The use of mycorrhized seedlings has been critical in the success of modern truffle cultivation, which nowadays supplies most black truffles 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 with black truffle, with two nursery assays and a full factorial design. Three inoculation methods were sequentially applied: radicle inoculation, inoculation of the substrate in seedling trays and inoculation of the substrate in the final pot. The sequential application appeared as an effective and realistic alternative for commercial inoculation of holm oak seedlings with black truffle. The increase in the amount of inoculum applied with each inoculation method improved the mycorrhizal colonization of seedlings, although separately none of the inoculation methods appeared clearly superior to the other ones. The depth distribution of mycorrhizae levels pointed to the inoculation in the final pot substrate as being 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. However, the difference of results between both assays suggests that cultivation conditions and/or the timing of the nursery operations may influence the relative effectiveness of these inoculation methods.

The success of truffle cultivation is especially dependent on the quality of truffle-mycorrhized seedlings, which are typically produced in nurseries under aseptic conditions to avoid root colonization by undesired ectomycorrhizal fungi. However, such practices may also eliminate beneficial microorganisms that could support truffle symbiosis and improve seedling quality. In this study, twelve endophytic bacterial and fungal strains, isolated from the Tuber melanosporum environment (gleba tissue, mycorrhizae and truffle brûlé), were tested for their effect on T. melanosporum mycorrhization levels in inoculated Quercus ilex seedlings under nursery conditions. Co-inoculation with a strain of Agrobacterium tumefaciens significantly enhanced root colonization by T. melanosporum, supporting its potential role as mycorrhizal helper bacterium. In contrast, a strain of Trichoderma harzianum negatively affected mycorrhization. The remaining strains did not show significant effects on seedling mycorrhization or seedling growth. Our findings support the hypothesis that specific bacterial strains associated with truffles can act as mycorrhizal helper bacteria, highlighting the potential for co-inoculation strategies to enhance quality of truffle-inoculated seedlings in nurseries. However, further research is needed to gain a deeper understanding of the interactions within the mycorrhizosphere that could contribute to improving nursery seedling quality.