Nephelostar galaxy

Germination of P. chrysosporium RP78 conidia was measured using a nephelometric reader (NEPHELOstar Galaxy, BMG Labtech, Offenburg, Germany). For inocula, suspensions of spores were obtained from 8 day-old mycelia grown on sporulation medium (Glucose (10 g.L^-1), malt extract (10 g.L^-1), peptone from potato (2 g.L^-1); yeast extract (2 g.L^-1), asparagine (1 g.L^-1), KH₂PO₄ (2 g.L^-1), MgSO₄.7H₂O (1 g.L^-1), thiamine HCl (1 mg.L^-1), Agar (30 g.L^-1), all chemicals used were purchased from Sigma Aldrich. Spores were collected with gentle scraping of the agar plates and filtration through Miracloth. The number of spores per 1 mL of suspension was determined with optical density (OD) measurement at 650 nm, and calculated as previously described [24 (link)]. For each microplate well, 200 μl of sample were prepared: 10,000 spores were resuspended in 198 μL of malt 1% and 2 μL of rapamycin (LC laboratories, R-5000) were added for treatment or 2 μl of DMSO as mock for control. Equipment was set up with the following parameters: temperature of incubation was 37°C, cycle time 1 hour and the sum of cycles was 72 hours. Relative nephelometric unit (NRU) values were calculated as previously described [25 (link)].

A. brassicicola and A. alternata were grown at 25 ℃ in the dark respectively on potato dextrose agar medium (Becton Dickinson, Franklin Lakes, NJ, USA) and on potato carrot agar medium (HiMedia Laboratories, Einhausen, Germany). For inoculum preparation, conidia were collected from 7–8 d-old solid cultures by adding sterile milli-Q water and then gently scraping the agar plates. They were then counted in a Thoma’s chamber (Glaswarenfabrik Karl Hecht, Sondheim vor der Rhön, Allemagne, 0,05 × 0,05 mm, depth of chamber 0.100 mm) and the conidial suspensions were diluted in sterile milli-Q water at the indicated concentrations. To a 96-well plate, 300 µl of one of the following solutions were added in triplicates: solution a) potato dextrose broth and inoculum solution (90:10 v:v); solution b) potato dextrose broth, inoculum solution, exudate (80:10:10 v:v) and solution c) potato dextrose broth and exudate (90:10 v/v). Fungal growth was recorded using a nephelometric reader at 635 nm (NEPHELOstar Galaxy, BMG Labtech, Offenburg, Germany) according to Joubert et al. [30 (link)] with the following modifications: run time 66 h with 20 min stepwise measurements at 20 ℃.