Quantifying Aflatoxin Biosynthesis Genes

The conidia of A. flavus or A. parasiticus were harvested from 3-day-old PDA cultures and then spread on a cellophane film placed on PDA in a Petri dish (9 cm in diameter) with 200 μL conidial suspension (1 × 10⁷ conidia/mL). Another Petri dish was loaded with 10 g the 7-day-old AWG culture of strain 3-10 or 10 g fresh AWG (CK). Then, the two dishes were face-to-face sealed by parafilm to form a DDS. After co-culturing at 28°C for 72 h, the mycelia on the film were collected and immediately frozen in liquid nitrogen. Total RNA in the mycelial sample was extracted using E.Z.N.A^® Fungal RNA Kit (Omega Bio-tek, Inc., Norcross, GA, United States) according to the provided manual. Expression of eleven important genes (aflR, AccC, aflCa, aflA, aflS, aflO, aflD, aflF, aflP, aflQ, and aflX) in the aflatoxins biosynthesis pathway in A. flavus and A. parasiticus were determined by quantitative real-time PCR (qRT-PCR) using the method described by Gong et al. (2019) (link). The primers used for the qRT-PCR are listed in Supplementary Table S3.