*2.3. DNA Extraction and PCR Conditions*

Isolate A1021B was grown in PDB on a rotary shaker at 120 rpm for 96 h at 25 ◦C. Fresh mycelium was collected after vacuum filtration through No. 4 Whatman filter paper (Whatman Biosystems Ltd., Maidstone, UK), then frozen in liquid nitrogen, ground to a fine powder and stored at −80 ◦C until further processing. Total genomic DNA was extracted from 10 mg of ground mycelium by using the NucleoSpin® Soil kit (Macherey-Nagel, Düren, Germany) according to the manufacturer's protocol. Sequences of the housekeeping genes calmodulin (*CAL1*), translation elongation factor (*TEF1*), β-tubulin (*TUB2*) and internal transcribed spacer 1–4 (ITS) were amplified using the following PCR program: denaturation at 96 ◦C for 2 min; 35 cycles of denaturation at 94 ◦C for 30 s, annealing at 55 ◦C for 30 s; extension at 68 ◦C for 75 s; and final extension at 68 ◦C for 10 min. Before sequencing, PCR products were purified using PureLink PCR purification kit (Invitrogen, Paisley, UK) following the manufacturer's instructions. Furthermore, the presence of amplicons related to the trichothecene biosynthetic genes *TRI1*, *TRI4*, *TRI5*, *TRI8*, *TRI11* was investigated. All primers used in this work are reported in Table 1.


**Table 1.** Primers used in this study for DNA sequence amplifications.

## *2.4. Species Identification and Phylogenetic Analysis*

Phylogenetic relationships of strain A1021B were investigated on account of *CAL1*, *TEF1* and *TUB2* sequences as reported in [15]. DNA sequences were blasted against the NCBI GenBank database using default parameters and then aligned with isolates belonging to the FIESC [15,41] and *Fusarium babinda* by the Clustal W algorithm [42] with MEGA7 software [43]. Phylogenetic trees were inferred using the maximum likelihood method based on Tamura-Nei model applied to the whole set of manually edited aligned sequences. The confidence of the branching was estimated by bootstrap (BP) analysis (1000 BP). A strain of *Fusarium concolor* was used as outgroup for rooting the phylogenetic tree. DNA sequences of the three loci were submitted to GenBank, with the following accession numbers: MK968883 (ITS), MK984207 (*CAL1*), MK984206 (*TEF1*), and MK984208 (*TUB2*).
