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 <i>Botryosphaeria</i> spp. isolated from apple with dieback symptoms, <i>Diplodia mutila</i>, <i>Diplodia seriata</i>, <i>Neofusicoccum arbuti,</i> and <i>Lasiodiplodia theobromae</i>, 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 <i>Botryosphaeriaceae</i> 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 (<i>p</i> < 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.