22 μm filter, dried under nitrogen gas, and re-dissolved in 200 μL chloroform before being analyzed by TLC as described previously [18]. The AFB1 content was measured by HPLC (Agilent 1200, Waldbronn, Germany) using a reverse phase C18 column (150 mm in length and 4.6 mm in internal diameter, 5 μm particle size, Agilent), eluted initially with 25% methanol/20% acetonitrile water solution for 3 min, and then with 38% methanol for 2.9 min, detected by a DAD analyzer at 360 nm. Quantifications were performed by measuring peak areas and

comparing with an AFB1 standard calibration curve. Spore counting Three mL of sterile water with 0.05% Tween-20 was added to the surface of PDA plates on which A. flavus were grown for 3 d. Spores were scraped with a cell scraper before being counted with a haemacytometer. qRT-PCR Mycelia grown in GMS media with or without 40 mg/mL Selleckchem VX-689 D-glucal find more for 3 d were collected and ground in liquid nitrogen, and total RNA was extracted using a Trizol solution (Invitrogen, CA, USA). PolyA mRNA was purified from mycelia with the PolyAT Rack mRNA isolation system (Promega, Madison, WI). Template cDNA was synthesized by reverse transcription with ReverTra Ace-α-®

(Toyobo, Japan) at 42°C AZD1390 clinical trial for 1 h, followed by incubation at 85°C for 15 min to terminate the reaction. qRT-PCR was performed using SYBR Green I (Takara, Japan) and a Rotor-Gene 3000 (Corbett, Australia) with primers described in Additional file 2: Table S1. PCR programs used are 94°C for 30 sec, 40 cycles at 94°C for 30 sec, followed by annealing (55°C for aflO, aflR, aflS, aflD and β-tubulin; 62.5°C for aflU and nadA; 58°C for kojA, Protein kinase N1 kojR and kojT; 61°C for hxtA, glcA and sugR; 60°C for aflC, aflM and aflP) for 30 sec, and 72°C for 30 sec.

The relative expression levels were quantified by comparing the expression level of β-tubulin. Kojic acid and glucose measurements A. flavus A3.2890 was cultured in a GMS liquid medium plus 40 mg/mL D-glucal for 5 d. Media samples were harvested by centrifugation at 12,000 rpm for 10 min before kojic acid was quantified according to Bentley [19]. Glucose contents in media were measured by using a glucose determination kit (Applygen, Beijing). The absorbance was measured at 550 nm using a multimode plate reader (Tecan Infinite M200 PRO, Switzerland), and calculated against a glucose standard curve. Metabolomics analyses Metabolites in mycelia of A. flavus A3.2890 cultured in a GMS liquid medium with or without 40 mg/mL D-glucal for 5 d were purified, silyl-derivatized and analyzed with GC-TOF MS as described previously [18], with minor modifications. The column temperature was held at 100°C for 3 min, and raised to 150°C at a rate of 10°C/min, then to 250°C at 5°C/min, finally to 300°C at 10°C/min, and held for 15 min at 300°C. PLS analysis was performed using SIMCA-P V12.0 (Umetrics, Sweden). NOR analyses A. flavus Papa 827 was cultured for 4 d on PDA media containing 0, 5, 10, 20, or 40 mg/mL D-glucal.