The cultivation of the ectomycorrhizal fungus Tuber melanosporum has considerably spread in recent years throughout the world. During the first years of truffle cultivation, weed control is a key practice to improve the establishment of host trees and the proliferation of the fungus in the soil. Glyphosate is nowadays the most commonly used herbicide in Spanish truffle orchards. We explored the effect of glyphosate on the proliferation of T. melanosporum mycorrhizae, on extraradical mycelium, and on the inoculum potential of T. melanosporum spores in greenhouse experiments. No detrimental effect on the secondary infection of T. melanosporum was found after three sequential glyphosate applications in young seedlings during one vegetative period. Instead, a change in the distribution of fine roots and T. melanosporum mycorrhizae along soil depth was observed. On the other hand, results indicate that high application rates of glyphosate hinder the infectivity of T. melanosporum spore inoculum, without apparent impact on the host performance. Our results suggest that glyphosate has the potential to jeopardise the role of the soil spore bank as inoculum source for the colonisation of new roots, also raising the question of whether glyphosate could hinder the presumed role of spores in sexual mating.

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.

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