9,10-dehydro-FA (177.0799 Da) were detected. Other putatively identified SMs were bikaverin (382.1126 Da), a tetracyclic benzoxanthone whose genetic base is reported to be clustered to FA [44,45], and culmorin (238.1446 Da), a sesquiterpenoid which is often associated with trichothecene production [46,47].

**Figure 1.** (**A**) Principal component analysis (PCA) score plot of secondary metabolites (SMs) produced by A1021B under different growth conditions. (**B**) Heat map illustrating the abundance of the main SMs in A1021B cultures, visualized through the color scale reported on the right. Each row represents differentially abundant products ordered by their mass (Da), while columns correspond to the different culturing conditions. MK = maize kernels; CDB = Czapek-Dox broth; PDB = potato dextrose broth; d = darkness; l = light; 1 w = 1 week; 2 w = 2 weeks.

Among the identified compounds, the LC-MS Q-TOF analysis revealed that fusarinol was predominantly produced in CDB, in dark as well as in light conditions, while 9,10-dehydro-FA accumulated in PDB and MK. However, both compounds were found only after two weeks of growth. The production of FA was mostly observed in CDB maintained in darkness, or in light exposure after two weeks only. A similar biosynthetic course is displayed by culmorin in PDB, while the production of bikaverin was mainly detected in CDB cultures regardless to the presence/absence of light. Furthermore, among the unidentified molecules, compound A and compound B (Figure 1B) were particularly affected by medium composition. In fact, compound A was detected exclusively in MK while compound B was produced only in PDB, and their production was not related to specific growth condition (1–2 w; light/darkness).

Our analysis did not show the production by A1021B of 8-O-methylbostrycoidin, a polyketide pigment which has been reported in association with FA [48]. Furthermore, no trichothecenes were detected in any cultivation condition.

Venn diagrams showed that A1021B was able to synthesize specific compounds in the different media, and that only a small part of them was in common among the three conditions (Figure 2). In darkness, the growth on MK enhanced the production of specific SMs at both time points considered (81 and 75 compounds, respectively, after 1 or 2 weeks of growth), while CDB was the least inductive medium (6 and 11 compounds, respectively). Moreover, very few compounds (3 and 4, respectively, after 1 or 2 weeks of growth) accumulated constitutively in darkness regardless to the medium. A similar distribution was observed when A1021B was cultivated under light exposure. In fact, MK represented the most inductive substrate, while in CDB few compounds accumulated. No specific SMs were detected in CDB at the first time point. Overall, SM production was higher in darkness (Figure 2), and MK was more effective in enhancing the production of certain compounds.

**Figure 2.** Venn diagrams showing the number of unique and overlapping products in A1021B cultures under the different growth conditions. (**A**) Secondary metabolites (SMs) produced in darkness (d) after one (1 w) or two weeks (2 w). (**B**) SMs produced under light exposure (l) after one (1 w) or two weeks (2 w). MK = maize kernels; CDB = Czapek-Dox broth; PDB = potato dextrose broth.
