Czapek dox broth

Two major fungal pathogens causing important crop losses worldwide were tested: F. oxysporum f.sp. radicis-lycopersici as soil pathogen and the necrotrophic shoot pathogen B. cinerea strain B05.10.
Fusarium oxysporum was grown on PDA at 25°C for 4 days. For spore production, 25 plugs of 4 mm diameter with new growing mycelia were removed from the PDA plates and transferred to 500 ml Erlenmeyer containing 200 ml of Czapek Dox Broth (Oxoid, Basingstoke, United Kingdom) and placed in a rotary shaker (110 rpm) at room temperature. After 4 days of incubation, the liquid culture was filtered using a sterile miracloth filter, and the spore concentration was quantified using a Bürker-Türk counting chamber. The resulting spore suspension was centrifuged at 9,500 rpm for 15 min and after discarding the supernatant, the pellet containing the spores was resuspended in sterile tap water to a final concentration of 1 × 10⁸ spores/ml.
Botrytis cinerea was cultured on PDA at 20°C. Spores were collected from sporulating 14 days old plates in potato dextrose broth (PDB, Difco, Le Pont de Claix, France), and the concentration of the spore suspension was quantified using a Bürker-Türk counting chamber and adjusted to 1 × 10⁶ spores/ml.

Mycelial plugs from actively growing cultures of strain AN4 were inoculated in 1 L Erlenmayer flasks containing 500 mL of Czapek-Dox broth (Oxoid, Hampshire, UK). The liquid cultures (1 L) were incubated in the dark at 25 ± 2 °C on stationary phase. After 14 days, the fungal biomass was removed through filtration (Whatman No. 4 filter paper), and the culture filtrate was extracted at a native pH (4.2) with ethyl acetate. The organic extracts were combined, dried with Na₂SO₄, and evaporated under reduced pressure at 37 °C to give a yellow oily residue (29.4 mg). The crude organic extract was submitted to fractionation on a silica gel column (1.0 × 40 cm ID), eluted with CHCl₃/iso-PrOH (95:5). The last fraction was eluted with MeOH. Six homogeneous fractions were collected and pooled on the basis of similar TLC profiles (A 1.6, B 5.2, C 1.5, D 1.6, E 7.2, and F 11.9 mg). The residue of fraction B after purification by TLC on silica gel eluted with n-hexane/ethyl acetate (6:4, v/v), produced the yellow solid identified as 1 (Figure 1, 3.5 mg, R_f 0.85). The residue from fraction E was further purified by preparative TLC on silica gel eluted with CHCl₃/i-PrOH (92:8, v/v), producing mycophenolic acid (8, Figure 1, 5.8 mg, R_f 0.48).

Millet seeds were used for spawn stock used for all the experiments. Three kilograms of millet for each isolate were weighed and washed with tap water and soaked in water plus 10 g L⁻¹ of Czapek dox broth (Oxoid) for 24 h at 4 °C. Furthermore, 500 g of millet seeds was packed in autoclavable bags and sterilized in an autoclave at 121 °C for 90 min. Bags containing the sterile substrate were cooled and inoculated aseptically by transferring 10 plugs, 1 cm in diameter, of 10-day-old fungal cultures and then incubating them in a chamber at 22 °C until the millet was completely colonized by mycelia (approximately 20 days). These preparations were stored at 4° C and used for all the experiments performed.