*2.6. qPCR Analysis*

A quantitative PCR (qPCR) assay was used to quantify the total *F. graminearum*, *F. verticillioides* and *F. culmorum* DNA content in the maize samples from 2017 and 2018. In 2016, no samples for qPCR were taken. These three species were selected based on the known fungal species composition in temperate climates and in Belgium in particular [21,35,73,74], and to cover most *Fusarium* producers of mycotoxins that were included in the LC-MS/MS analysis [15,35,77]. Each subsample (5 g) was crushed with liquid nitrogen using a pestle and mortar and approx. 150 mg (the exact amount was weighted) was transferred to a 1.5 ml Eppendorf tube for DNA extraction. DNA was extracted from harvested maize samples using a CTAB method modified for use with fungi [78]. The total amount of DNA was quantified with a Quantus fluorometer (Promega, Leiden, The Netherlands), and stored at –20 ◦C. Then qPCR analysis was performed. The qPCR mix consisted of 6.25 μL of GoTaq ®qPCR Master Mix (Promega, Leiden, The Netherlands), the corresponding primers (0.625 μL primer, 5 μM), 2 μL of DNA, 0.208 μL CXR reference dye (Promega, Leiden, The Netherlands), and watered to 12 μL. The used primers were FgramB379 forward (CCATTCCCTGGGCGCT), FgramB411 reverse (CCTATTGACAGGTGGTTAGTGACTGG), FculC561 forward (CACCGTCATTGGTATGTTGTCACT), FculC614 reverse (CGGGAGCGTCTGATAGTCG), Fver356 forward (CGTTTCTGCCCTCTCCCA), and Fver412 reverse (TGCTTGACACGTGACGATGA) [79]. The qPCR analysis was performed using a CFX96 system (Bio-Rad, Temse, Belgium), including the following thermal settings: 95 ◦C for 3 min; 40 cycles of 95 ◦C for 10 s, and 60 ◦C for 30 s, followed by dissociation curve analysis at 65 to 95 ◦C.
