Sequential application of inoculation methods improves mycorrhization of Quercus ilex seedlings by Tuber melanosporum

The use of mycorrhized seedlings has been critical to the success of modern truffle cultivation, which nowadays supplies most European black truffles (Tuber melanosporum) to markets. Ascospore inoculation has been traditionally used to produce these seedlings, but little scientific information is publicly available on the inoculation methods applied or on the possibility of combining them. We evaluated the potential of sequential inoculation for the controlled colonization of holm oak fine roots by T. melanosporum, with two different nursery assays and a full factorial design. Three inoculation methods were sequentially applied: radicle inoculation, inoculation of the substrate in seedling trays and inoc-ulation of the substrate in the final pot. Despite the differences in the results of the two assays, which suggest that cultivation conditions and/or the timing of nursery operations may influence the relative effectiveness of inoculation methods, the sequential application appeared as an effective and realistic alternative for commercial inoculation of holm oak seedlings with T. melanosporum. The increase in the amount of inoculum applied with each inoculation method improved the mycorrhizal colonization of seedlings, whereas separately none of the inoculation methods appeared clearly superior to the other ones. The depth distribution of truffle mycorrhizae pointed that the inoculation in the final pot was more effective than other methods in lower parts of the root system, whereas the early inoculation appeared more effective to reduce the occurrence of the opportunist ectomycorrhizal fungus Sphaerosporella brunnea. (c) 2023 British Mycological Society. Published by Elsevier Ltd. All rights reserved.