Freeze-Drying Strategies for the Conservation of Phytopathogenic Fungi (Diplodia mutila, Diplodia seriata, Lasiodiplodia theobromae and Neofusicoccum arbuti) Isolated from Apple Trees with Canker and Dieback Symptoms

Four Botryosphaeria spp. isolated from apple with dieback symptoms: Diplodia mutila, Diplodia seriata, Neofusicoccum arbuti and Lasiodiplodia theobromae, were subjected to differ-ent conditions to induce sporulation, then freeze-dried and stored in glass vials and am-poules at a temperature of 4 °C using two protective media. Viability was assessed after storage periods of 1, 90, 180 and 365 days. Low-nutrient growth conditions on 2% water agar supplemented with pine needles, incubated under UV light (λ = 350 nm) and at 24 °C, effectively stimulated sporulation of all four Botryosphaeriaceae species. The survival rate of the isolates was similar. Overall viability showed slight but significant differences de-pending on the type of protective medium and storage container used for the freeze-dried cultures (p< 0.001). Among the tested media, the highest viability was maintained in vac-uum-sealed glass ampoules using either a medium containing 20% skimmed milk pow-der with 5% inositol or 20% skimmed milk powder alone.