Methods to Stimulate Sporulation and Freeze-Drying Strategies for the Conservation of 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 different conditions to induce sporulation, then freeze-dried and stored in glass vials and ampoules at a temperature of 4 ° C using two protective media (skimmed milk powder in water at 20% and a mixture of skimmed milk powder at 20% plus 5% inositol). 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 depending 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 vacuum-sealed glass ampoules using either a medium containing 20% skimmed milk powder with 5% inositol or 20% skimmed milk powder alone.