*4.2. In Vitro Growth and Sporulation of Fusarium oxysporum f. sp. radicis-lycopersici in the Presence of Biosolid Leachates*

Anaerobically digested and dehydrated sludge [12] was subjected to the one-step static leaching method EN 12457-2 as described in Giannakis et al. [107] and was sterilized by filtration through 0.2 μm pore antimicrobial filters (Whatman PuradiskTM, Buckinghamshire, UK) before incorporation into PDA at a temperature of 40 ◦C, just before pouring plates. Growth substrates of biosolid leachates and PDA mixtures were prepared at concentrations of 0% (control), 2%, 5%, and 10% *v*/*v* in Petri dishes. Forl discs, 4 mm in diameter, taken from the periphery of a fresh colony on PDA, were used to inoculate the different mixtures of PDA-biosolid leachates and cultures were incubated for 6 days. After incubation, fungal growth was expressed as colony diameter in centimeters, as described by Bardas et al. [108]. More specifically, two vertical lines were drawn at the bottom of each plate with an intersection point at the center of the inoculation point. The diameter of the colony was measured on each line and an average value of 10 measurements derived from 5 replications (Petri dishes) was calculated.

Sporulation of the fungus in the PDA-biosolid leachates media of different concentrations was assessed as follows: 10 pieces of 0.5 cm2, from the periphery of each colony, were added to 20 mL sterile distilled water in screw-capped sterile tubes. Conidia were detached from mycelium by vortexing each tube for 1 min and were harvested by filtering through sterile Miracloth (Calbiochem, San Diego, CA, USA) followed by a 103-fold dilution with sterile distilled water under sterile conditions. Then, 100 μL of the diluted mixtures were placed on PDA plates and incubated for 2 days at 20–25 ◦C. The number of single spore colonies obtained was counted and the initial number of conidia per cm2 of colony was calculated for the different media. The fungal growth and sporulation assays were repeated 5 times.
